SARS-CoV-2 and nervous system: From pathogenesis to clinical manifestation.

Since the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a growing body of evidence indicates that besides common COVID-19 symptoms, patients may develop various neurological manifestations affecting both the central and peripheral nervous systems as well as skeletal muscles. These manifestations can occur prior, during and even after the onset of COVID-19 general symptoms. In this Review, we discuss the possible neuroimmunological mechanisms underlying the nervous system and skeletal muscle involvement, and viral triggered neuroimmunological conditions associated with SARS-CoV-2, as well as therapeutic approaches that have been considered for these specific complications worldwide.

Effect of Region of Interest Size on the Repeatability of Quantitative Brain Imaging Biomarkers.

In the repeatability analysis, when the measurement is the mean value of a parametric map within a region of interest (ROI), the ROI size becomes important as by increasing the size, the measurement will have a smaller variance. This is important in decision-making in prospective clinical studies of brain when the ROI size is variable, e.g., in monitoring the effect of treatment on lesions by quantitative MRI, and in particular when the ROI is small, e.g., in the case of brain lesions in multiple sclerosis. Thus, methods to estimate repeatability measures for arbitrary sizes of ROI are desired. We propose a statistical model of the values of parametric map within the ROI and a method to approximate the model parameters, based on which we estimate a number of repeatability measures including repeatability coefficient, coefficient of variation, and intraclass correlation coefficient for an ROI with an arbitrary size. We also show how this gives an insight into related problems such as spatial smoothing in voxel-wise analysis. Experiments are conducted on simulated data as well as on scan-rescan brain MRI of healthy subjects. The main application of this study is the adjustment of the decision threshold based on the lesion size in treatment monitoring.

In Vitro Effects of Propranolol on T Helper Type 1 Cytokine Profile in Human Leukemic T Cells.

INTRODUCTION: Cytokines are a large group of proteins play a key role in inflammation. Down-regulation of pro-inflammatory cytokines has beneficial effect on heart function. Propranolol, as a non selective beta-adrenergic blocker, has been extensively used for treatment of many cardiovascular problems such as arrhythmias and heart malfunction. In addition anti-inflammatory effects of propranolol have been revealed. In this study the propranolol effect on T helper type 1 cytokine profile in human leukemic T cells has been assessed in vitro. MATERIALS AND METHODS: Human leukemic T cells (Molt-4 and Jurkat) were cultured in complete RPMI medium. The cells were then incubated with different concentrations of propranolol (0.03- 30 µM) in the presence or absence of PHA (10 µg/ml) for 48 hours. The supernatants of cell culture media were collected and used for cytokines assay. RESULTS: Propranolol significantly decreased the T helper type 1 cytokine profile [Interleukin-2 (IL-2) and Interferon- γ (IFN-γ)] production in PHA stimulated Molt-4 and Jurkat cells, after 48 hour of incubation time, dose-dependently compared to untreated control cells. CONCLUSION: Our data showed a dose dependent inhibitory effect of propranolol on the IL-2 and IFN-γ production in human leukemic Molt-4 and Jurkat cells. The anti- inflammatory effect of propranolol reported by other investigators may be in part due to its suppressive effect on production of inflammatory cytokines such as IL-2 and IFN-γ. So, propranolol along with its chronic long-term usage in cardiovascular problems may have potential implication in treatment of inflammatory-based disorders.

Lipopolysaccharide Effect on Vascular Endothelial Factor and Matrix Metalloproteinases in Leukemic Cell Lines In vitro.

BACKGROUND: Angiogenesis, the process of new vessels generation, plays a critical role in tumor invasion and metastasis. Vascular Endothelial Growth Factor (VEGF), as a cytokine, and Matrix Metalloproteinases (MMPs), has been the important factors that involved in angiogenesis. Lipopolysaccharide (LPS) has an essential effect on angiogenesis. OBJECTIVES: In this study the effect of LPS on VEGF production and MMP-2/MMP-9 activity in two leukemic cell lines has been assessed in vitro. MATERIALS AND METHODS: Human leukemic U937 and THP1 cells were cultured in complete RPMI medium. Then the cells at the exponential growth phase were incubated with different concentrations of LPS (0 - 4 µg/mL) for 48 hours. Then the level of VEGF production and MMP-2/MMP-9 activity in cell culture supernatants were evaluated with the ELISA standard kits and gelatin zymography respectively. RESULTS: U937 cells have produced a large amount of VEGF without any stimulus and LPS has not shown any substantial effect on VEGF production by these cells. However THP1 cells have produced a small amount of VEGF without stimulation and LPS significantly has increased VEGF production in these cells dose-dependently. Moreover LPS significantly has augmented the MMP-2/MMP-9 activity in the both leukemic cell lines in a dose-dependent manner. CONCLUSIONS: Our results have shown that LPS might be a potential inducer/enhancer of VEGF production and MMP-2/MMP-9 activity (angiogenic factors) in leukemia. Moreover the LPS effect on angiogenesis might be in part, due to its stimulatory effects on VEGF and MMPs. Overall LPS-stimulated leukemic cells might be good models for study and planning the useful therapeutic approaches for angiogenesis- dependent diseases.

