[Detection rates and high concentration of herpesvirus (Orthoherpesviridae) DNA in autopsy materials from patients with COVID-19 fatal outcome].

INTRODUCTION: SARS-CoV-2 infection causes immune disorders that create conditions for the reactivation of human herpesviruses (HHVs). However, the estimates of the HHVs effect on the course and outcome of COVID-19 are ambiguous. Аim - to study the possible relationship between the HHV reactivation and the adverse outcome of COVID-19. MATERIALS AND METHODS: Postmortem samples from the brain, liver, spleen, lymph nodes and lungs were obtained from 59 patients treated at the Moscow Infectious Diseases Hospital No.1 in 2021-2023. The group 1 comprised 39 patients with fatal COVID-19; group 2 (comparison group) included 20 patients not infected with SARS-CoV-2 who died from various somatic diseases. HHV DNA and SARS-CoV-2 RNA were determined by PCR. RESULTS: HHV DNA was found in autopsy samples from all patients. In group 1, EBV was most often detected in lymph nodes (94%), HHV-6 in liver (68%), CMV in lymph nodes (18%), HSV in brain (16%), VZV in lung and spleen (3% each). The detection rates of HHVs in both groups was similar. Important differences were found in viral load. In patients with COVID-19, the number of samples containing more than 1,000 copies of HHV DNA per 100,000 cells was 52.4%, in the comparison group - 16.6% (p < 0.002). An association has been established between the reactivation of HSV and HHV-6 and the severity of lung damage. Reactivation of EBV correlated with increased levels of liver enzymes. CONCLUSION: Reactivation of HHVs in patients with fatal COVID-19 was associated with severe lung and liver damages, which indicates a link between HHV reactivation and COVID-19 deaths.

[Determination of cobalt in plasma blood samples by the ICP-MS method after oral intake of dietary supplements containing low doses of cobalt].

Salts of inorganic cobalt (Со) prevent the degradation of the alpha subunit of the hypoxia-inducible factor (HIF), imitating the state of hypoxia in the body and increasing the production of the endogenous hormone erythropoietin (EPO), and are used as doping substances that increase blood oxygen capacity and endurance, which give competitive advantages in sports. Currently, a large number of dietary supplements, including Co-containing ones, are offered on free sale. Their uncontrolled intake can affect not only the professional career of athletes, but also their health, due to the fact that this trace element and its salts are the strongest inorganic poisons and carcinogens. Despite this, their availability on the pharmaceutical market, a noticeable effect of erythropoiesis stimulation and a convenient oral form of administration lead to the need for their detection in modern doping control. The purpose of this research was to develop an approach to differentiate cobalt from vitamin B12, present in the body in its natural state, from the intake of cobalt salts by quantifying and comparing blood levels of vitamin B12 and total cobalt. Methods. The study involved 9 healthy volunteers (women and men) aged 25 to 45 years, leading an active lifestyle. Three of them took 2500 µg/day of cobalamin for 20 days (comparison group), three - dietary supplement containing cobalt asparaginate (100 µg/day in terms of pure cobalt), and the rest - dietary supplements with cobalt sulfate heptahydrate (100 µg/day in terms of pure cobalt) (administration groups) at the same time after meals. Blood samples were taken at baseline and on days 5, 9, 14 and 20. The concentrations of total cobalt in blood plasma samples of volunteers were measured by inductively coupled plasma mass-spectrometry (ICP-MS), the levels of cobalamin were determined on a Cobas 6000 immunochemical analyzer using the Elecsys Vitamin B12 II Assay ELISA kits. Results. It was found that oral intake of of cobalamin at a therapeutic dose significantly exceeding the recommended daily intake (3 µg), there was a regular slight increase in the blood concentration of total cobalt (1.1 times). At the same time intake of dietary supplements containing cobalt in the form of sulfate or asparaginate (about 100 µg per day in terms of pure cobalt) was accompanied by 4-6.7 fold increase in the concentration of total cobalt while unchanged vitamin B12 plasma concentration was observed. The detection of such changes can reliably indicate the use of prohibited salts and, of course, will be in demand for anti-doping control. Conclusion. Long-term monitoring of vitamin B12 and total cobalt levels, similar to hematological module of the Athlete Biological Passport program, will unambiguously detect possible abuse of cobalt salts and can be an additional evidence of the presence of these doping substances to other analytical methods, such as a combination of liquid chromatography and ICP-MS (LC-ICP-MS).

