Prevalence of organ impairment in Long COVID patients 6 and 12 months after initial symptoms

Authors:  Pooja Toshniwal PahariaMar 24 2022Reviewed by Danielle Ellis, B.Sc.

In a recent study posted to the medRxiv* preprint server, researchers assessed the prevalence of organ impairment in long coronavirus disease 2019 (COVID-19) six months and a year post-COVID-19 at London and Oxford.

Multi-organ impairment associated with long COVID-19 is a significant health burden. Standardized multi-organ evaluation is deficient, especially in non-hospitalized patients. Although the symptoms of long COVID-19, also known as post-acute sequelae of COVID-19 (PASC), are well-established, the natural history is poorly classified by symptoms, organ impairment, and function.

About the study

In the present prospective study, researchers assessed organ impairment in long COVID-19 patients six months and a year after the onset of early symptoms and correlated them to their clinical presentation.

The participants were recruited based on specialist referral or the response to advertisements in sites such as Mayo Clinic Healthcare, Perspectum, and Oxford from April 2020 to August 2021, based on their COVID-19 history.

The study was conducted on COVID-19 patients who recovered from the acute phase of the infection. Their health status, symptoms, and organ impairment were assessed. The symptoms assessed comprised cardiopulmonary, severe dyspnoea, and cognitive dysfunction. Biochemical and physiological parameters were analyzed at baseline and post-organ impairment. The radiological investigation comprised multi-organ magnetic resonance imaging (MRI) performed in the long COVID-19 patients and healthy controls.

Over a year, the team prospectively investigated the symptoms, organ impairment, and function, especially dyspnea, cognitive dysfunction, and health-related quality of life (HRQoL). They also evaluated the association between organ impairment and clinical symptoms.

Patients with symptoms of active pulmonary infections (body temperature >37.8°C or ≥3 coughing episodes in a day) and hospital discharges in the previous week or >4 months were excluded from the study. Asymptomatic patients and those with MRI contraindications such as defibrillators, pacemakers, devices with metal implants, and claustrophobia were removed.

Participants with impaired organs, as diagnosed by blood investigations, incidental findings, or MRI, were included in the follow-up assessments. Every visit comprised blood investigations, MRI scanning, and online questionnaire surveys, which were to be filled out beforehand. In addition, a sensitivity analysis was performed that excluded patients at risk of metabolic disorders (including body mass index (BMI) ≥30 kg/m2, diabetes, and hypertension)

Results

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Out of 536 participants, the majority were middle-aged (mean age 45 years), female (73%), White (89%), and healthcare workers (32%). About 13% of the COVID-19 patients hospitalized during the acute phase of the infection completed the baseline evaluation. A total of 331 patients (62%) had incidental findings, organ impairment, or reduction in the symptoms from the baseline at both the time points.

Cognitive dysfunction (50% and 38%), poor HRQoL (EuroQOL <0.7 in 55% and 45%), and severe dyspnea (36% and 30%) were observed at six months and one year, respectively. On follow-up, the symptoms were reduced, especially cardiopulmonary and systemic symptoms, whereas fatigue, dyspnea, and cognitive dysfunction were consistently present. The greatest impact on quality of life was related to pain and difficulties performing routine activities. Almost every patient took time off work due to COVID-19. The symptoms were largely associated with obese women, young age, and impairment of a single organ.

At baseline, fibrous inflammation was observed in the pancreas (9%), heart (9%), liver (11%), and kidney (15%). Additionally, increased volumes of the spleen (8%), kidney (9%), and liver (7%) were observed. Moreover, reduced lung capacity (2%), excess adipose deposits in pancreatic tissues (15%) and liver (25%) were observed. High liver fibro-inflammation was associated with cognitive dysfunction at follow-up in 19% and 12% of patients with and without cognitive dysfunction, respectively. Low liver fat was more likely in those without severe dyspnoea at both time points. Increased liver volumes at follow-up were associated with lower HRQoL scores.

The prevalence of multi and single-organ impairment was 23% and 59% at baseline, respectively, and persisted in 27% and 59% of the participants on follow-up assessments. Most of the organ impairments were mild. However, they did not improve substantially between visits. Notably, participants without organ impairment had the lowest symptom burden.

Most biochemical parameters were normal except creatinine kinase (8% and 13%), lactate dehydrogenase (16% and 22%), mean cell hemoglobin concentration (21% and 15%), and cholesterol (46% and 48%), at six months and a year post-COVID-19, respectively. These biochemical markers increased from the baseline on follow-up assessments.

Conclusion

To summarize, organ impairment was detected in 59% of the patients at six months post-COVID-19 and persisted in 59% at one-year follow-up. This has significant implications on the quality of life, symptoms, and long-term health of the patients. These observations highlight the requirement for enhanced preventive measures and integrated patient care to decrease the long COVID-19 burden.

Journal reference:

Pancreatic damage in COVID‐19: Why? How?

Authors: Ferhat Bacaksız, 1 Berat Ebik, 1 Nazım Ekin, 1 and Jihat Kılıc 2

Int J Clin Pract. 2021 Aug 6 : e14692.doi: 10.1111/ijcp.14692 [Epub ahead of print] PMCID:  PMC8420122PMID: 34331821

Abstract

Object

We aimed to evaluate the elevation of amylase and lipase enzymes in coronavirus disease 2019 (COVID‐19) patients and their relationship with the severity of COVID‐19.

Method

In this study, 1378 patients with COVID‐19 infection were included. Relation of elevated amylase and lipase levels and comorbidities with the severity of COVID‐19 was analysed. The effects of haemodynamic parameters and organ failure on pancreatic enzymes and their relations with prognosis were statistically analysed.

Results

The 1378 patients comprised of 700 (51.8%) men and 678 (%49.2) women. Of all patients, 687 (49.9%) had mild and 691 (50.1%) patients had severe COVID‐19 infection. Amylase elevation at different levels occurred in 316 (%23) out of 1378 patients. In these patients, the amylase levels increased one to three times in 261 and three times in 55 patients. Pancreatitis was detected in only six (%1.89) of these patients according to the Atlanta criteria. According to univariate and multivariate analyses, elevated amylase levels were significantly associated with the severity of COVID‐19 (odds ratio [OR]: 4.37; P < .001). Moreover, diabetes mellitus (DM; OR: 1.82; P = .001), kidney failure (OR: 5.18; P < .001), liver damage (OR: 6.63; P < .001), hypotension (OR: 6.86; P < .001) and sepsis (OR: 6.20; P = .008) were found to be associated with mortality from COVID‐19.

Conclusion

Elevated pancreatic enzyme levels in COVID‐19 infections are related to the severity of COVID‐19 infection and haemodynamic instability. In a similar way to other organs, the pancreas can be affected by severe COVID‐19 infection.

What’s known

  • It has been suggested that COVID‐19 can cause pancreatic damage.
  • There are a limited number of studies related to the possibility of an increase in the level of pancreatic enzymes in COVID‐19 patients.

What’s new

  • COVID‐19 does not directly cause pancreatic damage.
  • Pancreatic enzyme elevation in patients with COVID‐19 develops in the advanced stages of the disease caused by multiple organ dysfunction and shock.

1. INTRODUCTION

Coronavirus disease 2019 (COVID‐19) infection was initially considered to attack only the upper respiratory tract, but was later found to potentially affect almost all systems. This is caused by the angiotensin‐converting enzyme 2 (ACE2) receptors that coronavirus binds to in order to enter the cells. These receptors are also commonly available in the gastrointestinal system such as in hepatic, pancreatic and colonic cells.12

Recent studies have shown that COVID‐19 infection can cause damage to the pancreas caused by the high expression of ACE2 receptors from the pancreatic tissue.3 Additionally, it has also been reported that hyperglycaemia can occur because of pancreatic islet cell damage in patients with COVID‐19 and that severe patients with COVID‐19 should be followed up closely in terms of pancreatic damage.45

In this study, we evaluated the amylase and lipase elevations in patients with COVID‐19 in order to investigate the relationship between pancreatic enzyme elevations and the severity of COVID‐19 infection and to identify the underlying conditions.Go to:

2. PATIENTS AND METHODS

The study included 1378 patients with COVID‐19 infection who presented to our hospital between March and December 2020. Clinical characteristics including temperature, blood pressure, laboratory parameters, treatments and comorbidities were monitored throughout hospitalisation. In addition to other laboratory parameters, amylase and lipase levels were also studied in order to determine the ratio of patients with elevated pancreatic enzymes. Values above 105 U/L for amylase and 65 IU/L for lipase were considered high.6 Patients with pancreatitis were identified according to the Atlanta criteria.7

Additionally, pancreatic enzyme elevation in COVID‐19 infection was investigated with regard to the severity of disease. Patients were divided into two groups based on the severity of their COVID‐19 symptoms: mild (n = 687) and severe (n = 691). Patients with fever, headache, loss of taste and smell and generalised myalgia without tachypnoea (oxygen saturation >92%) were considered to have a mild infection, whereas patients on invasive or non‐invasive respiratory support or with deteriorated haemodynamic conditions were considered to have severe COVID‐19 infection.8

The causes of pancreatic enzyme elevation were compared between patients with mild and severe COVID‐19 infection and between surviving and non‐surviving patients. Relation between elevated pancreatic enzymes and metabolic parameters, haemodynamic findings, single and multiple organ failures was also examined.910

Hypotension was evaluated based on mean arterial pressure (MAP). A MAP value of 60‐110 mmHg was accepted as normal, <60 mmHg as hypotensive and >110 mmHg as hypertensive.11

Liver damage was determined according to the 2019 European Association for the Study of the Liver (EASL) guidelines, based on the upper limits of normal (ULN) serum alanine aminotransferase activity (ALT) and serum alkaline phosphatase activity (ALP), as follows: ALT ≥5 × ULN or ALP ≥2 ULN [in the absence of known bone pathology] or ALT ≥3 ULN with simultaneous increase of total bilirubin concentration ≥2 ULN.12 Kidney injury was determined according to the RIFLE (Risk, Injury, Failure, Loss of kidney function and End‐stage kidney disease) criteria.13

The study was conducted in accordance with the Helsinki Declaration and the study protocol was approved by the local ethics committee (No: 611, Date: 16 October 2020).

2.1. Statistical analysis

Data were analysed using SPSS 26.0 for Windows (Armonk, NY: IBM Corp.). Normal distribution of data was assessed using Kolmogorov‐Smirnov, Shapiro‐Wilk test, coefficient of variation, skewness and kurtosis. Continuous variables were expressed as mean and standard deviation (SD), and categorical variables were expressed as percentages (%). Student t test and Mann‐Whitney U‐test were used in paired groups to compare pancreatic enzymes and disorders of other organs between patients with severe and mild COVID‐19 infection. ANOVA test was used for parameters homogeneously distributed in triple groups. Bonferroni correction was used to determine the significant results in groups. Welch’s ANOVA and Kruskal‐Wallis tests were performed for non‐homogeneous parameters. Pearson and Spearman correlation coefficients were used to analyse the relationship between pancreatic enzyme elevation and other parameters. Univariate and multivariate analyses were performed to determine the factors associated with pancreatic enzyme elevation. All tests were bilateral and a P‐value of <.05 was considered significant.Go to:

3. RESULTS

The 1378 patients comprised of 700 (51.8%) men and 678 (%49.2) women. The prevalence of kidney failure, DM, ischaemic hepatitis and sepsis was significantly higher in patients with severe COVID‐19 compared with patients with mild disease. Moreover, amylase and lipase levels were also higher in patients with severe COVID‐19 (Table 1).

TABLE 1

Demographic data and biochemical parameters of patients with mild and severe COVID‐19

Mild COVID‐19±SDSevere COVID‐19±SDP
N687 (49.9%)691 (50.1%)
Age60.2 (29‐84)65 (51‐86)<.001
Gender F/M356/331322/369.053
Amylase (U/L)82.6 ± 50.4264.7 ± 292.0<.001
Lipase (IU/L)59.7 ± 51.279.0 ± 24.2.045
ALT (IU/L)70.4 ± 60.282.7 ± 56.4<.001
AST (IU/L)61.6 ± 40.7180 ± 135.5<.001
ALP (IU/L)84.1 ± 35.4133.2 ± 107.2.295
GGT (IU/L)54.2 ± 51.579.4 ± 58.9.099
T.Bil (mg/dL)0.67 ± 0.331.95 ± 1.53<.001
LDH (IU/L)411.9 ± 2101137 ± 248.7<.001
Urea (mg/dL)45.7 ± 26.9187.0 ± 92.8<.001
Creatinine (mg/dL)0.89 ± 0.493.74 ± 1.96<.001
Glucose (mg/dL)138 ± 85.0290 ± 135<.001
WBC (cell/µL)9840 ± 448518 422 ± 6039<.001
Lymphocyte (cell/µL)1993 ± 6651655 ± 946<.001
CRP (mg/L)107.8 ± 67.2217.9 ± 69.1<.001
Procalcitonin (ng/mL)1.04 ± 4.658.03 ± 19.7<.001

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Abbreviations: ALP, alkaline phosphatase, GGT, gamma glutamyl transpeptidase WBC, white blood cell, CRP, C reactive protein; ALT, alanine transaminase; AST, aspartate transaminase; LDH, lactate dehydrogenase; SD, standard deviation.

Amylase elevation at different levels occurred in 316 (%23) out of 1378 patients. In these patients, the amylase levels increased one to three times in 261 and three times in 55 patients. Pancreatitis was detected in only six (%1.89) of these patients according to the Atlanta criteria. Amylase and lipase elevation was found to be related to the severity of COVID‐19 infection in the remaining patients. The development of DM, kidney failure, hypotension and ischaemic hepatitis was found to be related to mortality from COVID‐19 infection. However, there was no relationship between lymphopenia and elevated amylase levels (Table 2). On the other hand, patients older than 65 years were more likely to have (1.89 times) elevated increased enzyme levels.

TABLE 2

Relationship between amylase level in COVID‐19 patients and gender, comorbid status, severity and consequence of COVID‐19, haemodynamic status, other organ failures and laboratory parameters

