Association of Myroides odoratimimus in immunocompromized piglets with post weaning multisystemic wasting syndrome.

AIM: To study the association of opportunistic infection due to Myroides odoratimimus in piglets immunocompromised by porcine circovirus type 2 (PCV2) infection. METHODS AND RESULTS: The clinical samples (n = 101) were analysed bacteriologically. The isolates were identified by their phenotypes and MALDI TOF-MS analysis as Myroides species. The phylogram constructed based on nucleotide sequences of the 16S rRNA gene showed identity (~99%) with the M. odoratimimus isolates. The minimum inhibitory concentration values for antibiotics revealed M. odoratimimus to be resistant against carbapenem, cephalosporins, aminoglycosides and fluoroquinolones. The presence of PCV2 in affected tissue samples was confirmed by amplification of the 565 bp region of ORF2 of the PCV2 genome. The topology of the phylogenetic tree grouped the PCV2 with cluster-2d. CONCLUSIONS: PCV2 being immunosuppressive in nature might have impaired the immunity thereby increasing the susceptibility of immunocompromised piglets to opportunistic pathogens such as M. odoratimimus leading to disease severity and high mortality. The M. odoratimimus isolates were found to be multidrug resistant and evidenced for uncertain clinical relevance and hence could act as hidden source of public health hazard. SIGNIFICANCE AND IMPACT OF THE STUDY: Myroides odoratimimus is a rarely reported human pathogen. We reported the incidence of infection due to seemingly rare isolates of M. odoratimimus causing an outbreak of pneumonia in piglets. This appears, to the best of authors' knowledge, to be the first outbreak due to Myroides recorded in animal clinical cases described in the literature.

Cerebral and renal phaeohyphomycosis in a dog infected with Bipolaris species.

Mycotic meningoencephalitis in dogs may manifest as a primary disease of the central nervous system or as a part of disseminated infection. Fungi belonging to the genus Bipolaris are saprophytic plant pathogens and can cause disease in humans. The authors report a case of Bipolaris infection in a dog with granulomatous meningoencephalitis, nephritis, and vasculitis. The clinical and histological features resembled those of the more common aspergillosis, thus warranting confirmation by molecular methods. Polymerase chain reaction and sequence analysis identified Bipolaris from the brain lesion, indicating its involvement in the disease. To the authors' knowledge, this is the first reported case of meningoencephalitis caused by this fungus in a domestic animal.

Giant cell glioblastoma in the cerebrum of a Pembroke Welsh corgi.

A 6-year-old, neutered female Pembroke Welsh corgi was presented with a 1-month history of ataxia and panting. The clinical signs progressed until the dog became anorexic, obtunded and exhibited circling to the left. At necropsy examination, a mass was detected in the left forebrain, impinging on the cribriform plate. Microscopically, the mass was composed of sheets of round to pleomorphic neoplastic cells with vacuolated cytoplasm. Nuclear atypia, anisocytosis and anisokaryosis were common. Numerous bizarre, multinucleated giant cells containing 60 or more nuclei and giant mononuclear cells were present. The matrix contained abundant reticulin. Immunohistochemistry revealed the neoplastic cells uniformly to express vimentin, and a small number of neoplastic cells expressed glial fibrillary acid protein. A diagnosis of giant cell glioblastoma was made. Although well recognized in man, this tumour has been documented rarely in the veterinary literature.

Enteric dysganglionosis resembling intestinal neuronal dysplasia in a foal with bacterial colitis.

A 5-day-old quarter horse colt with a history of hypothermia, agonal breathing, and diarrhea was euthanized. At necropsy, numerous slightly raised, discrete, closely approximated submucosal nodules were observed in the colon and small intestine. Histologically, these nodules were composed of expanded submucosal mesenchyme that contained numerous neurons either individually or in ganglia. Thirty-two percent of these ganglia included 8 or more neurons, in contrast to 6% in an age-matched foal. Some nodules had necrosuppurative inflammation with vasculitis, thrombosis, and bacterial colonization. A few heterotopic neurons were randomly distributed in the mucosa and the muscularis mucosa. Histologic changes were most consistent with intestinal neuronal dysplasia, a disease of the submucosal plexus described in humans.

