Inferences on Small Area Proportions.

Design-based methods are generally inefficient for making inferences about small area proportions for rare events. In this paper, we discuss an alternative hierarchical model and the associated hierarchical Bayes methodology. Sufficient conditions for propriety of the posterior distributions of relevant parameters are presented.

A study of platelet aggregation in patients with acute myocardial infarction at presentation and after 48 hrs of initiating standard anti platelet therapy.

AIMS & OBJECTIVES: Platelet aggregation is a key factor behind coronary artery disease. Various complications after an attack of acute coronary syndrome are often related to the platelet hyperactivity in the early hours following the event. There is a growing concern regarding aspirin & clopidogrel resistance, which has put the time-tested therapies under scrutiny. Time has come to address the issue of platelet hyperactivity in the early hours & whether to individualize therapy and drug doses in different patients. MATERIALS & METHODS: We prospectively enrolled 41 patients with a diagnosis of acute myocardial infarction (AMI) between July 2009 and July 2010 admitted to the cardiology ward and ICCU of Medical College, Kolkata, after fulfillment of inclusion & exclusion criteria. The study was reviewed and approved by the Institutional Ethical Committee. Platelet Aggregation (PA) with 10 microM epinephrine, 2 microg/ml collagen and 10 microM ADP was performed with light transmittance aggregometry in all patients according to the standard protocol. Tests were done within 3 hours of sampling with platelet-rich plasma (PRP) by the turbidometric method in a 2-channel aggregometer (Chrono-Log 490 Model, Chrono-Log Corp, Havertown, Pa). Aspirin & clopidogrel resistance were defined as per ACC/AHA guidelines. Platelet aggregation studies were done at presentation (zero hour) and 48 hours after instituting dual antiplatelet therapy in standard doses. RESULTS: Patients with first attack of AMI showed a high mean platelet aggregation at 0 hours of 77.4% +/- 18.8% with ADP, 77.5% +/- 26% with Epinephrine & 73.5% +/- 24.9% with Collagen. With all three agonists, the initial hyperactivity of platelets at 0 hours was significantly higher among diabetics & obese. Though reduced, significant platelet hyperactivity remained at 48 hours after initiating standard antiplatelet therapy; 50.3% +/- 14.3% with ADP, 56.5% +/- 21.6% with epinephrine & 38.4% +/- 22% with collagen. CONCLUSION: In the early hours after AMI there is a fairly high degree of platelet aggregation. Even after 48 hours of standard antiplatelet therapy the platelet aggregation though reduced, still remains significantly high. Since recurrent ischemic episodes frequently occur in this vulnerable period, time has come to assess platelet aggregation status in high risk groups, if not in all patients of acute coronary syndrome during this period so that therapy may be individualized. Further researches are required in this area.

Adjusted Maximum Likelihood Method in Small Area Estimation Problems.

For the well-known Fay-Herriot small area model, standard variance component estimation methods frequently produce zero estimates of the strictly positive model variance. As a consequence, an empirical best linear unbiased predictor of a small area mean, commonly used in the small area estimation, could reduce to a simple regression estimator, which typically has an overshrinking problem. We propose an adjusted maximum likelihood estimator of the model variance that maximizes an adjusted likelihood defined as a product of the model variance and a standard likelihood (e.g., profile or residual likelihood) function. The adjustment factor was suggested earlier by Carl Morris in the context of approximating a hierarchical Bayes solution where the hyperparameters, including the model variance, are assumed to follow a prior distribution. Interestingly, the proposed adjustment does not affect the mean squared error property of the model variance estimator or the corresponding empirical best linear unbiased predictors of the small area means in a higher order asymptotic sense. However, as demonstrated in our simulation study, the proposed adjustment has a considerable advantage in the small sample inference, especially in estimating the shrinkage parameters and in constructing the parametric bootstrap prediction intervals of the small area means, which require the use of a strictly positive consistent model variance estimate.

Abundance of siderophages in sputum: indicator of an adverse lung reaction to air pollution.

