von Willebrand Factor Multimer Formation Contributes to Immunothrombosis in Coronavirus Disease 2019.

OBJECTIVES: Prevention and therapy of immunothrombosis remain crucial challenges in the management of coronavirus disease 2019, since the underlying mechanisms are incompletely understood. We hypothesized that endothelial damage may lead to substantially increased concentrations of von Willebrand factor with subsequent relative deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13). DESIGN: Prospective controlled cross-over trial. SETTING: Blood samples of patients with confirmed coronavirus disease 2019 and healthy controls were obtained in three German hospitals and analyzed in a German hemostaseologic laboratory. PATIENTS: Seventy-five patients with confirmed coronavirus disease 2019 of mild to critical severity and 30 healthy controls. MEASUREMENTS AND MAIN RESULTS: von Willebrand factor antigen, ADAMTS13, and von Willebrand factor multimer formation were analyzed. von Willebrand factor antigen was 4.1 times higher in COVID-19 patients compared with healthy controls (p < 0.0001), whereas ADAMTS13 activities were not significantly different (p = 0.18). The ADAMTS13/von Willebrand factor antigen ratio was significantly lower in COVID-19 than in the control group (24.4 ± 20.5 vs 82.0 ± 30.7; p < 0.0001). Fourteen patients (18.7%) undercut a critical ratio of 10 as described in thrombotic thrombocytopenic purpura. Gel analysis of multimers resembled a thrombotic thrombocytopenic purpura pattern with loss of the largest multimers in 75% and a smeary triplet pattern in 39% of the patients. The ADAMTS13/von Willebrand factor antigen ratio decreased continuously from mild to critical disease (analysis of variance p = 0.026). Furthermore, it differed significantly between surviving patients and those who died from COVID-19 (p = 0.001) yielding an area under the curve of 0.232 in receiver operating characteristic curve curve analysis. CONCLUSION: COVID-19 is associated with a substantial increase in von Willebrand factor levels, which can exceed the ADAMTS13 processing capacity resulting in the formation of large von Willebrand factor multimers indistinguishable from thrombotic thrombocytopenic purpura. The ADAMTS13/von Willebrand factor antigen ratio is an independent predictor of severity of disease and mortality. These findings provide a rationale to consider plasma exchange as a therapeutic option in COVID-19 and to include von Willebrand factor and ADAMTS13 in the diagnostic workup.

Feasibility of non-invasive measurement of central blood pressure and arterial stiffness in shock.

BACKGROUND: Patients in haemodynamic shock are in need for an intensive care treatment. Invasive haemodynamic monitoring is state of the art for these patients. However, evolved, non-invasive blood pressure monitoring devices offer advanced functions like the assessment of central blood pressure and arterial stiffness. We analysed the feasibility of two oscillometric blood pressure devices in patients with shock. METHODS: We performed a monocentre prospective study, enrolling 57 patients admitted to the intensive care unit (ICU), due to septic and/or cardiogenic shock. We assessed invasive and non-invasive peripheral and central blood pressure <24 hours and 48 hours after admission on the ICU. Additional haemodynamic parameters such as pulse wave velocity (PWV), augmentation pressure and augmentation index were obtained through Mobil-o-Graph PWA (IEM) and SphygmoCor XCEL (AtCor Medical). RESULTS: A complete haemodynamic assessment was successful in all patients (48) with the Mobil-o-Graph 24 hours PWA and in 29 patients with the SphygmoCor XCEL (P = .001), when cases of death or device malfunction were excluded. Reasons for failure were severe peripheral artery disease, haemodynamic instability, oedema and agitation. Invasive blood pressure showed a sufficient correlation with both devices; however, large differences between invasive and non-invasive techniques were recorded in Bland-Altmann analysis (P < .05 for all parameters). PWV differed between the two devices. CONCLUSION: Non-invasive peripheral blood pressure measurement remains a rescue technique. However, non-invasive assessment of arterial stiffness and central blood pressure is possible in patients with septic or cardiogenic shock. Further studies are required to assess their clinical significance for patients in shock.

Immune monitoring facilitates the clinical decision in multifocal COVID-19 of a pancreas-kidney transplant patient.

The optimal management in transplant recipients with coronavirus disease 2019 (COVID-19) remains uncertain. The main concern is the ability of immunosuppressed patients to generate sufficient immunity for antiviral protection. Here, we report on immune monitoring facilitating a successful outcome of severe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated pneumonia, meningoencephalitis, gastroenteritis, and acute kidney and pancreas graft failure in a pancreas-kidney transplant recipient. Despite the very low numbers of circulating B, NK, and T cells identified in follow-up, a strong SARS-CoV-2 reactive T cell response was observed. Importantly, we detected T cells reactive to Spike, Membrane, and Nucleocapsid proteins of SARS-CoV-2 with majority of T cells showing polyfunctional proinflammatory Th1 phenotype at all analyzed time points. Antibodies against Spike protein were also detected with increasing titers in follow-up. Neutralization tests confirmed their antiviral protection. A correlation between cellular and humoral immunity was observed underscoring the specificity of demonstrated data. We conclude that analyzing the kinetics of nonspecific and SARS-CoV-2-reactive cellular and humoral immunity can facilitate the clinical decision on immunosuppression adjustment and allow successful outcome as demonstrated in the current clinical case. Although the antiviral protection of the detected SARS-CoV-2-reactive T cells requires further evaluation, our data prove an ability mounting a strong SARS-CoV-2-reactive T cell response with functional capacity in immunosuppressed patients.