FeatureAmylase (normal)Amylase (1‐3 times)Amylase (more than 3 times)P‐values
N/%1062 (77.0%)261 (19.0%)55 (4.0%)
Gender
Female (678%‐49.2%)565 (83.3%)100 (14.8%)13 (1.9%)<.001
Male (700%‐50.8%)497 (71.0%)161 (23.0%)42 (6.0%)
COVID‐19 severity
Mild COVID‐19 (687%‐49.9%)612 (89.1%)71 (10.3%)4 (0.6%)<.001
Severe COVID‐19 (691%‐50.1%)450 (65.1%)190 (27.5%)51 (7.4%)
COVID‐19
Healing (909%‐66.0%)793 (87.2%)109 (12.0%)7 (0.8%)<.001
Death (469%‐34.0%)269 (57.4%)152 (32.4%)48 (10.2%)
Diabetes
Absent (866%‐62.8%)703 (81.2%)143 (16.5%)20 (2.3%)<.001
Available (512%‐32.6%)359 (70.1%)118 (23.0%)35 (6.9%)
Kidney failure
Absent (934%‐67.8%)808 (86.5%)114 (12.2%)12 (1.3%)<.001
AKI (316%‐22.9%)186 (58.8%)101 (32.0%)29 (9.2%)
CRF (128%‐9.3%)68 (53.1%)46 (36.0%)14 (10.9%)
Blood pressure
Normal (810%‐58.8%)727 (89.7%)76 (9.4%)7 (0.9%)<.001
Hypotension (466%‐33.8%)260 (55.8%)161 (34.5%)45 (9.7%)
Hypertension (102%‐7.4%)75 (73.6%)24 (23.5%)3 (2.9%)
ALT
Normal (562%‐40.8%)488 (86.8%)65 (11.6%)9 (1.6%)<.001
1‐3 times (488%‐35.4%)389 (79.7%)86 (17.6%)13 (2.7%)
3‐5 times (135%‐9.8%)89 (65.9%)37 (27.4%)9 (6.7%)
5‐10 times (65%‐4.7%)36 (55.4%)20 (30.8%)9 (13.8%)
>10 times (61%‐4.4%)32 (52.5%)26 (42.6%)3 (4.9%)
>1000 (IU/L) (67%‐4.9%)28 (41.8%)27 (40.3%)12 (17.9%)
AST
Normal (468%‐34.0%)428 (91.4%)36 (7.7%)4 (0.9%)<.001
1‐3 times (564%‐40.9%)454 (80.5%)98 (17.4%)12 (2.1%)
3‐5 times (121%‐8.8%)76 (62.8%)38 (31.4%)7 (5.8%)
5‐10 times (71%‐5.1%)35 (49.3%)27 (38.0%)9 (12.7%)
More than 10 times (45%‐3.3%)22 (48.9%)16 (35.6%)7 (15.5%)
>1000 (IU/L) (109%‐7.9%)47 (43.1%)46 (42.2%)16 (14.7%)
ALP
Normal (966%‐70.1%)737 (76.3%)191 (19.8%)38 (3.9%).092
1‐2 times (234%‐17.0%)176 (75.2%)45 (19.2%)13 (5.6%)
More than 2 times(178%‐12.9%)149 (83.7%)25 (14.0%)4 (2.3%)
GGT
Normal (909%‐66%)722 (79.4%)158 (7.4%)29 (3.2%).072
1‐2 times (254%‐18.4%)173 (68.1%)62 (24.4%)19 (7.5%)
More than 2 times (215%‐15.6%)167 (77.7%)41 (19.1%)7 (3.2%)
Total Bilirubin
Normal (1059%‐76.9%)860 (81.2%)171 (16.2%)28 (2.6%)<.001
1‐2 times (257%‐18.6%)172 (66.9%)68 (26.4%)17 (6.7%)
More than 2 times (62%‐4.5%)30 (48.4%)22 (35.5%)10 (16.1%)
LDH
Normal (<225 IU/L) (161%‐11.7%)157 (97.5%)4 (2.5%)0 (0.0%)<.001
Normal‐1000 (IU/L) (969%‐70.3%)788 (81.3%)158 (16.3%)23 (2.4%)
1000‐2250 (IU/L) (148%‐10.7%)79 (53.4%)55 (37.2%)14 (9.4%)
>2250 (IU/L)(100%‐7.3%)38 (38.0%)44 (44.0%)18 (18.0%)
Lymphocyte levels
Normal (724%‐52.5%)581 (80.3%)119 (16.4%)24 (3.3%).120
Mild lymphopenia (489%‐35.5%)360 (73.6%)111 (22.7%)18 (3.7%)
Severe lymphopenia (165%‐12.0%)121 (73.3%)31 (18.8%)13 (7.9%)

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Abbreviations: AKI, Acute kidney injury; ALP, alkaline phosphatase, GGT, gamma glutamyl transpeptidase; ALT, alanine transaminase; AST, aspartate transaminase; CRF, Chronic renal failure; LDH, lactate dehydrogenase.

The prevalence of elevated amylase was 2.04 times higher in men than that in women. Hypotension (odds ratio [OR]: 6.63), sepsis (OR: 6.20), ischaemia‐related liver damage (OR: 6.63) and renal failure (OR: 5.18) were found to be significantly associated with pancreatic enzyme levels (Table 3).

TABLE 3

Analysis of factors affecting enzyme elevation in COVID‐19 patients with elevated amylase and lipase

FeatureUnivariateMultivariate
OR95% ClP valueOR95% ClP value
Age1.891.46‐2.44.0011.721.40‐2.11.001
Gender2.041.57‐2.64.0011.861.50‐2.31.001
COVID‐19 Severity4.373.28‐5.81<.0013.762.90‐4.88<.001
Death from COVID‐195.083.89‐6.64<.0014.233.33‐5.36<.001
Diabetes1.821.41‐2.35.0011.721.40‐2.11.001
Kidney failure5.183.95‐6.79<.0013.783.00‐4.75<.001
Liver damage6.634.56‐9.64<.0013.092.43‐3.94<.001
Hypotension6.864.50‐10.40<.0015.673.90‐8.22<.001
Sepsis6.203.83‐10.05.0082.882.24‐3.70.003
Pancreatitis21.22.54‐166.7.0058.024.54‐86.3.026

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A very strong positive correlation was found between amylase and lipase levels in all patients (r: .828, P < .001), which implicates that the increased amylase in COVID‐19 patients is caused by the pancreas. A weak correlation was found between amylase level and age or gender. Likewise, a weak but statistically significant correlation was found between amylase level and DM. A strong correlation was detected between the amylase level and the severity of COVID‐19. Additionally, the presence of liver damage, renal failure, hypotension and multiple organ dysfunction syndrome (MODS) in these patients was moderately correlated with amylase level (Figure 1).FIGURE 1

Correlation between amylase level and risk factors in COVID‐19 patients with hyperamylasaemiaGo to:

4. DISCUSSION

We found that 23% of patients with COVID‐19 infection had pancreatic enzyme elevations, and we also detected a relationship between pancreatic enzyme elevation and the severity of COVID‐19 infection, haemodynamic instability and MODS.

Although 10.9% of patients with mild COVID‐19 infection had elevated amylase levels, this rate was 34.9% in patients with severe COVID‐19 infection. It was also revealed that the causes of pancreatic enzyme elevation were hypotension and ischaemia in patients with severe COVID‐19 infection. Elevated amylase levels were detected in 10.3% and 44.2% of patients with a normal MAP and low MAP (<60 mmHg), respectively. Out of 316 patients with a high amylase level, 36.7% of the patients recovered and 63.3% of them died. Moreover, 53% of patients with ischaemic hepatitis had both amylase and lipase elevations. We consider that after the development of shock in the body, pancreatic damage occurs in addition to hepatic and intestinal injury as a result of the decrease in blood flow to the gastrointestinal system.

A study investigating the relationship between COVID‐19 infection and pancreas reported pancreatic damage in 1%‐2% and 17% of patients with mild and severe infection, respectively. The authors suggested that pancreatic damage can be exacerbated by systemic inflammation.14151617 Amylase and lipase elevation suggestive of pancreatic damage has been reported in 8.5%‐17.3% of patients with COVID‐19. Moreover, higher enzyme levels have been reported in severe COVID‐19 patients.14151617 Likewise, in two previous autopsy studies, five of 11 (45.5%) and two of eight (25%) cases were detected with focal pancreatitis with haemorrhagic and necrotic changes in the pancreas. These changes had no clinical manifestations and were attributed to ischaemia and end‐organ damage.1819 In the light of our data, we consider that pancreatic damage is the most important cause of amylase and lipase elevations. The exact pathophysiology of pancreatic damage remains unclear, while the most widely accepted hypothesis points to pancreatic ischaemia.202122 If septicaemia progresses towards septic shock, not only in COVID‐19 but also in other infections, the resulting hypotension and vasodilation reduce blood flow to organs. To protect blood flow to vital organs such as the brain and heart, blood flow to the celiac, superior and inferior mesenteric arteries are reduced as a part of the protective mechanism. Afterwards, this is followed by renal and iliac arteries. This is the neurohormonal mechanism protecting vital organs. Gastrointestinal system is the target organ of shock and hypotension. As a result, the blood flow to the liver, pancreas and the entire gastrointestinal system is reduced, thereby causing symptoms such as nausea, vomiting, distension, ileus, or diseases such as ischaemic hepatitis.

Pancreas is supplied well by pancreatic arteries that stem from the splenic, gastroduodenal and superior mesenteric arteries. Amylase, lipase, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) are released into the bloodstream caused by the ischaemia resulting from decreased blood flow to the pancreas.23 This damage is mainly caused by haemodynamic deterioration, not by the virus itself. Similarly, in our study, elevated amylase and lipase levels were found to be associated with haemodynamic parameters and hypotension.

Although increased amylase and lipase levels might have clinical importance, it seems highly unlikely to use these parameters as prognostic indicators in clinical practice, mainly because enzyme elevation occurs during the intensive care period when the disease is severe and requires mechanical ventilation. At this stage, most patients have single or multiple organ failure and require vasopressor support.

In conclusion, although ACE2 receptors are expressed highly in pancreatic tissue, pancreatic enzyme elevations occurring in COVID‐19 infection might be associated with the severity of disease and haemodynamic instability. If the opposite was the case, we would have seen too many cases of pancreatitis, mainly because the pancreas has ACE2 receptors. As a matter of fact, despite the huge number of COVID‐19 cases, which has exceeded 100 million, pancreatitis has remained only at the level of case reports.2425Go to:

Notes

Bacaksız F, Ebik B, Ekin N, Kılıc J. Pancreatic damage in COVID‐19: Why? How? Int J Clin Pract. 2021;00:e14692. 10.1111/ijcp.14692 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

DATA AVAILABILITY STATEMENT

Data may be made available upon request to the corresponding author.

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Consequences of COVID-19 for the Pancreas

Authors: Urszula Abramczyk,1,*Maciej Nowaczyński,2Adam Słomczyński,2Piotr Wojnicz,2Piotr Zatyka,2 and Aleksandra Kuzan1 Int J Mol Sci. 2022 Jan; 23(2): 864.Published online 2022 Jan 13. doi: 10.3390/ijms23020864

Abstract

Although coronavirus disease 2019 (COVID-19)-related major health consequences involve the lungs, a growing body of evidence indicates that COVID-19 is not inert to the pancreas either. This review presents a summary of the molecular mechanisms involved in the development of pancreatic dysfunction during the course of COVID-19, the comparison of the effects of non-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on pancreatic function, and a summary of how drugs used in COVID-19 treatment may affect this organ. It appears that diabetes is not only a condition that predisposes a patient to suffer from more severe COVID-19, but it may also develop as a consequence of infection with this virus. Some SARS-CoV-2 inpatients experience acute pancreatitis due to direct infection of the tissue with the virus or due to systemic multiple organ dysfunction syndrome (MODS) accompanied by elevated levels of amylase and lipase. There are also reports that reveal a relationship between the development and treatment of pancreatic cancer and SARS-CoV-2 infection. It has been postulated that evaluation of pancreatic function should be increased in post-COVID-19 patients, both adults and children.

1. Effects of Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome-Related Coronavirus (MERS-CoV) on the Pancreas

Coronaviruses are enveloped, single- and positive-stranded RNA viruses that infect birds and mammals. In humans, coronaviruses cause respiratory tract infection, usually the common cold, but they can also cause severe respiratory illness including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), caused by severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), respectively [1]. Coronaviruses tend to cause epidemics and even pandemics. The first coronavirus pandemic was the SARS outbreak in 2002–2003 [2]. With the experience gained during the SARS pandemic, it was possible to more quickly identify subsequent outbreaks of the MERS epidemic in 2012 [3]. The pathomechanism of both viruses is very similar—they even both use transmembrane protease serine 2 (TMPRSS2), except SARS-CoV uses angiotensin-converting enzyme 2 (ACE2) as its receptor, whereas MERS uses dipeptidyl peptidase-4 (DPP4) [4,5]. Moreover, there is a difference in terms of the severity and frequency of symptoms, which was observed in MERS patients as more frequent hospitalization in the intensive care unit (ICU) compared to SARS patients [2] (Table 1). Diabetes was one of the significant and independent predictors for developing severe SARS-CoV and MERS-CoV [6,7,8]. In MERS, no viral antigen was detected in any tissue other than pneumocytes [7], despite multiple organ dysfunction syndrome in critically ill patients. In SARS-CoV, the presence of the virus was detected not only in respiratory epithelial cells, but also in small intestinal and colonic epithelial cells, in which it also revealed replication features [9]. It is known that the ACE2 receptor is also present in tissues such as the heart, kidney, and pancreas [8,9]. According to some authors, the presence of the receptor is sufficient for tissue entry and pathogenic activity, although other researchers do not support this thesis [9,10]. Yang et al. were some of the first researchers who hypothesized that SARS coronavirus enters islets using ACE2 as its receptor and damages islets causing acute diabetes [8]. Yang’s study revealed that SARS-CoV had a much higher affinity for pancreatic islet cells than for pancreatic exocrine cells, which was consistent with the hyperglycemia observed in some patients and rarely reported acute pancreatitis (AP) [8]. Furthermore, insulin-dependent diabetes mellitus (IDDM) and high fasting blood glucose values were observed in some inpatients [8]. A 3-year follow-up revealed that both abnormalities were transient, which may be indicative of only temporary damage to the pancreatic islets [8]. However, another reason (different from that given by Young et al.) for high fasting blood glucose value in patients may result from increased stress hormones release. Cortisol, catecholamines, growth hormone, and glucagon, which are released during infection, fever, and trauma, can lead to hyperglycemia to the same degree as SARS-CoV can [11]. No information was found in the literature about a direct impact of the MERS virus on the pancreas or on glycemia during or after infection. This may be due to an insufficiently detailed analysis of the available data during previous studies that oscillated primarily, for laboratory tests, between complete blood count (CBC), lactate dehydrogenase (LDH), urea, and creatinine analysis. A summary of SARS-CoV, MERS, and SARS-CoV-2 is shown in Table 1.Table 1. The summary of characteristics of SARS and MERS coronaviruses. Dipeptidyl peptidase-4 (DPP4), transmembrane protease serine 2 (TMPRSS2), hospitalization in the intensive care unit (ICU), and cathepsin L (CTSL).

Table

In 2019, a new coronavirus named SARS-CoV-2 was identified, causing COVID-19. This virus has many characteristics that are analogous to SARS-CoV, for example, ACE2 is also used as its receptor [12]. Patients with diabetes are among those with the most severe forms of COVID-19 and related mortality; insights from recent experience can guide future management [17], particularly for the consequences on the pancreas. As the COVID-19 pandemic has been ongoing for nearly two years, this study aims to collect data concerning the impact of SARS-CoV-2 on the pancreas and analyze them to estimate the future health consequences of COVID-19 in populations.

2. Pancreatic Damage during Diabetes Mellitus and COVID-19

Pancreas tissue damage may cause to the lack of control over normal blood glucose levels in the body. Type 1 diabetes (T1D) is caused by insulin deficiency due to βcell dysfunction of immunologic or idiopathic cause. In contrast, β pancreatic cells in type 2 diabetes (T2D) become depleted over time due to compensatory insulin secretion caused by insulin resistance. There is also type 3 diabetes (T3D), which is described as diabetes associated with the development of Alzheimer’s disease [18]. It should not be confused with type 3c (pancreatogenic) diabetes, which relates to the exocrine and digestive functions of the pancreas. The issue concerning the impairing effect of hyperglycemia (glucotoxicity) on the secretory function of the islets of Langerhans has also been increasingly raised. In addition to endocrine dysfunction, some diabetic patients may also develop moderate exocrine pancreatic insufficiency (EPI), in which pancreatic enzyme secretion is impaired. EPI can be observed in almost all patients with type 3c (pancreatogenic) diabetes (secondary to pancreatic pathology), whereas the prevalence of this dysfunction in patients with T1D or T2D is 40% and 27%, respectively [19].With the ongoing SARS-CoV-2 pandemic, patients with reduced normal pancreatic function are at high risk for COVID-19 requiring hospitalization. In particular, elevated blood glucose levels in patient with and without diabetes makes them at high risk of mortality [20]. Hyperglycemia impairs the immune response (e.g., by reducing the activity of macrophages and polymorphonuclear leukocytes), which in addition influences the excessive cytokine response, and thus has a strong proinflammatory effect.The receptors for ACE2, which are also present in the pancreas, are a target of SARS-CoV-2 in the body, which may result in acute failure of both the islets of Langerhans and exocrine cells [15]. Infection-induced, transient β cell dysfunction may cause an uncontrolled hyperglycemic state, especially in patients whose pancreas is already affected by diabetes mellitus. Persistent hyperglycemia usually predisposes to severe COVID-19 and to viral infection complicated by secondary infections. The aforementioned risk can be found in T1D, T2D, and gestational diabetes mellitus (GDM). In T2D patients, the much more frequent coexistence of other risk factors such as atherosclerosis, hypertension, and obesity should be taken into consideration, which usually implies a worse prognosis for the course of COVID-19 [21,22]. In GDM, SARS-CoV-2 infection not only increases the risk of more severe course of the disease in a patient, but may also result in diabetic fetopathy or, in more advanced pregnancies, increase the risk of future pathologies involving glucose metabolism (such as T2D) in a child [23].

