Immunomodulation by biphalin,dimeric synthetic opioid peptide,and its analog

The opioid pentapeptides called enkephalins were originally described as the endogenous ligands for the opioid receptors. Although their precise physiological significance still remains elusive, the enkephalins have been reported to exhibit analgesic, antidepressant, antianxiety and anticonvulsant activities. In addition, enkephalins have also been shown to act as immunomodulator. The first generation of dimeric peptides was derived from enkephalins. Biphalin [(Tyr-D-Ala-Gly-Phe-NH)2] is a bivalent opioid analog containing two tyrosine residues. We have evaluated the immunomodulatory properties of biphalin and its analogs in various in vitro tests. We report that biphalin and one of its analogs [Tyr-D-Ala-Gly-Phe-NH.NH-Phe(p-Cl)-H] stimulate human T cell proliferation, natural killer (NK) cell cytotoxicity in vitro and interleukin-2 (IL-2) production. Biphalin and its analog also released chemokine like factor in the culture supernatant that was responsible for increased chemotaxis of monocytes. Furthermore, these peptides inhibited tumor necrosis factor (TNF-alpha) production in lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMC) and nitric oxide (NO) production in mouse macrophage cells, RAW 264.7. Our observations suggest immunomodulatory property of biphalin and its analog.     

Outbreak of Pichia anomala infection in the pediatric service of a tertiary-care center in Northern India

An outbreak of nosocomial fungemia due to the unusual yeast, Pichia anomala occurred in the pediatric wards of our hospital over a period of 23 months (April 1996 to February 1998). A total of 379 neonates and children (4.2% admissions) were infected. The probable index case was admitted to the pediatric emergency ward, with subsequent transmission to the premature nursery, pediatric intensive care units, and other children wards. Carriage on the hands of health care personnel was likely to be responsible for dissemination of the fungus. The outbreak could only be controlled after a health education campaign to improve hand-washing practices was instituted and after nystatin-fluconazole prophylaxis to all premature neonates and high-risk infants was introduced. In a case-control study, we identified a lower gestational age, a very low birth weight (<1,500 g), and a longer duration of hospital stay as significant risk factors associated with P. anomala fungemia in premature neonates. We conducted a culture prevalence survey of 50 consecutive premature neonates and found that 28% were colonized with P. anomala at a skin or mucosal site on the date of delivery and that 20% of these neonates subsequently developed P. anomala fungemia. We performed multilocus enzyme electrophoresis on 40 P. anomala outbreak isolates (including patient and health care workers' hand isolates), and the results suggested that these isolates were identical. Our study highlights the importance of P. anomala as an emerging nosocomial fungal pathogen.     

Aspiration vs nonaspiration technique of cytodiagnosis--a critical evaluation in 160 cases

The two sampling techniques were studied in 160 randomly selected cases of superficial swellings in various sites of the body. They were sampled by fine needle aspiration (FNA) and by non-aspiration (NA) (a needle without application of aspiration pressure). Cell samples were cytologically assessed and critically evaluated using five objective parameters. Contamination with blood was more in lymphnode, thyroid and liver lesions in aspiration smears than NA smears and values were statistically significant. Similarly when compared for the degree of cellular trauma and cellular degeneration statistically significant better results were obtained by nonaspiration technique for lymphnode lesions. Regarding amount of cellular material obtained by FNA, statistical significant better results were found for breast lesions only. Statistically significant better maintenance of architecture was observed only for thyroid lesions by NA technique. Better average scores were observed by NA technique for lymphnode and thyroid only. Categorizing all the smears obtained by FNA & NA on the basis of their scores according to predetermined criteria, greater number of diagnostically adequate specimens were obtained by FNA than by NA but the number of diagnostically superior specimens obtained by NA technique was found to be more than that by FNA. The difference was found to be statistically significant. However the number of inadequate smears was also more by NA technique than by FNA technique.     

Antilymphoproliferative activity of ethanolic extract of Boerhaavia diffusa roots

Extracts of plants have been widely evaluated for possible antiproliferative and anticarcinogenic properties. The antiproliferative activity of ethanolic extract of Boerhaavia diffusa, a plant used in traditional medicine, was evaluated in several cells. It inhibited T cell mitogen phytohemagglutinin and concanavalin A-stimulated proliferation of human peripheral blood mononuclear cells (PBMC). It also inhibited purified protein derivative antigen-stimulated PBMC proliferation and human mixed lymphocyte culture. In addition, B. diffusa extract inhibited the growth of several cell lines of mouse and human origin, such as mouse macrophage cells (RAW 264.7), human macrophage cells (U937), human monocytic cells (THP-1), mouse fibroblast cells (L929), human embryonic kidney cells (HEK293), mouse liver cells (BNLCL.2), African green monkey kidney cells (COS-1), mouse lymphoma cells (EL-4), human erythroleukemic cells (K562), and human T cells (Jurkat). The present study has demonstrated the antiproliferative potential of ethanolic extract of B. diffusa in vitro.     