Allograft infiltration and meningoencephalitis by SARS-CoV-2 in a pancreas-kidney transplant recipient.

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) preferentially affects epithelia of the upper and lower respiratory tract. Thus, impairment of kidney function has been primarily attributed until now to secondary effects such as cytokine release or fluid balance disturbances. We provide evidence that SARS-CoV-2 can directly infiltrate a kidney allograft. A 69-year-old male, who underwent pancreas-kidney transplantation 13 years previously, presented to our hospital with coronavirus disease 2019 (COVID-19) pneumonia and impaired pancreas and kidney allograft function. Kidney biopsy was performed showing tubular damage and an interstitial mononuclear cell infiltrate. Reverse transcriptase polymerase chain reaction from the biopsy specimen was positive for SARS-CoV-2. In-situ hybridization revealed SARS-CoV-2 RNA in tubular cells and the interstitium. Subsequently, he had 2 convulsive seizures. Magnetic resonance tomography suggested meningoencephalitis, which was confirmed by SARS-CoV-2 RNA transcripts in the cerebrospinal fluid. The patient had COVID-19 pneumonia, meningoencephalitis, and nephritis. SARS-CoV-2 binds to its target cells through angiotensin-converting enzyme 2, which is expressed in a broad variety of tissues including the lung, brain, and kidney. SARS-CoV-2 thereby shares features with other human coronaviruses including SARS-CoV that were identified as pathogens beyond the respiratory tract as well. The present case should provide awareness that extrapulmonary symptoms in COVID-19 may be attributable to viral infiltration of diverse organs.

Stability of 12 T-helper cell-associated cytokines in human serum under different pre-analytical conditions.

Cytokines are soluble and readily analyzed signaling molecules which reveal vital cues about the state of the immune system. As such, they serve in diagnosis and monitoring of immune-related disorders, where strictly controlled handling of the samples including storage and freeze/thawing procedures are required. In basic research and clinical trials, human serum samples can be left for long-term storage before processing. Storage space is commonly limited in scientific laboratories, which require storage of fewer but larger aliquots of patient serum samples. There are also practical limitations to the number of analytes to be processed at the same time. Further, new findings and technological progress might prompt analysis of hitherto unconsidered or undetectable molecules. Repeated freeze/thawing of serum samples is therefore a likely scenario, raising the question of the stability of the measured analytes under such conditions. To address this question, we subjected serum samples with spiked-in T-helper cell associated cytokines to several cycles of freeze/thawing under different conditions, including storage at -20 °C or -80 °C and thawing at 4 °C, 22 °C, and 37 °C, respectively. The concentration of TNF-α, IL-4, IL-17F, and IL-22 decreased after storage at room temperature for 4 h before freezing. Generally, storage at -20 °C resulted in reduced cytokine concentrations. This contrasts storage at -80 °C, which gave stable analyte concentrations; unaffected by repeated freeze/thaw cycles. The study presented here highlights the need for sentinel samples with known cytokine concentrations as internal control for the freeze/thaw process.

Generation of potentially inhibitory autoantibodies to ADAMTS13 in coronavirus disease 2019.

It has recently been shown that von Willebrand factor (VWF) multimers contribute to immunothrombosis in Coronavirus disease 2019 (COVID-19). Since COVID-19 is associated with an increased risk of autoreactivity, the present study investigates, whether the generation of autoantibodies to ADAMTS13 contributes to this finding. In this observational prospective controlled multicenter study blood samples and clinical data of patients hospitalized for COVID-19 were collected from April to November 2020. The study included 156 individuals with 90 patients having confirmed COVID-19 of mild to critical severity. 30 healthy individuals and 36 critically ill ICU patients without COVID-19 served as controls. ADAMTS13 antibodies occurred in 31 (34.4%) COVID-19 patients. Antibodies occurred more often in critically ill COVID-19 patients (55.9%) than non-COVID-19 ICU patients and healthy controls (5.6% and 6.7%; p < 0.001), respectively. Generation of ADAMTS13 antibodies in COVID-19 was associated with lower ADAMTS13 activity (56.5%, interquartile range (IQR) 21.25 vs. 71.5%, IQR 24.25, p = 0.0041), increased disease severity (severe or critical in 90% vs. 62.3%, p = 0.019), and a trend to higher mortality (35.5% vs. 18.6%, p = 0.077). Median time to antibody development was 11 days after first positive SARS-CoV-2-PCR specimen. Gel analysis of VWF multimers resembled the constellation in patients with TTP. The present study demonstrates for the first time, that generation of ADAMTS13 antibodies is frequent in COVID-19, associated with lower ADAMTS13 activity and increased risk of an adverse disease course. These findings provide a rationale to include ADAMTS13 antibodies in the diagnostic workup of SARS-CoV-2 infections.

