Biomedical subjects
Publications and source records attributed to S B Sharma.
Protein content of 60 batches of Yellow Fever Vaccine was measured by Biuret method and was compared to the values obtained by Kjeldahl method. Statistical analysis did not show any difference between the two methods. The Biuret method is specific, easy to carry out and takes little time for protein estimation during production of Yellow Fever Vaccine.
A widely recognized problem in the mental health services delivery system is that of patients' early discontinuance of therapy. But less attention has been given to the high rate of nonattendance at initial appointments in outpatient mental health settings. The purpose of the study discussed in this article was to determine if particular predischarge interventions had an effect on whether psychiatric patients attended their initial aftercare appointments at a Veterans Administration mental health clinic. Two groups of patients were given predischarge interventions and one was not (a control group). Results showed a trend of greater attendance at initial aftercare appointments by those who received a predischarge intervention; however, the differences were not statistically significant. Suggestions for clinicians and researchers are provided.
A broad-host-range bidirectional promoter reporter vector, pSPV4, has been constructed to analyse the activity of cloned divergent regulatory regions. Plasmid pSPV4 contains a pair of divergent promoterless reporter genes (lacZ and gusA) that are bisected by an extensive multiple cloning site. [corrected] The transcriptional fusion vector pSPV4 has a distinct advantage over unidirectional promoter probe vectors in that it can determine the activity of a cloned bidirectional regulatory region simultaneously in both directions using the same set of cells. The relative activity of each of the divergent promoters, and hence the reporter genes, is therefore not a function of the particular growth state of the culture. To demonstrate the utility of pSPV4, the promoter activity of two Rhizobium loti nod regulatory regions were examined. Although the nod gene organisation in this species is unusual, the promoter activity of these two divergent nod regulatory regions is consistent with the conventional mode of nod expression (constitutive towards the regulatory nodD gene and inducible in the divergent nod gene direction). The construction of the bidirectional reporter vector, pSPV4, involved two intermediary plasmids, pSPV1 and pSPV2. Both of these constructs will also be useful for other researchers, especially given the trend towards the utilisation of gusA as a reporter gene.
Twenty one batches of whole cell inactivated cholera vaccine manufactured at Central Research Institute, Kasauli were evaluated for potency by mouse protection assay (MPA) and antibody induction method. In the antibody induction method the sera of immunized mice were screened for the presence of antibodies against Vibrio cholerae by microagglutination (MA) test and IgG ELISA. The number of organisms estimated by MPA were correlated with agglutinating and neutralizing antibodies against individual serotypes by MA and ELISA respectively. Correlation coefficient(r) of 0.692 and 0.815 were observed for the titres evaluated by MA and ELISA when compared with standard MPA method for the serotype Ogawa. Similarly r values of 0.925 and 0.849 were observed for titres evaluated by MA and ELISA when compared with standard MPA method for the serotype Inaba. Antibody induction method can be as an alternative method for determining the potency of inactivated cholera vaccine.
To monitor the parameters controlling yellow fever vaccine production, eight different lots produced without stabilizers were studied. It was found that the freeze-thaw cycle did not have any adverse effect on virus infectivity and the mean loss in virus titre during lyophilization was 0.51 log10 ml-1. A thermodegradation study after storage of vaccine at different temperatures showed that the vaccine did not pass the accelerated stability test. To stabilize the 17D-204 substrain vaccine, four stabilizers with different sugars and amino acids were examined. The optimum time for addition of the stabilizers was found to be during homogenization of infected embryos. An accelerated stability test at 37 degrees C indicated that stabilizers S1, S2 and S3 kept the vaccine stable for up to 4 weeks, whereas the vaccine was only stable for up to 3 weeks in stabilizer S4. Stabilizer S3 was found statistically to be the best. Reproducibility in production methodology was established by preparing and testing more batches of the vaccine using stabilizer S3.
The stability of pertussis component (glutaraldehyde or heat inactivated pertussis vaccine) of the adsorbed diphtheria-pertussis-tetanus (DPT) vaccine preserved in thiomersal or benzethonium chloride was studied at 4-8 degrees C and 35 degrees C for 30 days. The potency of pertussis component of adsorbed DPT vaccine preserved with benzethonium chloride was lower than that preserved with thiomersal. After the initial loss of potency of pertussis component in the benzethonium chloride during blending, the stability of potency of pertussis component at 4-8 degrees C and 35 degrees C for 30 days was similar for vaccines preserved with either benzethonium chloride or thiomersal. The stability of both types of pertussis components inactivated with glutaraldehyde or heat was also similar at both the temperatures for 30 days.
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Partially purified acellular pertussis vaccine was prepared from Bordetella pertussis strains 10536, 134, Tohama and 509 using simple indigenously available techniques. The Stainer-Scholte (SS) medium with methylated-beta-cyclodextrin was the most suitable for production of acellular pertussis vaccine. For preparation of the vaccine, 5 day cultures of B. pertussis grown under stationary conditions at 35 degrees C were treated twice with ammonium sulphate and prospective protective antigens were extracted. The extracts contained pertussis toxin (PT), filamentous hemagglutinin and agglutinogens. These extracts were treated with formaldehyde and glutaraldehyde separately for detoxification of PT. The formaldehyde treatment of acellular preparations affected the potency and did not destroy the toxic effects of PT completely. Active PT was found in formaldehyde detoxified acellular pertussis vaccine (FDAPV) preparations by the Chinese hamster ovary (CHO) cell assay, the test for leucocytosis promoting factor (LPF) and the histamine sensitization (HS) test. The FDAPV preparations did not pass the mouse weight gain test (MWGT). The glutaraldehyde treatment had lesser adverse effects on potency than the formaldehyde treatment and the glutaraldehyde detoxified preparations did not show active PT by CHO cell assay, the test for LPF and the HS test. The mice tolerated high doses (up to four human doses) of GDAPV which passed the MWGT showing higher weight gains. Both FDAPV and GDAPV showed immunogenicity against agglutinogens and PT in mice. The GDAPV is a safe and potent vaccine. The total protein content of GDAPV was about 5 times lesser than that of whole cell pertussis vaccine.
