Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr Viral Infection

Infection of Epstein-Barr virus (EBV) is about 90% among people over the age of 40. The EBV causes a chronic infection that is characterized by chronic or recurrent symptoms and persists for a long time. Recombinant interferon-gamma (IFN-γ) has high clinical and antiviral efficacy in the treatment of herpesvirus infections. 110 patients with chronic EBV infection were examined. The patients were divided into three groups for different treatment regimens: Group 1—IFN-γ therapy (15 injections of Ingaron i/m, 500,000 IU every other day); Group 2—valaciclovir (Valtrex 500 mg × 2 times/day, orally for 2 months); Group 3—valganciclovir (Valcyte 450 mg × 2 times/day, orally for 2 months) and IFN-γ (10–20 injections of Ingaron i/m, 500,000 IU every other day). The best results were obtained in group 3–73.07% negative PCR. In this group, the combination of valganciclovir + IFN-γ was different. We showed that the efficacy of therapy in patients with chronic EBV is determined by the duration of INF-γ administration. We also determined spontaneous and induced production of IFN-α and -γ cytokines in serum and in lymphocyte culture. We demonstrated that in patients with an initially low level of induced IFN-γ, the production of this cytokine significantly increased in three months after the end of therapy.

Interleukin-5 (IL-5) Therapy Prevents Allograft Rejection by Promoting CD4+CD25+ Ts2 Regulatory Cells That Are Antigen-Specific and Express IL-5 Receptor

CD4+CD25+Foxp3+T cell population is heterogenous and contains three major sub-groups. First, thymus derived T regulatory cells (tTreg) that are naïve/resting. Second, activated/memory Treg that are produced by activation of tTreg by antigen and cytokines. Third, effector lineage CD4+CD25+T cells generated from CD4+CD25- T cells’ activation by antigen to transiently express CD25 and Foxp3. We have shown that freshly isolated CD4+CD25+T cells are activated by specific alloantigen and IL-4, not IL-2, to Ts2 cells that express the IL-5 receptor alpha. Ts2 cells are more potent than naïve/resting tTreg in suppressing specific alloimmunity. Here, we showed rIL-5 promoted further activation of Ts2 cells to Th2-like Treg, that expressed foxp3, irf4, gata3 and il5. In vivo, we studied the effects of rIL-5 treatment on Lewis heart allograft survival in F344 rats. Host CD4+CD25+T cells were assessed by FACS, in mixed lymphocyte culture and by RT-PCR to examine mRNA of Ts2 or Th2-like Treg markers. rIL-5 treatment given 7 days after transplantation reduced the severity of rejection and all grafts survived ≥60d whereas sham treated rats fully rejected by day 31 (p<0.01). Treatment with anti-CD25 or anti-IL-4 monoclonal antibody abolished the benefits of treatment with rIL-5 and accelerated rejection. After 10d treatment with rIL-5, hosts’ CD4+CD25+ cells expressed more Il5ra and responded to specific donor Lewis but not self. Enriched CD4+CD25+ cells from rIL-5 treated rats with allografts surviving >60 days proliferated to specific donor only when rIL-5 was present and did not proliferate to self or third party. These cells had more mRNA for molecules expressed by Th2-like Treg including Irf4, gata3 and Il5. These findings were consistent with IL-5 treatment preventing rejection by activation of Ts2 cells and Th2-like Treg.

Cytogenetic Characterisation of Nattukuttai - A Non-descript Cattle Population of Tamil Nadu

Background: Nattukuttai is a small-sized cattle population, native to north-eastern districts of Tamil Nadu. In light of the ongoing research on genetic characterisation of cattle genetic resources of India, the present study was undertaken with the objective of cytogenetic characterisation of Nattukuttai cattle, which is imperative for its conservation and genetic implications to breeding programs. Methods: Blood samples from ten Nattukuttai cattle (five males and five females) were utilized to study the chromosome profile through short-term lymphocyte culture method. Good metaphase spreads were selected for estimation of the relative length, arm ratio, centromeric index and morphological index. Result: The diploid number was 60. All the 29 pairs of autosomes and Y-chromosome were acrocentric while X-chromosome was sub-metacentric. The mean relative length of autosomes ranged from 5.24 ±0.08 to 1.90±0.06. X-chromosome was the largest in the karyotype (5.64±0.12), while the Y-chromosome was the smallest (1.85±0.03). The arm ratio, centromeric index and morphological index were 1.98±0.02, 0.33±0.03 and 4.06±0.4 respectively. The study revealed that the chromosome architecture of Nattukuttai cattle was similar to that of other breeds of Zebu cattle.

