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Biomedical subjects

R K Gupta

Publications and source records attributed to R K Gupta.

At least 271 records · Page 15Linked to original sources
PubMed1996

Development of a guinea-pig model for potency/immunogenicity evaluation of diphtheria, tetanus acellular pertussis (DTaP) and Haemophilus influenzae type b polysaccharide conjugate vaccines.

We have evaluated a guinea pig model for assessing the immunogenicity of Haemophilus influenzae type b (Hib) polysaccharide-protein conjugate vaccines, acellular pertussis vaccine and combination vaccines-consisting of tetanus toxoid (TT), diphtheria toxoid (DT), acellular pertussis vaccine and Hib-TT (Hib-T) conjugate vaccine. The model was based on the United States (US) potency test for TT and DT which requires injection of guinea pigs with a single dose of undiluted vaccine. Guinea pigs showed dose-dependent antibody responses to pertussis toxoid (PTxd) and filamentous haemagglutinin (FHA), two important components of acellular pertussis vaccine. Antibody response of guinea pigs to commercially available Hib conjugate vaccines qualitatively resembled those of human infants. Unconjugated polyribosylribitolphosphate (PRP) was not immunogenic; PRP-D conjugate produced a low antibody response, HbOC, PRP-T (Merieux) and Hib-T (MPHBL) produced a low response to the first dose and a strong anamnestic response to the booster dose. PRP-OMP uniquely produced a strong response after the first dose which was boosted by the second dose. In preliminary experiments, injection of guinea pigs with the combined vaccine formulations consisting of TT, DT, whole cell or acellular pertussis vaccine (Ptxd and FHA) and Hib-T conjugate showed that these vaccines were immunogenic when combined, with some effects on the antibody responses of certain components. This model for testing potency/immunogenicity of combined vaccines substantially reduces the number of animals needed to test each lot of vaccine. To reduce the use of animals in testing vaccines further, we propose the use of a Vero cell assay for titrating diphtheria antitoxin and ELISA for measuring IgG antibody to tetanus toxin. The guinea pig model may also be useful for evaluating combination vaccines.

Animal Testing Alternatives↗
PubMed1996

Strategies for stabilising tetanus toxoid towards the development of a single-dose tetanus vaccine.

The stability of tetanus toxoid (TT) has been investigated for the purpose of enhancing its immunogenicity when encapsulated in TT-polylactide-co-glycolide (PLGA) microspheres. In this paper we describe our simulations of various potential inactivating events during microsphere processing and release of the antigen. These include: i) processing aqueous TT solutions in the presence of organic solvents, ii) exposing lyophilized TT to moisture, and iii) incubating vaccine with the degrading PLGA. At 37 degrees C, TT began to aggregate in solution a few hours after the addition of either methylene chloride or ethyl acetate to form a single oil-water interface. Similarly, exposure of the lyophilized vaccine to elevated humidity caused the antigen to lose solubility. Previous analysis of moisture-induced aggregates has revealed that formaldehyde, which is stored in labile linkages in the vaccine following its detoxification, is the precursor to the reactive species in the principal aggregation pathway [1]. Methods to inhibit this mechanism, such as blocking nucleophilic amino groups of TT with succinic anhydride, were verified. Succinylation of TT resulted in the incorporation of 10-fold greater antigenically active vaccine in PLGA microspheres relative to the unmodified TT following processing by double-emulsion/solvent evaporation with ethyl acetate, strongly suggesting the formaldehyde-mediated aggregation pathway also occurs during the deleterious conditions of microsphere processing. Incubation of solutions of TT in the presence of excess blank (unloaded) poly (D,L) lactide (mol. wt. 2000) microspheres led to a dramatic reduction in pH (approximately 3.2 units after one day at 45 degrees C) and simultaneous precipitation of TT. Stabilisation in the presence of the degrading polymer is likely to be the final obstacle before controlled-release preparations can be formulated to release antigenically active TT over extended time periods. Hence, mechanistic analyses as described here may be crucial for the development of effective single-dose vaccines.