Studies on pathogenesis of fowl pox: virological study

One-month-old WLH chickens were inoculated with a field isolate of fowl pox virus (FPV) by intradermal (i.d.) and intratracheal (i.t.) routes. In intradermally infected chickens, the virus in titrable amounts was first detected in the skin at the inoculation site on day 2 and in lungs on day 4 followed by viraemia on the day 5 post-infection (p.i.). Subsequently the virus was recovered from liver, spleen, kidney and brain, but not from the heart. The chickens infected by i.t. route showed an almost similar outcome with minor differences as the virus was first demonstrated in the lungs on day 2 p.i., viraemia occurred on day 4 p.i. Initiation of pocks at the inoculation site in i.d. infected birds was observed on days 3 to 4 p.i., generalized cutaneous pock lesions appeared from 7 to 8 days p.i.     

Increased expression of osteopontin gene in atypical teratoid/rhabdoid tumor of the central nervous system.

The atypical teratoid/rhabdoid tumor, primary to the central nervous system, is a highly malignant and aggressive neoplasm of infancy and childhood. Although having distinct biological features and clinical outcomes, it is frequently misdiagnosed as primitive neuroectodermal tumor/medulloblastoma. To further distinguish the underlying pathogenesis and to identify biological markers for clinical use, an atypical teratoid/rhabdoid tumor-derived cell line was established and its gene expression pattern analyzed in comparison to the human astrocyte SVG12 cell line and the human DAOY medulloblastoma cell line using a complementary DNA microarray method. The osteopontin gene was found specifically upregulated in atypical teratoid/rhabdoid tumor cells. This specificity was confirmed by immunohistochemistry in pathological sections of tissues from atypical teratoid/rhabdoid tumor patients. Even though the role of osteopontin in the cytopathogenesis of atypical teratoid/rhabdoid tumor still needs to be determined, our data support that overexpressed osteopontin is a potential diagnostic marker for atypical teratoid/rhabdoid tumor.     

Loss of heterozygosity in normal breast epithelial tissue and benign breast lesions in BRCA1/2 carriers with breast cancer

Loss of heterozygosity (LOH) of the wild-type BRCA1/2 allele is a reproducible event in breast tumors of BRCA1/2 mutation carriers, but it is unknown if this allelic loss occurs only in association with recognizable histopathologic abnormalities. We evaluated the early genomic changes that occur in the mammary glands of patients with increased predisposition to breast cancer due to germline mutations in the BRCA1/2 genes. We tested the hypothesis that these genomic changes may be detected, not only in histologically abnormal and malignant breast tissues, but also in morphologically normal tissues and in areas with pathologically benign changes. Samples were obtained from five breast cancer patients: four BRCA1 carriers and one BRCA2 carrier. In each case, nontumor tissue areas surrounding the tumor or from other locations of the breast were isolated using laser capture microdissection. We evaluated 29 areas showing normal terminal ductal lobular units (TDLUs) or histopathologically benign changes (in particular, sclerosing adenosis), using a panel of polymorphic dinucleotide microsatellite markers for the BRCA1 gene and other chromosome 17 loci, for the BRCA2 gene and other chromosome 13 loci, and for the FHIT gene on 3p14.2. Overall, we analyzed a total of 105 samples of nontumor tissues; LOH was detected in 59 of the 105 (56%). In the normal TDLUs, 15 of 30 samples (50%) showed LOH; in the tissues with benign proliferative changes, such as sclerosing adenosis, 44 of 75 samples showed LOH (59%). Our results suggest that there is a field effect of early genetic events preceding morphologic changes in the mammary glands of BRCA mutation carriers.     