Profiling of Chromosomal Complement in Nandidurga Goats of Karnataka

Background: To characterize the chromosomal complement of Nandidurga goats. Methods: Blood samples for short term lymphocyte culture were collected from 5 bucks and 5 does from its breeding tract and then mitotic chromosomal spreads were accomplished. Result: The diploid chromosome number was found to be 60, consisting of 58 acrocentric autosomes and 2 sex chromosomes (X and Y). The X chromosome was found to be the longest acrocentric and Y chromosome was found to be sub metacentric. The mean mitotic drive was 64.5±2.01 and 63.15±1.30 percent in bucks and does, respectively. The mean relative length of autosomes varied from 2.08±0.24 to 4.81±0.12 in does and 2.09±0.09 to 4.70±0.11in bucks. The relative length of X chromosome in does was 5.14±0.17 and that in bucks was 4.92±0.21, whereas Y chromosome had a relative length of 1.99±0.20. The mean Arms Ratio, Centromeric Index and Morphological Index were 2.47, 31.62 and 396.14, respectively. This cytogenetic analysis indicates the normal chromosomal complement in the studied Nandidurga goats.

POS0673 TOLEROGENIC DENDRITIC CELLS IN RHEUMATOID ARTHRITIS PATIENTS: NEWS AND PROMISES

Background:Dendritic cells (DCs) are known to contribute to the pathogenesis of rheumatoid arthritis (RA) through presentation of cartilage glycoprotein, production of proinflammatory cytokines and activation of Th1/Th17 responses. Along with stimulating activity, DCs may exhibit suppressive functions via capacity to induce T cell apoptosis/anergy and to generate regulatory T cells. Since these DCs have potential to control autoreactive T-lymphocytes, the enhancing of tolerogenic properties of DCs seems to be a new important strategy in treatment of RA. Dexamethasone is widely used in clinical practice and can be used as a tolerogenic substance. Therefore, the properties of DCs generated in presence of dexamethasone are of great clinical interests.Objectives:The aim of our study is to describe the properties of tolerogenic DCs, generated with dexamethasone in patients with RA and their influence on autologous T-cells.Methods:Sixty five patients with RA with high and moderate activity of disease were recruited in this study. All patients follow ACR/EULAR criteria (2010). All studies were performed after receiving informed consent. All patients received conventional synthetic DMARDs. DCs were generated from blood monocytes culturing for 5 days with GM-CSF and IFN-α in the presence dexamethasone (dexDCS), applied on third day. LPS as maturation stimuli was added on fourth day. The expression of CD14, CD83, HLA-DR, TLR-2 on the surface of DCs was measured by flow cytometry. The functions of DCs were evaluated by measuring cytokine production and DCs allostimulatory activity in mixed lymphocyte culture. Mature DCs generated in absence of dexamethasone used as control.Results:We revealed that dexDCs are characterized by enhanced expression of CD14+cells and decreased number of CD83+cells but percent of HLA-DR+cells were constant (about 85). DexDCs show high expression of TLR-2 is seen as tolerogenic molecule (75%vs51%, p=0.05 compared to control). DexDCs also have marked prominent increase of TNFα/IL-10 ratio in contrast to control (0.59 vs 1.8, p=0.03). DexDCs suppressed proliferation of allogenic T-cells (2005 vs 7980 cpm, p=0.0002). To assess the stability of the DC in the proinfflamatory micro-environment after assessing stimulatory activity dexDCs were then cultivated with LPS and allostimulatory activity were evaluated one more. The stimulation activity dexDCs after incubation with LPS were not increase (4692 vs 6053 cpm, p=0.7). Also earlier we showed possibility of dexDCs induse apoptosis of autologous T-cells, activation of CD4+IL10+Tr1 and possession of antigen-specific suppression.Conclusion:The data obtained indicate that dexDCs from RA patients have the main tolerogenic features and stable in inflammatory environment that proves their potential in the treatment of rheumatoid arthritis.Disclosure of Interests:None declared

Biallelic Variants in the COLGALT1 Gene Causes Severe Congenital Porencephaly

ObjectiveWe describe a third patient with brain small vessel disease 3 (BSVD3), being the first with a homozygous essential splice site variant in the COLGALT1 gene, with a more severe phenotype than the 2 children reported earlier.MethodsAnalysis of whole exome sequencing (WES) data of the child and parents was performed. We validated the missplicing of the homozygous variant using reverse transcription PCR and Sanger sequencing of the mRNA in a lymphocyte culture.ResultsThe patient presented antenatally with porencephaly on ultrasound and MRI. Postnatally, he showed a severe developmental delay, refractory epilepsy, spastic quadriplegia, and a progressive hydrocephalus. WES revealed a homozygous canonical splice site variant NM_024656.3:c.625-2A>C. PCR and Sanger sequencing of the mRNA demonstrated that 2 cryptic splice sites are activated, causing a frameshift in the major transcript and in-frame deletion in a minor transcript.ConclusionsWe report a third patient with biallelic pathogenic variants in COLGALT1, confirming the role of this gene in autosomal recessive BSVD3.

