Scanning and transmission electron microscopic analysis of ampullary segment of oviduct during estrous cycle in caprines.

The ampullary segment of the mammalian oviduct provides suitable milieu for fertilization and development of zygote before implantation into uterus. It is, therefore, in the present study, the cyclic changes in the morphology of ampullary segment of goat oviduct were studied during follicular and luteal phases using scanning and transmission electron microscopy techniques. Topographical analysis revealed the presence of uniformly ciliated ampullary epithelia, concealing apical processes of non-ciliated cells along with bulbous secretory cells during follicular phase. The luteal phase was marked with decline in number of ciliated cells with increased occurrence of secretory cells. The ultrastructure analysis has demonstrated the presence of indented nuclear membrane, supranuclear cytoplasm, secretory granules, rough endoplasmic reticulum, large lipid droplets, apically located glycogen masses, oval shaped mitochondria in the secretory cells. The ciliated cells were characterized by the presence of elongated nuclei, abundant smooth endoplasmic reticulum, oval or spherical shaped mitochondria with crecentric cristae during follicular phase. However, in the luteal phase, secretory cells were possessing highly indented nucleus with diffused electron dense chromatin, hyaline nucleosol, increased number of lipid droplets. The ciliated cells had numerous fibrous granules and basal bodies. The parallel use of scanning and transmission electron microscopy techniques has enabled us to examine the cyclic and hormone dependent changes occurring in the topography and fine structure of epithelium of ampullary segment and its cells during different reproductive phases that will be great help in understanding major bottle neck that limits success rate in vitro fertilization and embryo transfer technology.

Topographic and ultrastructural variations in isthmus segment of oviduct during oestrous cycle in Caprines.

In the present study, the morphological variations in the isthmus segment of goat (Capra hircus) oviduct were studied using scanning and ultrastructural techniques during luteal and follicular phases of oestrous cycle. Topographic analysis of the isthmus epithelium in the follicular phase demonstrated irregular distribution of ciliated cells on the epithelium. A few secretory cells possessed bulbous apical processes which were concealed by the cilia. Cyclic changes were noticed in the secretory cells of isthmus. Under electron microscopy the isthmus region of the oviduct during the follicular phase revealed presence of almost blunt processes at their apical surfaces. The secretory cells were attached to the basal lamina and were characterized by the presence of secretory granules, numerous ribosomes, extensive smooth endoplasmic reticulum, well-developed golgi zones in the cytoplasm, and a microvillus luminal surface. The parallel use of these two methods in isthmus segment of the oviduct has enabled us to analyze the marked cyclic variations in the topography and fine structure during follicular and luteal phases of the oestrous cycle. The results of the present investigation on topography and ultrastructure will be of great help in explaining different bottlenecks of gamete interaction, maturation, fertilization, and early embryo development.

Scanning electron microscopic changes in granulosa cells during follicular atresia in Caprine ovary.

During this study, topographic changes in healthy and atretic granulosa cells have been investigated during follicular atresia in goat ovary. Under scanning electron microscopy atresia was marked by asymmetrical shrinkage and vacuolization of cytoplasm. The specific topographical alterations observed in atretic cells were loss of micro extensions, disruption of cell-cell interaction, and smooth-textured membrane with a number of uneven depressions and ruffles. Some portions of the cell membrane were marked by extensive shrinkage due to condensation of cytosol. Irregular membrane at occasions was studded with blunt microextensions. The findings of present investigation will help in understanding the cellular changes in granulosa cells during follicular atresia and will find applications in screening of follicles for in vitro culture, in vitro fertilization and Embryo transfer technology.

Potential of Complementary and Alternative Medicine in Preventive Management of Novel H1N1 Flu (Swine Flu) Pandemic: Thwarting Potential Disasters in the Bud.

