Vitamin D supplementation and calcium: Many-faced gods or nobody in fighting against Corona Virus Disease 2019.

In December 2019, Corona Virus Disease 2019 (COVID-19) was first identified and designated as a pandemic in March 2020 due to rapid spread of the virus globally. At the beginning of the pandemic, only a few treatment options, mainly focused on supportive care and repurposing medications, were available. Due to its effects on immune system, vitamin D was a topic of interest during the pandemic, and researchers investigated its potential impact on COVID-19 outcomes. However, the results of studies about the impact of vitamin D on the disease are inconclusive. In the present narrative review, different roles of vitamin D regarding the COVID-19 have been discussed to show that vitamin D supplementation should be recommended carefully.

Epitope specificity of anti-factor VIII antibodies from inhibitor positive acquired and congenital haemophilia A patients using synthetic peptides spanning A and C domains.

Development of antibodies (Ab) that either block the function of coagulation factor VIII (FVIII) (inhibitors) or clear it from circulation, seriously complicate the treatment of haemophilia A patients with FVIII products. Autoantibodies which develop in subjects without congenital FVIII defects, cause acquired haemophilia, a rare but life-threatening coagulopathy. Identification of the FVIII epitopes to which inhibitor Abs bind will help understanding the mechanisms of inhibitor activity, and perhaps development of new therapies. Here, we examined the FVIII peptide sequence regions recognised by anti-FVIII Ab in the plasma of six congenital and one acquired haemophilia patients with high inhibitor titers (24.4-2000 BU/ml). We used indirect ELISA and overlapping synthetic peptides, 20 residues long, spanning the sequence of the A and C FVIII domains. None of the plasma samples reacted with A1, A3 or C1 domain peptides. Six plasmas reacted with A2 and/or C2 peptides. Peptides spanning residues A2-521-690 and C2-2251-2332 were recognised most frequently and strongly. They include residues that contribute to the binding sites for activated factor IX and phosphatidyl serine/von Willebrand factor. These results suggest that anti-FVIII Abs share a pattern of antigen specificity in our panel of patients, and that exposed regions of the FVIII molecule that form functionally important binding sites elicit an intense Ab response.

Effective CpG immunotherapy of breast carcinoma prevents but fails to eradicate established brain metastasis.

PURPOSE: Breast cancer patients with brain metastasis have a dismal prognosis. We determined the ability of immunostimulatory CpG oligodeoxynucleotides (ODN) to treat or prevent brain metastasis in a mouse model. EXPERIMENTAL DESIGN: Mice bearing orthotopic breast carcinoma with or without concurrent i.c. tumors were treated by injections of CpG ODN at the primary tumor. Immunologic memory was tested by tumor rechallenge and immune responses were assessed by flow cytometry, delayed-type hypersensitivity, and CTL assays. RESULTS: Orthotopic tumors regressed in treated mice regardless of whether concurrent i.c. disease was present. In mice bearing only orthotopic tumors, CpG ODN rendered 50% tumor-free and they rejected tumor rechallenge in breast and brain. In mice with concurrent i.c. disease, there was no difference in brain tumor growth compared with saline controls, despite regression of the primary tumor. Flow cytometry revealed that treated mice that died from i.c. disease exhibited a significant increase in brain-infiltrating T and natural killer cells relative to saline controls. CTLs from these mice were able to kill tumor in vitro and extend survival of naive mice bearing less-established brain tumors by adoptive transfer. CONCLUSIONS: The lack of survival benefit in mice with appreciable brain metastasis was not explained by a deficit in lymphocyte trafficking or function because CTLs from these mice killed tumor and inhibited microscopic brain metastasis by adoptive transfer. These results indicate that CpG ODN might be beneficial as a preventative adjuvant to initial therapy preceding brain metastasis or to inhibit progression of microscopic brain metastases.

In vivo vaccination with tumor cell lysate plus CpG oligodeoxynucleotides eradicates murine glioblastoma.

Dendritic cell (DC) vaccines have shown antitumor activity in experimental glioma models and in human glioma patients. The typical approach has been to generate the vaccine ex vivo, by pulsing DCs with tumor lysate or peptides, then administering the DCs back into the patient. This process requires significant expertise and expenses in DC generation. Immature DCs which present antigens to T cells in the absence of appropriate costimulatory signals can lead to induction of immune tolerance. Recent studies have shown that coadministration of toll-like receptor 9 agonists, CpG oligodeoxynucleotides, can promote DC vaccines to break immune tolerance to tumor antigens. We investigated the therapeutic efficacy of in vivo DC activation, by directly administering glioma cell lysate with CpG oligodeoxynucleotides (CpG/lysate), in glioma-bearing mice. Subcutaneous vaccination with CpG/lysate induced a significant increase (P<0.05) in the number of total T cells and activated DCs in lymph nodes draining the vaccination site as compared to mice treated with CpG or tumor lysate alone. Mice vaccinated with CpG/lysate exhibited over 2 times greater median survival than mice in the control groups (P<0.05). Up to 55% of mice vaccinated with CpG/lysate were rendered tumor-free as assessed by survival and bioluminescent imaging. Splenocytes taken from mice vaccinated with CpG/lysate elaborated significantly more IFN-gamma production and displayed greater tumor cell lysis activity compared with the control groups (P<0.05). These results suggest direct vaccination with CpG/lysate provides an alternative and effective approach to induce host antitumor immunity and warrants clinical investigation in the immunotherapy of cancer.