OBJECTIVE: To investigate the lung response to traffic-related air pollution by enumerating hemosiderin-laden alveolar macrophages (AM) in sputum. STUDY DESIGN: Sputum samples were collected from 103 urban adult males from Calcutta chronically exposed to automobile exhaust. Forty-nine rural individuals served as controls. AM were identified by nonspecific esterase staining. Perl's Prussian blue technique was employed for the detection of hemosiderin-laden AM (siderophages). RESULTS: The urban group, consisting of 31 traffic officers, 25 automobile service station workers and 47 street hawkers, had seven times more AM in their sputum than did the matched controls. Besides, a remarkable rise (27-fold) in the number of siderophages in sputum was observed in urban individuals. Smoking further elevated the AM count and number of siderophages. CONCLUSION: Abundant siderophages in the urban group may indicate the toxic effect of airborne pollutants on the lung, leading to phagocytosis of destroyed cells, including erythrocytes, and accumulation of iron in AM. Enumeration of siderophages in sputum appears to be a simple, noninvasive, inexpensive cytochemical technique well suited to preliminary assessment of the adverse effects of air pollution on the lungs in large, population-based studies, especially in developing countries.

Air pollution in Calcutta elicits adverse pulmonary reaction in children.

BACKGROUND & OBJECTIVE: Pulmonary responses of children chronically exposed to ambient air pollution in Calcutta have been investigated. METHODS: A total number of 153 children from Calcutta and 116 from rural West Bengal in the age group of 6-17 yr were included in this study. Respiratory symptom complex, sputum cytology and micronucleus (MN) count of buccal epithelial cells were evaluated. Blood smears were examined for WBC differential count and RBC morphology. RESULTS: Marked rise in respiratory symptoms (43% in urban vs 14% in rural) and sputum alveolar macrophage (AM) number was observed in urban children compared to their rural counterparts (14.2 +/- 1.4 AM/hpf vs 6.7 +/- 1.4 AM/hpf, mean +/- SE, P < 0.001). The urban group also demonstrated increased numbers of neutrophils, eosinophils and iron-laden AM in their sputum. Besides, buccal epithelial cells of urban children exhibited higher MN frequency than their rural counterparts (0.22 vs 0.17%, P < 0.05). While sputum neutrophilia and eosinophilia suggest inflammatory and allergic lung reactions, elevated MN count is indicative of greater genotoxic effect on the exposed tissues of urban children. Hypochromic red cells in peripheral blood smear was a common finding in both urban and rural groups, but eosinophils and monocytes were present in elevated frequencies in the rural children. INTERPRETATION & CONCLUSION: The study demonstrated that children inhaling grossly polluted air of Calcutta suffer from adverse lung reactions and genetic abnormality in the exposed tissues.

Studies on the platelet alpha-adrenoceptor subtypes in diabetes mellitus.

Studies on the platelet alpha-adrenoceptor subtypes were carried out in 19 subjects with treated diabetes mellitus and 15 normal age and sex-matched controls. By utilising selective antagonists, it was noted that all the normal human platelets exhibited the alpha-2 adrenoceptor. 7 diabetics (37%) expressed both the alpha-1 and alpha-2 adrenoceptors and diabetic complications along with hypertension were most common in this group. Another 7 diabetics (37%) expressed the alpha-2 receptor only and diabetic complications were minimum in this group. Interestingly, 5 diabetics (26%) did not express either the alpha-1 or alpha-2 receptor and these patients occupied an intermediate position with regard to diabetic complications. Thus, it was concluded that platelet alpha-1 adrenoceptors perhaps indicated a poor prognosis in diabetes mellitus, opening up future scope for work in this area.

Some aspects of the causes of enhanced immune response of in vitro frozen ascites fibrosarcoma tumor cells in mice.

Estimation of 3 M KCl-extracted ascites fibrosarcoma (AFS) tumor cell membrane peripheral proteins in native and frozen tumor cells showed approximately a three-, four-, and fivefold increase per 1 x 10(6) cells of single-, three-, and programmed three-cycle frozen AFS tumor cells, respectively, compared to the same number of native cells, indicating an increase in surface membrane protein concentration with freezing. The 10% gel (homogeneous) electrophoretic (SDS-PAGE) study of 3 M KCl-extracted native and frozen cell membrane proteins showed (i) membrane proteins of native cells resolve into many more components compared to those of any frozen membranes, i.e., single, three, and programmed three cycle, the components decreasing in that order; (ii) the concentration of larger-molecular-weight protein fractions (> or = 75 kDa) decreases while those of smaller fractions (14 to 24 kDa) increase in frozen cells, with the maximum being in the programmed three-cycle frozen group. In contrast, the native cell membrane is rich in higher-molecular-weight proteins (> or = 75 kDa) with concentrations slowly decreasing toward lower-molecular weight fractions. Thus, the probable reasons for increased immune response of animals immunized with frozen AFS tumor cells are (i) absolute increase in cell surface protein concentrations as given by 3 M KCl extraction of cell membrane peripheral protein estimation in AFS tumor cells postfreeze; (ii) cell-surface protein pattern which is heterogeneous before freezing becomes relatively more homogeneous following freezing of AFS tumor cells; and (iii) depolymerization and breaking of higher-molecular-weight components which increase the concentration of terminal-sequence antigenic determinants and increase accessibility of determinant grouping by removing steric hindrance following freezing.

