Therapy of a refractory idiopathic thrombocytopenic purpura by vinblastine-loaded thrombocytes (author's transl)

A 15-year-old patient with ITP which was refractory to corticosteroids, splenectomy, and immunosuppressive therapy with vincristine was twice treated with platelets loaded with vinblastine. Five days after the application of the platelets vinblastine complex the platelets began to rise up to 600 X 10(9)/l. The remission has lasted until now for more than 15 weeks. The therapy showed no major side effects except for a transient granulocytopenia.     

Lipid-protein interactions at the erythrocyte membrane. Different influence of glucose and sorbose on membrane lipid transition

When observed over a temperature range, erythrocyte membrane lipids undergo a transition at 18–20 °C (Zimmer, G. and Shirmer, H. (1974) Biochim. Biophys. Acta 345, 314–320). This observation has prompted an investigation of the effects that substrate binding has on the transition of the red cell membrane. Glucose and sorbose were compared, since transport kinetics of these sugars still pose unresolved questions.In membranes, preloaded with glucose, the break at the transition temperature was intensified, while it was abolished or reversed in membranes preloaded with sorbose.These results were corroborated using different solubilization procedures (sonication, sodium dodecyl sulfate treatment) of the membranes, and also different techniques (viscosimetry, 90° light scattering, 1-anilino-naphthalene-8-sulfonate fluorescence).In extracted membrane lipids, viscosimetry indicated a break at transition temperature after preloading with either glucose or sorbose.Disc electrophoresis revealed a different binding pattern of the two sugars.It is suggested, that the amplification of the discontinuity in red cell membranes by glucose and the abolition or reversal of the break by sorbose are mediated by membrane protein- and/or membrane lipid-protein interaction.     

Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

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Cutting edge: a role for B lymphocyte stimulator in systemic lupus erythematosus

Increased levels of B lymphocyte stimulator (BLyS) are associated with systemic autoimmunity in animal models of spontaneous autoimmune disease, and transgenic animals expressing BLyS develop typical autoimmune disease. Here, we demonstrate significant elevations of BLyS in the patients with systemic lupus erythematosus (SLE). The BLyS isolated from the sera of SLE patients had the same m.w. as the natural soluble form and was able to stimulate B cell activation in vitro. Increased BLyS in SLE patients was partially associated with higher levels of anti-dsDNA Ab of the IgG, IgM, and IgA classes, but not associated with the disease activity. Our results suggest that BLyS may be a useful marker for early activation of an autoimmune diathesis and likely plays a critical role in triggering activation of self-Ag-driven autoimmune B cells in human SLE. BLyS may provide an effective therapeutic target in systemic autoimmunity.     

The jamming avoidance response in gymnotoid pulse-species: A mechanism to minimize the probability of pulse-train coincidence

Summary Gymnotoid electric fish with pulse-type electric organ discharges (EODs) shorten (lengthen) their EOD intervals as pulses of a slightly slower (faster) train scan their EODs (Figs. 1, 2). They thus minimize the chance of pulse coincidence by transient accelerations (decelerations) of their EOD rate.Studies in curarized preparations demonstrate that this Jamming Avoidance Response (JAR) is controlled by electroreceptive input alone and without reference to an internal electric organ pacemaker-related signal (Fig. 8). A sufficient stimulus input consists of a train of strong, EOD-like stimulus pulses (S₁), which mimic the animal's experience of its own EOD, and a train of small pulses (S₂) of slightly different repetition rate, which mimic EODs of a neighbor. Correct behavioral responses require S₁ pulses of sufficient intensity to recruit pulse-markertype receptors; also spatial and temporal patterns must closely resemble those of the animal's EOD. These features are of little significance for S₂ pulses which, while scanning S₁ pulses, only provide a small perturbation of electroreceptive feedback from S₁ pulses. Inappropriate S₁ stimulation impairs and sometimes reverses (Fig. 7) the behavioral discrimination of scan directions. The JAR is explained in terms of excitatory and inhibitory processes (Fig. 3) which are triggered by S₂ stimulation, at specific phases within the S₁ cycle (Figs. 4–6).The JAR in pulse species strongly resembles the JAR in wave-species (Bullock et al., 1972) and could be considered an evolutionary ancestor of the latter. It is a response to a particular novelty in electroreceptive feedback.We thank Drs. T.H. Bullock, C. Hopkins and an anonymous referee for most helpful criticism. This research was supported by NSF grand BMS74-18640 and NIMH grant PHSMH-2614901 to W.H. and NIH grant/ROI NS 12337-01 to J.B.     