Age-dependent renal accumulation of 4-hydroxy-2-nonenal (HNE)-modified proteins following parenteral administration of ferric nitrilotriacetate commensurate with its differential toxicity: implications for the involvement of HNE-protein adducts in oxidative stress and carcinogenesis.

In this study, we show that the toxicity of ferric nitrilotriacetate (Fe-NTA) can be correlated with the tissue accumulation of 4-hydroxy-2-nonenal (HNE)-modified protein adducts. It is observed that the toxic manifestations of Fe-NTA gradually increase with the increasing age of animals. A dose of Fe-NTA which produces almost 100% mortality in aged rats causes 70% mortality in adults, 30% in pups, 20% in litters, and less than 10% in neonates. The age-dependent increase in its toxicity is also evident from the data of renal microsomal lipid peroxidation and hydrogen peroxide generation. No significant difference in the generation of H2O2 and induction of renal microsomal lipid peroxidation between saline- and Fe-NTA-treated neonates, litters, and pups could be observed. However, in adult rats, a significant increase in both of the parameters was observed which was even greater in aged rats. On the contrary, renal glutathione levels in these animals show an inverse relationship with the oxidant generation. In neonates, litters, and pups the maximum decrease of glutathione was up to 22%, whereas in adult and aged rats, the depletion was more than 60% of their respective saline-treated controls. Parallel to this data, blood urea nitrogen and creatinine, the indicators of renal damage, show a significant increase in Fe-NTA-treated adult and aged rats only, whereas no significant alterations were observed in other groups. Similarly, the magnitude of ODC induction and [3H]thymidine incorporation was much higher in aged and adult rats in comparison to other groups of animals after Fe-NTA treatment. Additionally, the immunohistochemical localization studies show a significant increase in HNE-modified protein adducts in kidney of adult and aged rats, whereas no significant staining was observed in other groups. A similar increase in the level of protein carbonyls has also been observed with the increasing age of rats. These data suggest that the toxicity of Fe-NTA increases with the increasing age of rats and correlates with the accumulation of HNE-modified protein adducts. It may also be speculated that Fe-NTA-mediated renal toxicity leading to carcinogenesis may be related to the tissue accumulation of HNE-modified protein adducts. However, further studies are needed to establish a definite role of HNE-modified proteins in Fe-NTA-mediated carcinogenesis.

Cellular resistance to vincristine suppresses NF-kappa B activation and apoptosis but enhances c-Jun-NH2-terminal protein kinase activation by tumor necrosis.

Tumor necrosis factor (TNF) is a pleiotropic cytokine that potentiates the cytotoxic effects of chemotherapeutic drugs. Although emergence of resistance to chemotherapeutic drugs is a major problem in cancer therapy, its mechanism is incompletely understood. Recently, activation of a nuclear transcription factor NF-kappa B has been reported to be a signal for anti-apoptosis. In this report, we investigated the effect of TNF on activation of NF-kappa B, c-Jun N-terminal kinase (JNK), and apoptosis in vincristine-resistant human histiocytic lymphoma U937-VR cells. Unlike the parent clone (U937-VS), no activation of caspase-3, known to be required for apoptosism was found in vincristine-resistant cells on exposure to vincristine. These cells were also more resistant than U-937-VS cells to doxorubicin, daunomycin, and taxol. TNF-induced NF-kappa B activation, I kappa B alpha degradation, and nuclear translocation of p65 were all found to be highly suppressed in the U-937-VR cells. NF-kappa B activation by LPS, H2O2, and okadaic acid was also suppressed. However, vincristine resistance enhanced TNF-induced JNK activation. When examined for apoptosis, vincristine resistance suppressed the cytotoxic effects and caspase-3 activation by TNF. The resistant phenotype in U937-VR cells was independent of the expression of the apoptosis-suppressor, Bcl-2. Thus, overall these results indicate that vincristine resistance correlates with suppression of NF-kappa B activation, cytotoxicity, and caspase-3 activation but enhancement of JNK activation by TNF.