The emergence of novel H1N1 has posed a situation that warrants urgent global attention. Though antiviral drugs are available in mainstream medicine for treating symptoms of swine flu, currently there is no preventive medicine available. Even when available, they would be in short supply and ineffective in a pandemic situation, for treating the masses worldwide. Besides the development of drug resistance, emergence of mutant strains of the virus, emergence of a more virulent strain, prohibitive costs of available drugs, time lag between vaccine developments, and mass casualties would pose difficult problems. In view of this, complementary and alternative medicine (CAM) offers a plethora of interesting preventive possibilities in patients. Herbs exhibit a diverse array of biological activities and can be effectively harnessed for managing pandemic flu. Potentially active herbs can serve as effective anti influenza agents. The role of CAM for managing novel H1N1 flu and the mode of action of these botanicals is presented here in an evidence-based approach that can be followed to establish their potential use in the management of influenza pandemics. The complementary and alternative medicine approach deliberated in the paper should also be useful in treating the patients with serious influenza in non pandemic situations.

Ultrastructural characterization of apoptotic granulosa cells in caprine ovary.

The unique phenomenon of cell proliferation and apoptosis is encountered in the ovarian follicles undergoing early stages of atresia. The aim of this study was to verify the morphological variations in these two physiologically distinct processes operating in antral follicles of caprine ovaries using histological and ultrastructural techniques. Histologically the degenerating granulosa cells were characterized by condensed cytoplasm, and nucleus fragmentation in hazy cytosol. The pyknotic nuclei of degenerating cells stained darkly with haematoxylin and giemsa while the cytoplasm was eosinophilic. Under electron microscopy, apoptosis was marked by asymmetrical shrinkage, vacuolization of cytoplasm, swollen and vacuolated mitochondria, increased irregularity and/or fragmentation of nucleus, chromatin condensation and finally, production of membrane enclosed nuclear fragments containing intracellular material, the apoptotic bodies. The parallel use of these two methods on caprine ovaries has enabled us to analyse the decline in the frequency of granulosa cells during follicular atresia due to apoptosis.

Identification of Human Immunodeficiency Virus Type-1 Subtypes by Heteroduplex Mobility Assay.

BACKGROUND: Human immunodeficiency virus type-1 (HIV-1) has developed marked genomic sequence differences over the course of an epidemic because of an error prone reverse transcriptase (RT), which rapidly incorporates mutations resulting in genomic diversity, altered cell tropism, immune escape and variable resistance to antiretroviral drugs. The best preventive strategy for HIV control is development of an efficacious prophylactic vaccine using the most appropriate (antigenically related) subtypes. On the basis of phylogenetic analysis, HIV strains can be separated into major group "M" consisting of genetic subtypes A-K, "N", the new group and "O", the outlier group. METHODS: Heteroduplex mobility assay (HMA) is a rapid, economical and reliable technique of subtyping HIV-1. It is based on the principle of determining the genomic relatedness and divergence of the unknown sample with the known reference plasmid HIV-1 subtypes by studying the mobility patterns of the resulting heteroduplexes formed on the polyacrylamide gel. RESULT: A total of 70 HIV-1 seropositive samples obtained from service personnel, their families and civilians from service hospitals were analyzed and their subtype distribution studied. 66 (94.28%) were HIV-1 subtype C and two (2.85%) subtype B. In two (2.85%) samples, the subtype distribution was homotypic recombinant, one each of subtype C1 & C2 and C2 & C4 respectively. CONCLUSION: Service personnel and their families represent a divergent population from different regions of India. An analysis of subtypes in these HIV-1 seropositive individuals will help in understanding the geographical distribution and evolution of the virus. Determination of HIV-1 subtypes has significant implications for development of candidate vaccine for India.

Needle necropsy in AIDS.

