Targeted MRM-analysis of plasma proteins in frozen whole blood samples from patients with COVID-19: a retrospective study.

OBJECTIVES: The COVID-19 pandemic has exposed a number of key challenges that need to be urgently addressed. Mass spectrometric studies of blood plasma proteomics provide a deep understanding of the relationship between the severe course of infection and activation of specific pathophysiological pathways. Analysis of plasma proteins in whole blood may also be relevant for the pandemic as it requires minimal sample preparation. METHODS: The frozen whole blood samples were used to analyze 203 plasma proteins using multiple reaction monitoring (MRM) mass spectrometry and stable isotope-labeled peptide standards (SIS). A total of 131 samples (FRCC, Russia) from patients with mild (n=41), moderate (n=39) and severe (n=19) COVID-19 infection and healthy controls (n=32) were analyzed. RESULTS: Levels of 94 proteins were quantified and compared. Significant differences between all of the groups were revealed for 44 proteins. Changes in the levels of 61 reproducible COVID-19 markers (SERPINA3, SERPING1, ORM1, HRG, LBP, APOA1, AHSG, AFM, ITIH2, etc.) were consistent with studies performed with serum/plasma samples. The best-performing classifier built with 10 proteins achieved the best combination of ROC-AUC (0.97-0.98) and accuracy (0.90-0.93) metrics and distinguished patients from controls, as well as patients by severity. CONCLUSIONS: Here, for the first time, frozen whole blood samples were used for proteomic analysis and assessment of the status of patients with COVID-19. The results obtained with frozen whole blood samples are consistent with those from plasma and serum.

Association between convalescent plasma treatment and mortality in COVID-19: a collaborative systematic review and meta-analysis of randomized clinical trials.

BACKGROUND: Convalescent plasma has been widely used to treat COVID-19 and is under investigation in numerous randomized clinical trials, but results are publicly available only for a small number of trials. The objective of this study was to assess the benefits of convalescent plasma treatment compared to placebo or no treatment and all-cause mortality in patients with COVID-19, using data from all available randomized clinical trials, including unpublished and ongoing trials (Open Science Framework, https://doi.org/10.17605/OSF.IO/GEHFX ). METHODS: In this collaborative systematic review and meta-analysis, clinical trial registries (ClinicalTrials.gov, WHO International Clinical Trials Registry Platform), the Cochrane COVID-19 register, the LOVE database, and PubMed were searched until April 8, 2021. Investigators of trials registered by March 1, 2021, without published results were contacted via email. Eligible were ongoing, discontinued and completed randomized clinical trials that compared convalescent plasma with placebo or no treatment in COVID-19 patients, regardless of setting or treatment schedule. Aggregated mortality data were extracted from publications or provided by investigators of unpublished trials and combined using the Hartung-Knapp-Sidik-Jonkman random effects model. We investigated the contribution of unpublished trials to the overall evidence. RESULTS: A total of 16,477 patients were included in 33 trials (20 unpublished with 3190 patients, 13 published with 13,287 patients). 32 trials enrolled only hospitalized patients (including 3 with only intensive care unit patients). Risk of bias was low for 29/33 trials. Of 8495 patients who received convalescent plasma, 1997 died (23%), and of 7982 control patients, 1952 died (24%). The combined risk ratio for all-cause mortality was 0.97 (95% confidence interval: 0.92; 1.02) with between-study heterogeneity not beyond chance (I2 = 0%). The RECOVERY trial had 69.8% and the unpublished evidence 25.3% of the weight in the meta-analysis. CONCLUSIONS: Convalescent plasma treatment of patients with COVID-19 did not reduce all-cause mortality. These results provide strong evidence that convalescent plasma treatment for patients with COVID-19 should not be used outside of randomized trials. Evidence synthesis from collaborations among trial investigators can inform both evidence generation and evidence application in patient care.

Conformational Dynamics of the Single Lipopolysaccharide O-Antigen in Solution.

The O-antigen is the most variable and highly immunogenic part of the lipopolysaccharide molecule that covers the surface of Gram-negative bacteria and makes up the first line of cellular defense. To provide insight into the details of the O-antigen arrangement on the membrane surface, we simulated its behavior in solution by molecular dynamics. We developed the energetically favorable O-antigen conformation by analyzing free-energy distributions for its disaccharide fragments. Starting from this conformation, we simulated the behavior of the O-antigen chain on long timescales. Depending on the force field and temperature, the single molecule can undergo reversible or irreversible coil-to-globule transitions. The mechanism of these transitions is related either to the rotation of the carbohydrate residues around O-glycosidic bonds or to flips of the pyranose rings. We found that the presence of rhamnose in the O-antigen chain crucially increases its conformational mobility.

Comparative study of ex vivo probe-based confocal laser endomicroscopy and light microscopy in lung cancer diagnostics.