Survivability and leucocyte migration inhibition in mice immunized with cryotreated ascites fibrosarcoma cells using different freeze-thaw cycles.

Attempts have been made to assess as to what extent in vitro assay of cellular immunity, e.g. leucocyte migration inhibition (LMI) in mice immunized with different freeze-thaw cycles could reflect host resistance in vivo. While survivability of animals improved significantly by immunization with single cycle (P < 0.05) to three cycle (P < 0.001) and programmed three cycle (P < 0.001) cryo-treated tumor cells compared to controls, the percentage LMI in the same groups of animals decreased progressively. The KCl(3M) extracted tumor cell protein (antigen) of both viable and cryo-treated cells showed a progressively increased protein concentrations per 1 x 10(6) tumor cells with viable cells being least and programmed three cycle cryo-treated cells highest. The apparent discrepancy observed between percentage migration inhibition and survivability may be due to the fact that (1) survivability is a function of body's total immune response while LMI represents the response of one effector limb only; (2) immuno-regulatory mechanisms depend on a balance between activation and suppression and suppressor cells being more sensitive and of shorter life span, affect migration inhibition but not the survivability; (3) cryo-treatment alters tumor cell surface antigen affecting immunological balance; and (4) suppressor and antitumor activities against antigenic stimulation develop simultaneously in different organs and LMI performed with sensitized splenic cells, where, perhaps, suppressor cell activity dominates.

Human immunodeficiency virus infection related to blood transfusion service.

The prevalence of transmissible viruses, human immunodeficiency (HIV) and hepatitis B (HBV) in blood donors, recipients and blood bank staff in a Calcutta (eastern India) based blood bank and transfusion centre has been studied from 1987-93. HIV seropositivity of blood donors was of recent emergence and was low. Recipients of blood and blood components frequently i.e., haemophilics showed a progressive increase in HIV seropositivity since 1988 whereas in thalassaemics the emergence of HIV seropositivity was noted only in 1992. Blood bank staff were seronegative. HBV which has a similar portal of entry as HIV, had a higher prevalence in blood donors, recipients of blood/components and blood bank staff.

Hepatitis B vaccination and the rate of seroconversion in high risk contacts.

Twenty-seven employees of a blood transfusion centre, Calcutta were vaccinated with hepatitis B vaccine (recombinant DNA technology), 20 micrograms/1 ml intramuscularly on 0, 8, 32 weeks interval. The seroconversion rate at 8, 32, 40 weeks were 29.6%, 55.5% and 100% respectively. The recombinant hepatitis B vaccine is acceptable and safe. The seroconversion rate is comparable to plasma derived hepatitis B vaccine.

Immunosurveillance of transfusion dependent thalassaemia and hepatitis B vaccination.

The study evaluates the probable alteration of the immune system in multitransfused thalassaemics and the modification of their immune response following administration of hepatitis B virus (HBV) vaccine. B-thalassaemics (n = 50) and EB-thalassaemics (n = 30) who received multiple blood transfusion for the chronic anaemic status had significant iron overload. They had high prevalence for hepatitis B carrier state and almost all were exposed to hepatitis B virus during the course of transfusion as shown by the positivity of hepatitis B virus markers. Thalassaemics in presence of iron overload have altered immune status in terms of depressed T-lymphocytes and raised immunoglobulins G and M. However, they showed 100 per cent seroconversion with production of antibody to hepatitis B surface antigen following Hepatitis B vaccination.

Thyroidal influence on the cell surface GM1 of granule cells: its significance in cell migration during rat brain development.