Delayed-Action Preparations↗
PubMed1996

The prevalence of transfusion transmitted diseases in renal transplant recipients.

Majority of renal transplant patients have history of blood transfusion. Out of a total of 120 renal transplant patients, 20 (16.6%) patients showed raised alanine aminotransferase (ALT) levels (> 45 IU) on 2 or more occasions at 2 weeks interval. This study was undertaken to estimate the risk of transfusion transmitted diseases (TTD) in these 20 renal transplant patients. Another 200 voluntary blood donors were also included as control. Both subject groups were screened by ELISA for HBsAg, anti HBc (IgG & IgM), anti HCV, anti CMV (IgM), anti HIV and VDRL tests for syphilis. A total of 11 (55%) patients and 3 (1.5%) blood donors showed the evidence of HBsAg positivity (p < 0.01). Anti HBc antibody was present in 12 (60%) patients and 39 (19.5%) donors which was significantly (p < 0.01) different. Anti HCV antibody was detected in 5 patients and in one blood donor (p < 0.01). However, anti CMV antibody was present in 4 (20%) patients and 3 (1.5%) donors, respectively. There was no evidence of HIV and syphilis infection in both these groups. High incidence of hepatitis markers were observed in renal transplant patients.

Biomarkers↗
PubMed1996

Neonatal cholestasis syndrome: an appraisal at a tertiary center.

OBJECTIVE: To know the magnitude, etiology and clinical profile, the efficacy of various investigations and the outcome in patients with neonatal cholestasis syndrome (NCS). DESIGN: Prospective evaluation of 60 consecutive infants with NCS (mean age 3.9 +/- 1.9 months; 49 males) over a period of 3.5 years. SETTING: Tertiary level referral gastroenterology center in North India. METHODS: Liver function tests, urine examination, serum antibodies against Cytomegalovirus (CMV), Rubella and Toxoplasma; abdominal ultrasonography, hepatobiliary scintigraphy and liver biopsy were done. In appropriate setting, laparotomy and surgical corrections were done for biliary tract disorders. RESULTS: NCS constituted 19% of pediatric liver diseases. Considerable delay in presentation was observed [mean delay, extrahepatic biliary atresia (EHBA) = 4 +/- 2.0 months, neonatal hepatitis (NH) = 2.2 +/- 1.3 months]. Thirty three (55%) infants had EHBA, 14 (23%) NH (4 CMV, 2 galactosemia, 1 urinary tract infection and 7 idiopathic), 2 (3%) paucity of intralobular bile ducts and 11 (18%) were of indeterminate etiology. Liver biopsy was the most accurate (96.4%) investigation in discriminating between EHBA and NH. Of the 18 operated infants with EHBA (portoenterostomy-15 and hepatico-jejunostomy-3), 10 were alive (mean follow up = 22.8 +/- 8.6 months) of which 4 were completely asymptomatic. CONCLUSIONS: (i) NCS is an important cause of liver disease in Indian children. (ii) It requires prompt referral, quick investigative approach and targeted management. (iii) Liver biopsy is highly accurate in differentiating EHBA and NH. (iv) infants with EHBA and compensated status of liver should undergo corrective surgery irrespective of age at presentation.

Biliary Atresia↗
PubMed1995

NMR studies of the effect of Mg2+ on post-ischemic recovery of ATP and intracellular sodium in perfused kidney.