BACKGROUND AND OBJECTIVE: Probe-based confocal laser endoscopy (pCLE) allows for real-time non-invasive histological imaging via bronchoscopy. Interpreting CLE images and correlating with traditional histopathology remains challenging. We performed an ex vivo study to evaluate the correlation between light microscopy findings and pCLE imaging of primary lung carcinoma. METHODS: Post-lobectomy specimens for lung cancer nodules were examined ex vivo by pCLE. The examined areas were marked with brilliant green dye, and the surrounding tissues were stained by methylene blue dye. Lung tissue segments were resected and histopathological specimens were generated with 50-µm thickness from the marked areas and stained with haematoxylin and eosin. Pathologists and pulmonologists reviewed the images for correlating features. RESULTS: Eighteen lobectomy specimens from 18 different patients were collected. Three primary features were observed in all samples using pCLE in the cancer surroundings: alveolar dystelectasis with thickening of alveolar walls, alveolar edema and a large amount of macrophages. The stromal and parenchymal components of the studied subtypes of non-small-cell lung cancer differed from each other. The stromal component for all nine adenocarcinoma specimens had a highly fluorescent field penetrated by dark hollows. All six squamous cell carcinoma specimens had the stromal component appeared as 'biparously' branching, highly fluorescent fibres. No stromal component was observed in any small-cell carcinoma specimen, and at low power field, the cellular component was dominant with an observed light scattering pattern. CONCLUSIONS: pCLE can identify lung carcinoma in ex vivo samples. Certain light microscopy features of lung carcinoma can be visualized with pCLE.

Disease modifying treatment of spinal cord injury with directly reprogrammed neural precursor cells in non-human primates.

BACKGROUND: The development of regenerative therapy for human spinal cord injury (SCI) is dramatically restricted by two main challenges: the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing. Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge. The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI. AIM: To investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells (drNPCs). METHODS: Seven non-human primates with verified complete thoracic SCI were divided into two groups: drNPC group (n = 4) was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury, and lesion control (n = 3) was injected identically with the equivalent volume of vehicle. RESULTS: Follow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways. Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation. Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk, migrating to areas of axon growth cones. CONCLUSION: Our data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI, based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation. The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs. Instead, directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support, thereby further supporting the regeneration processes.

First-in-human high-cumulative-dose stem cell therapy in idiopathic pulmonary fibrosis with rapid lung function decline.

Previous phase I studies demonstrated safety and some beneficial effects of mesenchymal stem cells (MSCs) in patients with mild to moderate idiopathic pulmonary fibrosis (IPF). The aim of our study was to evaluate the safety, tolerability, and efficacy of a high cumulative dose of bone marrow MSCs in patients with rapid progressive course of severe to moderate IPF. Twenty patients with forced ventilation capacity (FVC) ≥40% and diffusing capacity of the lung for carbon monoxide (DLCO) ≥20% with a decline of both >10% over the previous 12 months were randomized into two groups: one group received two intravenous doses of allogeneic MSCs (2 × 108 cells) every 3 months, and the second group received a placebo. A total amount of 1.6 × 109 MSCs had been administered to each patient after the study completion. There were no significant adverse effects after administration of MSCs in any patients. In the group of MSC therapy, we observed significantly better improvement for the 6-minute walk distance in 13 weeks, for DLCO in 26 weeks, and for FVC in 39 weeks compared with placebo. FVC for 12 months in the MSCs therapy group increased by 7.8% from baseline, whereas it declined by 5.9% in the placebo group. We did not find differences between the groups in mortality (two patients died in each group) or any changes in the high-resolution computed tomography fibrosis score. In patients with IPF and a rapid pulmonary function decline, therapy with high doses of allogeneic MSCs is a safe and promising method to reduce disease progression.

European screening for alpha1 -antitrypsin deficiency in subjects with lung disease.

BACKGROUND AND AIMS: Alpha1 -antitrypsin deficiency (AATD) predisposes individuals to early-onset emphysema. Despite its prevalence, especially among patients with chronic obstructive pulmonary disease, AATD is still underdiagnosed. The aim of this study is to identify individuals with lung disease and severe AATD in central-eastern Europe. METHODS: Subjects with respiratory symptoms that could be indicative of AATD provided blood samples as dried blood spot. The alpha1 -antitrypsin (AAT) concentration was determined by nephelometry and, if lower than 1.70 mg/dL in dried blood spot (equivalent to 1.04 g/L in serum), polymerase chain reaction was used to detect the PiS and PiZ alleles. Isoelectric focusing was used for confirmation of doubtful genotype results. RESULTS: From 13 countries, 11 648 subjects were included. Genotyping of 1404 samples with AAT levels <1.70 mg/dL revealed 71 (5.06%) PiS, 151 (10.8%) PiZ, 1 (0.071%) PiSS, 8 (0.57%) PiSZ and 32 (2.28%) PiZZ. Phenotyping of 1363 samples negative for the S and Z alleles or with PiS and PiZ genotype showed two (0.147%) PiZ(rare) and two (0.147%) Pi(null)(null). The countries with the highest rate of severe AATD were Croatia, Russia and Slovakia. By regions, the Baltic countries area showed the highest rate of both PiZ and severe AATD (2.45% and 1.20%, respectively) while the lowest rates were observed in the Balkan Peninsula (0.48% and 0.31%, respectively). CONCLUSION: This study confirms the need for targeted testing of symptomatic patients and provides AATD genotype data from countries for which only some estimates of prevalence were available until now.