3. Pancreatic Damage in Patients without Pre-Existing Diabetes Infected with SARS-CoV-2

It has been postulated that, either by direct invasion of pancreatic cells by the virus or by indirect mechanisms described below, SARS-CoV-2 has a destructive effect on the pancreas and can lead to insulin deficiency and development of T1D [24].If the hypothesis that SARS-CoV-2 infection causes hyperglycemia is true, increased statistics of new T1D cases should be observed. Indeed, there are publications that describe such a phenomenon. For instance, Unsworth et al. and Kamrath et al. describe an increase in new-onset T1D in children during the COVID-19 pandemic [16,25]. Although pancreatic β cell damage induced transient hyperglycemia in SARS-CoV, it is still unclear whether β cell damage is transient or permanent in SARS-CoV-2 [22]. This information appears to be of great importance because COVID-19 in children is frequently considered “harmless”. Therefore, it is reasonable to sensitize parents to the fact that the consequences of COVID-19 may be potentially dangerous for their children.Below you will find the proposed molecular mechanisms that may participate in pancreatic damage that causes carbohydrate metabolism disorders.

4. Etiology Associated with ACE2, TMPRSS2, and Na+/H+ Exchanger

As previously mentioned, SARS-CoV infection of host cells is facilitated by ACE2, but also by the transmembrane protease serine 2 (TMPRSS2) and other host cell proteases such as cathepsin L (CTSL) [13].ACE2 is an enzyme that is expressed to varying degrees in most cells of the human body [14,26,27]. This enzyme catalyzes the conversion of angiotensin II to angiotensin 1–7, taking part in the maintenance of body homeostasis by influencing the regulation of blood pressure and water–electrolyte balance through the renin–angiotensin–aldosterone (RAA) system [28]. Moreover, ACE2/angiotensin (1–7) stimulates insulin secretion, reduces insulin resistance, and increases pancreatic βcell survival [27,28].In addition to the key role it plays in maintaining body homeostasis, ACE2 is now also the best-studied target for SARS-CoV-2 S glycoprotein, enabling infection of host cells [27,29]. ACE2 in the pancreas is expressed mainly within the pericytes of pancreatic microvessels and to a lesser extent on the surface of the islets of Langerhans, including pancreatic β cells [30]. SARS-CoV-2 shows 10–20 times more activity against ACE2 than SARS-CoV, which significantly increases the infectivity of SARS-CoV-2 [31,32]. Furthermore, studies indicate that SARS-CoV may also downregulate ACE2 expression in cells. This causes an imbalance between ACE and ACE2, consequently leading to blood pressure disorders and systemic inflammation [27,33,34]. Due to the 79% genetic similarity between SARS-CoV and SARS-CoV-2 [35], it is speculated that ACE2 expression may also be downregulated during SARS-CoV-2 infection, causing i.a. MODS observed in COVID-19 [27].During cell infection by SARS-CoV-2, in addition to the role played by ACE2, it is also appropriate to consider the significant pathogenic role of TMPRSS2 that is necessary for the preparation of S glycoprotein by its cleavage, thereby enabling fusion of the virus with the host cell [36,37]. The S1 and S2 domains can be distinguished in the SARS-CoV-2 S glycoprotein. The S1 domain is involved in binding to the ACE2 receptor and then TMPRSS2 intersects with the S protein, including at the boundary of the S1 and S2 domains and within the S2 domain, which enables the virus–cell fusion [38,39]. According to studies, TMPRSS2 expression is significantly increased in obese patients, which may contribute to the poorer prognosis that is observed during COVID-19 in this patient group [40]. Moreover, obese patients are frequently already burdened with problems such as insulin resistance at baseline, while the presence of ACE2 and TMPRSS2 within the pancreas as a binding site for SARS-CoV-2 may exacerbate insulin resistance causing problems in terms of diabetes management in COVID-19 patients.There are also other mechanisms by which COVID-19 may affect the development of hyperglycemia. It is reported that the virus may also affect the glucose regulation through the Na+/H+ exchanger and lactate pathways. The mechanism is that angiotensin II, which accumulates during infection, contributes to insulin resistance and—by activating the Na+/H+ exchanger in the pancreas—it leads to hypoxia and extracellular acidification, which, through the accumulation of calcium and sodium ions in the cells and the production of reactive oxygen species, damages pancreatic tissue [41]. Simultaneously, the concentration of lactate increases, which in COVID-19 infection is intensively released, among other things, from adipose tissue, and then monocarboxylate transporters transport lactate and H+ ion inward in the cell, which increases Na+/H+ exchanger activation, further disrupting pancreatic homeostasis [41].

5. The Etiology Associated with a Systemic Proinflammatory Environment, Immune System Aggression, and Production of Novel Autoantigens

A broad spectrum of proinflammatory cytokines, such as IL-2, IL-6, IL-7, IL-8, interferon-γ, and Tumor Necrosis Factor α (TNF-α), is released during, in particular severe, COVID-19 infection [42,43,44]. Based on current studies, it is reasonable to suspect that these cytokines are released in response to the binding of the virus to ACE2 receptors that are also located in the pancreas [9,42]. The cause of pancreatic damage during COVID-19 is the cytokine storm that plays a key role in this case, because in both acute pancreatitis (AP) and severe COVID-19, elevated levels of the aforementioned interleukins are associated with the severity of these both disease entities. Particular attention should be paid to IL-6, because it is suspected to play a key role in the pathogenesis of AP as well as acute respiratory distress syndrome (ARDS) that is the most common and most severe clinical manifestation of COVID-19. In COVID-19-induced ARDS, IL-6 levels are correlated with disease-related mortality [45,46,47]. At the same time, high IL-6 levels correlate with an increased risk of developing severe pancreatitis [48,49].The production of neutralizing antibodies is also an important response of the body in the course of COVID-19 [50,51,52]. It has been observed that early seroconversion and very high antibody titers occur in patients with severe SARS-CoV-2 infection [53,54]. The available literature details a mechanism called antibody-dependent enhancement (ADE), which is associated with a pathological response of the immune system [53]. ADE exploits the existence of FcRS receptors located on various cells of the immune system, for example, macrophages and B lymphocytes [53]. This relationship may lead to a likely bypass of the classical viral infection pathway by ACE2, and virus–antibody complexes may stimulate macrophages to overproduce cytokines including significant IL-6 [53,55].Molecular mimicry may be also one of potential causes of pancreatic cell damage [56]. There are similarities in the protein structure of the virus and β-pancreatic cells, which may induce cross-reactivity and lead to autoimmunity [56]. Furthermore, viral infection may also lead to increased cytokine secretion by surrounding dendritic cells and activation of naive T cells in genetically predisposed individuals [56].

6. Pancreatitis in COVID-19

Although the impact of the discussed coronavirus-induced disease on exocrine function is not fully understood, available literature is not able to unambiguously determine whether the tissue damage leading to AP occurs as a result of direct SARS-CoV-2 infection [57] or as a result of systemic MODS with increased levels of amylase and lipase [42]. Liu et al.’s study involving 121 COVID-19 patients with a mean age of 57 years and a variable course of infection proved above-normal levels of amylase and lipase in 1–2% of patients with moderate COVID-19 infection and in 17% of patients with severe COVID-19 infection. This may support the hypothesis that SARS-CoV-2-induced disease has a destructive effect not only on the endocrine portion of this gland, but also on the exocrine one [15].However, elevated levels of pancreatic enzymes in question do not have to mean the destruction of pancreatic cells—after all, such a situation may occur during kidney failure or diarrhea in the course of COVID-19. Furthermore, there remains the question of the effect of drugs administered during SARS-CoV-2 infection on changes in pancreatic function [42], discussed further in this article.According to the International Association of Pancreatology (IAP) and the American Pancreatic Association (APA), the diagnosis of AP is based on meeting two out of three of the following criteria: clinical (epigastric pain), laboratory (serum amylase or lipase > 3 × upper limit of normal), and/or imaging criteria (computed tomography, magnetic resonance imaging, ultrasound) [58]. Pancreatic lipase is considered as a potential marker of SARS-CoV-2 severity with concomitant AP. In Hemant Goyal et al.’s study, as many as 11.7% out of 756 COVID-19 patients had hyperlipidemia and they were three times more likely to have severe COVID-19 [59]. Those with higher lipase levels—17% out of 83 patients—required hospitalization [60]. However, it is difficult to distinguish whether these patients required hospitalization for severe systemic COVID-19 infection or for pancreatitis in the course of COVID-19 infection.AP in the course of COVID-19 was analyzed in different age groups; however, some studies only involve children [61]. Compared to pancreatic islet cells, cells of the exocrine pancreatic ducts are more abundant in ACE2 and TMPRSS2 that are necessary for the virus to penetrate the cell [62]. Infection of these cells may be one of the causes of AP [63]. Infections, both bacterial and viral, are one of the causes of AP. The definitive mechanism of how viral infections affect pancreatic cells is not known; however, a study by Maria K Smatti et al. found that there is infection of pancreatic islet cells and replication of the virus within them, ultimately resulting in autoimmune reactions that eventually affect both diabetes and AP in a negative way [64]. For non-SARS-CoV-2 patients, the etiology of AP is known and confirmed in most cases, although 69% of those undergoing infection do not have definite etiology of AP while meeting the AP-Atlanta criteria for diagnosis [65].Hegyi et al. show the mechanism of MODS formation during COVID-19 infection and AP [66]. This is lipotoxicity, involving an interstitial increase in pancreatic lipase levels, which leads to the breakdown of triacylglycerols contained in adipose tissue cells and the release of unsaturated fatty acids. These in turn exert a toxic effect on mitochondria causing the release of cytokines, which results in a cytokine storm.There is also a hypothesis, which claims that AP can develop because of blood circulatory centralization resulting from uncontrolled cytokine storm created by SARS-CoV-2 infection [67]. There exist reports that say that pancreatic ischemia may be the cause of different degrees of acute pancreatitis [68,69]. This statement can be supported by the reports that state that pancreatic blood reperfusion inhibits the development of AP and accelerate pancreas recovery [70].Another mechanism of developing AP during COVID-19 may be a coagulation cascade activation caused by active inflammatory process due to SARS-CoV-2 infection [71]. The ongoing inflammatory process causes not only hemostasis imbalance for blood clotting, but it also leads to intensification of coagulation by removing epithelial cell protein C receptor (EPCR) from epithelial by the means of inflammatory mediators and thrombin [71]. This means that both processes intensify each other. Simultaneously, it was proved that COVID-19 predisposes patients to venous thromboembolism resulting from excessive inflammation, platelet activation, and endothelial dysfunction [72]. It is also important to notice that AP is inherently connected with a coagulation cascade activation, increased fibrinolysis and, hence, higher level of D-dimers [73]. Acute pancreatitis severity may depend on hemostasis imbalance; local coagulation results in mild AP whereas, in more severe AP cases, the imbalance may lead to development of disseminated intravascular coagulation (DIC) [74]. These observations have been supported by the results of experimental studies showing that the inhibition of coagulation reduces the development of AP [75,76,77] and exhibits therapeutic effect in this disease [78,79]. Additionally it is worth noticing that infection-related hyperglycemia has powerful inflammation-promoting effects on the organism (especially when organism is under stress), thus increasing the number of inflammatory mediators [74]. Unfortunately, it is impossible to decide which process is dominant in causing AP in COVID-19 patients: local inflammation caused by SARS-CoV-2 or systemic hemostasis imbalance.Clinical reports on low molecular weight heparin (LMWH) treatment in AP seem to emphasize a more significant role of hemostasis imbalance in causing AP [74,80,81]. Heparin is extremely significant in the treatment of COVID19 patients due to its properties, mainly its similarity to heparan sulphate, which appears in a respiratory tract, its interactions with SARS-CoV-2 S protein, leading to viral adhesion inhibiting to the cell membrane [82], and its anti-inflammatory effects. Thanks to these properties, heparin may not only show its therapeutic effect as the anticoagulant, but also its protective role in acute pancreatitis or respiratory inflammations [83,84,85].

7. Drugs Used against SARS-CoV-2 Infection (Glucocorticoids, Lopinavir, Ritonavir, Remedesivir, Interferon-β1 (IFN-β1), and Azithromycin) Induce Pancreatic β Cell Damage

Statistical analyses revealed a significantly higher incidence of AP with the concomitant systemic use of glucocorticosteroids (GCS) [86]. In one study analyzing the development of drug-induced AP, dexamethasone, was classified as type IB—there was one case report in which administration of this drug-induced AP occurred; however, other causes of pancreatitis such as alcohol consumption could not be excluded [87]. Other GCS such as hydrocortisone, prednisone, and prednisolone were used in patients with mild to moderate AP; however, they cannot be classified into any group because they are frequently used together with other drugs that cause AP [86,87]. However, it has been determined that GCS independently increase the risk of AP, and patients with residual AP risk factors during GCS treatment should be more monitored for the development of AP [23]. Javier A. Cienfuegos et al. additionally observed that one of mechanisms of AP formation in COVID-19 patients may be GCS administered at the time of admission to the ICU with severe respiratory failure [88]. Because GCS were used in severe COVID-19 cases, it is difficult to say what true reason for AP was—either a severe course of COVID-19 or GCS application or both.GCS are used in the treatment of many diseases due to their immunosuppressive and anti-inflammatory nature. They induce diabetes in previously healthy patients as well as significantly exacerbate diabetes in diabetic patients [89,90]. Diabetes develops in these patients likely due to pancreatic β cell dysfunction, decreased insulin secretion, and increased insulin resistance in other tissues, which may depend on the timing and the dose of GCS used [89,91]. Long-acting or intermediate-acting insulin alone or combined with short-acting insulin should be used during the treatment [90]. At the same time, no advantage was found over the use of oral hypoglycemics [92]. Certainly, patients after long-term GCS therapy will need further observation for diabetes.Lopinavir/ritonavir was classified in the previously mentioned study as a type IV drug—medications reported with little information [87]. Both drugs are included in the group of antiretrovirals that act as protease inhibitors, and they are primarily used for HIV infection. Although Lopinavir is an active drug, it is not used alone. There have been reports about the occurrence of AP during the use of protease inhibitors in question, which is also described in the Summary of Product Characteristics (SmPC) of products approved by Committee for Medicinal Products for Human Use (CHMP). It has been proved that the use of lopinavir/ritonavir causes hyperglycemia [93,94].Remdesivir is an adenosine analogue with antiviral activity. There are single reports about the occurrence of pancreatitis as a result of the use of the aforementioned medication [95,96]. At the same time, it should be noted that other nucleoside-derivative drugs may cause pancreatitis [97].The current state of knowledge does not clearly indicate the therapeutic benefit of interferon-β in the treatment of COVID-19 patients [98,99]. To date, only single cases suggesting induction of pancreatitis by interferon-β have been reported. Based on this, Badalov et al. classified interferon into type III [87].There are few reports about the development of AP due to the use of azithromycin [100]. In the previously mentioned study by Badalov et al., two macrolide antibiotics were classified as type II and III. Unfortunately, there are no direct data concerning azithromycin. Interestingly, there were cases of patients with concomitant symptoms of AP and viral pneumonia caused by SARS-CoV-2 who were treated with azithromycin, which resulted in complete resolution of symptoms for both conditions [96,101]. Based on available data, the risk of azithromycin-induced AP is low.There is no clear evidence that azithromycin affects blood glucose levels in humans. However, it is known for its prokinetic effects, which may be helpful in patients who suffer from diabetic gastroparesis [102]). The incidence of hypo- and hyperglycemic episodes was not proved to be significant for azithromycin [103]; however, the risk of dysglycemia is emphasized [94]. In the SmPC, where azithromycin is the main ingredient, it is not possible to establish a causal relationship between the occurrence of pancreatitis and taking medications (Zithromax) based on the available data. In contrast, glycemic disturbances were not indicated as side effects (Zithromax) [104].Hydroxychloroquine has been extensively promoted for COVID-19 due to its anti-inflammatory and antiviral action; yet, the use of this agent in diabetes deserves particular attention for its documented hypoglycemic action, and its benefit on COVID-19 is controversial, although there is large usage [105].Table 2 shows a comparison of the side effects of medications in question.Table 2. Side effects of medications used in SARS-CoV-2 infection in the area of pancreatic effects and hyperglycemia.

