Viral Load and Patterns of SARS-CoV-2 Dissemination to the Lungs, Mediastinal Lymph Nodes, and Spleen of Patients with COVID-19 Associated Lymphopenia.

Lymphopenia is a frequent hematological manifestation, associated with a severe course of COVID-19, with an insufficiently understood pathogenesis. We present molecular genetic immunohistochemical, and electron microscopic data on SARS-CoV-2 dissemination and viral load (VL) in lungs, mediastinum lymph nodes, and the spleen of 36 patients who died from COVID-19. Lymphopenia <1 × 10/L was observed in 23 of 36 (63.

Impact on N-glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties.

Prophylaxis of hemolytic disease of newborns is based on the ability of polyclonal anti-D antibodies for suppressing maternal immune response against D-positive fetal red blood cells. The immunosuppressive effect of anti-D antibody is mediated by interaction between its Fc-fragment and low-affinity IgG Fc-receptor (FcγR) on the immune cell. No clinically effective monoclonal anti-D antibody (mAb) that can replace polyclonal anti-D immunoglobulin has been developed yet.

Effect of producer cell line on functional activity of anti-D monoclonal antibodies destined for prevention of rhesus sensitization.

The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies.

TSC-22 contributes to hematopoietic precursor cell proliferation and repopulation and is epigenetically silenced in large granular lymphocyte leukemia.

Aberrant methylation of tumor suppressor genes can lead to their silencing in many cancers. TSC-22 is a gene silenced in several solid tumors, but its function and the mechanism(s) responsible for its silencing are largely unknown. Here we demonstrate that the TSC-22 promoter is methylated in primary mouse T or natural killer (NK) large granular lymphocyte (LGL) leukemia and this is associated with down-regulation or silencing of TSC-22 expression.

Long-term persistence of a nonintegrated lentiviral vector in mouse hematopoietic stem cells.

Objective: Lentiviral transduction is an established method for efficiently modifying the gene expression program of primary cells, but the ability of the introduced construct to persist as an episome has not been well studied.

Material And Methods: Here we investigated this issue in lethally irradiated female mice injected with 300 or 3000 doubly sorted male lin(neg), Sca-1(high), c-kit(high), Thy-1.1(low) mouse bone marrow cells that had been exposed in vitro to self-inactivating lentivirus vector encoding a green fluorescence protein (GFP) cDNA.

Lentivirus vector can integrate in the genome and exist and replicate in the cell as an episome.

Lethally irradiated mice were reconstituted with few purified primitive hemopoietic stem cells containing sequences of a gene encoding green fluorescent protein. The gene was transferred using a lentivirus vector. The presence of the marker gene in splenocyte colonies derived from the bone marrow of reconstituted mice and in cells of other hemopoietic and non-hemopoietic organs was studied during the life.

Dynamics of precursor cell composition in bone marrow culture derived from mice deficient by tumor necrosis factor.

Hemopoiesis in a long-term bone marrow culture derived from mice deficient for tumor necrosis factor was maintained for more than 130 weeks, which was 4 times longer than in cultures from wild type mice. The dynamics of hemopoietic precursor cells of different maturity was studied in the suspension fraction of the culture. The incidence of granulocyt-macrophage precursor cells and cell forming cobblestone areas was studied by the method of limiting dilutions in culture.

Developmental fate of hematopoietic stem cells: the study of individual hematopoietic clones at the level of antigen-responsive B lymphocytes.

We have shown previously that hematopoiesis in mice reconstituted with retrovirally marked hematopoietic stem cells (HSCs) is provided by multiple, mainly short-lived clones, as measured by retroviral insertion site analysis of individual spleen colony-forming unit (CFU-S)-derived colonies. However, the CFU-S is the relatively early progenitor and the contribution of each CFU-S in the steady-state hematopoiesis is uncertain. Here, we have studied the fate of individual mature B cells, as well as CFU-S, representing the progeny of retrovirally transduced marrow-repopulating cells (MRC).

Neoplastic transformation is not the cause of extremely long (more than 100 weeks) hematopoiesis maintenance of long-term bone marrow culture from TNF-deficient mice.

Total cell production and longevity of hematopoiesis in long-term bone marrow culture of tumor necrosis factor (TNF)-deficient mice (LTBM-TNFko) are increased. The rate of apoptosis is decreased during the first 40 weeks in culture, then the level of apoptosis reaches levels of wild-type cultures. Extended lifespan of primary cultures usually is the consequence of the neoplastic transformation.

Long-term self-maintenance of hemopoietic precursors in bone marrow culture derived from tumor necrosis factor-deficient mice is not a result of neoplastic transformation.

In long-term bone marrow cultures derived from tumor necrosis factor-deficient mice the total cell production and the total duration of hemopoiesis are increased (the latter is comparable with mouse life span). Telomerase activity in cells of nonadherent fraction of long-term bone marrow cultures from tumor necrosis factor-deficient mice increases with time and peaks after 1-year culturing. Karyotyping of nonadherent and adherent cells of long-term bone marrow cultures revealed instability of nonadherent cells and hyperploidy of the stromal sublayer cells, which attested to the presence of a neoplastic transformation.

Radiation-induced hemopoietic cell growth factor: detection in a culture.

A simple, rapid, and easily reproducible method was developed for testing activity stimulating the growth of hemopoietic microenvironment in long-term bone marrow culture. It was found that in irradiated mice this activity is produced by bones.

Abnormalities in long-term bone culture from mice deficient in tumor necrosis factor: induction of apoptosis and cell proliferation.

The total duration of hemopoiesis and the total cell production in long-term bone marrow cultures from mice deficient by tumor necrosis factor are increased, while proliferation of granulocyte-macrophage precursor cells, the main cell populations in long-term bone marrow cultures, did not differ from that in wild-type mice. In bone marrow cultures from knockout mice the intensity of apoptosis remained low during 40-week culturing and was similar to that in early wild-type cultures (10 weeks). Then, the intensity of apoptosis in bone marrow cultures from knockout mice did not differ or even surpassed that in wild-type cultures.

Genomic structure and alternative splicing of the murine bhk/ctk/ntk gene.

Recently, we and others have cloned cDNAs encoding a second member of the Csk family of inhibitory protein kinases, which we termed Bhk [M.A. Ershler et al.

Sequence of the cDNA encoding murine CRK4 protein kinase.

A cDNA clone encoding murine CRK4 protein kinase [Ershler et al., Gene 124 (1993) 305-306] has been isolated and sequenced. The deduced amino acid (aa) sequence shows 85% overall identity with the Xenopus laevis MO15 PK (the catalytic subunit of CAK).

Cell cycle regulation of the p34cdc2/p33cdk2-activating kinase p40MO15.

A key component of Cdc2/Cdk2-activating kinase (CAK) is p40MO15, a protein kinase subunit that phosphorylates the T161/T160 residues of p34cdc2/p33cdk2. The level and activity of p40MO15 were essentially constant during cleavage of fertilised Xenopus eggs and in growing mouse 3T3 cells, but serum starvation of these cells reduced both the level and activity of p40MO15. Although the level and activity of endogenous p40MO15 did not vary in the cell cycle, we found that bacterially expressed p40MO15 was activated more rapidly by M-phase cell extracts than by interphase cell extracts.

Novel CDC2-related protein kinases produced in murine hematopoietic stem cells.

The polymerase chain reaction with degenerate primers was used for the amplification of cDNA encoding CDC2-related protein kinase (PK) sequences from murine hematopoietic stem cells. In total, nine different PK-encoding sequences were obtained. At least four of them encode previously unknown PKs.