The role of leukotrienes in immunopathogenesis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder of joints for which there is no strict cure. However, conventional medications can reduce inflammation, relieve pain, and slow joint damage. Leukotrienes are a family of paracrine agents derived from oxidative metabolism of arachidonic acid. Synthesis of lipid mediators and subsequent induction of receptor activity are tightly regulated under normal physiological conditions, so that enzyme and/or receptor dysfunction can lead to a variety of clinical signs and symptoms of disease, such as local pain and tissue edema. In these tissues, immunocompetent cells accumulate at the site of injury, contributing to tissue damage and perpetuation of the disease process. Leukotrienes (often leukotriene B4) as potent chemotactic agents can provoke most signs and symptoms in rheumatoid arthritis by initiating, coordinating, sustaining, and amplifying the inflammatory response, through recruitment of leukocytes. A number of studies have reported that pharmacological modulation in this field can significantly attenuate clinical manifestations associated with different inflammatory pathologies.

Suppression of gelatinase activity in human peripheral blood mononuclear cells by verapamil.

OBJECTIVE: Gelatinases are a large group of proteolytic enzymes that belong to the matrix metalloproteinases (MMPs). MMPs are a broad family of peptidases, which proteolyse the extracellular matrix and have an important role in inflammation. Verapamil is a calcium channel blocker extensively used in the treatment of numerous cardiovascular diseases such as arrhythmia and hypertension. The anti-tumor and anti-inflammatory effects of verapamil have also been shown. In this study, the effect of verapamil on gelatinase activity in human peripheral blood mononuclear cells (PBMCs) has been assessed in vitro. MATERIALS AND METHODS: In this experimental study, PBMCs from healthy adult volunteers were isolated by ficoll-hypaque-gradient centrifugation. The cells were then cultured in complete RPMI-1640 medium and after that incubated with different concentrations of verapamil (0-200 µM) in the presence or absence of phytoheamagglutinin (PHA) (10 µg/ml) for 48 hours. The gelatinase A (MMP-2)/gelatinase B (MMP-9) activity in cell-conditioned media was then evaluated by gelatin zymography. Statistical comparisons between groups were made by analysis of variance (ANOVA). RESULTS: Verapamil significantly decreased the MMP-2/MMP-9 activity in human PBMCs after 48 hours incubation time compared with untreated control cells. The association was dose-dependent. CONCLUSION: In this study verapamil exhibited a dose-dependent inhibitory effect on gelatinase A and gelatinase B activity in human PBMCs. It seems that the anti-inflammatory properties of verapamil may be in part due to its inhibitory effects on gelatinase activity. Regarding the beneficial effects of MMPs- inhibitors in the treatment of some cardiovascular diseases, the positive effect of verapamil on such diseases may be in part due to its anti-MMP activity. Verapamil with its inhibitory effects on gelatinases activity may be a useful MMP-inhibitor. Given the beneficial effect of MMP-inhibitors in some cancerous, inflammatory and autoimmune disorders, it seems likely that verapamil could also be used to treat these diseases.

The role of leukotrienes in immunopathogenesis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder of joints for which there is no strict cure. However, conventional medications can reduce inflammation, relieve pain, and slow joint damage. Leukotrienes are a family of paracrine agents derived from oxidative metabolism of arachidonic acid. Synthesis of lipid mediators and subsequent induction of receptor activity are tightly regulated under normal physiological conditions, so that enzyme and/or receptor dysfunction can lead to a variety of clinical signs and symptoms of disease, such as local pain and tissue edema. In these tissues, immunocompetent cells accumulate at the site of injury, contributing to tissue damage and perpetuation of the disease process. Leukotrienes (often leukotriene B4) as potent chemotactic agents can provoke most signs and symptoms in rheumatoid arthritis by initiating, coordinating, sustaining, and amplifying the inflammatory response, through recruitment of leukocytes. A number of studies have reported that pharmacological modulation in this field can significantly attenuate clinical manifestations associated with different inflammatory pathologies.

