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Biomedical subjects

S Ratnam

Publications and source records attributed to S Ratnam.

At least 55 records · Page 3Linked to original sources
PubMed1995

Comparison of commercial enzyme immunoassay kits with plaque reduction neutralization test for detection of measles virus antibody.

Four commercially available enzyme immunoassay (EIA) kits were evaluated in comparison with the plaque reduction neutralization (PRN) test for detection of measles virus antibody. The EIA kits, Enzygnost (Behring), Diamedix, Vidas (bioMerieux Vitek), and Measlestat (Biowhittaker), were assessed with two PRN cutoff titers: a PRN titer of 8, the lowest detectable antibody level by the PRN test under the test conditions, and a titer of 120, which has been shown to be the minimum protective antibody titer. At a PRN cutoff titer of 8, the sensitivity was 88.2, 91.1, 74.6, and 69.8% for Behring, Diamedix, Vidas, and Biowhittaker EIA tests, respectively, with negative predictive values ranging from 22.7 to 45.5%. The specificity was 93.8% for Diamedix and 100% for the rest. At a PRN cutoff titer of 120, the sensitivity and specificity, respectively, were 100 and 90.7% (Behring), 98.2 and 58.8% (Diamedix), 90.6 and 94.5% (Vidas), and 85.7 and 96.4% (Biowhittaker). At this PRN cutoff titer, the negative predictive values of all EIA tests improved considerably, ranging from 70.7 to 100%. The EIA results showed an excellent association with PRN results when the PRN titers of the test samples were either < 8 or > 1,052. Discrepancies occurred especially when testing samples having PRN titers in the range of 8 to 120, indicating lack of sensitivity of the EIA tests in detecting measles virus antibody at low levels. Maternally derived measles virus antibody at this level has been shown to interfere with measles vaccine response in children and hence has implications from the standpoint of measles immunization. The ready availability, ease of operation, and rapid turnaround time are strong plus points of the EIA kits, and they could be useful in a clinical laboratory setting for routine application, but they may have limited use in vaccine-related studies and seroepidemiological surveys.

Antibodies, Viral↗
PubMed1994

Occurrence of Listeria in hot and cold smoked seafood products.

Over a one-year period, 258 samples of smoked fish products were obtained from retail outlets in Newfoundland and processed for Listeria. Of these, 142 were hot smoked and 116 cold smoked, and the samples comprised of nine species of fish. The Canadian FDA listeria isolation protocol consisted of a two-stage enrichment followed by plating on selective isolation media, Oxford, and LPM. An additional selective medium, PALCAM, was also used. Listeria spp. were isolated from 43 of 258 (16.7%) samples processed in all, with hot smoked products yielding 25.4% (36/142) of the isolates, and cold smoked products yielding 6% of the isolates (7/116). Among the nine species tested, cod had the highest rate of Listeria contamination at 46.7%. Of the 43 Listeria spp. isolated, 18 (41.9%) were L. innocua, 13 (30.2%) were L. welshimeri and 12 (27.9%) were L. monocytogenes.

Food Microbiology↗
PubMed1994

Complete androgen insensitivity due to a splice-site mutation in the androgen receptor gene and genetic screening with single-stranded conformation polymorphism.

OBJECTIVE: To characterize the genetic defect in a family with complete androgen insensitivity syndrome and to determine whether single-stranded conformation polymorphism (SSCP) can be used to detect subtle mutations in the androgen receptor (AR) gene. DESIGN: Amplification, subcloning where appropriate, and sequencing of the AR gene in members of the affected family and to use SSCP to differentiate rapidly mutant from normal alleles. SETTING: Reproductive endocrinology clinic and laboratory in a university hospital. PATIENTS: A family of which two sisters (46XY) have complete androgen insensitivity syndrome. RESULTS: A novel single base (G --> A) mutation in the exon G-intron 7 junction of the AR gene caused an abnormal donor splice site leading to complete androgen insensitivity in both affected siblings. Their mother was demonstrated to be the heterozygous carrier of this mutation while the other two males in the family carried the normal allele. Single-stranded conformation polymorphism proved useful for defining the normal, mutant, and heterozygous carrier status of each member of this family. CONCLUSIONS: This new mutation of the human AR gene illustrates the importance of exon G in receptor function. Single-stranded conformation polymorphism is a simple and rapid screening technique that can be used to detect unknown subtle mutations in the AR gene.

Adolescent↗
PubMed1994

Human embryonic behavior in a sequential human oviduct-endometrial coculture system.