The value of autopsy in understanding the natural course of any disease is beyond any argument. The reluctance of pathologists to perform autopsy in HIV infected cadavers is justified due to the risks involved to the prosector and the morgue attendants. A relative low risk needle necropsy protocol is proposed using fine needle aspiration cytology, tru-cut biopsies and microbiological examination. Diagnosis could be offered in all the forty-four needle necropsies performed. Disseminated tuberculosis in 18/44 (40.9%) cases, disseminated cryptococcosis in 12/44 (27.2%) cases, poly-microbial infections in 27.2% cases and non-Hodgkin's lymphoma in 9% cases were detected in the study. Infectious agents like Histoplasma capsulatum, Isospora belli, tachyzoites of Toxoplasma gondii, Candida sp and Cryptococcus sp could be demonstrated in the samples obtained in the study. Lack of material for study of gross pathology, inaccessibility of deep-seated lesions and risk of needle stick injury to the prosector though low are the limitations of this procedure.

DIAGNOSING DISSEMINATED INTRAVASCULAR COAGULATION IN ACUTE INFECTION : CAN WE DO WITHOUT FDP & D-DIMER.

Alterations in coagulation profile viz. platelet count, prothrombin time (PT), partial thromboplastin time with kaolin (PTTK), thrombin time (TT) and fibrinogen were studied in 96 patients (73 males and 23 females) of acute infections. Fibrin/fibrinogen degradation products (FDP) level >25µg fibrinogen equivalent unit (FEU)/ml along-with D-dimer >1.0µg FEU/ml was considered criteria for diagnosis of disseminated intravascular coagulation (DIC). Normal values were established using plasma from 12 healthy voluntary blood donors. Out of these 96 patients, 15 had infection with Gram positive bacteria, 23 with Gram negative bacteria and 38 with Dengue. In 20 patients, nature of infection was not defined. Mean platelet count per cubic millimetre was 2.14 lac in Gram positive infection and 1.74 lac in Gram negative infection (p=0.07). There was no significant difference in other coagulation parameters in Gram positive and Gram negative infection. Platelet counts were low in 71% of Dengue patients but there was no significant alteration in PT, PTTK and TT. None of the Dengue patients had hypofibrinogenemia or DIC though hyperfibrinogenemia was present in 21% of Dengue patients. 20 patients had features of septicemia (Gram +ve 7, Gram -ve 8, undefined 5); 10 had concomitant DIC. DIC was present in additional 4 patients of acute infection without septicemia. PTTK was raised in 60% of the septicemia patients. 20 out of 82 non-DIC acute infection patients had subnormal PTTK. Commonest alteration in 14 DIC patients was raised PTTK with a sensitivity of 78.6% and specificity of 81.7%. Low fibrinogen levels though specific for DIC, were present in only 21.4% of the DIC patients. Combinations of PTTK >38 sec with PT >15 sec or platelet count < 1.5 lac/mmm(3) were good screening tests for DIC and detected 11 and 10 patients out of 14 with three and two false positives respectively.

THE CD 10 MARKER IN THE INVESTIGATION OF ACUTE LYMPHOBLASTIC LEUKAEMIA.

Immunophenotyping of leukaemias is presently well established. It is invaluable for proper case management [1]. The most useful information on the management of cases of Acute Lymphocytic Leukaemia (ALL) is provided by the detection of the CD 10 marker on the leukaemic cell. The CD 10 positive ALL has since long been associated with a favourable prognosis as compared to the CD 10 negative ALL [1, 2]. As immunophenotyping is resource intensive, the monoclonal antibodies (MO ABS), are not in widespread use in our country. The expenses involved make this procedure prohibitive in most institutions. For this reason, a limited panel of MO ABS has been used in this study, with a special emphasis on the CD 10 marker. A total of 25 cases of ALL were studied. 17 cases were found to be positive for the CD 10 marker (68%). These cases were associated with a favourable prognosis as compared to the CD 10 negative group. Although, the diagnosis of ALL and leukaemias in general, is essentially based on the study of Romanowsky stained smears [3, 4], the additional information provided by the cell surface marker study results in better case management [1].

EARLY DIAGNOSIS IN POST RENAL TRANSPLANT OPPORTUNISTIC INFECTIONS: A FRESH LOOK.