Assessment of in vitro cytokine response in hemophilia A patients with or without factor VIII inhibitory antibody.

Factor VIII (FVIII) inhibitor antibodies are produced in a proportion of hemophilia A patients. Development of anti-FVIII inhibitor antibodies is a T cell-dependent response, mediated by FVIII specific CD4(+) T cells. This study was performed to investigate the contribution of T helper (Th) cell-mediated cytokine response in inhibitor production. Peripheral blood mononuclear cells (PBMCs) were obtained from hemophilia A patients with (n = 14) or without inhibitor (n = 14) and from normal individuals (n = 14). Following stimulation of PBMCs with rFVIII and phytohemagglutinin (PHA) mitogen, the secreted cytokines, interferon-gamma (IFN-gamma), interleukin-10 (IL-10), and transforming growth factor-beta1 (TGF-beta1), in culture supernatant and the proliferative response were assessed using sandwich ELISA and (3)H-thymidine incorporation, respectively. No significant proliferative response to FVIII was observed, whereas PHA induced a strong response in all groups. No cytokine secretion was observed in response to FVIII stimulation. Although PHA induced IL-10, TGF-beta1 and IFN-gamma secretion in all groups, the level of IFN-gamma was significantly lower in hemophilia A patients than in normal individuals (p < 0.0001). The levels of TGF-beta1 and IL-10 were similarly higher in patients compared with normal subjects, but the difference was not statistically significant. Lack of FVIII-induced proliferative response and cytokine production together with reduced secretion of PHA-induced IFN-gamma in both groups of patients suggest involvement of nonspecific immunosuppression possibly due to hepatitis C virus (HCV) infection observed in the majority of patients.

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.

Comparative measurement of anti-factor VIII antibody by Bethesda assay and ELISA reveals restricted isotype profile and epitope specificity.

Factor VIII (FVIII) inhibitor antibodies are produced against functional epitopes of FVIII in about 30% of severe hemophilia A patients leading to inhibition of its procoagulant activity. The Bethesda assay, the most commonly used method to measure FVIII inhibitors, based on inhibition of coagulant activity of FVIII, is neither able to detect noninhibitory antibodies nor their isotype. In this study we employed an indirect enzyme-linked immunosorbent assay (ELISA) to measure dif ferent isotypes and IgG subclasses of anti-FVIII anti body in the plasma of hemophiliacs (with and without inhibitor) and normal individuals using recombinant FVIII-coated microtiter plates. The results showed a predominance of IgG and IgG4, though IgA was slightly elevated in a few inhibitor-positive patients and IgM was hardly detectable. A highly significant correlation was found between the Bethesda titer and the optical density values of total Ig, IgG and IgG4 anti-FVIII antibodies obtained by ELISA (p<0.0001). These findings suggest a restricted specificity of anti-FVIII response in hemophiliacs towards functional epitopes of the molecule. Furthermore, high specificity and reasonable sensitivity of the ELISA, together with other technical advantages, suggest this method as a suitable supplementary technique for rapid large-scale screening of inhibitor-positive samples, though ELISA-negative samples need to be rechecked by the Bethesda assay to identify patients with a low inhibitor titer.

Molecular analysis of the heavy chain variable region genes of human hybridoma clones specific for coagulation factor VIII.

HemophiliaA is a X-linked hematologic disorder characterized by undetectable or low amounts of functional coagulation factor VIII (FVIII). Replacement therapy induces FVIII neutralizing antibody (Ab) (inhibitor) in a proportion of patients which makes further treatment of these patients ineffective and costly. To envisage mechanisms underlying inhibitor development, seven hybridoma clones specific for FVIII were generated from two hemophilia A patients with high titer of inhibitor. Specificity and isotype of the monoclonal antibodies (mAbs) were determined by ELISA. Immunoglobulin (Ig) variable region heavy (VH) chain gene family usage was identified by RT-PCR using VH1-6 specific primers. Nucleotide sequences of the VH gene of FVIII specific clones were determined and aligned to the most homologous germ line genes in the GenBank. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized either the VH1 (71%) or the VH3 (29%) gene family. Three VH domains were encoded by V1-69 (DP-10),V1-2 (DP-8), and V1-8 (DP-15) genes and two by V1-18 (DP-14) gene, all from the VH1 gene family. Of theVH3-gene family expressing clones, one belonged to V3-66 (DP-86) and the other one to V3-21 (DP-77) germline genes. The CDR3 length was found to be highly different amongst these clones ranging from 11 to 22 amino acid residues. These data suggest that FVIII-specific Abs preferentially use VH gene segments derived from VH1 gene family. Diversity of the expressed VH genes and their CDR3 length implies that different epitopes are recognized by these mAbs.