Mycobacterium avium-intracellulare complex activates nuclear transcription factor-kappaB in different cell types through reactive oxygen intermediates.

Mycobacterium avium-intracellulare complex (MAC) is one of the most common opportunistic pathogens in HIV-infected patients. Their synergistic interaction leads to a rapid deterioration of the host defense. In vivo, MAC manifests as a disseminated granulomatous disease that produces a massive inflammatory tissue response perhaps through its activation of inflammatory cytokines. The intracellular signaling following interaction of the mycobacterium with host cells is incompletely understood. Because the response is dependent, in part, on the activation of NF-kappaB, we investigated the effect of MAC on this nuclear transcription factor in cells of macrophage and nonmacrophage lineage. We demonstrate that both high and low virulence strains of MAC potently and rapidly activated NF-kappaB. In supershift assays, using specific Abs against the NF-kappaB subunits, we identified a p50/p65 heterodimer that was formed within 5 min after incubation with the bacterium too rapidly for cytokines to be involved in the activation. This activation was instead mediated through the generation of reactive oxygen intermediates, inasmuch as preincubation of cells with a variety of antioxidants inhibited NF-kappaB activation. Likewise, the transfection of cells with Mn-superoxide dismutase blocked the NF-kappaB activation induced by the bacterium. These data suggest that NF-kappaB activation is a consequence of interaction of host cells with the bacterium and that the interaction may play a pivotal role in the pathogenesis of the disease.

Induction of matrix metalloproteinase-9 requires a polymerized actin cytoskeleton in human malignant glioma cells.

Alterations in cytoskeleton and subsequent cell shape changes exert specific effects on the expression of various genes. Our previous results suggested that malignant human gliomas express elevated levels of matrix metalloproteinases compared with normal brain tissue and low grade gliomas. To understand the role of cell shape changes on matrix metalloproteinase expression in human glioma cells, we treated SNB19 cells with cytochalasin-D, an inhibitor of actin polymerization, and colchicine-B, a tubulin inhibitor, in the presence of phorbol 12-myristate 13-acetate. Cytochalasin-D treatment of SNB19 cells resulted in the loss of phorbol 12-myristate 13-acetate-induced matrix metalloproteinase-9 (also known as gelatinase-B) expression and coincided with inhibition of actin polymerization, resulting in cell rounding. Moreover, compared with monolayers, cells grown as spheroids or cell aggregates failed to express matrix metalloproteinase-9 in the presence of phorbol 12-myristate 13-acetate. Matrix metalloproteinase-9 expression was also inhibited by calphostin-C, a protein kinase inhibitor, suggesting the involvement of protein kinase C in matrix metalloproteinase-9 expression. Phorbol 12-myristate 13-acetate-induced invasion of SNB19 cells through Matrigel was inhibited by cytochalasin-D and calphostin-C. These results suggest that the actin polymerization transduces signals that modulate the expression of matrix metalloproteinase-9 expression and the subsequent invasion of human glioma cells.

Constitutive activation of NF-kappaB causes resistance to apoptosis in human cutaneous T cell lymphoma HuT-78 cells. Autocrine role of tumor necrosis factor and reactive oxygen intermediates.

