Characterization of monoclonal antibodies and epitope mapping of the NS4 protein of hepatitis C virus

Recombinant DNA containing sequences of HCV NS4 protein was expressed in Escherichia coli cells. Six hybridoma clones producing monoclonal antibodies (MAB) to recombinant NS4 protein (rNS4), aa 1677-1756, were developed. Mapping with a panel of 33 peptides and reciprocal competitive EIA have shown that MAB obtained revealed five antigen determinants, not described earlier: MAB 3F11 and 3F12-one genotype-independent epitope of NS4A (aa 1700-1707) common for genotypes 1, 2 and 3; MAB 1D11-genotype-independent epitope (aa 1713-1728) and MAB 1D3-genotype (subtype 1b)-specific epitope of NS4B (aa 1711-1731); MAB 6B11 and C1-two conformation-dependent determinants in 5-1-1 region. These data indicate that the 5-1-1 region of NS4 protein has a complex antigenic structure and contains at least eight epitopes, including five, revealed in the present work. MAB obtained recognized native viral protein in the cytoplasm of liver cells of patients with chronic hepatitis C. The positive rates of the immunostaining for NS4 antigen using MAB 6B11, 1D11 and 3F12 were 64, 59 and 50%, respectively. It was found that 6B11 MAB to a conformation-dependent epitope much more actively interacts with native NS4 than with the recombinant protein to which MAB was developed. The epitope recognized by 6B11 MAB is highly immunogenic since it induces the B-cell response in all patients investigated with identified anti-NS4 antibodies in blood serum. The MAB panel obtained in this study may become a useful tool for the diagnostic purposes, for the investigation of NS4B function and for the host-viral interactions at the cell level.     

Hepatitis C virus proteins in the liver cells in acute hepatitis C: their association with liver damage and with the outcome of the disease

The accumulation of individual hepatitis C virus (HCV) proteins in the liver cells of patients with acute hepatitis C (AHC) and their association with the course and outcome of the disease were studied. AHC protein expression in the cryostat liver sections from 20 patients with AHC was estimated by immunohistochemical assay using original monoclonal antibodies to 5 HCV proteins (core, NS3, NS4A, NS4B, and NS5A). The results of HCV detection in the patients were compared with their biochemical, clinical, and morphological findings. HCV proteins were totally revealed in the livers of all the patients, individual proteins were identified with a frequency of 89-95%, which is significantly more than those in patients with chronic hepatitis C. The hepatic expression of core protein was shown to inversely correlate with the duration of an acute period. There was a direct relationship between the accumulation of core, NS3, and NS5A proteins and the liver tissue damage caused by stepwise necrosis rather than intralobular necrosis. The presumed convalescence was ascertained to be associated with the larger count of hepatocytes containing the proteins NS4A and NS3 early after AHC manifestation.     

Development of monoclonal antibodies to highly pathogenic avian influenza H5N1 virus and their application to diagnostics, prophylaxis, and therapy

A panel of 17 monoclonal antibodies (MAbs) against highly pathogenic avian influenza virus (HPAIV) A/Duck/Novosibirsk/56/05 A/H5N1 (subclade 2.2) isolated in Russian Federation was developed. Immunoblot analysis showed that 12 MAbs were specific for the hemagglutinin (HA) and 5 MAbs for nucleoprotein (NP). All anti-HA MAbs were reactive in ELISA and immunofluorescence (IF) test and 10 of them were reactive in hemagglutination-inhibition (HI) and neutralization tests. Quantitative competitive ELISA revealed that anti-HA MAbs recognized at least 4 non-overlapping antigenic determinants and anti-NP MAbs recognized at least 3 non-overlapping antigenic determinants. Four sandwich ELISA procedures were developed using the obtained MAbs. These procedures are useful for 1) identification of avian, human, and swine influenza A viruses, 2) differentiation of avian influenza virus (AIV) from human and swine influenza viruses, 3) differentiation of AIV H5 from other AIV subtypes, and 4) differentiation between 2.2 and 2.3.2 subclades of H5N1 influenza viruses. Prophylactic and therapeutic efficacy of anti-HA MAbs with high neutralization activity was tested in BALB/c mice. A complete protection was achieved by single injection of MAbs (20 mg/kg) 24 hrs before challenge with 10 LD50 of HPAIV H5N1. Therapeutic efficacy was 90% that was similar to those of Rimantadine and Tamiflu.     

Delta glycophorin (glycophorin B) gene deletion in two individuals homozygous for the S--s--U-- blood group phenotype

Blood cells from two unrelated individuals whose erythrocytes exhibit respectively N S-s-U- and MN S-s-U- blood group phenotypes were examined by immunoblotting, periodic acid-Schiff (PAS) staining, and Southern blotting. Protein bands characteristic of delta glycophorin (glycophorin B) were absent from the immunoblots of whole erythrocyte lysates when probed with polyclonal glycophorin antisera and from isolated erythrocyte membranes stained with PAS reagents. Genomic DNA from the two individuals' leukocytes was digested with a panel of restriction enzymes and probed with alpha M glycophorin cDNA obtained from human K562 leukemic cell line. The EcoRI, PstI, and KpnI restriction site patterns were identical to those of S+s+U+ controls in fragment numbers and relative size but differed from controls in band intensities. Restriction mapping with HindIII, PvuII, SacI, MspI, and BamHI revealed that S-s-U- individuals lack fragments that are reproducibly observed in S+s+U+ controls, and most likely encode delta glycophorin. Using truncated 5' and 3' cDNA segments as probes and comparing, in control individuals, hybridization intensities of fragments with amino acid sequence homologies, we have inferred the assignment of restriction fragments to the alpha and delta glycophorin genes. Our results suggest that the absence of delta glycophorin in the two S-s-U- individuals is a result of deletion of the entire delta glycophorin gene. This is the first report of a glycophorin gene deletion.     

