Immunoinformatic analysis of crimean congo hemorrhagic fever virus glycoproteins and epitope prediction for synthetic peptide vaccine

Objective: To determine the Crimean Congo Hemorrhagic Fever [CCHF] virus M segement glycoprotein's immunoinformatic parameters, and identify Human Leukocyte Antigen [HLA] class I binders as candidates for synthetic peptide vaccines

Study Design: Cross-sectional study

Place and Duration of Study: Combined Military Hospital, Khuzdar Cantt, in May 2015

Methodology: Data acquisition, antigenicity prediction, secondary and tertiary structure prediction, residue analysis were done using immunoinformatics tools. HLA class I binders in glycoprotein's sequence were identified at nanomer length using NetMHC 3.4 and mapped onto tertiary structure. Docking was done for strongest binder against its corresponding allele with CABS-dock

Results: HLA A*0101, 0201, 0301, 2402, 2601 and B*0702, 0801, 2705, 3901, 4001, 5801, 1501 were analyzed against two glycoprotein components of the virus. A total of 35 nanomers from GP1, and 3 from GP2 were identified. HLA B*0702 bound maximum number of peptides [6], while HLA B*4001 showed strongest binding affinity

Conclusion: HLA specific glycoproteins epitope prediction can help identify synthetic peptide vaccine candidates

Evolution of DNA sequencing

Sanger and coworkers introduced DNA sequencing in 1970s for the first time. It principally relied on termination of growing nucleotide chain when a dideoxythymidine triphosphate [ddTTP] was inserted in it. Detection of terminated sequences was done radiographically on Polyacrylamide Gel Electrophoresis [PAGE]. Improvements that have evolved over time in original Sanger sequencing include replacement of radiography with fluorescence, use of separate fluorescent markers for each nucleotide, use of capillary electrophoresis instead of polyacrylamide gel electrophoresis and then introduction of capillary array electrophoresis. However, this technique suffered from few inherent limitations like decreased sensitivity for low level mutant alleles, complexities in analyzing highly polymorphic regions like Major Histocompatibility Complex [MHC] and high DNA concentrations required. Several Next Generation Sequencing [NGS] technologies have been introduced by Roche, Illumina and other commercial manufacturers that tend to overcome Sanger sequencing limitations and have been reviewed. Introduction of NGS in clinical research and medical diagnostics is expected to change entire diagnostic approach. These include study of cancer variants, detection of minimal residual disease, exome sequencing, detection of Single Nucleotide Polymorphisms [SNPs] and their disease association, epigenetic regulation of gene expression and sequencing of microorganisms genome

Bone marrow and peripheral blood stem cell transplant: a bioinformatics approach for mismatched donor recipient pairs

Bone marrow and peripheral blood stem cell transplants when performed outside the family require high resolution matching of donor and recipient for human leukocyte antigen loci. Marrow registries like National Marrow Development Program in developed countries maintain record of donors and provide most suitable donor when a recipient needs a transplant. Being outside families and due to lack of shared haplotypes, these are not fully matched. Depending upon condition of patient and time available, several times one or two loci mismatched marrow has to be transplanted. This matching can be further enhanced by introduction of a recently introduced branch of science known as Bioinformatics. Combining the knowledge of computer softwares and transplant biology, it is possible to place any protein [in this case specific human leukocyte antigen alleles of potential donors and recipient] against any other, for exact amino-acids match/mismatch in user defined region and thus choosing the better-matched donor. In this write-up, an introduction of few programs available that can be used for the said purpose is given with a brief discussion of approach already used by other scientists

Laboratory evidence of autoimmunity in HCV infected patients after interferon treatment