Production and Characterization of Mouse Monoclonal Antibodies Recognizing Human Pan-IgG Specific Conformational or Linear Epitopes.

BACKGROUND: Pan-IgG specific monoclonal antibodies (MAbs) are essential tools for assessment of humoral immunity, immune deficiency and gammopathy. In this study, four hybridoma clones producing MAbs with different specificities for human IgG isotypes were established. METHODS: Splenocytes from Balb/c mice immunized with Fc fractions of human IgG were fused with SP2/0 myeloma cells. Hybridoma cells were selected in HAT selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed using a panel of purified myeloma IgG proteins by ELISA and immunoblotting. Cross-reactivity to immunoglobulins (Igs) of other species was studied by indirect ELISA using serum samples collected from 9 animals. RESULTS: Immunoblotting studies revealed recognition of either linear or conformational epitopes by these MAbs. The most abundant cross-reactivity (100%) was observed with monkey Igs, while no cross-reactivity was detected with rabbit, guinea pig, sheep, goat, cat and hen sera. Two of the MAbs cross-reacted with either dog or horse sera. The affinity constant of two MAbs was measured by ELISA and found to be 0.74×10(8)M(-1) and 0.96×10(7)M(-1). CONCLUSION: Our results indicate that these pan-IgG specific MAbs recognize restricted linear or conformational epitopes located on all human IgG subclasses with no cross-reactivity to IgG from most species studied. These MAbs are potentially useful tools for quantification of total or antigen-specific IgG levels.

Profiles of MMP-2 expression in Jurkat, Molt-4 and U937 cells.

BACKGROUND: Leukemia is a malignant proliferative disorder of the hematopoietic cells. The important role of angiogenesis in leukemia has been reported by several studies. Matrix metalloproteinases (MMPs) are a large group of endopeptidases which degredate the extracellular matrix and play an important role in angiogenesis. OBJECTIVE: The present study was conducted to evaluate the patterns of MMP-2 activity in three leukemic cell lines. METHODS: Human leukemic monocyte (U937) and T cells (Molt-4 and Jurkat) were cultured in complete RPMI-1640 medium. The cells were then seeded at a density of 106 cells/ml and were incubated with different concentrations of phorbol myristate acetate (PMA) (1-25 ng/ml) or phytoheamagglutinin (PHA) (2-10 µg/ml) for 24 hours. The MMP-2 activity in cell-conditioned media was then evaluated by gelatin zymography. Statistical comparisons between groups were made by analysis of variance (ANOVA). RESULTS: PHA/PMA significantly and dose-dependently increased MMP-2 activity in U937 cells after 24 hours of incubation compared with untreated control cells. Moreover, PHA/PMA significantly induced MMP-2 activity in Molt-4 and Jurkat cells after 24 hours of incubation in a dose-dependent manner compared with untreated control cells. CONCLUSION: We conclude that human leukemic Jurkat, U937 and Molt-4 cells could potentially display MMP-2 activity with different degrees. Thus, these cell lines could provide an appropriate system to study the mechanisms regulating MMPs production in leukemia patients.

Production and characterization of mouse monoclonal antibodies recognizing multiple subclasses of human IgG.

Different IgG subclass profiles are produced in response to different antigenic stimuli in a variety of diseases. IgG subclass levels may reflect disease severity. Quantification of IgG subclasses depends on the availability of specific Monoclonal antibodies (MAbs). In the present study seven hybridoma clones producing MAbs reactive with multiple subclasses of human IgG were established. Splenocytes from Balb/c mice immunized with Fc fractions of human IgG1 or IgG2 myeloma proteins were fused with mouse myeloma cells. Fused cells were selected and cloned by limiting dilution assay. Antibody secreting cells were screened by Enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified human myeloma paraproteins of different IgG subclasses by ELISA and immunoblotting. Cross-reactivity to immunoglobulins (Igs) of other species was studied by indirect ELISA using serum samples collected from 9 animals. The MAbs were found to react with triple IgG subclasses, including IgG1,2,4 (n=4) and IgG1,2,3 (n=3). Immunoblotting studies revealed recognition of linear (n=4) or conformational (n=3) epitopes by these MAbs. The most abundant cross-reactivity (71.4%) was observed with monkey Ig while no cross-reactivity was detected with hen and cat sera. The MAbs mostly displayed a restricted pattern of cross-reactivity and one of them did not bind to any of the animal sera tested. The affinity constant of 3 MAbs was measured by ELISA. Based on the data obtained from this study, mouse MAbs reactive with multiple human IgG subclasses are directed to a variety of immunogenic epitopes, mostly shared with IgG of other species. These MAbs are valuable tools for purification of non-reactive IgG subclasses through negative affinity chromatography. These MAbs could also provide an opportunity for epitope mapping of the Fc region of IgG, as well as serological phylogenetic studies.