1. No difference was observed in the in vitro growing ability of granule cells isolated from hypothyroid or normal rat brain. When granule cells were taken from hypothyroid rat brain and grown in normal culture medium containing 10% fetal calf serum, they behaved similarly to the granule cells obtained from normal rat brain. 2. In both cases there were progressive losses of in vitro growing ability of the granule cells with the age of the animal and it became impossible to grow them when derived from 21 days or older animals. 3. A marked decrease in cell surface GM1 was observed when the cells were maintained under thyroid hormone-deficient conditions in culture. 4. Anti-GM1 antibody was found to inhibit significantly the migration of granule cells along the astrocyte fibers. 5. These results indicate that GM1 has an important role in thyroid hormone-dependent postnatal brain maturation in rat.

Sero-surveillance of transmissible hepatitis B & C viruses in asymptomatic HIV infection in haemophilics.

In a group of 37 haemophilics, 9 (24.3%) were seropositive for human immunodeficiency virus (HIV), while 9 (24.3%) and 10 (27%) were positive for hepatitis B virus (HBV) and hepatitis C virus (HCV) respectively. Haemophilics who were HIV seropositive had higher prevalence of HBV and HCV. Seropositivity for HIV was more in patients with severe haemophilia A who required frequent factor VIII replacement. The need for long term surveillance of voluntary blood donors for transfusion associated viruses like HIV, HBV and HCV, is emphasized.

Prenatal diagnosis of bilirubin-UDP-glucuronosyltransferase deficiency in rats by genomic DNA analysis.

Hepatic bilirubin excretion requires UDP-glucuronosyltransferase-mediated glucuronidation. Patients with type I Crigler-Najjar syndrome and mutant rats (Gunn strain) inherit deficiency of UDP-glucuronyltransferase activity toward bilirubin as an autosomal recessive trait and, as a result, exhibit marked nonhemolytic unconjugated hyperbilirubinemia throughout postnatal life. Heterozygous carriers of the trait have normal serum bilirubin levels. Because of placental excretion of unconjugated bilirubin, type 1 Crigler-Najjar syndrome patients and Gunn rats are not jaundiced in utero, making prenatal diagnosis difficult. Here we report a diagnostic method in Gunn rats based on genomic DNA analysis for prenatal recognition of deficiency of UDP-glucuronyltransferase activity toward bilirubin in Gunn rats and identification of heterozygous carriers. We and others have shown that two distinct messenger RNA species (UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible phenol-UDP-glucuronyltransferase messenger RNA) in Gunn rat liver contain identical deletions of a single guanosine residue in their common 3' regions. Loss of the restriction site for the endonuclease BstNI, which results from this deletion, was used as the basis for a diagnostic test. Female heterozygous Gunn rats were mated with male homozygous Gunn rats. Genomic DNA was extracted from the chorionic aspect of placenta of 17-day fetuses or from leukocytes from normal rats, obligate heterozygotes and homozygous Gunn rats. The DNA was sequentially digested with the restriction enzymes EcoRI and BstNI and subjected to Southern-blot analysis with a double-stranded DNA probe for the common region of UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible UDP-glucuronyltransferase messenger RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of inherited deficiencies of multiple UDP-glucuronosyltransferase isoforms in two patients with Crigler-Najjar syndrome, type I.

Crigler-Najjar syndrome, type I (CN-I) is a potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from a recessively inherited deficiency of hepatic UDP-glucuronosyl-transferase (UGT) activity toward bilirubin (B-UGT). Two forms of B-UGT exist in human liver. mRNAs for these two forms and that for another isoform with activity toward simple phenols (P-UGT) have unique 5' regions, but their 3' regions are identical. The three mRNA species are derived from a single locus; the unique 5' regions are encoded by single unique exons and the identical 3' regions consist of four consecutive exons that are shared by all three isoforms. In this paper, we determined genetic lesions in two CN-I patients with deficiency of hepatic B-UGT and P-UGT activities. In one patient, there was a C----T substitution in exon 4 (common region) predicting the substitution of a serine residue with a phenylalanine residue; this mutation was present in the identical region of B-UGT and P-UGT mRNAs. In the other patient, a C----T substitution in exon 2 (common region) of the B-UGT/P-UGT locus resulted in a premature stop codon. This exon (132 nt) was absent in heptic B-UGT and P-UGT mRNAs of this patient due to exon skipping during pre-mRNA processing. Sequence abnormality of three distinct mRNA species explains the abnormality of multiple UGT isoforms in these patients. Presence of identical abnormalities in the common regions of the three mRNAs is consistent with the finding that the common 3' regions of the two B-UGT mRNAs and the P-UGT mRNA are encoded by four shared exons.