The impact of calibration approaches on the accuracy of oscillometric central aortic blood pressure measurement.

BACKGROUND: Two fully automated oscillometric devices have become available for the noninvasive assessment of central aortic blood pressure (BP). They tend, however, to underestimate SBP. It has been proposed that calibration by mean/diastolic instead of systolic/diastolic brachial BP may reduce this bias. The present work compares the accuracy of these two calibrations in the Mobil-O-Graph. METHODS: Post-hoc analysis of the largest validation study on noninvasive assessment of central BP so far. Data on both calibration approaches were available in 159 patients without atrial fibrillation, who underwent simultaneous invasive and noninvasive assessment of central BP. Noninvasive BP measurements were conducted using the SphygmoCor XCEL (calibration by systolic/diastolic brachial BP only) and the Mobil-O-Graph (calibration by both systolic/diastolic and mean/diastolic brachial BP). RESULTS: Measurements of both devices and both calibration methods revealed highly significant correlations for systolic and diastolic central BP with invasively assessed BP (P < 0.001 each). Calibration by mean/diastolic and systolic/diastolic BP yielded similar correlations for central DBP (R 0.56 vs. R 0.55, P = 0.919). Correlation of central SBP, however, was significantly lower using calibration by mean/diastolic brachial BP (R 0.86 vs. R 0.74, P = 0.002). Numerically, the SphygmoCor device revealed the highest correlation (R 0.92 for central SBP and 0.72 for central DBP; P < 0.001 each). Calibration by systolic/diastolic brachial BP was associated with an underestimation of central SBP using both the SphygmoCor and the Mobil-O-Graph. Calibration by mean/diastolic brachial BP, instead, was associated with an overestimation, which was numerically comparable (4.8 ±â€Š11.3 vs. -4.2 ±â€Š8.0). The calibration method had little effects on the biases of diastolic measurements. CONCLUSION: Calibration by mean/diastolic instead of systolic/diastolic brachial BP led to an overestimation instead of underestimation of central SBP without improving accuracy. Hence, mean/diastolic calibration is not necessarily superior to systolic/diastolic calibration and the optimal approach has to be determined in a device-specific manner.

Detection of SARS-CoV-2 pneumonia: two case reports.

BACKGROUND: Developing therapeutic strategies for a SARS-CoV-2 infection is challenging, but first the correct diagnosis has to be made. Unspecific upper and lower respiratory tract symptoms can be misleading; hence, a nasopharyngeal swab test with a real-time reverse-transcription-polymerase chain reaction is of great importance. However, early viral clearing jeopardizes a sound diagnosis of COVID-19. CASE PRESENTATION: We report on two Caucasian patients who had negative pharyngeal swab tests at the onset of SARS-CoV-2 pneumonia. In one patient, the virus was not even detectable in bronchoalveolar lavage despite typical radiomorphologic changes. CONCLUSIONS: Negative PCR findings in both the pharynx and bronchoalveolar lavage do not exclude COVID-19 pneumonia. Computed tomography is a crucial diagnostic prerequisite in this context.

Lessons for the clinical nephrologist: recurrence of nephrotic syndrome induced by SARS-CoV-2.

SARS-CoV-2 is characterized by a multiorgan tropism including the kidneys. Recent autopsy series indicated that SARS-CoV-2 can infect both tubular and glomerular cells. Whereas tubular cell infiltration may contribute to acute kidney injury, data on a potential clinical correlative to glomerular affection is rare. We describe the first case of nephrotic syndrome in the context of COVID-19 in a renal transplant recipient. A 35 year old male patient received a kidney allograft for primary focal segmental glomerulosclerosis (FSGS). Three months posttransplant a recurrence of podocytopathy was successfully managed by plasma exchange, ivIG, and a conversion from tacrolimus to belatacept (initial proteinuria > 6 g/l decreased to 169 mg/l). Six weeks later he was tested positive for SARS-CoV-2 and developed a second increase of proteinuria (5.6 g/l). Renal allograft biopsy revealed diffuse podocyte effacement and was positive for SARS-CoV-2 in RNA in-situ hybridation indicating a SARS-CoV-2 associated recurrence of podocytopathy. Noteworthy, nephrotic proteinuria resolved spontaneously after recovering from COVID-19. The present case expands the spectrum of renal involvement in COVID-19 from acute tubular injury to podocytopathy in renal transplant recipients. Thus, it may be wise to test for SARS-CoV-2 prior to initiation of immunosuppression in new onset glomerulopathy during the pandemic.