Effect of propranolol on angiogenic factors in human hematopoietic cell lines in vitro.

BACKGROUND: Beta-adrenergic blocking agents have been broadly used for treatment of many cardiovascular diseases such as arterial hypertension and ischemic heart failure. Anti-tumoral, anti-inflammatory and anti-angiogenesis effects of propranolol (a non-selective beta-adrenergic blocker) have been shown. Angiogenesis (replenish of the pre-existing vascular networks) plays a critical role in some pathological conditions such as tumor expansion and metastasis. In this study, we investigated the effects of propranolol on vascular endothelial growth factor (VEGF) production and matrix metalloproteinase-2 (MMP-2) activity (two important angiogenic factors) in human leukemic cell lines in vitro. METHODS: Two human leukemic T (Molt-4 and Jurkat) and one monocyte (U937) cell lines were used in this study. The cells were cultured in complete RPMI medium and then incubated with different concentrations of propranolol (0.3-30 microM) in the presence or absence of phorbol myristate acetate (PMA, 25 ng/ml) for 48 hours. The level of VEGF secreted in the cell culture supernatants was measured with enzyme-linked immunosorbent assay kits (R and D systems) and MMP-2 activity in cell-conditioned media was evaluated by gelatin zymography. RESULTS: Propranolol significantly decreased VEGF production and also MMP-2 activity in PMA-activated human leukemic cell lines Molt-4, Jurkat and U937 at 30 microM concentration of the drug compared to untreated control cells (P<0.05). CONCLUSION: Propranolol might be a useful anti-angiogenic agent in hematopoietic malignancies. Thus, propranolol along with its chronic long-term usage in cardiac problems may have potential implication in treatment of leukemia.

In vitro sensitivity of leukemia cells to propranolol.

BACKGROUND: Propranolol, as a beta-adrenergic blocker is used for treatment of a large number of cardiovascular diseases such as hypertension and arrhythmias. The inhibitory effects of propranolol on tumor cells growth and also its cytotoxicity on cancerous cells have been revealed by several studies. In this study the sensitivity of a number of human leukemic cell lines to propranolol was evaluated in vitro. METHODS: Two human leukemic T cells (Molt-4 and Jurkat) and a monocyte (U937) cell line were used in this study. The cells were cultured in complete RPMI medium and then incubated with different concentrations of propranolol (0.0004 -0.4 mM) in the presence or absence of phytoheamagglutinin (20 µg/ml) for 12, 24 and 48 hours. The cytotoxic effect of the drug was then assessed by trypan blue dye exclusion and also 3-(4,5-dimethyl thiazol-2,5-diphenyltetrazoliumbromide) (MTT) reduction methods. RESULTS: Propranolol induced a significant dose dependent cytotoxic effect at ≥ 0.2 mM concentration on all three human cell lines (Molt-4, Jurkat and U937) used in this study, after 12 hours incubation onwards, compared to untreated control cells. CONCLUSIONS: Our results demonstrated that leukemic cell lines used in this study were sensitive to propranolol at ≥ 0.2 mM concentration of the drug. These results suggest that propranolol may have potential implication in chemoprevention of lymphoproliferative disorders along with its chronic long-term usage in cardiac problems. KEYWORDS: Propranolol; Leukemia; Cell lines; Sensitivity.

Generation and Characterization of Mouse Hybridomas Secreting Monoclonal Antibodies Specific for Human IgG3.

