PD-1-Expressing SARS-CoV-2-Specific CD8+ T Cells Are Not Exhausted,but Functional in Patients with COVID-19

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Investigation on the Maternal-Infantile Infection with Human Parvovirus B19

With the investigations on pregnant women and newbornsinfected withToxoplasma, rubella virus, cytomegalovirus,herpes simplex virus (TORCH), it was found that humanparvovirus B19 (B19 virus), which belongs to the familyParvoviridae and the genus Erythrovir…     

Fluoropyrimidine chemotherapy in a rat model: comparison of fluorouracil metabolite profiles determined by high-field 19F-NMR spectroscopy of tissues ex vivo with therapy response and toxicity for locoregional vs systemic infusion protocols

A Sprague-Dawley rat model with DS sarcoma transplanted in the thigh was used to compare transcatheter locoregional i.a. and systemic i.v. administration of 5-fluorouracil (FU) at 12 dose-rate schedules: 25, 50 and 100 mg/kg; bolus, 1, 5 and 24 h infusions. In experiment A tumor (62/67 animals) as well as liver and kidney (56/67 animals) were excised 1 h after a single bolus or 1 h infusion or at the end of 5 and 24 h infusions. (19)F-NMR spectroscopy at 11.7 T was used to quantitate FU and its metabolites in ca. 1 g of tissue at 4 degrees C. In experiment B analogous FU treatments were repeated for 5 days (rats 80+11 controls). Tumor volumes vs time, various blood parameters and survival times were recorded, and a log growth rate parameter log GR, a response index RI, and a toxicity index TI were calculated. The i.a. vs i.v. ratios for tumor concentrations of FU and total anabolites (F-Nucl) were >1 for nearly all treatments and increased with infusion time at the higher doses. F-Nucl in tumor correlated linearly with total fluorine concentration (Tot. F range 30-1100 nmol/g) over all treatments (r=0.92, slope=0.45, p<0.0001). For non-bolus i.v. treatments [FU+F-Nucl] decreased linearly with decreasing FU dose rate (r(2)=0.74, zero intercept), while i.a. treatments showed non-linear behavior. For non-bolus treatments the mean log GR per treatment group showed a negative correlation (r=-0.87) with log[F-Nucl]. The most effective non-toxic treatments were 25 mg/kg over 5 or 24 h; the i.a. route was superior to i.v. on the basis of [FU+F-Nucl], RI, the reduction in log GR, and Kaplan-Meier survival statistics. For liver and kidney Tot. F (>83% FU catabolites) reached ca. 3-4 and 6-7 micromol/g, respectively, at the highest dose rates for either route; F-Nucl were detected only for Tot. F>500 nmol/g and increased exponentially as Tot. F increased (toxic treatments). The concentrations of the main catabolite (alpha-fluoro-beta-alanine, FBAL) in tumor did not correlate with Tot. F but rather with FBAL levels in kidney (r=0.90, all treatments), indicating that uptake of liver-derived FBAL from the circulation is the major source of FBAL in tumor.     

T helper cell-mediated interferon-gamma expression after human parvovirus B19 infection: persisting VP2-specific and transient VP1u-specific activity

Human parvovirus B19 is a small non-enveloped DNA virus with an icosahedral capsid consisting of proteins of only two species, the major protein VP2 and the minor protein VP1. VP2 is contained within VP1, which has an additional unique portion (VP1u) of 227 amino acids. We determined the ability of eukaryotically expressed parvovirus B19 virus-like particles consisting of VP1 and VP2 in the ratio recommended for vaccine use, or of VP2 alone, to stimulate, in an HLA class II restricted manner, peripheral blood mononuclear cells (PBMC) to proliferate and to secrete interferon gamma (IFN-gamma) and interleukin (IL)-10 cytokines among recently and remotely B19 infected subjects. PBMC reactivity with VP1u was determined specifically with a prokaryotically expressed VP1u antigen. In general, B19-specific IFN-gamma responses were stronger than IL-10 responses in both recent and remote infection; however, IL-10 responses were readily detectable among both groups, with the exception of patients with relapsed or persisting symptoms who showed strikingly low IL-10 responses. Whereas VP1u-specific IFN-gamma responses were very strong among the recently infected subjects, the VP1u-specific IFN-gamma and IL-10 responses were virtually absent among the remotely infected subjects. The disappearance of VP1u-specific IFN-gamma expression is surprising, as B-cell immunity against VP1u is well maintained.     