Catalytic antibodies in the bone marrow and other organs of Th mice during spontaneous development of experimental autoimmune encephalomyelitis associated with cell differentiation

Exact mechanisms of autoimmune disease development are still yet unknown. However, it is known that the development of autoimmune diseases is associated with defects in the immune system, namely, the violation of the bone marrow hematopoietic stem cells (HSCs) differentiation profiles. Different characteristics of autoimmune reaction development in experimental autoimmune encephalomyelitis (EAE) prone Th mice characterizing T-lymphocytes response were analyzed using standard approaches. Profiles of several HSCs differentiation of bone marrow (BFU-E, CFU-E, CFU-GM, CFU-GEMM, T- and B-lymphocytes) of Th male and female mice during spontaneous development of EAE were noticeably different. Patterns of total lymphocytes, B- and T-cells proliferation in several different organs (bone marrow, blood, spleen, thymus, and lymph nodes) were also remarkably different. In addition, there were in time noticeable differences in their changes for some organs of male and female mice. Characters of changes in the profiles of CD4 and CD8 cells proliferation in some organs not always coincide with those for total T lymphocytes. The changes in the differentiation profiles of HSCs and the level of lymphocytes proliferation in the bone marrow and other organs were associated with the increase in the concentration of antibodies against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, and catalytic antibodies hydrolyzing these substrates. Despite some differences in changes in the analyzed parameters, in general, the spontaneous development of EAE in male and female mice occurs to some extent in a comparable way.

     

Signal Peptide Cleavage from GP5 of PRRSV: A Minor Fraction of Molecules Retains the Decoy Epitope,a Presumed Molecular Cause for Viral Persistence

Porcine reproductive and respiratory syndrome virus (PRRSV) is the major pathogen in the pig industry. Variability of the antigens and persistence are the biggest challenges for successful control and elimination of the disease. GP5, the major glycoprotein of PRRSV, is considered an important target of neutralizing antibodies, which however appear only late in infection. This was attributed to the presence of a “decoy epitope” located near a hypervariable region of GP5. This region also harbors the predicted signal peptide cleavage sites and (dependent on the virus strain) a variable number of potential N-glycosylation sites. Molecular processing of GP5 has not been addressed experimentally so far: whether and where the signal peptide is cleaved and (as a consequence) whether the “decoy epitope” is present in virus particles. We show that the signal peptide of GP5 from the American type 2 reference strain VR-2332 is cleaved, both during in vitro translation in the presence of microsomes and in transfected cells. This was found to be independent of neighboring glycosylation sites and occurred in a variety of porcine cells for GP5 sequences derived from various type 2 strains. The exact signal peptide cleavage site was elucidated by mass spectrometry of virus-derived and recombinant GP5. The results revealed that the signal peptide of GP5 is cleaved at two sites. As a result, a mixture of GP5 proteins exists in virus particles, some of which still contain the “decoy epitope” sequence. Heterogeneity was also observed for the use of glycosylation sites in the hypervariable region. Lastly, GP5 mutants were engineered where one of the signal peptide cleavage sites was blocked. Wildtype GP5 exhibited exactly the same SDS-PAGE mobility as the mutant that is cleavable at site 2 only. This indicates that the overwhelming majority of all GP5 molecules does not contain the “decoy epitope”.     

A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA

Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR) or RIG-I like helicases (RLH) are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA) that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4) was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC) based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand) in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.     

Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.