Table

8. COVID-19, Pancreas, and Glycation

In T2D diabetics, oxidative stress leading to pancreatic damage may be stimulated by, among other things, the intense glycation that accompanies hyperglycemia [24]. Glycation is a non-enzymatic process involving reducing sugar and amino groups of proteins, which contributes to the formation of advanced glycation end products (AGEs). These products have significantly altered biochemical properties relative to the substrates, including proteins that have altered conformation, increased rigidity, resistance to proteolysis, etc. [106,107].Part of the pathomechanism involved in facilitating coronavirus infection in diabetics may be due to glycation of ACE2 and SARS-CoV-2 spike protein [108,109].An interesting hypothesis is that COVID-19 has a worse prognosis in patients with intense glycation, and thus high tissue AGE content. Glycated hemoglobin (HbA1c) is a commonly used diagnostic tool that estimates intensity of glycation. The parameter is not only a marker of long-term persistent hyperglycemia, but an active participant in immune processes, as HbA1c levels are associated with NK cell activity [110].Zhang et al.’s retrospective cohort study concerning COVID-19 patients revealed that glycated hemoglobin correlates negatively with saturation (SaO2) and positively with C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and fibrinogen (Fbg). It was concluded that determination of HbA1c levels may be helpful in assessing inflammation, hypercoagulability, and prognosis of COVID-19 patients [111].According to the meta-analysis by Chen et al. (2020), Hba1c levels were slightly higher in patients with severe COVID-19 compared to patients with mild COVID-19; however, this correlation was not statistically significant. However, it is of great importance to note that only two studies analyzing HbA1c in COVID-19 patients were included in this analysis because only these studies were available in May 2020 [112].Glycation plays its physiological effects not only directly by changing the properties of various proteins, but also indirectly through various receptors. RAGE is the most common receptor for AGEs. Binding of RAGE to its ligands activates a proinflammatory response primarily by mitogen-activated protein kinase (MAPK) and nuclear factor κβ (NFκβ) pathways. This interaction was proved to be significant in the pathogenesis of cancer, diabetes mellitus, and other inflammatory disorders [113]. RAGE was found to be expressed in the pancreas, and S100P-derived RAGE antagonistic peptide (RAP) reduces pancreatic tumor growth and metastasis [113]. The implications of this fact may also apply to the etiology and treatment of COVID-19. It has been postulated that targeting RAGE by various antagonists of this receptor may inhibit damage to various organs including the pancreas [114].

9. COVID-19 vs. Pancreatic Cancer

Immunosuppression as a treatment effect, elevated cytokine levels, altered expression of receptors for SARS-CoV-2, and a prothrombotic state in patients with various types of cancer may exacerbate the effects of COVID-19 [115].Focusing on pancreatic cancer, it can be observed that the pathomechanism of both diseases—COVID-19 and tumorigenesis in the pancreas—overlap in several molecular mechanisms. As mentioned above, SARS-CoV-2 infection of host cells is facilitated by ACE-2, TMPRSS2, and CTSL. Cathepsin L is upregulated in a wide variety of cancers, including pancreatic adenocarcinoma [13]. TMPRSS2 upregulation in pancreatic cancers is moderate, whereas ACE-2 is overexpressed in some cancers, including pancreatic carcinomas [115]. Interestingly, ACE2 upregulation seems to be associated with favorable survival in pancreatic cancer [116], and it is known that SARS-CoV-2 reduces ACE2 expression [22]. Furthermore, the above-mentioned RAGE may also participate in both pancreatic cancer development and SARS-CoV-2 infection. RAGE facilitates neutrophil extracellular trap (NET) formation in pancreatic cancer [117]. In conclusion, pancreatic cancer predisposes to an increased risk of COVID-19 and its more severe course, and coronavirus infection may contribute to pancreatic cancer.It also seems important how the COVID-19 epidemic has affected the treatment of patients with pancreatic cancer of SARS-CoV-2-independent etiology. According to the study by Pergolini et al., care of patients with pancreatic cancer can be disrupted or delayed, particularly in the context of treatment selection, postoperative course, and outpatient care [118].A separate issue is how patients after pancreatoduodenectomy respond to SARS-CoV-2 infection. A case series reported by Bacalbasa reveal that patients who develop SARS-CoV-2 infection postoperatively require re-admission in the ICU and a longer hospital stay; however, these infections are not fatal [119]. Although the analysis was performed on single cases, it is concluded that these results are an argument to perform elective oncological surgeries [119].There are also reports that chemotherapy in pancreatic cancer patients who become ill between treatment series can be successfully completed after a complete cure of the infection [120]. Guidelines for, e.g., prioritization and treatment regimens regarding pancreatic cancer treatment in the era of the pandemic, are developed and described, for example, by Catanese et al. or Jones et al. [121,122].

10. Conclusions

Evidence shows that SARS-CoV-2 infection contributes to damage within the pancreas. The mechanisms that are involved in this include but are not limited to direct cytopathic effect of SARS-CoV-2 replication and systemic and local inflammatory response [123]. At the current state of knowledge, it is certain that the virus attacks the endocrine portion of the pancreas as well as, to a much lesser extent, the exocrine portion. It has been shown that a bidirectional relationship between COVID-19 and diabetes exists; indeed, diabetes is associated with COVID-19 severity and mortality but, at the same time, patients with COVID-19 have shown new onset of diabetes [124]. SARS-CoV-2 virus infection not only directly affects glycemic levels, but also exacerbates already existing hyperglycemia through its negative impact on the functional competence of the islets of Langerhans. It cannot be excluded that the real cause of exocrine dysfunction of this gland is the negative effect of the drugs used for treatment of the infection. As the pandemic progresses, special attention should be given to the evaluation of chronic and acute pancreatic diseases, including pancreatic cancer, so that faster diagnosis enables faster implementation of treatment.

Author Contributions

Conceptualization, A.K.; investigation, U.A., M.N., A.S., P.W., P.Z. and A.K.; resources, U.A., M.N., A.S., P.W., P.Z. and A.K.; writing—original draft preparation, U.A., M.N., A.S., P.W., P.Z. and A.K.; visualization, U.A.; supervision, A.K. All authors have read and agreed to the published version of the manuscript.

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SARS-CoV-2 infects human pancreatic β cells and elicits β cell impairment

aUTHORS: Chien-Ting Wu,1,13Peter V. Lidsky,2,13Yinghong Xiao,2,13Ivan T. Lee,3,4,5,13Ran Cheng,1,6,13Tsuguhisa Nakayama,5,7,13Sizun Jiang,3,13Janos Demeter,1Romina J. Bevacqua,8Charles A. Chang,8,9,10,11Robert L. Whitener,8Anna K. Stalder,12Bokai Zhu,3Han Chen,3

Cell Metab. 2021 Aug 3; 33(8): 1565–1576.e5.Published online 2021 May18. doi: 10.1016/j.cmet.2021.05.013 :  PMC8130512PMID: 34081912

Abstract

Emerging evidence points toward an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While preexisting diabetes is associated with severe COVID-19, it is unclear whether COVID-19 severity is a cause or consequence of diabetes. To mechanistically link COVID-19 to diabetes, we tested whether insulin-producing pancreatic β cells can be infected by SARS-CoV-2 and cause β cell depletion. We found that the SARS-CoV-2 receptor, ACE2, and related entry factors (TMPRSS2, NRP1, and TRFC) are expressed in β cells, with selectively high expression of NRP1. We discovered that SARS-CoV-2 infects human pancreatic β cells in patients who succumbed to COVID-19 and selectively infects human islet β cells in vitro. We demonstrated that SARS-CoV-2 infection attenuates pancreatic insulin levels and secretion and induces β cell apoptosis, each rescued by NRP1 inhibition. Phosphoproteomic pathway analysis of infected islets indicates apoptotic β cell signaling, similar to that observed in type 1 diabetes (T1D). In summary, our study shows SARS-CoV-2 can directly induce β cell killing.Keywords: SARS-CoV-2, COVID-19, ACE2, type 1 diabetes, neuropilin 1, phosphoproteomics, apoptosis, SARS-CoV-2 spike protein, insulin, pancreatic beta cell

Introduction

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic infection caused by the positive-sense RNA virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Zhu et al., 2020b). Although initial studies focused on lung injury and cardiovascular manifestations (Yang et al., 2020Zheng et al., 2020), other organ dysfunctions have been observed, notably in the kidney, pancreas, intestine, and olfactory epithelia (Fang et al., 2020Giacomelli et al., 2020Lamers et al., 2020Puelles et al., 2020). With regard to diabetes, several recent clinical studies suggested a significant increase in new-onset hyperglycemia, diabetic ketoacidosis (DKA), and diabetes in patients with COVID-19 (Chee et al., 2020Ebekozien et al., 2020Hollstein et al., 2020Naguib et al., 2021Rubino et al., 2020Singh and Singh, 2020Unsworth et al., 2020), although some studies question the statistical significance of the effect (Boddu et al., 2020). Conceptually, β cell damage could be a consequence of direct virally induced cell death or T cell autoreactivity. Therefore, the clinical association between COVID-19 and diabetes raises the first question of whether SARS-CoV-2 can infect pancreatic islet endocrine cells, particularly insulin-secreting β cells, and cause cell death or dysfunction to initiate diabetes. The binding of SARS-CoV-2 to the host cell membrane is mediated primarily by the interaction between the viral spike glycoprotein (S) and its main entry host receptor, angiotensin-converting enzyme 2 (ACE2) (Hoffmann et al., 2020). Accordingly, many recent studies have focused on analyzing the expression levels of ACE2 in pancreatic endocrine cells. Recent RNA and protein expression studies suggested low ACE2 expression levels in α, β, and δ cells in the pancreas, leading some to postulate that SARS-CoV-2 is unable to infect β cells (Arda et al., 2016Baron et al., 2016Blodgett et al., 2015Coate et al., 2020Kusmartseva et al., 2020Segerstolpe et al., 2016). However, these characterizations are incomplete, and a more direct evaluation of cellular SARS-CoV-2 tropism is needed. Here, we suggest that the clinical severity of diabetes in patients with COVID-19 may be notably influenced by showing direct viral infection of endocrine cells, particularly β cells.Go to:

Results

Pancreatic β cells selectively express SARS-CoV-2 entry factor proteins

Recently published studies have been discordant in terms of whether the SARS-CoV-2 receptor, ACE2, is present within insulin-secreting β cells of the pancreas (Coate et al., 2020Kusmartseva et al., 2020). While technical differences can explain the discrepancies, several of the studies found low levels of ACE2 mRNA expression in pancreatic islets, leading to speculation that SARS-CoV-2 is unable to infect β cells. However, SARS-CoV-2 entry is thought to be not only mediated by ACE2 but also by transmembrane serine protease 2 (TMPRSS2), neuropilin 1 (NRP1) (Cantuti-Castelvetri et al., 2020Daly et al., 2020), and transferrin receptor (TFRC) (Tang et al., 2020). We first evaluated the mRNA expression level of ACE2TMPRSS2NRP1TFRC, and FURIN in three previously published single-cell RNA sequencing (RNA-seq) datasets (Arda et al., 2016Blodgett et al., 2015Kim et al., 2020) in order to assess their expression within the two major pancreatic islet cell populations: insulin-secreting β cells and glucagon-secreting α cells (Figures S1A–S1C). We observed that ACE2 and TMPRSS2 transcripts, while expressed at low levels, are nonetheless readily measurable within both β cells and α cells. Additionally, the transcripts of other SARS-CoV-2 entry factors, NRP1TFRC, and FURIN, are expressed abundantly in pancreatic islets. We next investigated the protein expression of these SARS-CoV-2 entry factors by co-staining ACE2 (Lee et al., 2020), TMPRSS2 (Suárez-Fariñas et al., 2021), NRP1 (Cantuti-Castelvetri et al., 2020Daly et al., 2020), and TFRC (Haberger et al., 2020) in combination with insulin (INS), a β cell marker, or glucagon (GLU), an α cell marker, in pancreatic autopsy samples from 5 non-COVID-19 donors negative for COVID-19 (by PCR test). The characteristics of these donors are summarized in Table 1 . Consistent with recent mRNA work, ACE2 and TMPRSS2 were generally expressed within β cells and α cells but at low protein levels (Figures 1 A and S1D) (Coate et al., 2020Kusmartseva et al., 2020). Strikingly, we found robust NRP1 and TFRC protein expression within β cells, but not α cells, suggesting a potential mechanism for SARS-CoV-2 tropism for β cells (Figures 1A and S1D). For orthogonal confirmation of this result, we utilized a different anti-NRP1 antibody to confirm the NRP1 expression in the pancreas and arrived at the same conclusion (Figure S2A). Pre-incubation of the anti-NRP1 and anti-TFRC antibodies with the immunizing peptides drastically reduced staining in the pancreas, further validating the specificity of the result (Figures S2B and S2C). To further explore this, we compared the differential protein expression of SARS-CoV-2 entry factors within β cells and α cells. Similar to the mRNA data, no major differences in ACE2 and TMPRSS2 protein expression were observed between β cells and α cells (Figure 1B), suggesting that the expression levels of these receptors are unlikely to be singularly responsible for a propensity for SARS-CoV-2 to infect β cells. Importantly, though, NRP1 and TFRC proteins were significantly increased in β cells as compared with α cells (Figure 1B). NRP1 and TFRC were recently found to facilitate ACE2-mediated SARS-CoV-2 entry (Cantuti-Castelvetri et al., 2020Daly et al., 2020Tang et al., 2020), whereas stable SARS-CoV-2 spike-ACE2 interactions depended on both NRP1 and TFRC. These results indicate that β cells contain the necessary molecular components for SARS-CoV-2 viral entry and that the higher expression of NRP1 and TFRC entry factors may in part explain the tropism of SARS-CoV-2 for β cells.

Table 1

Non-COVID-19 pancreatic tissue donor characteristics

DonorGenderAgeBMIDiabetesCause of deathCOVID-19
1male7826noacute cardiac arrestnegative
2male5029noadvanced cancernegative
3male8218noleft-sided cardiac failurenegative
4female8624nocombined hemorrhagic and cardiogenic shocknegative
5male8128.4noprogressing coronary insufficiencynegative

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SARS-CoV-2-associated receptors are expressed in pancreatic β cells

(A) Representative double immunofluorescence staining of ACE2, TMPRSS2, NRP1, and TFRC with the β cell marker, insulin (INS), and α cell marker, glucagon (GLU), in the normal human pancreas, donor 1. See Table 1.

(B) Quantification of ACE2, TMPRSS2, NRP1, and TFRC in β cells (INS +) and α cells (GLU +) from a normal pancreas. No statistically significant changes in ACE2 and TMPRSS2 expression were detected between β and α cells. NRP1 and TFRC expression was statistically significantly higher in β cells compared with α cells. Rabbit anti-NRP1 (Abcam, ab81321, 1:200) and mouse anti-TFRC (Thermo Fisher, # 13-6800, 1:200) were used for the experiments shown here.

Error bars represent mean ± SD (~10–15 islets from the pancreas of 5 non-COVID-19 donors; see Table 1). ∗∗p < 0.001, one-way ANOVA with Tukey’s post-test. Each dot represents one donor. Scale bars, 5 μm (A) and 2 μm (insets). See also Figures S1 and S2 and Table 1.

SARS-CoV-2 infects β cells ex vivo and requires NRP1

To test our hypothesis regarding the increased tropism of SARS-CoV-2 for pancreatic β cells, we isolated human islets from healthy donors and infected them with SARS-CoV-2 ex vivo. The characteristics of the islet donors are summarized in Table 2 . Two or 6 dpi, infected pancreatic islets were fixed and stained with antibodies against the SARS-CoV-2 nucleocapsid protein (NP) in combination with antibodies against cell-type-specific markers: insulin (β cells), glucagon (α cells), somatostatin (δ cells), or CD31 (endothelial cells). Interestingly, SARS-CoV-2 NP was primarily observed in insulin-positive β cells at both 2 and 6 dpi (Figures 2A, 2C, and 2D), indicating preferential infection of β cells by SARS-CoV-2. Similar results were obtained using an antibody raised against the SARS-CoV-2 spike protein (SP) (Figures 2B–2D). In contrast, the presence of SARS-CoV-2 was notably lower in other pancreatic cell types, namely α and δ cells, and endothelial cells (Figures 2A–2D). These results strongly support the increased susceptibility of human pancreatic β cells for SARS-CoV-2.