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The immunogenicity of different types of glutaraldehyde inactivated pertussis vaccine (GIPV) preparations made by inactivation of Bordetella pertussis organisms with different concentrations of glutaraldehyde for variable periods at room temperature and conventional heat inactivated pertussis vaccine (HIPV) was evaluated with regard to production of agglutinins and neutralizing antibodies against pertussis toxin (PT). The different types of GIPV preparations had variable intracerebral mouse potency which depended upon the conditions of inactivation with glutaraldehyde. The agglutinin production in mice against various types of GIPV preparations and HIPV were very similar irrespective of the potency of the preparations. The agglutinins were produced against all the three major agglutinogens 1, 2 and 3 as the preparations were made from B. pertussis strain 10536 (serotype 1,2,3). The titres of agglutinins produced against adjuvanted preparations were slightly higher than those against non-adjuvanted preparations. Neutralizing antibodies against PT were produced for eight out of nine types of GIPV preparations while these antibodies were not produced for conventional HIPV.
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A total of 112 samples of adsorbed DPT vaccine were tested in mice for freedom from abnormal toxicity by three procedures: (a) single human dose by intraperitoneal route; (2) single human dose by subcutaneous route; and (3) half the single human dose by intraperitoneal route. The percentage of samples passing by the three methods was 25, 90 and 54.5, respectively. Method 3, which is being recommended now by the World Health Organization, is supported by the results of this study to be neither very stringent nor very lenient to pass the adsorbed DPT vaccine for freedom from abnormal toxicity.
Tn5-gusA promoter/probe transposons have been constructed that fuse the Escherichia coli gusA reporter gene transcriptionally or translationally with a target promoter. These have been used to monitor expression of Rhizobium meliloti symbiotic genes within alfalfa nodules. Fusions in all 11 nod genes studied show the same pattern of expression: first on the root surface, then throughout the developing nodule, then mainly in the nodule meristem, falling off progressively through the central region, and then disappearing. In contrast, fusions in all five nif genes studied, all four fix genes, and syrM show a second, different pattern: expression beginning later, first throughout the nodule except for the meristem, strongest just behind the meristem, and falling off progressively through the central region. Novel features revealed by these studies include nod expression in the meristem, regulated in planta expression of control genes nodD1 and nodD3, disappearance of nod expression late in organogenesis, and properties of syrM.
The hemagglutinating (HA) activities of purified pertussis toxin (PT) and filamentous hemagglutinin (FHA) were evaluated against unfixed and glutaraldehyde-fixed erythrocytes from ox, goose, horse, monkey, sheep, chicken, and rabbit. Both PT and FHA showed HA activities against fixed and unfixed erythrocytes from all the animals studied. The HA titers of FHA were higher than those of PT. The HA activities of FHA and PT were not destroyed completely even after heating these preparations at 56 C for 30 min. A simple test for the assay of PT in culture supernatants of Bordetella pertussis on the basis of HA activity has been described.
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The Rhizobium meliloti nod region IIb is involved in host-range determination: (i) the presence of region IIb is necessary for transfer of alfalfa root hair curling ability to Rhizobium leguminosarum biovar trifolii; (ii) a mutation in region IIb extends the R. meliloti infection host range to Vicia sativa nigra; (iii) dominance of R. meliloti nod genes over R. leguminosarum biovar viciae nod genes is abolished by mutations in region IIb. The nucleotide sequence of this region has been determined. Genes corresponding to the two open reading frames identified are designated nodP and nodQ. The predicted amino acid sequence of the NodQ protein shows homology with translation initiation and elongation factors. The consensus sequence involved in the GTP-binding domain is conserved.
A good correlation was observed between mouse intracerebral potency in terms of International Units and 50 per cent histamine sensitizing dose (HSD50) of various types of pertussis vaccine. The correlation was significant irrespective of the inactivating agent used for the preparation of pertussis vaccine. If the potency of the vaccine increased, the HSD50 decreased and vice versa showing a negative correlation between potency and HSD50. The relative histamine sensitizing (HS) activities of various types of pertussis vaccines in comparison to that of heat inactivated pertussis vaccine (HIPV) obtained in different experiments were very similar although the HSD50 values of same types of vaccines obtained in these experiments varied greatly. The conventional HIPV preparations having potency around 8 IU per 40 x 10(9) organisms had HSD50 values in the range of 1.0 to 2.2 x 10(8) organisms. The estimation of HSD50 of pertussis vaccine has been found to be a useful measure of the toxicity of the vaccine and also correlates well with the potency of the vaccine.
A mouse model to estimate the potency of the diphtheria toxoid component in diphtheria-tetanus vaccines and diphtheria-tetanus-pertussis vaccines has been developed as an alternative to the conventional method of testing in guinea-pigs. Optimal conditions with regard to dose, route and period of immunization have been standardized. The maximum levels of antitoxin were detected five weeks after vaccination and the s.c. route was found to be optimal. Potency data have been compared with other studies in mouse models and with those obtained by the conventional method in guinea-pigs.