Embryonic behavior to blastocyst, hatching, and hatched stages were evaluated in 77, four-cell human embryos that were first grown in oviductal cell coculture and then equally allotted at the eight-cell stage to two coculture systems in a serum-free medium (34 continued on oviductal monolayers, 32 on endometrium monolayers). Sixty-three percent and 40% of embryos expanded and hatched in the sequential oviductal-endometrial coculture system when compared with 41% and 9% in the oviductal system alone, respectively. The sequential coculture system appears to be an improved system over the single human oviductal coculture system.

Culture Techniques↗
PubMed1994

Isolation and culture of inner cell mass cells from human blastocysts.

Totipotent non-committed inner cell mass (ICM) cells from human blastocyts, if demonstrated to be capable of proliferating in vitro without differentiation, will have several beneficial uses, not only in the treatment of neurodegenerative and genetic disorders, but also as a model in studying the events involved in embryogenesis and genomic manipulation. Nine patients admitted to an in-vitro fertilization programme donated 21 spare embryos for this study. All 21 embryos were grown from the 2-pronuclear until blastocyst stages on a human tubal epithelial monolayer in commercial Earle's medium (Medicult, Denmark) supplemented with 10% human serum. The medium was changed after blastocyst formation to Chang's medium supplemented with 1000 units/ml of human leukaemia inhibitory factor (HLIF) and the embryos left undisturbed for 72 h to allow the hatched ICM and trophoblast to attach to the feeder monolayer. Nineteen of the 21 embryos from nine patients produced healthy ICM lumps which could be separated and grown in vitro. Two of the lumps differentiated into fibroblasts while the remaining 17 (eight patients) produced cells with typical stem cell-like morphology, were alkaline phosphatase positive and could be maintained for two passages. It was possible to retain the stem cell-like morphology, alkaline phosphatase positiveness and normal karyotype through the two passages in all of them using repeated doses of HLIF every 48 to 72 h. This is the first report on the successful isolation of human ICM cells and their continued culture for at least two passages in vitro.

Adult↗
PubMed1993

Mitogenic and cytogenetic evaluation of transforming growth factor-beta on murine preimplantation embryonic development in vitro.

Slow cleavage rate has been a major contributory factor influencing embryo morphology in in vitro fertilization (IVF) programs. The role of transforming growth factor-beta (TGF beta 1) in improving this characteristic was evaluated using the murine model. Replicate batches of eight-cell compacting embryos from superovulated mice were divided into three groups. Group A were treated with 0.3 ng/ml TGF beta 1 at the initial compacting stage, followed by a second treatment of 0.1 ng/mL 22 h later at the cavitating stage; group B received 0.3 ng/ml TGF beta 1 at the cavitating stage; group C were controls. The percentages of treated embryos reaching fixed embryonic stages, total cell number (TCN), mitotic index, and incidence of chromosome anomalies were monitored. The percentage of embryos reaching the cavitating, expanded, hatching, and hatched stages in both treatment groups were not significantly different from control (96.6% +/- 4.2% to 37.7% +/- 12.7% vs. 95.3% +/- 7.3% to 47.0% +/- 3.5%; P > 0.05). Values between the two treatment groups were also not significantly different. Embryos in groups A and B produced significantly greater TCN at expanded blastocyst and hatching stages compared to controls (Group A: 107.0 +/- 18.9 vs. 89.9 +/- 17.4, P < 0.05 and 125.5 +/- 16.4 vs. 113.9 +/- 12.1, P < 0.05; Group B: 107.9 +/- 14.0 vs. 89.9 +/- 17.4, P < 0.05 and 124.9 +/- 17.4 vs. 113.9 +/- 12.1, P < 0.05). Values, however, were not significantly different between treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Prevalence of leptospiral agglutinins among conservancy workers in Madras City, India.

In a study of 584 Corporation conservancy (sanitation) workers who lived mostly in slums, and who worked in four Corporation Circles of Madras City, India, 192 (32.9%) were found to be positive for agglutinins to Leptospira interrogans. Seropositivity prevalence increased with age, but was similar in males and females except in the youngest age group, where males predominated. Prevalence in the four study areas ranged between 17.8 and 40.5% (P < 0.01). Among 152 sera in which one serogroup predominated, Autumnalis was the most commonly recorded (33.6%), followed by Icterohaemorrhagiae (15.1%), Panama (15.1%), Sejroe (14.5%) and others (21.7%). Forty sera reacted to two or more serogroups at the same (highest) titre, most frequently to the first three serogroups above. The titre range was 1:50-1:3200 (geometric mean titre 149). Among a group of 46 male automobile industry workers who lived in middle-class housing, seropositivity prevalence (17.4%) was approximately half that of the sanitation workers (P < 0.05), and the titre range was lower (1:50-1:200, GMT 84). The predominating serogroups were those found in the sanitation workers. Bearing in mind that sanitation workers are the urban group probably at highest risk of leptospiral infection, the prevalence rate (< 33%) found in our study is not considered to be particularly high.