Molecular testing for detection of in vitro infectivity of plasma pools contaminated with B19 virus

B19 virus can be transmitted by contaminated blood or blood products. Recent observations, in healthy volunteers, suggest that active B19 infection can follow the administration of plasma pools with a concentration > or =10(7) genome equivalents/ml (geq/ml) of B19 DNA. However, patients receiving batches with levels of virus DNA lower than 10(4) geq/ml do not show any evidence of transmission of the virus. The aim of the study was to show, by in vitro assays, a threshold of viral load in B19 contaminated plasma pools over which the infection can be transmitted. Twenty plasma pools, each containing 960 single donations, were tested to correlate the viral load and the level of antibodies anti-B19 with the in vitro infectivity and expression of B19 virus. All the plasma pools, titrated for B19 viral load by competitive PCR, were inoculated into KU812Ep6 erythroid human cell line. Five of the nine contaminated plasma pools, with a B19 DNA concentration > or =3.60 x 10(6) geq/ml, were able to infect KU812Ep6 cells. In vitro infectivity was shown in KU812Ep6 cells at 24 h post-infection by in situ hybridisation and amplification assays for viral DNA and RNAs. Plasma pools with a viral load in the range of 6.00 x 10(3)-8.96 x 10(4) geq/ml did not show infectivity when inoculated into KU812Ep6 cells. Medium-high titres of IgG antibodies anti-B19 were detectable in all the plasma pools and the neutralising activity associated with specific IgG anti-B19 may explain the lack of infectivity of plasma pools contaminated with a low viral load. In conclusion, in situ hybridisation and amplification assays for viral DNA and RNAs in KU812Ep6 cells inoculated with plasma pools can be valid assays to test for the presence of infectious virus in the production of B19-safe material.     

Fibre type specific expression of Leu19-antigen and N-CAM in skeletal muscle in various stages after experimental denervation

Leu-19 antigen, which seems to be identical with neural cell adhesion molecule (N-CAM), plays a major role in the innervation of muscle cells, and in adult muscle appears after denervation and during regeneration of muscle fibres, where it acts as part of a signalling system increasing the probability of re-innervation. This combined enzyme-histochemical and immunohistochemical study examined whether this signalling process was regulated in a uniform or differential pattern for type 1 and type 2 muscle fibres. The subscapular nerve of 18 rabbits was transsected with subsequent complete denervation of the supraspinatus muscle. Leu19 and N-CAM immunohistochemistry was performed 2 to 64 days after surgery. Whereas in normal muscle there are virtually no Leu-19/N-CAM positive muscle fibres; from day 2 after denervation an increasing proportion of fibres expressed Leu-19/N-CAM, prior to any neurogenic atrophy. In the early stage of denervation Leul9/N-CAM expression was confined to type 1 fibres. After 11 days nearly all fibres were Leul9/N-CAM positive irrespective of their fibre type. Sixty-four days after denervation type 1 fibres became Leu19/N-CAM negative, while atrophic type 2 fibres showed intensive staining. Thus, expression of Leu-19 antigenity is differently regulated in both fibre types.     

The CR2/CD19 complex on human B cells contains the src-family kinase Lyn

The complement receptor 2 (CR2 or CD21) can be found in non-covalentassociation with the Blymphocyte specific CD19 complex at thesurface of mature human B cells. Upon ligation of the B cellantigen receptor complex (BCR), members of the CR2-CD19 complexmay associate with membrane immunoglobulin (mlg). Moreover,CD19 and CD21 ligands, either murine mAb, C3d fragments or Epstein—Barrvirus, are known to have profound effects on B cell activation.We here show that CD19 is tightly linked to the non-receptorsrc kinase Lyn and that the CD19 glycoprotein itself servesas a substrate for a yet undefined serine/threonine kinase presentwithin the complex. In the process of antigen recognition, mlgand the CR2-CD19 complex may bind different sites of a complement-opsonizedantigenic particle. We hypothesize that in this process, approximationto the BCR allows CD19-associated Lyn kinase to phosphorylatepotential substrates within the antigen—receptor complex,thereby effecting its coupling to the intracellular compartment.     

Impact of anxiety,stress and depression related to COVID-19 pandemic on the course of hereditary angioedema with C1-inhibitor deficiency

Background

Hereditary angioedema (HAE) attacks can be provoked with psychological factors. The aim of this study was to assess the effects of anxiety, depression and stress related to COVID-19 pandemic on disease activity of HAE patients during the quarantine period (QP) and the return to normal period (RTNP).

Methods

This study was conducted between March 2020 and September 2020 in four allergy centres. Demographic, clinical features and mental health status were evaluated in QP (from March to the beginning of June) and RTNP (from June to the beginning of September) applied by the government. The 10-point visual analogue scale (VAS10) was used to define the severity of HAE attacks. Depression, Anxiety and Stress Scales-21 (DASS-21) and Fear of COVID-19 (FC-19) scale were performed to assess mental health status.

Results

139 HAE patients were included in the study. In QP, median attack numbers and median VAS10 scores were 5 (min-max: 0–45) and 6 (min-max: 0–10), respectively. HAE attack numbers, DASS-21 stress, anxiety, depression and total DASS-21 scores, and FC-19 scores were higher in QP than RTNP (p = 0.001, p < 0.001, p = 0.001, p < 0.001, p < 0.001, p < 0.001, respectively). However, there was no difference in attack severity scores between the two periods (p > 0.05).

Conclusions

This study revealed that the restriction measures during COVID-19 outbreak cause an increase in the number of HAE attacks in relation to anxiety, depression, stress and fear of COVID-19 pandemic. Therefore, it is important to provide psychological support to HAE patients during the pandemic.