To determine the significance of interferon alpha therapy in emergence of autoantibodies in HCV infected patients in a local population Quasi experimental study Department of Immunology, Armed Forces Institute of Pathology [AFIP] Rawalpindi from Mar 2007 to Oct 2007. A total of 106 HCV infected individuals [not on any antiviral therapy], were screened for laboratory evidence of autoimmunity [ANA, SMA, AMA, Anti LKM, Anti GPC, Anti Thyroid microsomal and RA factor]. HCV infected patients without any laboratory evidence of autoimmunity were included in the study and this population was divided in to a test group [36] who were treated with interferon alpha and a control group [32] who did not receive anti viral treatment during this period. All were retested for the same autoantibodies after a period of 3 months. All autoantibodies were tested by indirect immunofluorescence except RA factor which was tested by agglutination method. Out of 106 patients scarred, 32 showed autoimmunity that were excluded from study. Six denied to participate in study. After 3 months, 61% of the patients showed autoimmunity in study group and frequencies of ASMA, anti-TPO [p 0.036], ANA [p 0.14] and RA were 36%, 25%, 25% and 33% respectively. Control group showed autoimmunity in 37.5% of the patients with frequency of 28%, 15.6%, 6.3% and 3% for ASMA, RA, TPO and ANA respectively. AMA anti-LKM antibodies were not found in both groups. ANA and anti thyroid antibodies emerge in increased frequency in HCV infected patients after treatment with IFN-alpha

Human leukocyte antigen class II susceptibility conferring alleles among non-insulin dependent diabetes mellitus patients

To determine the frequency of Human Leukocyte Antigen [HLA] class II susceptibility conferring alleles among type 2 Diabetes mellitus patients, in comparison with healthy controls. Cross-sectional comparative study. Department of Immunology, Armed Forces Institute of Pathology, Rawalpindi, from January 2009 to April 2010. Patients with non-insulin dependent Diabetes mellitus meeting World Health Organization criteria were studied. These were compared with age and gender matched healthy control subjects. For each subject [patients as well as controls], DMA was extracted from ethylene diamine tetra-acetate sample and HLA class II DRB1 typing was carried out at allele group level [DRB1*01-DRB1*16] by sequence specific primers. Human leukocyte antigen DRB1 type was determined by agarose gel electrophoresis and results were recorded. Frequencies were determined as number of an allele divided by total number of alleles per group; p-value was computed using Pearson's chi-square test. Among the 100 patients, there were 63 males and 37 females with 68 controls. A total of 13 different HLA DRB1 alleles were detected, with DRB1*15 being the commonest in both the groups. The allele DRB1*13 had statistically significant higher frequency in patient group as compared to controls [p = 0.005]. HLA DRB1*13 was found with a significantly increased frequency in non-insulin dependent Diabetes mellitus

Frequency of antinuclear autoantibodies among healthy volunteers

To determine the frequency of antinuclear autoantibodies [ANA] among healthy individuals. Descriptive study. Combined Military Hospital, Chunian Cantt and Armed forces Institute of Pathology, Rawalpindi, from September to December 2009. serum samples were collected from healthy volunteers [after informed consent] through non probability convenience sampling. Antinuclear antibodies were detected by indirect immunofluorescence. Titer of a positive sample was determined by serial dilution. Data was analyzed for frequency and percentage of positive samples. A total of 100 volunteers [50 males and 50 females] were inducted in study. Their age ranged from 2 years to 75 years. Out of these, 3 [3%] volunteers were found to be positive for ANA, though in low titers, i.e., less than 1:10. Antinuclear autoantibodies are found in approximately 3% of healthy individuals. In the absence of symptoms, a low titer of ANA may not be of much significance as it may be found in healthy people as well. But in appropriate clinical settings, a positive ANA in high titer should be further investigated

Clinical, histopathological and immunofluorescent findings of IgA nephropathy

IgA nephropathy, a prevalent disease in Asia, is considered the main cause of end stage renal disease among primary glomerular disease. To determine the frequency of different clinical, histopathological and immunofluorescent characteristics of IgA nephropathy. Renal biopsies of 376 patients were received for immunofluorescent and for histopathological studies. Biopsies were stained with fluorescene isothyocyanate [FITC] labeled antibodies against IgG, IgA, IgM, C3, C4 and fibrinogen for fluorescent microscopy. For histopathological examination, the specimens were stained with hematoxylin and eosin, periodic acid schiff and methanamine silver stains for light microscopy. IgA nephropathy was diagnosed in 39 cases [10.4%] with a mean age 31.5 years and a male to female ratio of 2.8:1. The disease was observed in 11[29.7%] patients aged 21-30 years, followed by 8 patients [21.6%] aged 11-20 years group. Nephrotic range proteinuria was the most common laboratory finding which was detected in 11 patients [37%]. Mesangioproliferative glomerulonephritis was the most common histopathological finding which was found in 7 patients [35%]. IgA with other immunoglobulins and complements were deposited in 28 specimens [71.8%] as detected by immunofluorescence. IgA nephropathy is common in young people and one third of it results in end stage renal disease. We suggest that Immunofluorescent assay can be considered for the conclusive diagnosis of IgA nephropathy in young patients presenting with proteinuria/hematuria