A total of 86 renal transplant patients who were transplanted with live related donor (LRD) and live unrelated donor (LURD) kidneys were studied for opportunistic infections. Immune diagnosis of Toxoplasma, Cytomegalovirus (CMV), Herpes-simplex virus type II (HSV-2), Aspergillosis and Tuberculosis was carried out in these patients along with sputum examination, CSF studies and biopsy of lymphnode and other tissues in few cases. A high degree of Toxoplasma, CMV & HSV-2 positivity was seen in transplanted patients. However sensitivity of serological diagnosis of tuberculos was found to be low with standard criteria, which increased significantly when modified criteria were used. It is concluded that regular immunological monitoring should be carried out in transplanted patients so as to reach an early diagnosis and management of opportunistic infections.

IMPORTANCE OF PROSTATIC SPECIFIC ANTIGEN (PSA) IN CLINICAL PRACTICE : OUR EXPERIENCE.

A total of 89 patients in the age group of 50-92 years having different histopathologically proven prostatic conditions were analysed with prostate specific antigen (PSA) and clinical co-relation undertaken. PSA levels were found to be significantly higher (p < 0.05) in 48 carcinoma prostate cases (mean 93.16 ± 50.75 ng/ml) as compared to 32 benign prostatic hyperplasia (mean 4.66 ± 3.85 ng/ml). Similarly, levels were considerably reduced in 15 post-operative adenocarcinoma cases (mean 10.77 ± 9.65 ng/ml) as compared to their pre-operative samples (mean 93.16 ± 60.75 ng/ml). PSA levels were moderately higher in 9 cases of prostatitis (mean 13.28 ± 4.53 ng/ml). A very high degree of sensitivity, specificity and positive predictive value of PSA e.g. 95.8%, 75.6% and 82.2% respectively was found in adenocarcinoma cases, when levels of PSA were > 10 ng/ml. Thus it necessitates a detailed and thorough examination in such cases to come to final conclusion and early management of the cases.

HELICOBACTER PYLORI AND GASTRIC CANCER: IS THERE A LINK?

This paper examines the correlation between prevalence of H pylori, gastritis and the histological type of gastric cancer. 50 gastrectomy specimens from the patients with gastric cancers were studied for the prevalence of H pylori, type of gastritis as classified by the Sydney system, presence of intestinal metaplasia, and the histological type of gastric cancer. H pylori was detected in 27 (54%) stomachs. The common site for finding this bacterium was either antrum or body of stomach (16 and 17 sections respectively), Gastritis was found in 48/50 specimen (96%) from non-neoplastic areas. However, 15 of the 18 patients with atrophic gastritis had H pylori in their stomach specimens either in the body or in antrum. Intestinal metaplasia was detected in 19 (38%) specimen. Of these, 14 (73.7%) specimens had type 1 and remaining 5 (26.3%) had type 2 metaplasia. The distribution of metaplasia was significantly more in H pylori positive patients. Prevalence of H pylori positivity was highest in intestinal type of cancer (66.7%) and lowest in diffuse variety (45.8%). In two third specimens of intestinal type of cancer, gastritis was related to H pylori while such association with H pylori was seen in approximately half (45.8% & 57.1% respectively) the specimens of other types of cancers. There is strong association between the prevalence of H pylori and gastritis on one hand and the intestinal metaplasia on the other, in patients with gastric cancer. These findings, indirectly support Correa's hypothesis that H pylori is a causal factor in the intestinal form of gastric cancer.

IMMUNOLOGICAL MONITORING IN TRANSPLANTATION.