Mammalians express several subclasses of the IgG molecule. In human being there are four homologous IgG subclasses, each of which is structurally unique and has different functions. Quantification of IgG subclasses is fundamental to clinical assessment and diagnosis of many diseases as such assessments depends on the availability of subclassspecific antibodies (Abs), particularly monoclonal antibodies (MAbs). In the present study, we produced and characterized two murine MAbs specific for human IgG3 molecule. These MAbs were obtained by the fusion of myeloma cells with splenocytes from Balb/c mice immunized with heavy chain of a human IgG3 myeloma protein. Fused cells were selected in hypoxanthine, aminopterine and thymidine (HAT) medium and cloned by limiting dilution assay. Ab-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. Two stable hybridomas designated 1F18G7 and 1F18A11 were obtained secreting MAbs specific for Fc fragment of human IgG3. None of these MAbs showed cross-reactivity with other immunoglobulin isotypes derived from human and nine other animals, except 1F18A11 which displayed a weak cross-reactivity with only dog serum. Immunoblotting results indicate that these MAbs react with linear epitope(s) located in the heavy chain of human IgG3 molecules. The affinity constant of 1F18G7 and 1F18A11 MAbs was found to be 0.81×10(9) Mol (-1) and 0.71×10(9) Mol (-1), respectively, as measured by ELISA. These two MAbs with relatively high affinity can be useful tools for quantification of IgG3 subclass levels in human serum.

Development of two murine monoclonal antibodies recognizing human nG1m(a)-like isoallotypic markers.

Antigenic determinants of immunoglobulin molecules are categorized into three groups: idiotypes, isotypes, and allotypes. An isoallotype is defined as an isotypic determinant in one or more subclasses and an allotype in another subclass of a given isotype. The isoallotype nG1m(a), formerly called non-a, is an allotype located on human IgG1 molecules lacking the G1m(a) allotype. This marker, however, is also detectable on all human IgG2 and IgG3 molecules. Anti-isoallotypic antibodies are useful tools for structural studies of immunoglobulins, forensic science, and epidemiological demographic investigations. In this study, two murine monoclonal antibodies (MAbs) recognizing nG1m(a)-like epitope(s) were generated against a human IgG myeloma protein. These MAbs are produced by hybridoma clones (4F18B12 and 6F18D1) obtained by fusion of SP2/0 myeloma cells with splenocytes from BALB/c mice immunized with heavy chain of a human IgG3 myeloma protein. These MAbs reacted with Fc, but not Fab fragment of the immunizing IgG3 paraprotein. Specificity of the MAbs was further analyzed using a panel of purified myeloma proteins and polyclonal IgG3, including IgG1 (n = 9), IgG2 (n = 4), IgG3 (n = 8), and IgG4 (n = 6) subclasses. Our results demonstrated that these MAbs reacted with linear epitope(s) located on heavy chain of all IgG2 and IgG3 molecules tested and some paraproteins of IgG1 subclass, but none of the IgG4 molecules. So these MAbs seem to recognize nG1m(a)-like isoallotypic marker. These MAbs showed no cross-reactivity with serum of other species tested. Both MAbs belonged to IgG1 subclass with affinity constants of 4.5 x 10(9) mol(-1) (4F18B12) and 3.46 x 10(9) mol(-1) (6F18D1), respectively. These MAbs might be used as a tool to detect nG1m(a) + IgG molecules.

Private idiotypes located on light and heavy chains of human myeloma proteins characterized by monoclonal antibodies.

Idiotypic determinant, an epitope located on the variable region of the heavy or light chain of an immunoglobulin molecule, could be classified into private and public forms. The private idiotype is a marker unique to a single clone of B cell and hence a fingerprint of an individual clone. It could therefore be exploited to monitor expansion of normal or malignant B cells and to target clonally expanded tumorous B cells specifically. In the present study, five murine monoclonal anti-idiotypic antibodies were generated against two human immunoglobulin G (IgG) myeloma proteins. These monoclonal antibodies (MAbs) are produced by hybridoma clones obtained by the fusion of myeloma cells with splenocytes from BALB/c mice immunized with either human IgG1 (three clones) or IgG2 (two clones) myeloma proteins. All MAbs reacted only with the immunizing antigens and had no reactivity with a panel of purified myeloma proteins of four IgG subclasses with different light chains, including IgG1 (n = 9), IgG2 (n = 4), IgG3 (n = 4) and IgG4 (n = 5). They reacted with the Fab, but not the Fc fraction of the immunizing antigen and displayed no reactivity with normal human serum or polyclonal IgG. Immunoblotting analysis demonstrated that two of the MAbs react with linear idiotypes on light chain, whereas the remaining three MAbs recognize heavy chain associated idiotopes, either conformational (n = 2) or linear (n = 1). Such MAbs with specificity for private idiotypes could have potential implications for monitoring and specific immunotherapy of B cell malignancies. They also are useful tools to study structural correlates of idiotypes.