Diagnostic application of smooth muscle antibodies in autoimmune hepatitis

To measure the level of C-reactive protein and find its association with the glycaemic status [fasting plasma glucose, glycosylated haemoglobin] of metabolically normal and diabetic albino rats. Total 60 Albino rats were included [normal n=30; diabetic n=30]. Plasma glucose levels were determined by using glucose oxidase method while determination of total Hb and glycosylated hemoglobin [HbA[1]c] was done by diagnostic kit that uses weak cation-exchange resin to bind Hb. The% HbA[1]c was determined by measuring the absorbance at 415 nm of the glycosylated hemoglobin fraction and the total hemoglobin fraction. The ratio of the two absorbances gave the% of HbA[1]c. C-reactive protein was measured by the ELISA kit. Significant difference was found in the values of fasting glucose, of the normal and diabetic groups [p<0.05] but no significant difference was present in the values of HbA[1]c of both groups. There was no significant difference in the values of C-reactive protein of the normal and diabetic groups. Short duration hyperglycemia has no role in producing inflammation and raising the levels of bioinflammatory marker C-reactive protein

CD5-positive acute lymphoblastic leukemia

CD5-positive B-ALL is a rare variant of Acute Lymphoblastic Leukenia [ALL]. In literature, only three cases have been reported so far. This fourth case report describes a young lady who was diagnosed as ALL [L-2] ib bibe marrow examination and was found to be CD5 positive B-cell acute lymphoblastic leukemia on immunophenotyping. Cytogenetic analysis revealed translocation t[9:22]

Suppression of humoral immune response due to psychological stress in survivors of large scale natural disaster

To study the impact of psychological stress following a natural disaster on specific immunological parameters. The study was carried out over a 3 month period [Feb 2006 to May 2006] at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan. Physically uninjured male adults between 15-60 years of age, with no prior history of an active physical or psychological disorder, who witnessed the earthquake on 8[th] Oct, 2005 in Pakistan but escaped physical injury, were included in the study. Age and gender matched healthy adults were also studied as control group. Analysis of haemoglobin, total leucocyte count, lymphocyte count, lymphocyte subsets, IgG, IgA, IgM levels, C reactive protein and nitrobluetetrazolium [NBT] dye reduction test was carried out on both the groups. Psychologically distressed individuals had increased CD 3+ cells [p=0.02], increased CD4:CD8 ration [p=0.04], reduced CD19+ cells count [p=0.03] and IgG levels [p=0.01]. Neutrophil oxidative burst activity without stimulation was increased [p=0.02]. Psychological stress consequent to exposure to a natural disaster can suppress humoral immune response

Validity of human leukocyte antigen class ii typing in-house assay in comparison with commercial kit

To determine the validity of in-house assay for DNA based HLA class II typing against commercial kit as gold standard. Validation Study. Department of Immunology, Armed Forces Institute of Pathology, Rawalpindi, from Feb 2006 to March 2007. 56 cases referred to Immunology Department for HLA class II typing were included in the study and each subject tested by both the assays. INNO-LiPA assay was done by commercial kits which are based on sequence specific oligonucleotide probes. For in-house assay, individual reagents were obtained from commercial sources and protocol of the assay was established. The results of in-house assay were compared with INNO-LiPA kit. The sensitivity and specificity of in-house assay as compared to commercial assay were calculated, for each of the individual alleles identified, and at the end, cumulative sensitivity and specificity were found to be 92% and 99% respectively. High sensitivity, specificity, cost effectiveness and being easy to perform, make in-house assay a suitable alternative approach for performing HLA-DRB1 typing, especially in renal transplant patients where it can replace serological techniques. In addition, with the addition of primers for new alleles, it can be used to resolve any ambiguity that arises during the course of commercial testing. However, further work is required to study and analyze primers for DRB1 03 and DRB115, to make in-house assay, a cost effective method for low resolution typing