Ischemia is often implicated as a cause of acute renal failure. We have investigated the effect of various concentrations of extracellular Mg2+ on the post-ischemic recovery of ATP and low intracellular Na+ in the isolated perfused rat kidney using 31P and triple-quantum filtered (TQ) 23Na-NMR spectroscopy. Following a 1 h period of stopped flow ischemia, the kidneys exposed to 0.3 mM Mg2+ throughout the experiment exhibited a significantly (p < 0.05) decreased post-ischemic fractional recovery of ATP (56 +/- 7%) as well as a significantly (p < 0.05) increased accumulation of P(i) (250 +/- 30%) as compared to kidneys exposed to 1.2 mM Mg2+ throughout (88 +/- 5% recovery of [ATP] and 158 +/- 8% accumulation of [P(i)]). Kidneys exposed to 0.3 mM Mg2+ during the pre-ischemic and ischemic periods but to 1.2 mM Mg2+ during reperfusion also showed better recovery of ATP (83 +/- 6%) and lower accumulation of P(i) (143 +/- 8%) compared to kidneys exposed to low Mg2+ (0.3 mM) throughout the experiment. Measurements of the 23Na TQ signal following ischemia-reperfusion revealed that kidneys exposed to 1.2 mM Mg2+ exhibited significantly improved maintenance of low intracellular Na+ as compared to those exposed to 0.3 mM Mg2+ ([Na+]i = 107 +/- 7% in 1.2 mM Mg2+ vs. 152 +/- 3% in 0.3 mM Mg2+). No significant difference was found in the pre-ischemic basal intracellular free Ca2+ level (as measured by 19F-NMR in combination with 5 FBAPTA) between kidneys perfused with 1.2 mM and 0.3 mM Mg2+, and comparable depletion of ATP occurred during ischemia under both experimental conditions. These data indicate that increased extracellular Mg2+ has a protective effect against post-ischemic damage, probably related to its role in resynthesis of ATP during post-ischemic reperfusion. Our results would imply a greater vulnerability of the kidney to ischemic damage in hypomagnesemic clinical conditions such as alcoholism and diabetes.

Adenosine Triphosphate↗
PubMed1995

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

The progress toward single-dose vaccines has been limited by the poor solid-state stability of vaccine antigens within controlled-release polymers, such as poly(lactide-co-glycolide). For example, herein we report that lyophilized tetanus toxoid aggregates during incubation at 37 degrees C and elevated humidity--i.e., conditions relevant to its release from such systems. The mechanism and extent of this aggregation are dependent on the moisture level in the solid protein, with maximum aggregation observed at intermediate moisture contents. The main aggregation pathway is consistent with formaldehyde-mediated cross-linking, where reactive electrophiles created and stored in the vaccine upon formalinization (exposure to formaldehyde during vaccine preparation) react with nucleophiles of a second vaccine molecule to form intermolecular cross-links. This process is inhibited by the following: (i) succinylating the vaccine to block reactive amino groups; (ii) treating the vaccine with sodium cyanoborohydride, which presumably reduces Schiff bases and some other electrophiles created upon formalinization; and (iii) addition of low-molecular-weight excipients, particularly sorbitol. The moisture-induced aggregation of another formalinized vaccine, diphtheria toxoid, is also retarded by succinylation, suggesting the generality of this mechanism for formalinized vaccines. Hence, mechanistic stability studies of the type described herein may be important for the development of effective single-dose vaccines.

Bacterial Vaccines↗
PubMed1995

Use of a multiparametric panel to target subpopulations in a heterogeneous solid tumor model for improved analytical accuracy.

The exclusion of non-tumor and dead cells from the analysis of live tumor cells can significantly improve the accuracy of prognostic indicators such as proliferative and DNA indexes. To target live breast tumor cells in a heterogeneous breast tumor model, we have designed a panel consisting of the DNA-specific dye DAPI and epithelial tissue-specific (cytokeratin), tumor-associated (MC5), proliferation-associated (proliferating cell nuclear antigen), and viability-associated (tubulin) markers. The breast tumor model consisted of a mixture of equal numbers of live and dead MDA-MB-175-VII (breast tumor) cells, live CEM (leukemic) cells, and live peripheral blood mononuclear cells. Targeting the live MDA cells in the mixture by gating on tubulin, cytokeratin, and MC5 resulted in a sevenfold increase in PCNA positivity (from 3% ungated to 22.3%), a 60% decrease in the %S-phase fraction (from 37.2% ungated to 15%), and elimination of extraneous hypodiploid and diploid components, enriching the tetraploid MDAs. These results are consistent with those obtained from unmixed live MDA cells. The combined utilization of this panel and "cumulative" electronic gating of the targeted population increases the number of relevant parameters that can be analyzed per sample and the accuracy of the resultant data.