Adult↗
PubMed1992

Use of two types of estradiol-releasing skin patches for menopausal patients in a tropical climate.

As part of a program to evaluate nonoral routes for delivery of estrogen replacement therapy at the National University Hospital, Singapore, we assessed two different 50 micrograms estradiol patches. The first study (patch A) was terminated because of a high rate of skin reactions. Tests of the components of this patch identified the alcohol carrier as the cause of these reactions when combined with an occlusive membrane in the tropical Singapore climate. A subsequent clinical trial with 21 female subjects was undertaken with a different 50 micrograms estradiol patch (patch B). Twenty patients completed the 6-month study. Patch B was as effective as patch A, caused fewer skin reactions, and adhered better.

Administration, Cutaneous↗
PubMed1992

Studies on cell-wall deficient non-acid fast variants of Mycobacterium tuberculosis.

While the host-parasite relationship in tuberculosis still remains incompletely understood, there has been recent renewed interest in indications that tubercle bacilli are converted into metabolically inactive, non-acid fast (NAF) granular forms in the presence of host defence mechanisms and antituberculosis drugs. The present study investigates the mechanism of induction of these NAF variants in vitro and in vivo, and their ultimate pathogenicity. Evidence is provided that appears to clearly indicate that acid-fast mycobacteria are converted into NAF, cell wall deficient variants which remain dormant, only to revert to the parent, acid-fast bacilli in immune-compromised hosts, thence ultimately producing disease. It is then suggested that this may be one of the causes of the observed persistence of the bacilli in hosts in spite of chemotherapy. In a typical study in experimental animals in the present investigation, NAF variants were separated from lung lavage by differential centrifuging. When these were then injected into animals made immune-deficient with Freund's adjuvant or cyclophosphamide, they reverted to parent acid-fast forms. The presence of these NAF forms as variants of M. tuberculosis, and not merely contaminants, was clearly established by a number of methods. These included phase contrast and electron microscopy, immunological studies employing antiserum and comparison with the parent organisms, and gel electrophoresis of the proteins of the parent organisms. Other evidence is also offered confirming the hypothesis of reversion of NAF forms. It is also shown in this study that NAF forms can be induced in vitro by hydrolases.

Animals↗
PubMed1992

Fertilization, cleavage, and cytogenetics of 48-hour zona-intact and zona-free human unfertilized oocytes reinseminated with donor sperm.

OBJECTIVE: To examine the fertilization rates of 48-hour unfertilized oocytes inseminated with fertile donor sperm and to evaluate the cleavage and cytogenetics of ensuing embryos. DESIGN: Prospective. SETTING: Assisted reproductive technology (ART) program. PATIENTS: Four hundred ninety-seven unfertilized oocytes from 97 ART patients were categorized into four groups. A (zona-intact) and B (zona-free) were from patients with partial fertilization failure, whereas C (zona-intact) and D (zona-free) were total fertilization failures. RESULTS: Fertilization rates in groups A and B were significantly higher than C and D (33.2% to 60.9% versus 20.0% to 48.1%; P less than 0.01). Zona-free oocytes had higher fertilization rates than zona-intact oocytes (48.1% to 60.9% versus 20.0% to 32.2%). Multiple pronuclei were high in zona-free oocytes (33.1% to 41.3%). Forty-eight to 54% of embryos generated after donor insemination had chromosome anomalies (mosaicism, aneuploidy, pulverization). CONCLUSIONS: One cause of total fertilization failure appears to lie in intrinsic oocyte problems confined to the zona and oolemma. The fertilization of 48-hour unfertilized oocytes may be of some value in diagnosing fertilization failure in ART patients.

Cell Division↗
PubMed1992

Improved pregnancy rate after transfer of embryos grown in human fallopian tubal cell coculture.