Stress load and neurodegeneration after gastrostomy tube placement in amyotrophic lateral sclerosis patients.

Dysphagia and progressive swallowing problems due to motoneuron death is one of amyotrophic lateral sclerosis (ALS) symptoms. Malnutrition and body weight loss result in immunological disturbances, fatigability and increase risk of secondary complications in ALS patients, percutaneous endoscopic gastrostomy tube (PEG) placement representing a well-recognized method for malnutrition correction and potentially increasing life expectancy. However, despite nutritional correction, occasional rapid neurological deterioration may develop after PEG placement. We have hypothesized that this decline can be a result of exteroceptive stress during PEG placement and promote neurodegeneration in ALS patients. Intravenous sedation may decrease stress during invasive procedures and it is safe during PEG placement in ALS patients. The aim of the study was comparing different PEG placement protocols of anesthesia (local anesthesia or local anesthesia plus intravenous sedation) in ALS from perspectives of stress load and neurological deterioration profile. During 1.5 years 94 ALS patients were admitted; gastrostomy was performed in 79 patients. After screening according to inclusion and exclusion criteria, 30 patients were included in the prospective consecutive study. All patients were divided in two groups, with local anesthesia and with combination of local anesthesia and intravenous sedation. Routine biochemical indices, neurodegeneration and stress markers were measured. The age of ALS patients was 61 ± 10 years; 20 patients were included at stage 4A and 10 at stage 4B (King's College staging). PEG was placed at average14 months after the diagnosis and 2.2 years after first symptoms. Mean ALS Functional Rating Scale-Revised was 27.8, mean forced vital capacity of lung 46.3% (19-91%). After one year of observation only 8 patients survived. Mean life duration after PEG was 5 months (5 days-20 months). Comparison of two PEG placement protocols did not reveal differences in survival time, stress load and inflammation level. Higher saliva cortisol levels, serum cortisol, glucose, C-reactive protein and interleukin-6 were detected after PEG placement, confirming considerable stress response. PEG is a stressful factor for ALS patients, PEG placement representing a natural model of exteroceptive stress. Stress response was detected as increased cortisol, C-reactive protein, interleukin-6, and glucose levels. Intravenous sedation did not increase the risk of PEG placement procedure, however, sedation protocol did not affect stress load.

GMP production of anti-tumor cytotoxic T-cell lines for adoptive T-cell therapy in patients with solid neoplasia.

BACKGROUND: The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use. METHODS: Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way. RESULTS: Large amounts of anti-tumor CTL (range 14-20 x 10(9)), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3(+) CD8(+) T cells, while CD4(+) T lymphocytes and/or NK cells were less than 10%. DISCUSSION: Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches.

Early acute necrosis, delayed apoptosis and cytoskeletal breakdown in cultured cerebellar granule neurons exposed to methylmercury.

Cerebellar granule cells (CGCs) are a sensitive target for methylmercury (MeHg) neurotoxicity. In vitro exposure of primary cultures of rat CGCs to MeHg resulted in a time- and concentration-dependent cell death. Within 1 hr exposure, MeHg at 5-10 microM caused impairment of mitochondrial activity, de-energization of mitochondria and plasma membrane lysis, resulting in necrotic cell death. Lower MeHg concentrations (0.5-1 microM) did not compromise cell viability, mitochondrial membrane potential and function at early time points. Later, however, the cells progressively underwent apoptosis and 100% cell death was reached by 18 hr treatment. Neuronal network fragmentation and microtubule depolymerization were detected as early as within 1.5 hr of MeHg (1 microM) exposure, long before the occurrence of nuclear condensation (6-9 hr). Neurite damage worsened with longer exposure time and proceeded to the complete dissolution of microtubules and neuronal processes (18 hr). Microtubule stabilization by taxol did not prevent MeHg-induced delayed apoptosis. Similarly ineffective were the caspase inhibitors z-VAD-fluoromethylketone and z-DEVD-chloromethylketone, the L-type calcium channel inhibitor nifedipine, the calcium chelator EGTA and BAPTA, and the NMDA receptor antagonist MK-801. On the other hand, insulin-like growth factor-I partially rescued CGCs from MeHg-triggered apoptosis. Altogether these results provide evidence that the intensity of MeHg insult is decisive in the time of onset and the mode of neuronal death that follows, i.e., necrosis vs. apoptosis, and suggest that cytoskeletal breakdown and deprivation of neurotrophic support play a role in MeHg delayed toxicity.