Biomarkers, Tumor↗
PubMed1995

The magnetic resonance renogram in renal transplant evaluation using dynamic contrast-enhanced MR imaging.

To assess the role of the dynamic gadolinium DTPA (Gd-DTPA) magnetic resonance (MR) renogram in differentiating various causes of renal allograft dysfunction, contrast-enhanced MR imaging studies were performed in 5 normal renal allografts, 5 patients with acute rejection (AR), and 7 patients with cyclosporine nephrotoxicity. Time-versus-signal intensity (SI) curves were plotted. Normal renal allografts showed a rapid increase and slow decay, with a definite peak in cortical (CX) SI curves (peak mean signal intensity (Amax 338.6 +/- 46.5). The outer medullary (OM) (Amax 306.5 +/- 59) and inner medullary (IM) (Amax 263.4 +/- 47.4) curves did not show a definite peak. The OM curve slowly reached a steady state and caught up with the CX curve. AR episodes were characterized by a blunted uprise and delayed peak of CX (Amax 180 +/- 70.9) and a low-amplitude vascular phase with a slow constant uprise of the inner medullary curve (Amax 120.35 +/- 42.4). The OM signal intensity curve (Amax 150.73 +/- 78) failed to catch up with the CX curve. The maximum amplitude of SI curves in CsA-induced allograft dysfunction were low, with no definite peak, and CX, OM, and IM curves ran parallel to each other with a constant gap. Dynamic Gd-DTPA, MR imaging is a noninvasive technique that shows distinct characteristics in acute rejection, cyclosporine nephrotoxicity, and normally functioning renal allografts.

Adult↗
PubMed1995

Method for quantitation of IgG subclass antibodies in mouse serum by enzyme-linked immunosorbent assay.

One of the methods for calibration of antigen specific antibodies in a serum involves parallel titration of known concentrations of purified normal immunoglobulins (Igs) and a specific antiserum. We evaluated the effect of three methods for capturing known concentrations of purified normal mouse Igs (IgG, IgG subclasses and IgM) on the anti-tetanus toxoid antibody concentrations assigned to a reference mouse serum: (1) direct coating, (2) capture by pre-coated anti-mouse IgG (Fc) specific antibodies, and (3) capture by pre-coated anti-mouse IgG (Fab) specific antibodies. Direct coating of purified Igs onto plastic was the least efficient and would greatly overestimate antigen specific antibody concentrations. Equivalent concentrations of purified IgG subclasses and IgM produced higher absorbances when captured by anti-Fab antibodies pre-coated on plates than by anti-Fc antibodies. As a consequence, the anti-Fc capture method resulted in the assignment of IgG1, IgG2a and IgG2b tetanus specific antibody concentrations which exceeded the total IgG1, IgG2a and IgG2b concentrations. Additionally, the sum of tetanus specific IgG subclass antibodies exceeded the tetanus specific IgG antibody concentration by > 2-fold. In contrast, the anti-Fab capture method resulted in 2-9-fold lower assigned tetanus antibody concentrations which did not exceed the total Ig concentrations. We conclude that when using enzyme-linked immunosorbent assays, parallel titration of purified normal Ig standards captured by anti-Fab antibodies is superior to anti-Fc capture or direct coating for calibrating antigen specific antibodies in a reference serum.

Animals↗
PubMed1995

Aspiration cytodiagnosis of metastatic Merkel-cell carcinoma.

A case of metastatic Merkel-cell carcinoma to lymph nodes on the left side of the neck and left parotid is described. The primary was at the left side of the cheek and bridge of the nose. The cytologic features in needle aspirates of metastases included increased cellularity, discohesive or loose groups of small-to-medium size malignant cells with uniform, round-to-oval nuclei, delicate nuclear membranes, fine chromatin, multiple micronucleoli, and scanty cytoplasm. Additionally, rare intermediate filament "buttons" and frequent mitotic figures were found. The primary tumour showed similar features. On immunostaining, although a positivity for chromogranin was noted in the primary tumour, it was found to be negative in the cytologic material. All other histochemical and immunostains were found to be negative in both the primary and metastatic neoplasm. The problems in differential diagnosis from other small cell tumours and the features of this unique tumour that may be helpful in the diagnosis are discussed.