The effect of unilateral vasectomy on testosterone and testicular parameters in the adult male African giant rat (Cricetomys gambianus)

Background

The effects of vasectomy on spermatogenesis and reproductive parameters are recognized to be specie-dependent with marked differences in levels of perturbations observed.

Objectives

To assess the impact of unilateral vasectomy on testosterone level and other testicular parameters in the male African giant rat (AGR) (Cricetomys gambianus).

Methods

Sixteen adult male AGRs weighing 500–1300 g were recruited for the experiment. Animals were randomly divided into three experimental groups (1–3) and one control (sham operated) group with four rats per group. Experimental vasectomy was done by carefully ligating the vas deferens of the right testis of all the experimental groups (1, 2, and 3) and animals were allowed either 8, 6 and 2 weeks respectively before sacrifice. Sham-operated animals served as the control. Blood samples were collected and assayed for testosterone while testicular tissue was further processed for seminal fluid and histo-pathological analyses.

Results

Spermatogenic parameters indicate a pattern of decline in sperm count and motility between the experimental groups and the control and azoospermia in the eight-week group. Histological alterations were marked by atrophy of seminiferous tubules which was proportional to the duration of vasectomy. Serum testosterone levels were significantly reduced at eight weeks. There was no statistically significant difference between sperm counts of right and left testes except for group 3. Results suggest that unilateral vasectomy of the AGR may have negative impact on the contralateral testis in the male African giant rat.

Conclusion

These preliminary results reveal that unilateral vasectomy in the AGR may result in perturbations of the histo-architecture of the testes with possible decline in function.     

A Quantitative Analysis of a Probiotic Storage Media for Avulsed Teeth

Aim

The aim of the present in vitro study was to investigate the potential of a storage medium, probiotic yogurt (Bifidibacterium animalis DN 173010) in comparison with Hank''s balanced salt solution (HBSS), saline and milk in maintaining viable periodontal ligament (PDL) cells on simulated avulsed teeth.

Materials and methods

Thirty-six freshly extracted single-rooted human teeth with closed apices were divided into six experimental groups (N=6). The teeth were extracted as atraumatically as possible and washed in sterile saline solution to eliminate residual blood. Following extractions, the coronal 3 mm of PDL tissues were scraped with a #15 scalpel to remove cells that may have been damaged. The positive and negative controls corresponded to 0 minutes and an 8-hour dry time, respectively. After extraction, the positive control teeth were immediately treated with dispase and collagenase. The negative control teeth were bench-dried for 8 h, with no follow-up storage solution time, and then placed in the dispase and collagenase. The number of viable protective least significant difference PDL cells were counted under a light microscope with a hemocytometer at 20× magnification and analyzed. Statistical analysis of the data was accomplished using Nonparametric ANOVA complemented by Kruskal-Wallis Test and Dunn''s Multiple Comparisons Test.

Results

Positive control was found to be significantly better than the others, there were statistically significant differences between positive control and other test groups (p=0.000). The teeth stored in positive control demonstrated the highest number of viable PDL cells followed in order by probiotic yogurt, HBSS, saline and milk.

Conclusion

Bifidibacterium animalis DN 173010 seems to be an alternative for the temporary storage of avulsed teeth, due to high number of viable PDL cells. Probiotics may be suitable transport media for avulsed teeth, but further research is warranted using the commercially available products.Key words: dental trauma, probiotic, storage media     

Uncooked, lactobacilli-rich, vegan food and rheumatoid arthritis

We tested the effects of an uncooked vegan diet, rich in lactobacilli, in rheumatoid patients randomized into diet and control groups. The intervention group experienced subjective relief of rheumatic symptoms during intervention. A return to an omnivorous diet aggravated symptoms. Half of the patients experienced adverse effects (nausea, diarrhoea) during the diet and stopped the experiment prematurely. Indicators of rheumatic disease activity did not differ statistically between groups. The positive subjective effect experienced by the patients was not discernible in the more objective measures of disease activity (Health Assessment Questionnaire, duration of morning stiffness, pain at rest and pain on movement). However, a composite index showed a higher number of patients with 3-5 improved disease activity measures in the intervention group. Stepwise regression analysis associated a decrease in the disease activity (measured as change in the Disease Activity Score, DAS) with lactobacilli-rich and chlorophyll-rich drinks, increase in fibre intake, and no need for gold, methotrexate or steroid medication (R2=0.48, P=0.02). The results showed that an uncooked vegan diet, rich in lactobacilli, decreased subjective symptoms of rheumatoid arthritis. Large amounts of living lactobacilli consumed daily may also have positive effects on objective measures of rheumatoid arthritis.      

The Role of the Serotoninergic System in the Regulation of Thyroid Function in Old Rats

The effect of serotoninotropic substances with positive and negative effects on serum concentrations of thyrotropic hormone, total triiodothyronine, and total thyroxin was studied in old male rats. Chronic treatment with L-tryptophan and citalopram reduced serum level of total thyroxin in old intact animals and serum concentration of thyrotropic hormone in old thyrectomied rats. DL-p-chlorophenylalanine increased the serum concentrations of total thyroxin in old intact rats and in thyroidectomied animals treated with triiodothyronine. Chronic methysergide therapy was associated with reduction of serum concentration of thyrotropic hormone in old intact and old thyroidectomied rats and of total triiodothyronine in old intact animals.