OBJECTIVE: To evaluate the embryonic behavior in vitro and the pregnancy and implantation rates of embryos grown in a human ampullary cell coculture system. DESIGN: In a prospective study, two pronuclei embryos were cultured on human ampullary feeder layers up to the two to six-cell and blastocyst stages and replaced either as tubal, uterine, or sequential transfers. SETTING: Assisted reproductive technology program in a university-based hospital. PATIENTS: Fifty women with a mean age of 35.6 years who went through a single coculture cycle. Thirty of the patients were admitted for in vitro fertilization (IVF) and 20 for tubal embryo transfer (TET). RESULTS: The overall clinical pregnancy rate (PR) for all 50 patients was 44% per cycle (IVF, 37%; TET, 55%) and the implantation rate was 31.8% (IVF, 31.0%; TET, 32.6%). Sixty-eight percent of pregnant patients were over 35 years, and 68% had two previously failed assisted reproduction cycles. Five of 9 patients who received sequential transfers became pregnant. Three of the 22 pregnancies aborted (2 after sequential transfer), and there was one ectopic. Overall, 88% of two to six-cell stage embryos were of good quality. CONCLUSIONS: The human ampullary coculture system produces better quality embryos, increased numbers of blastocysts with improved PRs and implantation rates. The beneficial effects of the feeder layer may be through the release of embryotrophic factors and detoxification of the medium by the cells. Coculture is a new concept in assisted reproduction and has tremendous potential in boosting conception rates by mimicking the in vivo environment.

Adult↗
PubMed1992

The evaluation of various culture media in combination with dimethylsulfoxide for ultrarapid freezing of murine embryos.

Bicarbonate-buffered HTF medium, Medicult, and T6 are as effective as PB1 medium when used in combination with DMSO in ultrarapid freezing of two-cell mouse embryos. However, the use of phosphate-buffered T6 results in reduced in vitro development and inner cell mass size as compared with bicarbonate- and Hepes-buffered T6 when used with 3.5 M of DMSO. Hence, the use of this media for ultrarapid freezing should be avoided when this concentration of DMSO is used.

Animals↗
PubMed1991

Heparin-binding serum protein(s) is required for the protection of sialyltransferase released during the incubation of rat jejunal slices.

Incubation of rat jejunal slices in Krebs-Ringer bicarbonate buffer (KRB) required the presence of heat-inactivated horse serum (HHS) in order to show time-dependent release of sialyltransferase into the medium. Sialyltransferase activity could not be detected in the medium when KRB alone or KRB supplemented with either albumin or glycerol was used in the incubations. The viability of the jejunal slices for up to 4 h of incubation was determined by studying the incorporation of glucosamine and leucine into acid-insoluble proteins. Supplementation of KRB with HHS had no beneficial effect on the rate of incorporation of leucine and glucosamine into proteins. KRB medium obtained after different periods of incubation contained higher trypsin-like activity than KRB medium containing HHS. Various antiproteases present as supplements to KRB resulted in the release of sialyltransferase activity from the jejunal slices. Among these antiproteases, alpha 1-proteinase inhibitor (alpha 1-PI) was the most effective. Also, HHS added to KRB immediately following incubation resulted in partial restoration of sialyltransferase activity in the medium, suggesting the presence of anti-proteolytic factors in HHS. The addition of increasing concentrations of heparin to incubations containing HHS caused a decrease in the medium sialyltransferase activity. The heparin-binding fraction (HBF) from HHS, when added to incubations, was able to protect the sialyltransferase released into medium. However, HHS depleted of its heparin-binding fraction by heparin-agarose affinity chromatography was unable to protect the sialyltransferase. HBF was separated into high- and low-molecular-mass fractions (fractions A and B respectively) by gel-filtration chromatography. The capacity to protect the released sialyltransferase was contained in fraction B. Fraction A contained multiple bands on SDS/PAGE and did not protect the enzyme. Fraction B contained a major protein band on the gel which corresponded to the migration of a similar band in human alpha 1-PI. HBF as well as fraction B isolated from HHS showed anti-trypsin-like activity. The results presented indicate that HHS contains a heparin-binding protein(s) similar to human alpha 1-PI which plays a role in the protection of sialyltransferase released from jejunal slices.

Animals↗
PubMed1991

Improved fertilization rates of human oocytes in coculture.

Passaged human tubal ampullary epithelial cells from cell lines were used to evaluate fertilization rates and the support of early human embryonic cleavage in vitro. A total of 225 mature oocytes from 32 patients was used in this study. Oocytes from each patient were equally allotted at random to two groups. One hundred thirty-two oocytes were inseminated in the presence of passaged human ampullary cells, while 123 oocytes were inseminated in culture medium alone. Fertilized oocytes were transferred to tubal ampullary cell monolayers and monitored for up to 48 hr. Eighty-five percent of oocytes fertilized in coculture, as compared to 67% in medium alone (P less than 0.01). A higher percentage of good-quality embryos was observed in cocultures than in controls (79 vs 67%; P less than 0.05). Binding of sperm to ampullary monolayer cells was observed. Human ampullary-cell cocultures may be useful to improve fertilization rates and embryonic viability and thus increase take-home baby rates for in vitro fertilization programs.

Adult↗