Aged↗
PubMed1995

Value of image guided fine-needle aspiration cytology in the diagnosis of pancreatic malignancies.

In the period between 1981-1993, 93 image guided fine-needle aspiration cytology (FNAC) of the pancreas were performed on 63 patients suspected of having a pancreatic carcinoma. From these, 42 patients were subsequently confirmed to have had a pancreatic malignancy by a tissue biopsy. However, FNAC was positive in 29 cases (69.04%) while seven cases (16.66%) were classified as suspicious. In six cases of malignancy (14.28%), cytologically diagnostic material was not sampled using FNAC; hence it was considered that FNAC evaluation gave false negative results in these cases. As a result of this study it was felt that while a cautious approach is necessary in view of certain false negative results, on the whole FNAC was a valuable investigation in the diagnosis of pancreatic carcinoma with no serious complications in this series. Also, FNAC under imaging assistance is a fairly reliable and safe procedure and the false negative results may be reduced by repeat aspirations especially in cases when insufficient diagnostic material is obtained in a patient clinically suspected of a pancreatic carcinoma.

Biopsy, Needle↗
PubMed1995

Adjuvant properties of aluminum and calcium compounds.

It is likely that aluminum compounds will continue to be used with human vaccines for many years as a result of their excellent track record of safety and adjuvanticity with a variety of antigens. For infections that can be prevented by induction of serum antibodies, aluminum adjuvants formulated under optimal conditions are the adjuvants of choice. It is important to select carefully the type of aluminum adjuvant and optimize the conditions of adsorption for every antigen since the degree of adsorption of antigens onto aluminum adjuvants markedly affects immunogenicity. The mechanism of adjuvanticity of aluminum compounds includes formation of a depot at the site of injection from which antigen is released slowly; stimulation of immune-competent cells of the body through activation of complement, induction of eosinophilia, and activation of macrophages; and efficient uptake of aluminum-adsorbed antigen particles by antigen-presenting cells because of their particulate nature and optimal size (< 10 microns). Limitations of aluminum adjuvants include local reactions, production of IgE antibodies, ineffectiveness for some antigens, and inability to elicit cell-mediated immune responses especially cytotoxic T-cell responses. Calcium phosphate, which has adjuvant properties similar to aluminum adjuvants, has the potential advantages of being a natural component of the body and of not increasing IgE production. There is a need for alternative adjuvants, particularly for diseases in which cell-mediated immune responses are important for prevention or cure.

Adjuvants, Immunologic↗
PubMed1995

Mitoxantrone and cytosine arabinoside as treatment for acute myeloblastic leukemia in older patients.

The majority of patients with acute myeloid leukemia (AML) are elderly, and their response to chemotherapy is poorer than that of younger patients. The combination of mitoxantrone (MTN) and cytosine arabinoside (Ara-C) is a possible alternative to an anthracycline/Ara-C combination for the treatment of AML in these patients. Of 52 older patients (> 59 years) referred over a 3.5-year period, 33 patients (age range 60-78 years, median 67 years) received MTN and Ara-C as therapy for newly diagnosed AML. MTN was administered at a dose of 12 mg/m2/day, intravenously, for 3 days (23 patients), or 10 mg/m2/day for 5 days (10 patients), and Ara-C at a dose of 100 mg/m2 twice daily, intravenously, for 7 days. Complete remission (CR) was achieved in 16/33 patients (48%). The median remission duration was 6 months (range 1-37 months). The median survival was 14 months for those who achieved CR compared with 9 months for those with resistant disease. Two patients remain in first CR after 13 and 37 months, but three patients died whilst receiving consolidation therapy. In selected elderly patients with AML, the combination of MTN and Ara-C provides an acceptable alternative to an anthracycline/Ara-C regimen, with a higher CR rate than historical controls. However, the CR rate and remission duration remain low compared with those of younger patients, supporting the need to investigate new approaches to treatment in this population.

Age Factors↗