How tumor cells develop resistance to apoptosis induced by cytokines and chemotherapeutic agents is incompletely understood. In the present report, we investigated apoptosis induction by tumor necrosis factor (TNF) in two human T cell lines, Jurkat and HuT-78. While TNF inhibited the growth of Jurkat cells and activated caspase-3, it had no effect on HuT-78 cells. It was further found that HuT-78 cells constitutively expressed the nuclear transcription factor NF-kappaB. TNF activated NF-kappaB in Jurkat cells but not in HuT-78 cells. HuT-78 cells were also resistant to NF-kappaB activation induced by phorbol ester, H2O2, ceramide, endotoxin, and interleukin-1. Despite the presence of preactivated NF-kappaB, HuT-78 cells also expressed high levels of IkappaB-alpha, the inhibitory subunit of NF-kappaB and, unlike Jurkat cells, were resistant to TNF-induced degradation of IkappaB-alpha. Its half-life in HuT-78 cells was 12 h as opposed to 45 min in Jurkat cells. Antibodies against TNF blocked the constitutive activation of NF-kappaB and proliferation of HuT-78 cells but had no significant effect on Jurkat cells, suggesting an autocrine role for TNF. The antioxidant pyrrolidine dithiocarbamate also suppressed constitutive NF-kappaB activation and it reversed the cell's sensitivity to TNF-induced cytotoxicity and activation of caspase-3. Overall, these results suggest that constitutive activation of NF-kappaB, TNF, and prooxidant pathway in certain T cell lymphomas causes resistance to apoptosis, and this can be reversed by antioxidants.

Evidence that Fe-NTA-induced renal prostaglandin F2 alpha is responsible for hyperplastic response in kidney: implications for the role of cyclooxygenase-dependent arachidonic acid metabolism in renal tumor promotion.

Oxidative stress in a tissue activates phospholipase A2 which releases free arachidonic acid. In addition, a low grade oxidative tone also stimulates the tissue cyclooxygenase activity. Cyclooxygenase-dependent arachidonic acid metabolites such as PGF 2 alpha are known to play an important role in the development and maintenance of hyperplasia in skin in response to the application of tumor promoters. In this study we show that Fe-NTA, an oxidant renal tumor promoter induces PGF 2 alpha which was maximum at 12 hours after Fe-NTA treatment. However, at all time points studied, the elevated levels of PGF 2 alpha have been observed. As a result of the induction of PGF 2 alpha, the hyperplastic response can also be observed in the histopathology of the tissue. Additionally, an increased incorporation of [3H]thymidine in renal DNA has also been observed. Pretreatment of animals with indomethacin suppresses Fe-NTA-mediated hyperproliferation suggesting a role of cyclooxygenase in Fe-NTA-mediated stimulation of hyperplastic activity. The pretreatment of animals with the chain breaking antioxidants, Vit. E, BHA and BHT were only partially effective in inhibiting Fe-NTA-mediated PGF2 alpha production, further suggesting a role of non-free radical-dependent mechanism in its production. Our data suggest that Fe-NTA-induced PGF2 alpha through the activation of cyclooxygenase is responsible for the development and maintenance of hyperplasia in kidney.

Embryotoxicity and teratogenicity studies of poly (DL-lactide-co-glycolide) microspheres incorporated tetanus toxoid in Wistar rats.

Tetanus Toxoid loaded biodegradable microspheres (MTT) (poly (DL-Lactide-co-Glycolide) were administered intramuscularly to pregnant Wistar rats from Days 6 to 15 of gestation, at 1, 5 and 10-times the human equivalent dose of TT. Developmental defects in relation to soft tissues and skeleton, weight and sex of live pups and early fetal deaths from treated and control rats were analysed. The findings in treatment groups were comparable to those in the controls. These observations show that MTT was safe for pregnant rats and developing pups.

Poly (D,L-lactide) glycolide polymer microsphere entrapped tetanus toxoid: safety evaluation in Wistar rats.

A vaccine preparation consisting of tetanus toxoid (TT) encapsulated in biodegradable poly (D,L-Lactide) glycolide polymer has been developed. The toxicity of the preparation was evaluated in adult male and female Wistar rats injected IM with 20, 100 and 200 Lf tetanus toxoid, in polymer in 0.5 ml vehicle. No adverse effect of the vaccine could be seen, except for a granulomatous reaction at the site of injection. The vaccine was considered safe based on the findings in rats of both sexes.