Prevalence of hepatitis C in transfusion dependent thalassaemics & haemophilics.

Seventy thalassaemics (B = 37, EB = 33) and 20 haemophilics (A = 18, B = 1, C = 1) were tested for antibodies to hepatitis C virus (anti-HCV) and markers for hepatitis B virus (HBV). The seropositivity for anti-HCV in thalassaemics and haemophilics was 14.3 and 25 per cent respectively. The subjects who were sero-positive to anti-HCV had had additional exposure to HBV. Anti-HCV positivity was not related to the age of the subject nor the number of units of blood and blood components transfused. Screening of blood donors for anti-HCV, apart from HBV, may minimise the hazards of post transfusion hepatitis in high risk recipients like transfusion dependent thalassaemics and haemophilics.

Use of high-performance liquid chromatography for assay of glutamic acid decarboxylase. Its limitation in use for post-mortem brain.

Rat brain, obtained 10 min after death, contained high levels of endogenous gamma-aminobutyric acid (GABA) and glutamic acid. Incubation of this brain homogenate at 37 degrees C indicated decrease of GABA with time due to degradation by GABA-transaminase. Reported high-performance liquid chromatographic (HPLC) methods for glutamic acid decarboxylase (GAD) assay depend on the difference between the GABA content of the reaction mixture after and before the incubation period. None of the methods considered the degradation of GABA during incubation. Furthermore, during determination of the Michaelis constant (KM) for the reaction none of them considered the endogenous substrate. Here we have focused on these factors which seriously affect the maximum velocity (Vmax) and KM values during GAD assay by the HPLC technique. By a simple and rapid HPLC technique we have measured GAD activity in post-mortem rat brain after removing endogenous glutamic acid by charcoal treatment and using gabaquline to prevent GABA degradation during incubation period. By this method a Vmax value of 46 +/- 4 nmol/h/mg protein and a KM value of 7.5 +/- 0.6 mM were observed for GAD activity of crude brain homogenate. For a comparative study, we have carried out radiometric assay of GAD activity from the same sample and observed a Vmax of 48 +/- 6 nmol/h/mg protein and KM of 6.9 +/- 0.4 mM.

Suppression of adrenocortical function in female mice by lindane (gamma-HCH).

Lindane (gamma-HCH) given to adult female mice orally at various doses and over varying periods adversely affected adrenocortical function. Adrenal weights decreased and both fasciculata and reticularis zones markedly regressed. Histopathological lesions were also noted and plasma and glandular glucocorticoid contents were significantly reduced. Simultaneously an increase in cholesterol and a decrease in ascorbic acid content of the adrenal glands were noticed. The adrenotoxic effect of this chlorinated insecticide possibly results from depressed corticoid production in situ and/or suppressed activity of enzyme(s) that catalyze the peripheral transformation of steroids.

Expression of specific UDP-glucuronosyltransferase isoforms in carcinogen-induced preneoplastic rat liver nodules.

The expression of specific UDP-glucuronosyltransferase isoforms in 2-acetylaminofluorane-induced rat liver preneoplastic nodules was studied; livers from pair-fed littermates were used as controls. For comparison, liver and kidney from 3-methylcholanthrene-treated or untreated (control) rats were used. Steady-state UDP-glucuronosyltransferase mRNA levels were determined by Northern blot analysis or in situ hybridization of tissue sections using a 30-mer oligonucleotide specific for the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase (which is active toward 4-nitrophenol) or a double-stranded cDNA probe specific for androsterone-UDP-glucuronosyltransferase. For 3-methylcholanthrene-inducible UDP-glucuronosyltransferase, the mRNA level was very low in control liver; there was a 15-fold increase after 3-methylcholanthrene treatment. This mRNA was present at relatively high concentration in the kidney and there was a threefold increase after 3-methylcholanthrene administration. In livers with preneoplastic nodules 1 mo after cessation of carcinogen administration, this mRNA concentration was approximately 15 times greater than in control liver. Similar changes in the level of the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase were also observed by in situ hybridization of tissue sections. Immunocytochemical studies using an antiserum that recognizes the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase showed a marked increase in the concentration of this isoform in preneoplastic nodules compared with the adjacent nonnodular liver.