Table 2

Non-COVID-19 pancreatic islet donor characteristics

DonorGenderAgeBMIDiabetesCause of deathCOVID-19Islet RRID
1male2725.3noanoxianegativeSAMN15314807
2female4830.9nocerebrovascular/strokenegativeSAMN15770453
3male5224.5nocerebrovascular/strokenegativeSAMN15850322
4female6326.9nocerebrovascular/strokenegativeSAMN15942269
5male3531.3nohead traumanegativeSAMN16191825
6female4231.2nocerebrovascular/strokenegativeSAMN16427178
7female5125.2nocerebrovascular/strokenegativeSAMN16515959
8male3725nohead traumanegativeUNOS ID: AHJX486
9female5327.4nocerebrovascular/strokenegativeUNOS ID: AHK2444
10male6029.9noanoxianegativeSAMN17528599
11female4423.8nocerebrovascular/strokenegativeUNOS ID: AIA3480
12male6225.9nohead traumanegativeSAMN17928660
13male5624.2noanoxianegativeSAMN18021384

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SARS-CoV-2 preferentially infects β cells of human pancreatic islets ex vivo

(A–D) Mock-treated or SARS-CoV-2-infected human pancreatic islets were stained after 2 or 6 dpi. (A) Representative double immunofluorescence staining of SARS-CoV-2 nucleocapsid protein (NP) in combination with β cell marker, insulin (INS); ɑ cell marker, glucagon (GLU); δ cell marker, somatostatin (SST); and endothelial cell marker (CD31).

(B) Representative double immunofluorescence staining of SARS-CoV-2 spike protein (SP) in combination with a similar combination of markers as (A). The nuclei were stained using DAPI (blue) as a counterstain.

(C) Quantified percentages of SARS-CoV-2 NP and SP within α, β, δ, and endothelial cells of pancreatic islets. Around 40% to 60% NP and SP staining, respectively, are present within β cells.

(D) Quantified percentages of SARS-CoV-2 NP- and SP-positive α, β, δ, and endothelial cells.

(C and D) Error bars represent mean ± SD (~500–1,000 cells were quantified from healthy isolated human islets from donors 1–5; see Table 2).

(E) Representative double immunofluorescence staining of SARS-CoV-2 NP in combination with insulin after pre-treating islets with dimethyl sulfoxide (DMSO) or 100 μM EG00229 for 1 h before infection with SARS-CoV-2. Islets were fixed at 2 dpi and stained for SARS-CoV-2 NP and β cell marker, insulin (INS). Quantification of the percentages of β cells containing NP-positive β cells (right).

Error bars represent mean ± SD (~500–1,000 cells were quantified from healthy isolated human islets from donors 10–13; see Table 2). p < 0.05, two-tailed Student’s t test. Each dot represents one donor. Scale bars, 5 μm (A, B, and E) and 2 μm (insets). See also Table 2.

Given the selectively high expression of NRP1 in β cells, we hypothesized that inhibition of NRP1 would be sufficient to block infection, even if other co-receptors were important. It has been shown that the treatment of the small molecule EG00229, a selective NRP1 antagonist, reduced the efficiency of SARS-CoV-2 infection in vitro (Daly et al., 2020). Here, we also found that incubation of ex vivo pancreatic islets with EG00229 notably reduced the efficiency of SARS-CoV-2 infection (Figure 2E). This result supports a critical role of NRP1 protein in the increased tropism of SARS-CoV-2 for pancreatic β cells. Additional studies will be needed to further establish the relationship between levels of NRP1 and the levels of other viral receptors and the efficiency of infection.

SARS-CoV-2 infects β cells in subjects with COVID-19

Next, we determined whether SARS-CoV-2 tropism for β cells is also observed in patients with COVID-19. We obtained pancreatic autopsy samples from 9 patients who died from severe COVID-19-related complications. The characteristics of these patients are summarized in Table 3 . Histological analysis revealed lipomatosis, fibrosis, or autolysis in some of the samples, whereas acute or chronic pancreatitis was not observed in any patient (Table 1), tending to exclude that broad pancreatic damage is a universal feature. The pancreas of 7 out of 9 of these patients had SARS-CoV-2 viral positivity as measured by RT-PCR. We observed SARS-CoV-2 NP staining selective to insulin-positive β cells in 4 of 7 patients, while the remaining 3 pancreatic samples and healthy control samples were negative for NP staining (Figure 3 A). The specificity of the anti-NP antibody was validated through peptide blocking assays (Figure S2D). The 3 negative samples (staining not shown) from patients with COVID-19 had extensive autolysis/atrophy (Table 3), which may explain the lack of NP signal due to rapid proteolysis of tissue by digestive enzymes. As an orthogonal confirmation of our observations of viral presence in β cells, we performed in situ hybridization (ISH) using a validated SARS-CoV-2 spike mRNA probe in combination with an antibody targeting insulin on the four positive SARS-CoV-2-infected human pancreatic tissues (see Figures S3A and S3B for SARS-CoV-2 probe validation) (Lee et al., 2020). Similar to the NP staining results, SARS-CoV-2 spike transcripts were detected in β cells of these autopsied pancreatic islets (Figure 3B). These results confirm SARS-CoV-2 tropism for β cells, supporting a model in which SARS-CoV-2 infects and replicates in β cells to induce pancreatic dysfunction, thus leading to hyperglycemia or diabetes.

Table 3

COVID-19 patient characteristics, pancreas viral load, percentage of NP+ islets, and histological analysis

PatientAgeBMIDiabetesViral load PCR (mean)Ct values (ORF, S, N)NP-positive islets (%)Histological analysis
16735no2933/32/354well preserved, mild fibrosis
28526type II10835/35/3531mild fibrosis, lipomatosis, and autolysis
39523no2434/33/335well preserved, lipomatosis, and mild fibrosis
47124no1535/35/indet12mild lipomatosis and autolysis
58828no631/32/320extensive atrophy, lipomatosis, and moderate fibrosis
68530no1138/37/360extensive autolysis
75847no1638/35/360extensive autolysis
86629noindeterminateindeterminate0mild lipomatosis, fibrosis, autolysis
95430noindeterminateindeterminate0extensive autolysis

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Pancreatic autopsies were obtained from 9 patients who died from COVID-19-related complications. The pancreas of 7 out of 9 of these patients had SARS-CoV-2 viral positivity by RT-PCR. Ct values of RT-PCR targeting three different SARS-CoV-2 genomic regions (ORF1ab, spike [S], and nucleocapsid [N]) are shown. Ct values of the pancreas from patients 8 and 9 were between 37 and 40 and therefore considered “indeterminate” and not positive. The percentages of the nucleocapsid (NP)-positive islets by immunofluorescence staining as described in the manuscript are also shown for each patient sample. Pancreas from patients 5–7 did not have NP positivity, possibly due to extensive autolysis/atrophy. Histological analysis was performed by a board-certified pathologist (M.S.M.).

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Open in a separate windowFigure 3

SARS-CoV-2 infects pancreatic β cells of patients with COVID-19

(A) Representative double immunofluorescence staining of pancreatic islets from patients with COVID-19 and healthy controls using antibodies against SARS-CoV-2 NP and INS.

(B) Representative multiplexed images of in situ hybridization against the SARS-CoV-2 spike mRNA, in combination with immunofluorescence staining of insulin (INS). SARS-CoV-2 spike mRNA expression (red dots) was detected within pancreatic β cells. The nuclei were stained using DAPI (blue) as a counterstain.

Scale bars, 5 μm (A and B) and 2 μm (insets). See also Figure S3 and Table 3.

We next investigated whether ACE2 and NRP1 are differentially expressed in the pancreatic β cells of patients with COVID-19 compared with non-COVID-19 donors as a potential explanation for why β cells are more susceptible to viral infection. ACE2 expression remained low in individuals with COVID-19 with no statistically significant difference compared with non-COVID-19 donors (Figures S3C and S3D). Conversely, NRP1 expression is upregulated in patients with COVID-19 compared with non-COVID-19 donors (Figures S3C and S3D). These results support a potential role of NRP1 in β cell susceptibility of viral infection. However, it is also possible that this is due to the increased NRP1 expression in the organ caused by SARS-CoV-2 infection, which in turn causes the cells to be more susceptible to infection. Further research is needed to establish the generality and mechanism by which SARS-CoV-2 may require either (1) preexisting or (2) virally induced upregulation of NRP1 levels.

SARS-CoV-2 infection suppresses insulin secretion and kills β cells ex vivo

To determine whether SARS-CoV-2 infection affected pancreatic islet function, we quantified the insulin content and glucose-stimulated insulin secretion (GSIS), a functional assay for β cell insulin release, in infected islets. We observed a dramatic decrease in insulin content and GSIS in SARS-CoV-2-infected human islets, compared with mock-treated islets (Figures 4A and 4B). Notably, this effect is partially reversed upon treatment with the NRP1 antagonist EG00229 (Figure S3E). In type 1 diabetes (T1D), virus-induced β cell damage can be a result of either virus-triggered cell death or immune-mediated loss of infected pancreatic β cell mass. Previous reports of SARS-CoV-1/2-induced apoptosis in ACE2-expressing A549 and Vero E6 cells (Diemer et al., 2008Li et al., 2020Zhu et al., 2020a) suggested a similar mechanism of virus-mediated cell death in pancreatic β cells ex vivo. To this end, we performed the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay on SARS-CoV-2-infected human islets 6 dpi. TUNEL signal was significantly increased in infected β cells, compared with mock-infected β cells (Figures 4C and 4D). Since SARS-CoV-2 also infects a small number of other cells, such as α cells, a population that accounts for the second-largest number of cells in the islet, we next investigated whether SARS-CoV-2 infection can cause apoptosis of α cells (Figure S3F). The increased TUNEL signal in infected α cells suggests that viral infection-induced cell death was agnostic to cell type, although the percentage of β cells undergoing apoptosis was higher due to higher susceptibility. SARS-CoV-2 spike protein treatment was sufficient to induce apoptosis in β cells, as indicated by an increase in TUNEL signal (Figures 4E and 4F). This observation is consistent with past findings that SARS-CoV-1 SP can induce apoptosis in Vero E6 cells (Chow et al., 2005). Altogether, these results support a model in which SARS-CoV-2-induced β cell apoptosis leads to dysregulation in insulin production and secretion.

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Figure 4

SARS-CoV-2 infection interferes with insulin content/secretion and induces β cell apoptosis

(A–F) Pancreatic islet functionality was analyzed by insulin content, glucose-stimulated insulin secretion (GSIS), and TUNEL staining ex vivo.

(A) Insulin content is decreased in SARS-CoV-2-infected islets compared with mock-treated islets.

(B) GSIS is decreased in SARS-CoV-2-infected islets compared with mock-treated islets.

(A and B) Error bars represent mean ± SD (data were collected from 7 healthy isolated human islets, donors 2–8; see Table 2). p < 0.05, two-tailed Student’s t test.

(C) Representative staining of β cell apoptosis by in situ TUNEL and DAPI staining in β cells (INS) of mock- or SARS-CoV-2-treated human islets. DNase-treated sections were used as a positive control in the TUNEL assay.

(D and F) Quantification of the percentages of islets containing TUNEL-positive β cells. Error bars represent mean ± SD (~500–1,000 cells were quantified from each of 3–5 separate healthy isolated human islets, donors 1–5 [D] and 7–9 [F]; see Table 2).

(E) Representative staining of β cell apoptosis by in situ TUNEL and DAPI staining in β cells (INS) of mock-treated versus SARS-CoV-2-SP-treated human islets.

p < 0.05, ∗∗p < 0.01, two-tailed Student’s t test. Scale bars, 5 μm (C and E). See also Figures S3–S5 and Tables 2S1S2, and S3.

Given the central role of regulatory kinases in the control of apoptosis, we next asked whether the binding of SARS-CoV-2 to its receptors is sufficient to trigger apoptosis-related signaling pathways independent of additional cellular stress as a result of viral infection and replication. We first leveraged assaying global phosphoproteomics to determine potential kinase regulatory roles of the SARS-CoV-2 SP, which directly binds the viral receptors. Isolated human islets were incubated with SARS-CoV-2 SP from SARS-CoV-2 for 15 or 30 min in parallel with vehicle control treatment. Cells were then harvested, and extracts were prepared for phosphoproteomic mass spectrometry and analysis of signaling (Figure S4A). We employed a substrate-based kinase activity prediction model to determine the activity levels of specific kinases from this large-scale phosphoproteomic data. This analysis is based on the underlying assumption that the activity levels of upstream kinases can be inferred through the measured abundance of known downstream target phosphorylation events (Hernandez-Armenta et al., 2017). Using kinase set enrichment analysis (KSEA) (Drake et al., 2012Ochoa et al., 2016), we were able to assign an enrichment score (ES) value (weighted Kolmogorov-Smirnov statistic; STAR Methods) to each kinase to reflect its activity in a manner analogous to that of gene set enrichment analysis (GSEA) (Subramanian et al., 2005). Differential expression of phosphosites in the 15- and 30-min SARS-CoV-2 SP-treated human islets was calculated by comparing them to vehicle control, and KSEA was performed using a kinase-substrate database created using PhosphoSitePlus (Hornbeck et al., 2015) and NetworKin (Linding et al., 2008).

Quantification of the activity of 67 kinases (Figure S4B; Tables S1 and S2) revealed an upregulation of stress-response MAP kinases, including JNK/p38 (MAPK8/11) (Wada and Penninger, 2004) and cytoskeleton reorganizing p21-activated kinases (PAK) (Manser et al., 1994), which are two classic pathways triggering cell death by the apoptosis pathways. Additionally, multiple members of the protein kinase C (PKC) family were downregulated in response to SARS-CoV-2 SP treatment. Through pathway analysis using gene ontology (GO) biological process over-representation analysis (ORA), apoptosis emerged as a recurring top hit (Figures S4C and S5A–S5D; Table S3) as early as 30 min post-SARS-CoV-2 spike protein incubation. Apoptotic kinases, including PAK, were upregulated, as was activation of GSK3β and the proapoptotic kinase JNK1, both previously linked to apoptosis in β cells (Dhanasekaran and Reddy, 2008Guo et al., 2016Tournier et al., 2000) (Figures S5E–S5I).

To understand whether the same pathways were activated by a viral infection, we repeated the phosphoproteomics analysis on cells infected with SARS-CoV-2 (24 h post-infection) to measure the activity levels of specific kinases triggered by the virus. As with SARS-CoV-2 SP treatment, we also observed upregulation of JNK and PAK in SARS-CoV-2-infected islet cells (Figure S4B). As before, GO analysis pointed to apoptosis and programmed cell death as the most significantly enriched categories (Figure S4D). To validate that JNK and PAK are indeed activated in virus-infected islets, we co-stained phosphorylated JNK1/2 (pJNK1/2) and phosphorylated PAK1/2 (pPAK1/2) in combination with the SARS-CoV-2 SP in infected pancreatic islets. Our results confirm that pJNK1/2 and pPAK1/2 were primarily observed in SARS-CoV-2 SP-positive cells at 24 h post-infection (Figure S4E), confirming that the SARS-CoV-2 infection induces the activation of JNK and PAK. The kinase activities induced by SARS-CoV-2 SP and SARS-CoV-2 infection support a mechanism through which SARS-CoV-2 induces apoptosis via the JNK-MAPK apoptosis pathway, allowing a potential window for therapeutic intervention.Go to:

Discussion

Emerging clinical reports have noted a significant increase in new-onset hyperglycemia, DKA, and diabetes in patients with COVID-19. Understanding how SARS-CoV-2 affects the normal function of the pancreas is an urgent unmet need with fundamental healthcare implications. In this study, we discovered that SARS-CoV-2 preferentially infects β cells in isolated human pancreatic islets ex vivo and in patients who succumbed to COVID-19. Building on recent studies that identified low-level ACE2 expression in pancreatic islets (Fignani et al., 2020Kusmartseva et al., 2020), we observed that ACE2 as well as TMPRSS2 are indeed modestly expressed in β cells. Importantly, we uncover the selective expression of other critical SARS-CoV-2 entry factors, NRP1 and TFRC, in β cells. We propose that this enrichment of NRP1, and possibly TFRC, is a potential mechanism underlying SARS-CoV-2 tropism for β cells. Further studies incorporating more robust reagents, such as well-validated antibodies, will be needed to better understand the additional roles of other factors implicated in SARS-CoV-2 entry, including TMPRSS4, Furin, and heparan sulfate (Clausen et al., 2020). Heparan sulfate, in particular, has been shown to be highly expressed in pancreatic β cells and plays an important role in regulating β cell survival (Simeonovic et al., 2018Ziolkowski et al., 2012) and would be another leading candidate in determining SARS-CoV-2 tropism for β cells.