Propolis protects against doxorubicin-induced myocardiopathy in rats.

Propolis (bee glue) is one of the major hive products of bees and is rich in flavonoids, which are known for antioxidant activities. Doxorubicin-induced myocardiopathy is the consequence of oxidative stress through the mediation of free radicals. The effect of intraperitoneal administration of propolis (50 and 100 mg/kg) was studied on cardiomyopathy produced by doxorubicin (10 mg/kg, i.v.) in rats. Serum creatine phosphokinase (CK), aspartate aminotransferase (AST), blood and tissue glutathione (GSH), and thiobarbituric acid reactive substances (TBARS) in heart were estimated to assess the status of heart muscle. An elevation of the levels of CK, AST, GSH, and TBARS was observed following doxorubicin treatment. Parallel experiments with a pretreatment of propolis significantly reduced the levels of these parameters . Biochemical observations were supplemented by histopathological examination of heart sections. The protective effect of propolis was compared with that of rutin, a known cardioprotective flavonoid. The study demonstrates the cardioprotective effect of propolis in doxorubicin-induced experimental cardiotoxicity.

Immunohistochemical localization of transforming growth factor-alpha, epidermal growth factor receptor and c-erbB-2 protein in hyperplastic human prostates.

Transforming growth factor-alpha (TGF-alpha) is a potent mitogenic factor which acts by binding to the epidermal growth factor (EGF). c-erbB-2 is a member of the EGF receptor family and is known to be associated with cellular growth and differentiation. The roles played by these factors in benign prostatic hyperplasia (BPH) are not clearly known. In the present study, expression of these factors was investigated immunohistochemically in frozen and formalin fixed prostate tissues from subjects suffering from BPH. Intracytoplasmic localization of TGF-alpha was observed in the epithelium of nine out of 39 (23.07%) cases. Twenty-six out of 36 (72.22%) frozen BPH tissues exhibited moderate to strong staining for immunoreactive EGF-receptor in the cell membrane. c-erbB-2 oncoprotein was localized in 35 out of 39 cases (89.74%) with the intensity of staining being variable. All nine cases positive for TGF-alpha were also positive for both EGF-receptor and c-erbB-2 protein. Staining reaction had no correlation with the serum testosterone, prostate specific acid phosphatase and prostate specific antigen levels. Immunohistochemical studies indicate the expression of TGF-alpha, EGF receptor and c-erbB-2 protein in BPH tissues. Further study is required to elucidate the precise roles played by these factors in benign growth of prostates.

Expression of NEU/HER-2 oncoprotein (p185neu) in prostate tumors: an immunohistochemical study.

The expression of the neu oncogene product was investigated immunohistochemically in 36 cases of benign prostatic hyperplasia (BPH) and seven cases of adenocarcinoma of prostate (CaP). c-neu oncogene encodes a transmembrane growth factor receptor that has partial structural homology with EGF receptor, and is overexpressed and amplified in a number of human tumors, specially, breast cancers. Using a monoclonal antibody, AB-3, which recognizes -COOH-terminal of neu oncoprotein, we have analyzed immunohistochemically the expression of this protein in buffered formalin and Zamboni fluid-fixed surgically removed tissues. Focal patchy and/or diffused cytoplasmic staining of varying intensity was observed in 34 of 36 BPH cases. Four cases showed cell membrane staining as well (4/36 = 11%). All seven cases of adenocarcinomas had moderate to strong c-neu immunoreactivity, and two gave a distinct cell membrane-positive reaction (100%). The available data indicate that prostatic tumors as well as a high percentage of prostatic hyperplasia tissues express c-neu protein; however, its role in cellular proliferation needs further study.

An epidemic of non-A, non-B hepatitis in south Delhi: epidemiological studies and transmission of the disease to rhesus monkeys.