Sixty eight patients who had undergone live related donor renal transplantation (LRD), were evaluated for soluble interleukin-2 receptors (sIL-2R), tumour necrosis factor alpha (TNF-α) and autoantibodies against IgG(Fab')2 and IgG(Fc), at pre- and various post-transplant intervals. Serum sIL-2R levels were significantly elevated in hemodialysed patients awaiting transplantation (mean 259.2 ± 90.5 pmol/L) as compared to healthy volunteers (mean 52.6 ± 16.7 pmol/L). In 96 samples obtained from patients with well-functioning grafts (WFG), the post-transplant sIL-2R levels (135.6 ± 65.4 pmol/L) were significantly lower (p < 0.001) than their pretransplant values. Eight patients with cyclosporin-A (CsA) nephrotoxicity, 14 with reversible acute tubular necrosis (ATN) and 4 patients with partial surgical obstruction, revealed moderate levels (99.0 ± 13.7, 184.1 ± 47.5, 156.7 ± 40.4 pmol/L respectively). On the other hand, 29 patients with acute rejection episodes, 11 with chronic rejection and 8 with infections had significantly higher levels (307.9 ± 89.3, 253.3 ± 68.6,345 ± 110.6 pmol/L), (p < 0.001). TNF-α levels were also raised in rejection and infective episodes but were not statistically significant. Serum anti-IgG(Fab')2 levels were found higher (0.407 OD) in WFG as compared to those with declining graft functions (0.279 OD). On the contrary high pre- and post-transplant anti-IgG(Fc) activity was associated with increased graft rejection and lower survival rate.

IMMUNOBIOLOGICAL MONITORING OF VARIOUS GASTROINTESTINAL AND PRIMARY HEPATIC MALIGNANCIES.

Carcinoembryonic antigen (CEA) and alpha fetoprotein levels (AFP) were assayed by enzyme-labelled immunoassay in 75 cases of gastrointestinal (GI) and primary hepatic malignancies. Mean CEA levels were found to be significantly higher (p<0.005) in metastatic non-operative group of GI malignancies (range 5.32 ng/mL to 175.2 ng/mL) as compared to early pre-operative cases (range 1.25 ng/mL to 33.2 ng/mL) and post-operative cases (range 1.41 ng/mL to 22.24 ng/mL). Variable levels of AFP were visualised in 12 cases of primary hepatic malignancies with eight cases having values less than 100 ng/mL. When both CEA and AFP were assayed simultaneously, the markers were helpful in differentiating cases of primary hepatic malignancies with low levels of CEA from 3 cases of secondaries in the liver where CEA levels were found to be highly raised (80.4 ng/mL to 146.4 ng/mL). To evaluate the variation of CEA and AFP levels in different patients having same stage of the disease, immunological monitoring for the functions of T and B cells was carried out by estimation of cytokine, i.e. interleukin-1 alpha (IL-1a), interleukin-2R (II-2R) and various immunoglobulins. IL-1a and 1L-2R levels were significantly higher (p<0.05) in the groups of patients having higher CEA and AFP. This indicates an important T cell (TH1 and TH2) function in the tumour antigen production.

HORMONAL MARKERS IN THE STUDY OF PITUITARY ADENOMAS.

Twenty pituitary adenomas were studied using light microscopy, immunohistochemistry and electron microscopy. Based on clinical manifestations of hormonal hypersecretion they were divided into two groups, i.e. group-I (12 patients) with no endocrine dysfunction and group-II (8 patients) with endocrine dysfunction. Group-I could be further sub-divided into two groups based on IHC : group LA in which no hormone could be localised in the tissue (null cell adenomas) and group IB where hormones could be localised in the tissue (1). Tumours in group II, with light microscopy, were acidophil (n=5), basophil (n=1) or mixed (n=2) adenomas. Eight of these 20 cases were plurihormonal. Growth hormone, and prolactin were the commonest combination. This study has helped in the reclassification of the pituitary adenomas according to the hormone they produce.

MICROWAVE STIMULATED FIXATION AND RAPID PROCESSING OF TISSUE FOR HISTOPATHOLOGY.

A rapid method for obtaining high quality paraffin sections is described. This method enables preparation of paraffin blocks in about 20 minutes and is based on microwave stimulated tissue fixation and processing. A total of 200 tissues were processed. Tissue blocks of size 1×1×0.5 were found ideal for processing. Three protocols were evaluated for tissue processing using different timings of exposure to graded alcohols, chloroform and wax. It was observed that utilizing an additional step of exposure to 70 per cent alcohol during tissue dehydration resulted in preparation of paraffin blocks which were light microscopically excellent and highly comparable to conventional methods.