Dual β-Catenin and γ-Catenin Loss in Hepatocytes Impacts Their Polarity through Altered Transforming Growth Factor-β and Hepatocyte Nuclear Factor 4α Signaling

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of β-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of β-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-β signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of β-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

A hallmark of epithelial cells is polarization, which is achieved by the orchestration of external cues, such as cellular contact, extracellular matrix, signal transduction, growth factors, and spatial organization.¹ Hepatocytes in the liver show a unique polarity by forming several apical and basolateral poles within a cell.² The apical poles of adjacent hepatocytes form a continuous network of bile canaliculi into which bile is secreted, whereas the basolateral membrane domain forms the sinusoidal pole, which secretes various components, such as proteins or drugs, into the blood circulation.³ Loss of hepatic polarity has been associated with several cholestatic and developmental disorders, including progressive familial intrahepatic cholestasis (PFIC) and primary sclerosing cholangitis (PSC).^4,5 Although the molecular mechanisms governing hepatocyte polarity have been extensively studied in the in vitro systems, there is still a significant gap in our understanding of how polarity is established within the context of tissue during development or maintained during homeostasis.^6,7 Similarly, the molecular pathways contributing to hepatic polarity are not entirely understood, and a better comprehension of hepatic polarity regulation is thus warranted.Previous studies have confirmed the role of hepatocellular junctions, such as tight and gap junctions, in the maintenance of hepatocyte polarity.^8,9 Studies done in vitro and in vivo have shown that loss of junctional proteins, such as zonula occludens protein (ZO)-1, junctional adhesion molecule-A, and claudins, lead to impairment of polarity and distorted bile canaliculi formation.10, 11, 12, 13 In addition, proteins involved in tight junction assembly, such as liver kinase B1, are also involved in polarity maintenance.¹⁴ Among adherens junction proteins, various in vitro cell culture models have confirmed the role of E-cadherin in the regulation of hepatocyte polarity, possibly through its interaction with β-catenin.^15,16 However, there is a lack of an in vivo model to study the role of adherens junction proteins in hepatocyte polarity and their misexpression contributing to various liver diseases.β-Catenin plays diverse functions in the liver during development, regeneration, zonation, and tumorigenesis.17, 18, 19 The relative contribution of β-catenin as part of the adherens junction is challenging to study because like in other tissues, γ-catenin compensates for the β-catenin loss in the liver.^20,21 To address this redundancy, we previously reported a hepatocyte-specific β-catenin and γ-catenin double-knockout (DKO) mouse model was reported.²² Simultaneous deletion of β-catenin and γ-catenin in mice livers led to cholestasis, partially through the breach of cell-cell junctions. However, more comprehensive understanding of the molecular underpinnings of the phenotype is needed.In the current study, prior preclinical findings of dual β-catenin and γ-catenin loss were extended to a subset of PFIC and PSC patients. In vivo studies using the murine model with hepatocyte-specific dual loss of β-catenin and γ-catenin showed complete loss of hepatocyte polarity compared to the wild-type controls (CONs). Loss of polarity in DKO liver was accompanied by epithelial-mesenchymal transition (EMT), activation of transforming growth factor (TGF)-β signaling, and reduced expression of hepatocyte nuclear factor 4α (HNF4α). Our findings suggest that β-catenin and γ-catenin and in turn adherens junction integrity, are critical for the maintenance of hepatocyte polarity, and any perturbations in this process can contribute to the pathogenesis of cholestatic liver disease.     

A Vero cell-attenuated Goatpox virus provides protection against virulent virus challenge

An Indian isolate of Goatpox virus (GTPV) was adapted and propagated in Vero cells for development of an attenuated virus. The virus was initially passaged in primary lamb testes cells and subsequently in Vero cells. At the 55th passage, the virus showed evidence of attenuation when tested for safety in seronegative goats. At this stage, the virus was found to be completely non-pathogenic. The virus was passaged further and the 60th passage was used for testing its immunogenicity in goats. The latter were inoculated with 10, 100 and 1000 TCID50 of the attenuated virus by intradermal (i.d.) route and challenged after 28 days with virulent GTPV. The attenuated virus produced no adverse reaction even at the highest dose and conferred complete protection even at the lowest dose against challenge with a high dose (2 x 10(6) of 50% skin-reactive dose SRD50) of virulent virus. Increased levels of virus-specific serum antibodies could be demonstrated by both indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test in all the immunized goats. No horizontal transmission of the virus from the immunized to in-contact animals took place. Our results suggest that this attenuated virus could be a safe, immunogenic and potent candidate for developing a vaccine against goatpox.     

Sclerosing stromal tumour of ovary--a clinicopathological and immunohistochemical study of five cases

We describe the clinicopathological and immunohistochemical findings in five cases of sclerosing stromal tumours of ovary and compare our findings with other reported cases of this uncommon tumour and with fibromas and thecomas which they may mimic.