Of note, while we found SARS-CoV-2 localization within β cells from 4 autopsied patients, SARS-CoV-2 NP was not detected in pancreatic islets from 3 autopsied patient samples from a separate report (Kusmartseva et al., 2020). This discrepancy is likely due to the issue that pancreatic tissues are highly prone to autolysis, resulting in the rapid proteolysis of proteins due to the abundance of digestive enzymes. Indeed, we also did not detect SARS-CoV-2 NP signal in 3 out of 7 pancreas tissues with extensive pathologist-verified autolysis/atrophy, further suggesting the importance of rapid tissue preservation and documentation during COVID-19 autopsies. For these samples, we were able to confirm viral genomic expression by ISH in β cells as evidence of viral infection. Finally, we showed that SARS-CoV-2 infection leads to dysregulation of insulin homeostasis, induction of apoptosis-associated signaling pathways, along with cell apoptosis, mainly in β cells. These key observations support a mechanism through which SARS-CoV-2 can directly drive β cell damage to cause clinical T1D linked to hyperglycemia. These effects of the virus are not mutually exclusive with the possibility that SARS-CoV-2 can also induce autoimmune-mediated β cell destruction and is the subject of further investigation. Nonetheless, at least our microscopic histological observations do not suggest ongoing insulitis. Additional limitations of our study are the small sample size of pancreas samples from patients with COVID-19 and the lack of pancreas from children due to challenges in procuring these tissues. Moreover, since we utilized the pancreas from patients who succumbed to severe COVID-19, we are unable to generalize the SARS-CoV-2 β cell tropism to all patients with COVID-19, particularly those with mild COVID-19 due to the invasiveness of such a biopsy. Indeed, only a minority of patients with COVID-19 develop hyperglycemia, DKA, or T1D (Chee et al., 2020Ebekozien et al., 2020Hollstein et al., 2020Naguib et al., 2021Rubino et al., 2020Singh and Singh, 2020Unsworth et al., 2020).

Although we identify a mechanism explaining β cell-selective cell death, many details of how the virus migrates to the pancreas in patients with severe COVID-19 remain unclear. We suspect that following the initial infection of the upper airway and secondary expansion of the virus to the lungs, viral particles can be taken up by the vasculature and propagated to vascularized organs including the pancreas, kidney, and brain. From there, the route of vascular exit and viral entry to the tissue itself may require additional steps or preconditions to favor viral attack of secondary tissues like the pancreas. It would be of value to evaluate patient records to determine the time of onset of COVID-19-induced pneumonia, marking severe lung infection, compared with the evolution of hyperglycemia as a marker of pancreatic damage and a diabetes-like effect on insulin secretion.

While this manuscript was under review, Müller and colleagues (Ulm University Medical Center, Germany) reported that SARS-CoV-2 infects human pancreatic endocrine cells ex vivo and in vivo and interferes with β cell functions ex vivo (Müller et al., 2021). While the general findings are similar to ours, there are some important key discrepancies. First, inconsistent with previous studies, these authors found that ACE2 and TMPRSS2 expression are higher in β cells than ɑ and δ cells. We and others found that ACE2 and TMPRSS2 expression are low in islets (Figure 1) (Coate et al., 2020Kusmartseva et al., 2020) both at the mRNA and protein levels. Furthermore, we identified the expression of two other SARS-CoV-2 entry receptors, NRP1 and TFRC, to be higher in β cells than ɑ cells (Figure 1). NRP1 expression, and not ACE2, was found to be upregulated in patients with COVID-19 compared with non-COVID-19 donors (Figure S3). Treatment of human islets with an NRP1 inhibitor reduces infection by SARS-CoV-2 and partially rescues GSIS in the islets, showing the critical role of NRP1 for enabling SARS-CoV-2 infection. Second, although the authors confirmed that SARS-CoV-2 infects β cells by staining pancreatic tissue sections from COVID-19 autopsies, the authors did not find β cells to be selectively infected by the SARS-CoV-2, but rather a large number of endocrine cells were infected in isolated islets. In contrast, we found that β cells are more susceptible to SARS-CoV-2 infection. Finally, the authors measured apoptosis via cleaved caspase-3 staining at 3 dpi and found no increased apoptosis in infected islets at 3 dpi. Our results indicate a significant increase in apoptotic β cells in infected islets at 2 and 6 dpi (Figure 4). This is further confirmed through phosphoproteomic analysis and subsequent identification of the specific upregulated kinases involved in apoptotic processes in isolated pancreatic islet post-SARS-CoV-2 SP treatment or virus infection (Figures S4 and S5).

There are a number of variations between our studies, one of which is the source of the SARS-CoV-2 used. Although we used the same SARS-CoV-2 D614G variant, the source is quite different. The clinically isolated virus used by Müller et al. originated from the Amsterdam University, Netherlands (010V-03903), while the clinical virus strain used in this paper was from UCSF, United States (CA-UCSF-0001C). Further studies are required to determine whether these different virus strains may affect pathological outcomes. In addition, our source of human islets is different. Our human islets are from patients of United States origin and may reflect different ethnicities. Given the scale of the ongoing epidemic, our findings emphasize the urgent need for the development of therapies to prevent COVID-19-induced diabetes, here informed through a combination of ex vivo tissue culture models, retrospective autopsy samples, and unbiased phosphoproteomic analysis.

Limitations of study

There are expected limitations to this study. First, the localization and quantification of pancreatic ACE2, TMPRSS2, NRP1, and TFRC are largely based on ex vivo and in situ analyses of protein (IHC and IF) expression in pancreatic tissues from a limited cohort of healthy or COVID-19 subjects. The histological analysis of these pancreatic tissue sections can only provide approximate predictions for ACE2, TMPRSS2, NRP1, and TFRC protein expression in isolated islets and islet cells and a general signature for their susceptibility to in vitro SARS-CoV-2 infection. This limitation is improved by our ability to infect human islet ex vivo and reconstitute several critical aspects of viral infection and pathogenesis, increasing our confidence in the results. For this study, we could not directly translate our ex vivo observations of the dramatic decrease in insulin content and GSIS in SARS-CoV-2-infected human islets to the COVID-19 clinical samples in this study, as the insulin and blood glucose levels of these patients were not measured. Additional studies correlating patient serum markers of islet secretion of insulin, glucose levels, and COVID-19 status would be of considerable benefit to better understand virus effects on patients with typical versus severe COVID-19 disease.

Finally, phosphoproteomics was performed across the entire population of islet cell types, since single-cell phosphoproteomics is currently technically challenging. Therefore, we are unable to deconvolute any diversity of signaling pathways caused by the treatment of SARS-CoV-2 SP or SARS-CoV-2 infection in individual cell types in this study. Given the measured selectivity of infection in β cells and preponderance of β cells, we can reasonably estimate that the strong effects we observe reflect β cell alterations, rather than bystander effects.Go to:

STAR★Methods

Key resources table

REAGENT or RESOURCESOURCEIDENTIFIER
Antibodies
Rabbit anti-SARS-CoV-2-NPGeneTexCat# GTX135361; RRID: AB_2887484
Mouse anti-SARS-CoV-2-NPThermo FisherCat# MA1-7403; RRID: AB_1018420
Mouse anti-SARS-CoV-2-SPGeneTexCat# GTX632604; RRID: AB_2864418
Rabbit anti-ACE2AbcamCat# ab15348; RRID: AB_301861
Mouse anti-TMPRSS2MilliporeCat# MABF2158
Mouse anti-NRP1Santa CruzCat# sc-5307; RRID: AB_2282634
Rabbit anti-NRP1AbcamCat# ab81321; RRID: AB_1640739
Rabbit anti-NRP1AtlasCat# HPA030278; RRID: AB_10601976
Mouse anti-TFRCThermo FisherCat# 13-6800; RRID: AB_2533029
Rabbit anti-TFRCAtlasCat# HPA028598; RRID: AB_10601599
Mouse anti-InsulinCell SignalingCat# 8138S; RRID: AB_10949314
Mouse anti-InsulinSanta CruzCat# sc-8033; RRID: AB_627285
Rabbit anti-glucagonProteinTechCat# 15954-1-AP; RRID: AB_2878200
Mouse anti-glucagonAbcamCat# ab10988; RRID: AB_297642
Mouse-somatostatinSanta CruzCat# sc-55565; RRID: AB_831726
Mouse-somatostatinSanta CruzCat# sc-74556; RRID: AB_2271061
Mouse anti-CD31BDCat# 550389; RRID: AB_2252087
Mouse anti-CD31NovusCat# NBP2-47785; RRID: AB_2864381
Rabbit anti- Phospho-JNK1/2Cell SignalingCat# 4668T
Rabbit anti- Phospho-PAK1/2Cell SignalingCat# 2601S; RRID: AB_330220
Bacterial and virus strains
SARS-CoV-2Joe DeRisi Lab, UCSFSARS-CoV-2/human/USA/CA-UCSF-0001C/2020
Biological samples
Human pancreatic isletsIntegrated Islet Distribution Program (IIDP)Table 2
Chemicals, peptides, and recombinant proteins
DMSOSigma-AldrichCat# 276855
EG00229Sigma-AldrichCat# SML1367
DAPIBio TrendCat# 40043
Pen/StrepThermo FisherCat# 15140163
ParaformaldehydeAlfaAesarCat# 433689M
Normal Donkey SerumJackson ImmunoResearchCat# 017-000-121
NP40SigmaCat# 11332473001
The unrelated mock peptide: KKHKNQRSRKKHKNQRSRGenscriptN/A
The NRP1 peptide for blocking assayAbcamCat# ab189308
The TFRC peptide for blocking assay: DQARSAFSNLFGGEPLSYTRFSLARQGenScriptN/A
The SARS-CoV-2 NP peptide for blocking assay: STGSNQNGERSGARSKGenScriptN/A
BSASigmaCat# A6003-25G
1X PBSCorningCat# 46-013-CM
Triton X-100USBCat# 22686
OCT compoundVWRCat# 25608-930
SaponinSigma-AldrichCat# S7900
Fluoromount-GSouthernBiotechCat# 0100-01
Dako Target Retrieval Solution, pH 9DAKO AgilentCat# S236784-2
ProLong Gold Antifade mounting medium with DAPIThermo FisherCat# P36931
HoechstThermo FisherCat# 33342
GlutaMaxlifeCat# 35050-079
Critical commercial assays
Human insulin ELISA kitMercodiaCat# 10-1113-01
In Situ Cell Death Detection Kit, TMR redSigma-AldrichCat# 12156792910
TaqMan 2019-nCoV Control Kit v1Thermo FisherCat# A47533
RNAscope Multiplex Fluorescent Reagent Kit v2Bio-TechneCat# 323100
TSA Cyanine 3Akoya BiosciencesCat# NEL744001KT
High-Select Fe-NTA Phosphopeptide Enrichment KitThermo ScientificCat# A32992
Deposited data
RNA-seq data for FACS purified human alpha and beta cells (Blodgett et al., 2015)NCBI Gene Expression OmnibusGEO: GSE67543
RNA-seq data for FACS purified human alpha and beta cells (Arda et al., 2016)NCBI Gene Expression OmnibusGEO: GSE57973
RNA-seq data for FACS purified human alpha, beta, and delta cells (Kim et al., 2020)NCBI Gene Expression OmnibusGEO: GSE143889
Phosphoproteomics data of SARS-CoV-2 infected and Spike protein treated human pancreatic isletsThis paperPRIDE: PXD025629
Oligonucleotides
SARS-CoV-2 spike mRNABio-TechneCat# 848561
Software and algorithms
ImageJNIHhttps://imagej.nih.gov/ij/
Zen blackCarl Zeisshttps://www.zeiss.com/microscopy/us/products/microscope-software/zen.html

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Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Peter Jackson (pjackson@stanford.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The analysis code used to support the findings of this study are available at https://github.com/bmyury/membrane_ACE2_quantitation.

Experimental model and subject details

Human pancreas tissue collection

De-identified human pancreatic islets were obtained from adult organ donors without a history of diabetes or glucose intolerance. Islets were procured through the Integrated Islet Distribution Program, Alberta Diabetes Institute IsletCore, and the International Institute for the Advancement of Medicine. SARS-CoV-2 infected pancreas tissue was obtained during autopsy and processed as previously described (Menter et al., 2020); the procedure was approved by the ethics commission of Northern Switzerland (EKNZ; study ID: 2020-00969). All patients with COVID-19 or their relatives consented to the use of tissue for research purposes. The characteristics of the autopsy and islet donors are summarized in Tables 1​,2,2, and ​and33.

Method details

Virus production, islet infection, and drug treatment

A549 cells stably expressing ACE2 under CMV promoter were infected with SARS-CoV-2 clinical isolate of the pandemic D614G variant (SARS-CoV-2/human/USA/CA-UCSF-0001C/2020, kindly provided by Sara Sunshine and Joe DeRisi) with MOI ~0.05 in MEM medium supplemented with 2% FBS and penicillin/streptavidin (Gibco). 3 days after infection, the medium was collected and cleared from cell debris by centrifugation at 3,000 g for 10 min at 4 C. The virus titers were measured by plaque assay. To infect the islets, 100 ul of virus suspension (5-10×106 PFU per ml) was added to 1 ml of the pancreatic islet culture and incubated at 37°C for the indicated time. Small molecules were dissolved in DMSO (276855, Sigma-Aldrich). Islets were first treated with 100 μM EG00229 (SML1367, Sigma-Aldrich) or DMSO for 1 hour before infection with SARS-CoV-2. Islets were fixed at 2 dpi.

Immunofluorescence immunohistochemistry (IF IHC) of cryosections

For cryosections, isolated human islets were fixed in 4% paraformaldehyde in 1X PBS for 1 h, embedded in collagen I (Becton-Dickinson, San Jose, CA, USA) 4% (wt/vol.) and then fixed on ice in 4% paraformaldehyde in 1X PBS for another 20 min, followed by three 5 min washes with 1X PBS and equilibration in 30% sucrose/1X PBS overnight. Tissue specimens were processed for OCT-embedded, 6-μm cryosections.

Cryosections were blocked with 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) in IF buffer (3% BSA and 0.4% saponin in PBS; for all else: 3% BSA and 0.1% NP-40 in PBS) at room temperature for 1 hour. Samples were incubated with primary antibody in IF buffer at room temperature for overnight at 4°C, followed by 5 washes with IF buffer. Samples were then incubated with fluorescent-labeled secondary antibody at room temperature for 1 hour, followed by a 5 min incubation with 4’,6-dia-midino-2-phenylindole (DAPI) in PBS at room temperature for 5min and 3 washes with IF buffer. Coverslips were mounted with Fluoromount-G (0100-01, SouthernBiotech) onto glass slides followed by image acquisition.

Immunofluorescence immunohistochemistry (IF IHC) of FFPE sections

Sections were cut to 4 μm thickness onto frosted glass slides at the Stanford University Histology Service Center and University Hospital Basel. H&E-stained sections were obtained from each formalin-fixed paraffin-embedded (FFPE) block. Deparaffinization, rehydration, and heat-induced epitope retrieval (HIER) were performed on a ST4020 small linear stainer (Leica). For deparaffinization, slides were baked at 70 °C for 1 h, followed by rehydration in descending concentrations of ethanol (100% twice, 95% twice, 80%, 70%, ddH2O twice; each step for 3 min). Washes were performed using a Leica ST4020 Linear Stainer (Leica Biosystems, Wetzlar, Germany) programmed to three dips per wash for 30 s each. HIER was performed in a Lab VisionTM PT module (Thermo Fisher) using Dako Target Retrieval Solution, pH 9 (S236784-2, DAKO Agilent) at 97 °C for 10 min and cooled down to 65 °C. After further cooling to room temperature for 30 min, slides were washed for 5-10 min three times in Tris-Buffered Saline (TBS), containing 0.1% Tween 20 (Cell Marque; TBS-T). Sections were then blocked in 5% normal donkey serum ((D9663, Sigma-Aldrich) in TBS-T at room temperature for 1 h, followed by incubation with primary antibodies in the blocking solution. After one overnight incubation of primary antibodies in 4 °C, sections were washed three times with TBS-T and stained with the appropriate secondary antibodies in PBS with 3% bovine serum albumin, 0.4% saponin, and 0.02% sodium azide at room temperature for 1 h. Following this, sections were washed three times with TBS-T and mounted with ProLong Gold Antifade mounting medium with DAPI (Invitrogen). Hoechst (33342, Thermo) was also used in the second to last TBS-T wash for additional nuclear staining. For the peptide blocking assay, the NRP1 (Abcam, ab81321), TFRC (Thermo, # 13-6800), and SARS-CoV-2 NP (GeneTex, GTX135361) antibodies were preincubated with a 20-fold molar excess of the immunizing peptide or an unrelated mock peptide for 3 hours with rotation at room temperature immediately before primary antibody staining. The peptides used are described below.