Siplizumab Induces NK Cell Fratricide Through Antibody-Dependent Cell-Mediated Cytotoxicity

The glycoprotein CD2 is expressed on T and NK cells and contributes to cell-cell conjugation, agonistic signaling and actin cytoskeleton rearrangement. CD2 has previously been shown to have an important function in natural NK cell cytotoxicity but to be expendable in antibody-mediated cytotoxicity. Siplizumab is a monoclonal anti-CD2 IgG1 antibody that is currently undergoing clinical trials in the field of transplantation. This study investigated the effect of CD2 binding and Fc γ receptor binding by siplizumab (Fc-active) and Fc-silent anti-CD2 monoclonal antibodies in allogeneic mixed lymphocyte reaction and autologous lymphocyte culture. Further, induction of NK cell fratricide and inhibition of natural cytotoxicity as well as antibody-dependent cytotoxicity by these agents were assessed. Blockade of CD2 via monoclonal antibodies in the absence of Fc γ receptor binding inhibited NK cell activation in allogeneic mixed lymphocyte reaction. In contrast, siplizumab increased NK cell activation in both mixed lymphocyte reaction and autologous lymphocyte culture due to FcγRIIIA binding. However, experiments using purified NK cells did not show an inhibitory effect of CD2 blockade on natural cytotoxicity or antibody-dependent cytotoxicity. Lastly, it was shown that siplizumab induces NK cell fratricide. Concluding, siplizumab is a promising biopharmaceutical drug candidate for depletion of T and NK cells with minimal off-target effects.

Genital ambiguity: a cytogenetic evaluation of gender

Background: Genital ambiguity is a complex genetic disorder of sexual differentiation into male or female. The purpose of the present study is to correlate the sex of rearing with the genetic sex and to find out the prevalence of chromosomal anomalies in patients with ambiguous genitalia. The findings can help in proper diagnosis, genetic counselling, and the reassignment of sex, if necessary. Methods: In this cross-sectional study, 22 patients from north Kerala, ranging in age from 17 days to 17 years, were included. All cases were subjected to the following: a detailed history, physical examination, evaluation of clinical data, and cytogenetic analysis. Based on the standard protocol, peripheral blood lymphocyte culture was done. Chromosomal analysis was carried out with the help of an automated karyotyping system after G-banding of chromosomes.Results: Out of the 22 patients with ambiguous genitalia, 12 patients were genetic females with karyotype 46, XX, and nine patients were genetic males with 46, XY karyotype. One was a rare variant of Klinefelter syndrome with karyotype 49, XXXXY. The most common diagnosis was congenital adrenal hyperplasia, followed by partial androgen insensitivity syndrome. Discrepancies between genetic sex and sex of rearing were noted in 27% of the cases.Conclusions: This study unfolds the variable etiology of ambiguous genitalia and emphasizes the importance of karyotyping in diagnosis, proper assignment of the sex, and appropriate management of patients with genital ambiguity.

Cytogenetic and oxidative effects of three lichen extracts on human peripheral lymphocytes

In the present study, we investigated cytogenetic and oxidative [total antioxidant capacity (TAC), total oxidant status (TOS)] effects of methanol and water extracts of Cladonia chlorophaea (Flörke ex Sommerf.) Sprengel, Dermatocarpon miniatum (L.) W.Mann and Parmelia saxatilis (L.) Ach. on cultured human lymphocytes. In addition, different phenolic compounds in the extracts were quantified by high performance liquid chromatography (HPLC) analysis. As a result of HPLC analysis, methanol extracts of all lichen species tested had higher phenolic compounds. Likewise, methanol extracts of each lichen increased TAC levels in lymphocytes more than water extracts. The TOS levels of the cells treated with different concentrations (1–100 mg/L) of the extracts decreased due to the increasing concentration of the extracts. Genotoxicity experiments revealed that the tested lichen extracts did not significantly increase (p > 0.05) the level of genotoxicity on human peripheral lymphocyte culture compared to the negative control group. The results showed that C. chlorophaea, D. miniatum and P. saxatilis lichens, which were found to be a rich source of phenolic compounds, might be of interest in the pharmaceutical and food industries.