In November 1987 an epidemic of NANB-hepatitis broke out in a residential colony of South Delhi which lasted for nearly two months. The epidemic was caused due to the sewage contamination of the drinking water supply. Analysis of the epidemiological data showed that the disease was more common in the younger age group of 11-20 years and that both sexes were equally prone to the disease. The disease could be transmitted to rhesus monkeys by intravenous inoculation of the stool extracts from the patients. Experimentally infected monkeys showed elevated levels of serum aminotransferases and excreted the infectious agent in the stools. Hepatic lesions characteristic of enteric non-A, non-B hepatitis were observed in an infected monkey.

Potentials of Shigella flexneri Y strain TSF21 as a candidate vaccine against shigellosis: safety, immunogenicity and protective efficacy in Bonnet monkeys.

A thymine-requiring and temperature-sensitive mutant of Shigella flexneri Y was tested in Bonnet monkeys for safety, immunogenicity and protective efficacy. A dose of 10(11) cells when fed orally mimicked natural infection in having invaded epithelial cells, but was otherwise clinically non-reactogenic. Animals immunized with two oral doses, each dose consisting of 1 x 10(11) mutant bacteria, were fully protected when challenged, with respect to the lack of any clinical symptoms or detectable histological abnormalities in the intestinal mucosa. Unimmunized animals when similarly challenged developed frank dysentery and the intestinal mucosa showed severe histological abnormalities. Titres of serum antibodies increased by about 11-fold of the base level in animals immunized with a dose of 10(11) cells, but not with lower doses. The challenge bacteria appeared to be phagocytised by macrophages. In some monkeys of a particular group, congestive patches were seen in the stomach, but not in any other part of the gut, after the animals were fed with the virulent parent strain. The lesions were relatively severe in the immunized groups of animals.

Prostatic hypoplasia in bonnet monkeys following active immunization with semisynthetic anti-LHRH vaccine.

An active immunization study using semisynthetic anti-luteinizing hormone releasing hormone (LHRH) vaccine was undertaken in adult male bonnet monkeys (Macaca radiata). Four male bonnet monkeys were immunized with LHRH-DT vaccine (100 micrograms/monkey). When the elevation of serum anti-LHRH titers reached an effective concentration, a remarkable decrease in serum testosterone level was observed. The sperm counts at 12 and 20 weeks following immunization in all immunized monkeys were reduced markedly. One representative animal with high antibody titer along with an age-matched control animal was sacrificed 20 weeks following primary immunization. The prostate weight of the immunized monkey was reduced to one-third (0.027) of the control (0.085). A reduction was also observed in other reproductive organs. Remaining animals are being studied for a reversible effect of immunization. Thus the study demonstrates that the size of the prostate can be reduced significantly by inhibition of hypothalamic-pituitary-testicular axis using this anti-LHRH vaccine.

Histopathological changes in reproductive organs of male Wistar rats following active immunization against LHRH.

Histopathological changes induced by immune responses generated against luteinizing hormone releasing hormone (LHRH) were studied in adult male Wistar rats. Active immunization with a semisynthetic anti-LHRH vaccine, LHRH D-Lys6, (10, 40, and 80 micrograms/animal) conjugated with diphtheria toxoid, produced bioeffective antibodies as indicated by significant reduction in circulating testosterone levels. At the 14th week after immunization the animals were sacrificed and reproductive organs were evaluated. These organs were studied for histopathological changes and compared with those of nonimmunized control rats. Marked hypoplastic changes were observed in the genital organs. Testicular changes such as arrest of spermatogenesis at the spermatocyte level and atrophic changes in the interstitial Leydig cells were noticed in treated animals. Similarly attenuation of secretory epithelial cells with substantial increase in the stromal tissues was observed in the prostate and seminal vesicles. The current observation suggests the possible usefulness of this anti-LHRH vaccine under clinical conditions where reduction in androgenic response is desired as in the case of hormone-dependent prostatic carcinoma.