SARS-COV-2 PCR

For detection of SARS-CoV-2, RNA was first isolated from formalin-fixed and paraffin embedded pancreas tissue by using the Maxwell RSC RNA FFPE Kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Afterwards, TaqMan reverse transcription polymerase chain reaction (RT-PCR) was performed by using the TaqMan 2019-nCoV Control Kit v1 (A47533, ThermoFisher Scientific) to target three different viral genomic regions (ORF1ab, S and N) and the human RPPH1 gene (RNAse-P). According to the manufacturer’s protocol, a Cт value below 37 in at least two out of three viral genomic regions was considered positive. A case was considered negative if Cт values were above 40. Values between 37 and 40 were considered indeterminate and the assay was repeated. Samples were always run as duplicates.

Antibodies and reagents

Antibodies used include the following: rabbit anti-SARS-CoV-2-NP (GeneTex, GTX135361, 1:2,000), mouse anti-SARS-CoV-2-NP (Thermo Fisher, MA1-7403, 1:200), mouse anti-SARS-CoV-2-SP (GeneTex, GTX632604, 1:500), rabbit anti-ACE2 (Abcam, ab15348, 1:200), mouse anti-TMPRSS2 (Millipore, MABF2158, 1:200), mouse anti-NRP1 (Santa Cruz, sc-5307, 1:200), rabbit anti-NRP1 (Abcam, ab81321, 1:200), rabbit anti-NRP1 (Atlas, HPA030278, 1:200), mouse anti-TFRC (Thermo Fisher, # 13-6800, 1:200), rabbit anti-TFRC (Atlas, HPA028598, 1:200), mouse anti-Insulin (Cell Signaling, 8138S, 1:4,000), mouse anti-Insulin (Santa Cruz, sc-8033, 1:1,000), rabbit anti-glucagon (ProteinTech, 15954-1-AP, 1:3,000), mouse anti-glucagon (Abcam, ab10988, 1:4,000), mouse-somatostatin (Santa Cruz, sc-55565, 1:6,000), mouse-somatostatin (Santa Cruz, sc-74556, 1:2,000), mouse anti-CD31 (BD, 550389, 1:100), mouse anti-CD31 (Novus, NBP2-47785, 1:200), rabbit anti- Phospho-JNK1/2 (Cell Signaling, 4668T, 1:100), and rabbit anti- Phospho-PAK1/2 (Cell Signaling, 2601S, 1:100). Spike protein (BPS Bioscience, 100688) was used in the apoptosis experiment in Figures 4E and 4F. The immunizing peptides for the peptide blocking assay were NRP1 (Abcam, ab189308), TFRC (custom synthesized through GenScript using sequences provided in confidence by Thermo Fisher), and SARS-CoV-2 NP (custom synthesized through GenScript using sequences provided in confidence by GeneTex). The sequence of the unrelated mock peptide used for the peptide blocking assay was KKHKNQRSRKKHKNQRSR (Genscript).

In situ hybridization staining

Rehydration and HIER of tissue sections were performed as described above and in Lee et al. (2020). After cooling to room temperature, slides were washed for 2 × 2 min ddH2O before a 15 min H2O2 block at 40 °C (322335, Bio-Techne). Slides were then washed for 2 × 2 min ddH2O before an overnight hybridization at 40 °C with probes against the SARS-CoV-2 spike mRNA (848561, Bio-Techne). Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for SARS-CoV-2 spike mRNA probe targets (NEL744001KT, Akoya Biosciences). Slides were then processed as described above for IF IHC staining for mouse anti-insulin (Cell Signaling, 8138S, 1:4000).

Microscopy

Fluorescence-immunolabeled images were acquired using a Zeiss AxioImager Z1 microscope or a Marianas spinning disk confocal (SDC) microscopy (Intelligent Imaging Innovations). Post-imaging processing was performed using ZEN (Carl Zeiss). Final figures were organized using Adobe Illustrator.

In vitro insulin secretion assays

GSIS was performed 6 days post infection. Batches of 25 islets were used for in vitro secretion assays. Islets were incubated at a glucose concentration of 2.8 mM for 1 h as an initial equilibration period. Subsequently, islets were incubated at 2.8 mM glucose concentration for 1 h. Supernatant was taken and stored for insulin quantification. Islets were incubated at 16.7 mM glucose concentration for another 1 h. Supernatant was taken and stored for insulin quantification. Islets were then lysed in an acid-ethanol solution (1.5% HCL in 75% ethanol) to extract the total cellular insulin or glucagon content. Secreted human insulin in the supernatants and islet lysates were quantified using a human insulin ELISA kit (Mercodia). Secreted insulin levels were divided by total insulin content and presented as a percentage of total insulin content and normalized to values obtained at 2.8 mM glucose. All secretion assays were carried out in RPMI 1640 (Gibco) and the above-mentioned glucose concentrations.

TUNEL staining

Cellular apoptosis was measured by TUNEL staining according to the manufacturer’s instructions (Roche, Berlin, Germany). The proportion of TUNEL-positive nuclei in pancreatic β cell was determined through image analysis of the cryosections. The number of TUNEL-positive cells in pancreatic islets was counted under an Everest deconvolution workstation (Intelligent Imaging Innovations) equipped with a Zeiss AxioImager Z1 microscope and a CoolSnapHQ cooled CCD camera (Roper Scientific).

Phosphopeptide shotgun proteomics

Isolated human islet cells were treated with SARS-CoV-2 spike protein for 15, 30 minutes or with vehicle (water) for 30 minutes. Cells were harvested, lysed, reduce, and alkylated using 100 μl of lysis buffer (6M Guanidine Hydrochloride, 100 mM Tris-HCl pH 8.0, 10 mM TCEP, 10 mM CAA) and boiled 60°C for 1 hour. Proteins were precipitated by adding 100 μl methanol, vortexed, 50 μl chloroform, vortexed, 100 μl water, vortexed, and centrifuged at 13,000g for 2 minutes. The top aqueous layer was removed, 200 μl of methanol was added, vortexed, and centrifuged at 13,000g for 3 minutes. Methanol was removed and dried proteins were resuspended using 200mM HEPES pH 8.5. Proteins were digested using Trypsin/Lys-C overnight at 37°C 250 RPM. Sample was acidified using TFA and cleaned using stage tips. Stage tips were created using 5 layers of C18 filters packed into a P200 tip. The stage tips were activated using methanol, equilibrated twice with equilibration buffer (5% ACN, 0.5% TFA). Sample were bound, washed twice with equilibration buffer, and eluted using elution buffer (50% ACN, 0.1% FA). Eluted samples were dried using a Speed-Vac and resuspended using Binding/Wash Buffer in High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific, A32992). The peptides were enriched for phosphopeptides according to the manufacturer’s instructions. Eluted samples were eluted and resuspended using Solution A (2% ACN, 0.1% FA).

Samples were analyzed using the timsTOF Pro (Bruker Daltonics) (Meier et al., 2018), an ion-mobility spectrometry quadrupole time of flight mass spectrometer. Specifically, a nanoElute (Bruker Daltonics) high pressure nanoflow system was connected to the timsTOF Pro. Peptides were delivered to a reversed phase analytical column (10 cm x 75 μm i.d., Bruker 1866154). Liquid chromatography was performed at 50 °C and peptides were separated on the analytical column using a 48 min gradient (solvent A: 2% ACN, 0.1% FA; solvent B: 0.1% FA, in ACN) at a flow rate of 500 nl/min. A linear gradient from 2-35 % B was applied for 45 min, followed by a step to 95% B for 1.5 min and 3 min of washing at 95% B. The timsTOF Pro was operated in PASEF mode with the following settings: Mass Range 100 to 1700m/z, 1/K0 Start 0.85 V·s/cm2, End 1.3 V·s/cm2, Ramp time 100ms, Lock Duty Cycle to 100%, Capillary Voltage 1700, Dry Gas 3 l/min, Dry Temp 200°C, PASEF settings: 4 MS/MS, charge range 0-5, active exclusion for 0.04 min, Scheduling Target intensity 20000, Intensity threshold 500, CID collision energy 10eV.

For analysis, Bruker raw data files were processed using Byonic software (Protein Metrics) to identify peptides and proteins using the NCBI Homo sapiens refseq protein database. Data were searched with 20 ppm error tolerance for precursor and 40 ppm for fragment ions using QTOF/HCD fragmentation type. Besides standard variable modifications, we searched for S/T/Y phosphorylation and set 1 % FDR for protein identifications.

Kinase set enrichment analysis (KSEA Analysis)

Counts for phosphosites between two technical replicates were summed after the total counts per sample were normalized to the median of total counts. The counts were then log2-transformed and quantile normalized. Batch correction was done using ComBat function from the sva package from Bioconductor and the log2-transformation was undone to obtain the counts for differential expression analysis. Differential expression analysis was conducted with the msms.glm.pois function from the msmsTests package from Bioconductor (Josep Gregori, Alex Sanchez and Josep Villanueva (2020). msmsTests: LC-MS/MS Differential Expression Tests. R package version 1.26.0.). KSEA was calculated with the ksea function from ksea package from GitHub (David Ochoa (2020). ksea: Kinase Activity Prediction based in Quantitative Phosphoproteomic Data. R package version 0.1.2.) using a kinase substrate database created from PhosphositePlus (Hornbeck et al., 2015) and NetworKin (Linding et al., 2008). p-values were adjusted using Benjamini-Hochberg procedure. GO ORA was conducted using enrichGO function from the clusterProfiler package from Bioconductor (Yu et al., 2012) and the database org.Hs.eg.db package from Bioconductor (Marc Carlson (2020). org.Hs.eg.db: Genome wide annotation for Human. R package version 3.11.4.).

Quantification and statistical analysis

For signal quantification, samples were stained simultaneously in batch with the primary antibodies (ex. ACE2, TMPRSS2, NRP1, TFRC, insulin, glucagon as described above) using the same master mixes and identical incubation times under similar staining conditions described above. Exposure times under confocal microscopy were identical for the quantified samples. Quantification was performed using a custom script developed in the FIJI package of ImageJ as previously described (Lee et al., 2020). Briefly, a binary mask was created by thresholding the insulin and glucagon channels using selected cutoff values to generate a comprehensive outline of each channel. Insulin- and glucagon-positive regions were segmented using continuity of high signal regions on a binary mask. Finally, the signals of proteins of interest within the segmented regions were computed. While the experimenters were not strictly blinded to the samples, all sample processing, staining, and data acquisition were performed in parallel under identical conditions without regard to the specific identity of the samples. Quantification used a custom script developed in the FIJI package of ImageJ as previously described (Lee et al., 2020). Experimental sample sizes were not predetermined given the exploratory nature of the work and the limited availability of tissue specimens. No pancreatic samples were excluded from experimentation/analyses unless otherwise stated in the manuscript (see Table 3). Mann-Whitney U test was used when the data were not normally distributed by Shapiro-Wilk normality test and were not at equal variance by F-test. When the normal distribution and equal variance were confirmed, Student’s t test were used. Kruskal-Wallis test and post-hoc Dunn’s multiple comparison test were used for comparisons of more than two groups. Analyses were performed with GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA) software and IBM SPSS Statistics version 23 (IBM, Armonk, NY).Go to:

Acknowledgments

The authors acknowledge members of the Kim laboratory, especially Jonathan Lam and Dr. Sangbin Park, for helpful discussions and assistance with islet experiments. We thank the Stanford Diabetes Research Center/Stanford Islet Research Core (supported by # P30DK116074). We thank the Stanford Diabetes Research Center/Stanford Islet Research Core (SDRC/SIRC), Alberta Diabetes Institute Islet (ADI) Research Core, IIDP, NDRI, and IIAM for islet and/or pancreas procurement, and especially the organ donors and their families. This work was supported by the National Institutes of Health R01DK127665 (P.K.J.), R01HD085901 (P.K.J.), R01GM121565 (P.K.J.), P30DK116074 (P.K.J.), R01AI149672-01 (G.P.N.), and U54-CA209971 (G.P.N.); Stanford Diabetes Research Center (SDRC) Pilot and Feasibility Research grant (P.K.J.); the Fast Grant Funding for COVID-19 Science (P.K.J. and G.P.N.); the Botnar Research Centre for Child Health Emergency Response to COVID-19 grant (S.J., M.S.M., G.P.N., and A.T.); a Bill and Melinda Gates Foundation COVID-19 Pilot Award (S.J. and G.P.N.); the Rachford & Carlotta A. Harris Endowed Chair (G.P.N.); California Institute for Regenerative Medicine (DISC2-09637) (J.V.N.); Defense Advanced Research Project Agency (HR001118S0037-PREPARE-FP-001) (J.V.N.); the Operndorf Foundation (J.V.N.); Stanford Respond. Innovate. Scale. Empower (RISE) COVID-19 crisis response trainee seed grant (C.-T.W., R.C., I.T.L., S.J., and T.N.); Stanford Translational Research and Applied Medicine (TRAM) pilot grant (I.T.L.); Thrasher Research Fund Early Career Award (I.T.L.); Stanford Maternal and Child Health Research Institute (MCHRI) Clinical (MD) Trainee Support Award (I.T.L., Ernest and Amelia Gallo endowed postdoctoral fellow); Leukemia & Lymphoma Society Career Development Program (S.J.); Cellular and Molecular Biology Training grant (NIH 5 T32 GM007276) (R.C.); and the Swiss National Science Foundation (SNSF; grant no. 320030_189275) (M.S.M.).

Author contributions

C.-T.W. conceived and coordinated the study. C.-T.W., P.V.L., Y.X., I.T.L., R.C., S.J., and T.N. designed and performed the experiments. C.-T.W., I.T.L., and T.N. performed the microscopy imaging. P.V.L. and Y.X. made virus and infected isolated human pancreatic islets. I.T.L., S.J., A.K.S., A.T., and M.S.M. obtained patient consent, and collected, processed, banked, and/or evaluated the human samples. R.C. and J.D. performed phosphopeptide shotgun proteomics. C.-T.W., I.T.L., R.C., J.D., T.N., and S.J. analyzed the data. C.-T.W., T.N., and S.J. conducted statistical analyses. R.J.B., R.L.W., and C.A.C. provided isolated pancreatic islets. B.Z. and H.C. assisted in experiments. Y.G. contributed novel tools that enabled the analysis. C.-T.W. and T.N. prepared the final figures. C.-T.W., I.T.L., S.J., and R.C. wrote the manuscript with contributions by P.V.L., J.V.N., A.T., M.S.M., and P.K.J. The co-first authors, C.-T.W., P.V.L., Y.X., I.T.L., R.C., S.J., and T.N. contributed equally and have the right to list their name first in their CV. Funding and supervision were provided by P.K.J. All authors reviewed and agreed with the content of this manuscript.