Association of polymorphic markers of genes FTO, KCNJ11, CDKAL1, SLC30A8, and CDKN2B with type 2 diabetes mellitus in the Russian population.

BACKGROUND: The association of type 2 diabetes mellitus (T2DM) with the KCNJ11, CDKAL1, SLC30A8, CDKN2B, and FTO genes in the Russian population has not been well studied. In this study, we analysed the population frequencies of polymorphic markers of these genes. METHODS: The study included 862 patients with T2DM and 443 control subjects of Russian origin. All subjects were genotyped for 10 single nucleotide polymorphisms (SNPs) of the genes using real-time PCR (TaqMan assays). HOMA-IR and HOMA-ß were used to measure insulin resistance and ß-cell secretory function, respectively. RESULTS: The analysis of the frequency distribution of polymorphic markers for genes KCNJ11, CDKAL1, SLC30A8 and CDKN2B showed statistically significant associations with T2DM in the Russian population. The association between the FTO gene and T2DM was not statistically significant. The polymorphic markers rs5219 of the KCNJ11 gene, rs13266634 of the SLC30A8 gene, rs10811661 of the CDKN2B gene and rs9465871, rs7756992 and rs10946398 of the CDKAL1 gene showed a significant association with impaired glucose metabolism or impaired ß-cell function. CONCLUSION: In the Russian population, genes, which affect insulin synthesis and secretion in the ß-cells of the pancreas, play a central role in the development of T2DM.

Efficacy and safety of Ergoferon versus oseltamivir in adult outpatients with seasonal influenza virus infection: a multicenter, open-label, randomized trial.

OBJECTIVES: Ergoferon is an antiviral complex drug containing released-active forms of antibodies to interferon gamma, CD4, and histamine. Its efficacy and safety in the treatment of acute respiratory viral infections has been reported previously. The aim of this study was to compare Ergoferon with oseltamivir. METHODS: A multicenter, open-label, randomized controlled trial of patients aged 18 to 65 years, who had tested positive for influenza A or B antigens, was performed. A total of 156 patients were enrolled as the intention-to-treat population; these patients were assigned randomly to receive either Ergoferon or oseltamivir (n=78 in each group). RESULTS: The percentage of patients achieving a normal body temperature (≤37.0°Ð¡) following 5 days of treatment did not differ significantly between the groups. The mean duration of fever in the Ergoferon and oseltamivir groups was 2.1±1.5 days and 2.3±1.6 days, respectively (p=0.01). The average time to the resolution of influenza symptoms was approximately 3 days, with no significant between-group difference. Total quality of life scores were similar in the two groups following 5 days of drug administration. The incidence of adverse events did not differ significantly between the groups, nor were there any serious adverse events. CONCLUSIONS: Ergoferon and oseltamivir were equally effective and safe in adult outpatients with seasonal influenza A or B virus infection. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifierNCT01804946.

Confocal laser endomicroscopy for diagnosis and monitoring of pulmonary alveolar proteinosis.

BACKGROUND: The diagnosis of pulmonary alveolar proteinosis (PAP) is based on computed tomography, histology, and antibodies to granulocyte-macrophage colony-stimulating factor. The role of a novel technique for imaging cells and elastin during endoscopy, probe-based confocal laser endomicroscopy (pCLE), has not yet been investigated in PAP patients. The aim of the present study was to estimate the value of pCLE in the PAP diagnosis and treatment in comparison with the findings of high-resolution computed tomography (HRCT) before and after whole-lung lavage. METHODS: In vivo pCLE was performed during bronchoscopy in 6 male patients with PAP before and after whole-lung lavage. In certain lung segments, pCLE was followed by HRCT. RESULTS: During the in vivo pCLE, we found characteristic signs of PAP: a fluorescent floating amorphous substance in the alveoli lumen sticking to conglomerates along with alveolar macrophages. These features were present to a lesser extent after a whole-lung lavage. pCLE revealed specific PAP features not only in segments with crazy-paving and ground-glass opacity, but also in segments without HRCT findings. CONCLUSIONS: The alveolar imaging in PAP patients is able to reveal characteristic changes, both in the presence and in the absence of HRCT findings. Therefore, pCLE may be a helpful tool for the diagnosis and whole-lung lavage therapy. Our data prove that accumulation of lipoproteinaceous substances within the alveoli at PAP is a diffuse but not a patchy process.

The case of diagnostics of invasive pulmonary aspergillosis by in vivo probe-based confocal laser endomicroscopy of central and distal airways.

We present a case of 41-year-old patient with invasive pulmonary aspergillosis (IPA) in which probe-based confocal laser endomicroscopy (pCLE) imaging of central and distal airways was first performed in vivo. pCLE imaging showed the signs of complete or partial destruction of elastin network of alveolar wall with fibrillar branching fluorescent structures in the zone with typical IPA changes on HRCT.