Declaration of interests

The authors declare no competing interests.Go to:

Notes

Published: May 18, 2021Go to:

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.cmet.2021.05.013.Go to:

Supplemental information

Document S1. Figures S1–S5:Click here to view.(22M, pdf)Table S1. Phosphoproteomic data, related to Figures 4 and S4:

Contains raw count of filtered phosphoproteomic data and fold change analysis of phosphosites for human islet cells treated with purified Spike protein (PhosphoSpike) or SARS-CoV-2 (PhosphoSars). Unfiltered phosphoproteomic raw count data is also included (UnfilteredPhospho).Click here to view.(5.6M, xlsx)Table S2. Predicted kinase activity, related to Figures 4 and S4:

Kinase activity as predicted by KSEA of various kinases in human islet cells treated with purified Spike protein (KSEA.Spike) or SARS-CoV-2 (KSEA.Sars). Kinase activity is reported as ES as calculated by weighted Kolmogorov-Smirnov statistics.Click here to view.(22K, xlsx)Table S3. Gene ontology pathway analysis, related to Figures 4 and S5:

Gene ontology enrichments for upregulated/downregulated kinases upon treatment by Spike protein or SARS-CoV-2.Click here to view.(397K, xlsx)Document S2. Article plus supplemental information:Click here to view.(26M, pdf)Go to:

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Review of COVID-19, part 1: Abdominal manifestations in adults and multisystem inflammatory syndrome in children

Authors: Devaraju Kanmaniraja,a,⁎ Jessica Kurian,a Justin Holder,a Molly Somberg Gunther,a Victoria Chernyak,b Kevin Hsu,a Jimmy Lee,a Andrew Mcclelland,a Shira E. Slasky,a Jenna Le,a and Zina J. Riccia

Abstract

The coronavirus disease 2019 (COVID -19) pandemic caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) has affected almost every country in the world, resulting in severe morbidity, mortality and economic hardship, and altering the landscape of healthcare forever. Although primarily a pulmonary illness, it can affect multiple organ systems throughout the body, sometimes with devastating complications and long-term sequelae. As we move into the second year of this pandemic, a better understanding of the pathophysiology of the virus and the varied imaging findings of COVID-19 in the involved organs is crucial to better manage this complex multi-organ disease and to help improve overall survival. This manuscript provides a comprehensive overview of the pathophysiology of the virus along with a detailed and systematic imaging review of the extra-thoracic manifestation of COVID-19 with the exception of unique cardiothoracic features associated with multisystem inflammatory syndrome in children (MIS-C). In Part I, extra-thoracic manifestations of COVID-19 in the abdomen in adults and features of MIS-C will be reviewed. In Part II, manifestations of COVID-19 in the musculoskeletal, central nervous and vascular systems will be reviewed.

Keywords: Abdominal imaging, COVID-19, Multisystem inflammatory syndrome

1. Abdominal findings of COVID019 in adults

The coronavirus 2019 disease (COVID-19), which originated in Wuhan, China, has quickly become a global pandemic, bringing normal life to a standstill in almost all countries around the world. The severe acute respiratory syndrome coronavirus (SARS-CoV-2) is a novel virus preceded by two other recent coronavirus infections, the severe acute respiratory syndrome coronavirus (SARS-CoV-1) and the Middle Eastern respiratory syndrome coronavirus (MERS–CoV), but it has more far-reaching and devastating consequences. As of March 2021, the COVID-19 pandemic has resulted in over 29 million cases in the United States and over 121 million cases globally. As of April 2021, it is responsible for the deaths of over half a million people in the United States and more than 2 ½ million worldwide [1]. As the disease has evolved over the past year, so has our understanding of the virus, including its pathophysiology, clinical presentation and imaging manifestations. Although COVID-19 is predominately a pulmonary illness, it is now established to have widespread extra-pulmonary involvement affecting multiple organ systems. The SARS-CoV-2 has a highly virulent spike protein which binds efficiently to the angiotensin converting enzyme 2 (ACE2) receptors which are expressed in many organs, including the airways, lung parenchyma, several organs in the abdomen, particularly the kidneys and GI system, central nervous system and the smooth and skeletal muscles of the body [2]. The virus initially induces a specific adaptive immune response, and when this response is ineffective, it results in uncontrolled inflammation, which ultimately results in tissue injury [2].

This article provides a comprehensive review of the pathophysiology and imaging findings of the extra-thoracic manifestations of COVID-19 with the exception of unique cardiothoracic features associated with multisystem inflammatory syndrome in children (MIS-C). In Part I, extra-thoracic manifestations of COVID-19 in the abdomen in adults and the varying features of multisystem inflammatory syndrome in children will be reviewed, with imaging findings summarized in Table 1Table 2 . In Part II, manifestations of COVID-19 in the musculoskeletal system, the central nervous system and central and peripheral vascular systems will be reviewed.

Table 1

Summary of abdominal imaging findings in COVID-19 in adults.

OrganImaging findings
Liver• Hepatomegaly
• Increased or coarsened echogenicity on US
• Hypoattenuation on non-contrast or contrast enhanced CT
• Periportal edema and heterogeneous enhancement on CT
• Loss of signal on opposed-phase sequences on MRI
• Portal vein thrombus
Pancreas• Features of acute interstitial pancreatitis
Biliary Tree• Biliary ductal dilatation
Kidney• Increased or heterogeneous parenchymal echogenicity on US
• Loss of corticomedullary differentiation on US
• Preserved cortical thickness
• Perinephric fat stranding and thickening of Gerota’s fascia on CT
• Wedge shaped perfusion defects on CT or MRI
• Thrombus in the renal artery or vein
Gallbladder• Distension
• Mural edema
• Sludge
• Acalculous cholecystitis
Urinary Bladder• Bladder wall thickening
• Mural hyperenhancement
• Perivesicular stranding
Bowel• Mural thickening
• Ileus
• Fluid-filled colon
• Pneumatosis intestinalis
• Portal vein gas
• Pneumoperitoneum
• Acute mesenteric ischemia
• Vascular occlusion (superior mesenteric artery, superior mesenteric vein, or portal vein)
• Mesenteric fat stranding, ascites
• Active gastrointestinal bleeding (duodenal or gastric ulcer) on CTA
• Clostridium difficile colitis
• Ischemic colitis
Spleen• Wedge shaped perfusion defects on CT or MRI
• Thrombus in the splenic artery or vein

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Table 2

Summary of imaging findings in Multisystem Inflammatory Syndrome in Children.

RegionImaging findings
Cardiothoracic• Bilateral symmetric diffuse airspace opacities with lower lobe predominance on CXR
• Diffuse ground glass opacity, septal thickening, and mild hilar lymphadenopathy on CT
• Bilateral pleural effusions
• Cardiomegaly
• Pericardial effusion
• Myocarditis pattern on cardiac MRI
Abdominal• Mesenteric lymphadenopathy, most common in right lower quadrant
• Mesenteric edema
• Ascites
• Bowel wall thickening
• Ileus
• Hepatosplenomegaly
• Gallbladder wall thickening

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2. Abdominal findings of COVID-19 in adults

2.1. Hepatobiliary derangement

Varying derangements of the liver, biliary system, gallbladder, portal vein and pancreas may occur in COVID-19 with hepatic parenchymal injury and biliary stasis reported with highest frequency. The mechanism of involvement of these structures appears to be multifactorial. The most direct form of injury results from SARS CoV-2 entry into host cells by binding to ACE2 receptors detected in several locations in the hepatobiliary system, including biliary epithelial cells (cholangiocytes), gallbladder endothelial cells and both pancreatic islet cells and exocrine glands [[3][4][5][6]].

2.1.1. Hepatic injury

Direct SARS CoV-2 entry into cholangiocytes may cause liver damage by disrupting bile acid transportation or by triggering acid accumulation resulting in liver injury [7]. Systemic inflammation, hypoxia inducing hepatitis and adverse drug reactions may incite liver injury [8]. Several drugs commonly used to treat COVID-19 patients, including acetaminophen, lopinavir and ritonavir can be hepatotoxic [9]. One study excluding COVID-19 patients receiving hepatotoxic drugs, still found patients with liver injury. Therefore, liver damage in COVID-19 patients is likely not entirely drug-induced but may also be due to acute infection [8,9]. Furthermore, since patients with chronic liver disease such as cirrhosis, autoimmune liver disease and prior liver transplantation are more susceptible to COVID-19 infection [9], underlying conditions may also contribute to liver injury.

The most frequent hepatic derangement is abnormal liver function tests reported in 16–53% of patients [10,11] and including raised levels of alanine aminotransferase, aspartate aminotransferase, and γ-glutamyl transferase with mild elevation of bilirubin. The majority of cases are mild and self-limited, with severe liver damage rare [7]. Liver injury is most prevalent in the second week of COVID-19 infection, and has a higher incidence in those with gastrointestinal symptoms and more severe infection [9]. Based on a meta-analysis of hepatic autopsy findings of deceased COVID-19 patients in 7 countries, hepatic steatosis (55%), hepatic sinus congestion (35%) and vascular thrombosis (29%) were the most common [10]. In a retrospective study of abdominal imaging findings of 37 COVID-19 patients, 27% who underwent ultrasound had increased hepatic echogenicity considered to represent fatty liver with elevated liver enzymes being the most frequent indication for ultrasound [4]. It should be noted that since obesity is a major risk factor for severe COVID-19 infection, it might contribute to the frequency of steatosis identified on imaging. In another retrospective abdominal sonographic study of 30 ICU patients with COVID-19, the most common finding was hepatomegaly (56%), with most cases having increased hepatic echogenicity and elevated liver function tests [12]. In the only retrospective case-control study of 204 COVID-19 patients who underwent non-contrast chest CT scan, steatosis was found in 31.9% of cases and only 7.1% of controls [13]. Steatosis was based on a single ROI measurement in the right lobe with an attenuation value ≤ 40 HU. However, underlying risk factors for steatosis such as diabetes, obesity, hypertension and abnormal lipid profile, were not available to exclude preexisting conditions leading to steatosis. Finally, unlike in the spleen and kidney where infarcts are reported in COVID-19, hepatic infarction is not a distinct feature. This is likely due to the liver’s unique dual blood supply.

On imaging the liver may be enlarged. On ultrasound, the liver of patients with abnormal liver function tests may be coarsened and/or increased in echogenicity (Fig. 1Fig. 2 ). On CT scan, the liver may be hypoattenuated on non-contrast or contrast-enhanced exam due to steatosis (Fig. 3 ). Periportal edema and heterogeneity of hepatic enhancement may be seen on contrast-enhanced CT or MRI due to parenchymal inflammation. On MRI, loss of signal on opposed-phase sequences (Fig. 4 ) may be seen due to steatosis and periportal edema may be conspicuous on T2-weighted images or on contrast-enhanced images [7,8,14]. Periportal lymphadenopathy, typical of chronic liver disease, is not reported in COVID-19 [8]. In patients with severe COVID-19 infection, ancillary manifestations of hepatic inflammation and injury, such as parenchymal attenuation changes and abscesses may be seen (Fig. 5 ).

This Article Presents a Detailed Overview with Imaging. To View the Rest of This Analysis Click Here:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8223038/

COVID-19 also attacks the pancreas; one vaccine dose may be enough for those previously infected

Authors: Nancy Lapid February 3, 2021

COVID-19 attacks the pancreas

The new coronavirus directly targets the pancreas, infecting and damaging its insulin producing cells, according to a new study. The findings may help explain why blood sugar problems develop in many COVID-19 patients, and why there have been reports of diabetes developing as a result of the virus. The pancreas has two jobs: production of enzymes important to digestion, and creation and release of the hormones insulin and glucagon that regulate blood sugar levels. In a paper published on Wednesday in Nature Metabolism, researchers report that lab and autopsy studies show the new coronavirus infects pancreas cells involved in these processes and changes their shape, disturbs their genes, and impairs their function. The new data “identify the human pancreas as a target of SARS-CoV-2 infection and suggest that beta-cell infection could contribute to the metabolic dysregulation observed in patients with COVID-19,” the authors conclude. (https://go.nature.com/36Cmtfy)

One vaccine dose might be enough for COVID-19 survivors

COVID-19 survivors might only need one shot of the new vaccines from Moderna Inc and Pfizer/BioNTech, instead of the usual two doses, because their immune systems have gotten a head start on learning to recognize the virus, according to two separate reports posted this week on medRxiv ahead of peer review. In one study of 59 healthcare workers who recovered from COVID-19 and received one of the vaccines, antibody levels after the first shot were higher than levels usually seen after two doses in people without a history of COVID-19. In a separate study, researchers found that 41 COVID-19 survivors developed “high antibody titers within days of vaccination,” and those levels were 10 to 20 times higher than in uninfected, unvaccinated volunteers after just one vaccine dose. “The antibody response to the first vaccine dose in individuals with pre-existing immunity is equal to or even exceeds” levels found in uninfected individuals after the second vaccine dose, the authors of that paper said. “Changing the policy to give these individuals only one dose of vaccine would not negatively impact on their antibody titers, spare them from unnecessary pain and free up many urgently needed vaccine doses,” they said. (https://bit.ly/3je4Zv4; https://bit.ly/2YG0EYf)

Gout drug shows promise for mildly ill COVID-19 patients

Colchicine, an anti-inflammatory drug used to treat gout and other rheumatic diseases, reduced hospitalizations and deaths by more than 20% in COVID-19 patients in a large international trial. COVID-19 patients with mild illness and at least one condition that put them at high risk for complications, such as diabetes or heart disease, received either colchicine or a placebo for 30 days. Overall, the risk of hospitalization or death was statistically similar in the two groups. But among the 4,159 patients whose coronavirus infections had been diagnosed with a gold-standard PCR test, death or hospital admission occurred in 4.6% of those on colchicine versus 60% of those who got a placebo. After taking patients’ other risk factors into account, colchicine was associated with a statistically significant 25% risk reduction, the researchers reported on medRxiv ahead of peer review. Patients taking colchicine also had fewer cases of pneumonia. “Given that colchicine is inexpensive, taken by mouth, was generally safe in this study, and does not generally need lab monitoring during use, it shows potential as the first oral drug to treat COVID-19 in the outpatient setting,” the researchers said. (https://bit.ly/3oDSDgY)

Oxford/AstraZeneca vaccine might work better with doses months apart

Among recipients of the COVID-19 vaccine from Oxford University and AstraZeneca, prolonging the interval between the first and second doses led to better results, researchers said in a paper posted on Monday ahead of peer-review by The Lancet on its preprint site. For volunteers aged 18 to 55, vaccine efficacy was 82.4% with 12 or more weeks between doses, compared to 54.9% when the booster was given within 6 weeks after the first dose. The longest interval between doses given to older volunteers was 8 weeks, so there were no data for the efficacy of a 12-week dosing gap in that group. Europe’s medicine regulator has said there is not enough data to determine how well the vaccine will work in people over 55. Given their findings, the authors say “a second dose given after a three-month period is an effective strategy … and may be the optimal for rollout of a pandemic vaccine when supplies are limited in the short term.”

Long covid: Damage to multiple organs presents in young, low risk patients

Authors: Gareth Iacobucci BMJ 2020; 371 doi: https://doi.org/10.1136/bmj.m4470 (Published 17 November 2020)Cite this as: BMJ 2020;371:m4470

Young, low risk patients with ongoing symptoms of covid-19 had signs of damage to multiple organs four months after initially being infected, a preprint study has suggested.1

Initial data from 201 patients suggest that almost 70% had impairments in one or more organs four months after their initial symptoms of SARS-CoV-2 infection.

The results emerged as the NHS announced plans to establish a network of more than 40 long covid specialist clinics across England this month to help patients with long term symptoms of infection.

The prospective Coverscan study examined the impact of long covid (persistent symptoms three months post infection) across multiple organs in low risk people who are relatively young and had no major underlying health problems. Assessment was done using results from magnetic resonance image scans, blood tests, and online questionnaires.

The research has not yet been peer reviewed and could not establish a causal link between organ impairment and infection. But the authors said the results had “implications not only for [the] burden of long covid but also public health approaches which have assumed low risk in young people with no comorbidities.”

The study enrolled participants at two UK sites in Oxford and London between April and August 2020. Two hundred and one individuals (mean age 44 (standard deviation 11.0) years) completed assessments after SARS-CoV-2 infection a median of 140 days after initial symptoms.

Participants were eligible if they tested positive for SARS-CoV-2 by random polymerase chain reaction swab (n=62), a positive antibody test (n=63), or had typical symptoms and were determined to have covid-19 by two independent clinicians (n=73).

The prevalence of pre-existing conditions was low (obesity: 20%, hypertension: 6%, diabetes: 2%, heart disease: 4%), and less than a fifth (18%) of individuals had been hospitalised with covid-19.

The most commonly reported ongoing symptoms—regardless of hospitalization status—were fatigue (98%), muscle ache (88%), shortness of breath (87%), and headache (83%). There was evidence of mild organ impairment in the heart (32% of patients), lungs (33%), kidneys (12%), liver (10%), pancreas (17%), and spleen (6%).

For More Information: https://www.bmj.com/content/371/bmj.m4470