Identifying novel Mycobacterium species in fish using multiple gene phylogenetic analysis

Mycobacteria have, for a long time, been suspected to be causing severe disease in ornamental and farmed fish in Trinidad and Tobago (T&T), however, up to now, these mycobacteria species have not been identified and characterised. Many piscine mycobacteria species are also known to be zoonotic, potentially affecting human health. Objective: To identify and characterize the species of mycobacteria affecting fish (and possibly man) in T&T. Design and Methodology: Homogenised internal organs were collected from a total of 13 fish showing clinical signs consistent with mycobacterial infection. Samples were analysed using Ziehl-Neelsen (acid-fast) staining and real-time Polymerase Chain Reaction (rPCR). The species of mycobacteria were further characterised using conventional PCR targeting the 16s rRNA (564 bp), rpoB (396 bp) and sod (408 bp) genes. PCR products were sequenced and the sequences were compared with those from known and recently identified mycobacteria species through phylogenetic analysis. Results: Acid-fast non-branching bacilli were detected in all samples. All samples were also positive for Mycobacterium sp. by real-time PCR. Multi-gene phylogenetic analysis revealed the presence of two distinct species of mycobacteria. One aligned closely with Mycobacterium marinum, a well known pathogen affecting fish and man, and a second aligned closely with a species also known to affect both fish and humans, Mycobacterium stomatepiae. Conclusions: Phylogenetic analysis demonstrated the presence of two mycobacterium species in organs from fish showing clinical signs of Piscine Mycobacteriosis in T&T. Further work is needed to characterise these mycobacteria species and investigate their zoonotic potential.

Ocular localization of mycobacterial lesions in tank-reared juvenile cobia, Rachycentron canadum.

Severe clinical mycobacteriosis with consistent ocular lesion localization was diagnosed in a population of 800 juvenile tank-reared Cobia (Rachycentron canadum) which experienced a sudden increase in mortality approximately 5 months after arriving into Trinidad and Tobago from Florida, USA. Moderate daily mortality (15-20 animals per day) persisted for just over 1 month. Moribund fish displayed circling behaviour and had an open-mouth gape upon death. Fish consistently presented with bilateral exophthalmia, corneal cloudiness and hyphema. Non-branching acid-fast rods were detected in aqueous humour touch preparations. Histological analysis revealed severe bilateral intra-ocular granulomatous responses in all specimens. Mycobacterium sp. was identified using a real-time PCR assay detecting the RNA polymerase ß-subunit (rpoB) gene in different tissue samples. Specimens did not present with characteristic granulomatous responses usually seen in viscera. To the best of our knowledge, this represents only the third documentation of piscine mycobacterial infection presenting with only localized ocular lesions, and the second documented case of mycobacteriosis in cobia. It is, however, the first documentation of an ocular presentation of mycobacteriosis in a marine species and is the first documentation of such a presentation in cobia.

Edwardsiella ictaluri infection in Pangasius catfish imported from West Bengal into the Southern Caribbean.

In response to a mortality event, seven Pangasius catfish (Pangasianodon hypophthalmus) were submitted to the University of the West Indies, School of Veterinary Medicine, Trinidad and Tobago, for diagnostic evaluation. These fish were part of a consignment that arrived from Kolkata two weeks earlier. Fish presented with perianal haemorrhage and blister-like swellings on the skin which ruptured to leave ulcers. Edwardsiella ictaluri was consistently recovered from the brain and skin. Repetitive sequence-mediated PCR analysis revealed genetic fingerprints consistent with E. ictaluri isolates from farm-raised channel catfish in Mississippi, USA. Plasmid analysis of the case isolates identified two unique plasmids that differ slightly in conformation and content from the pEI1 and pEI2 plasmids described for E. ictaluri from other fish hosts. The case isolates were also PCR negative for several E. ictaluri virulence factors. The biological implications of these genetic differences are unclear and warrant further study. This is the first report and documentation of E. ictaluri infection in Trinidad and Tobago, suggesting the pathogen may have been introduced concurrently with the importation of fish. This report emphasizes the importance of adequate health screenings of imported lots to minimize the threat of introducing E. ictaluri to non-endemic areas.

Cardiovascular reactivity patterns and pathways to hypertension: a multivariate cluster analysis.

Substantial evidence links exaggerated mental stress induced blood pressure reactivity to future hypertension, but the results for heart rate reactivity are less clear. For this reason multivariate cluster analysis was carried out to examine the relationship between heart rate and blood pressure reactivity patterns and hypertension in a large prospective cohort (age range 55-60 years). Four clusters emerged with statistically different systolic and diastolic blood pressure and heart rate reactivity patterns. Cluster 1 was characterised by a relatively exaggerated blood pressure and heart rate response while the blood pressure and heart rate responses of cluster 2 were relatively modest and in line with the sample mean. Cluster 3 was characterised by blunted cardiovascular stress reactivity across all variables and cluster 4, by an exaggerated blood pressure response and modest heart rate response. Membership to cluster 4 conferred an increased risk of hypertension at 5-year follow-up (hazard ratio=2.98 (95% CI: 1.50-5.90), P<0.01) that survived adjustment for a host of potential confounding variables. These results suggest that the cardiac reactivity plays a potentially important role in the link between blood pressure reactivity and hypertension and support the use of multivariate approaches to stress psychophysiology.

Depression and anxiety are associated with a diagnosis of hypertension 5 years later in a cohort of late middle-aged men and women.

The aim of this study is to examine the association between symptoms of depression and anxiety and hypertension status. Participants (n=455, 238 women) were drawn from the Dutch Famine Birth Cohort Study. In 2002-2004, they attended a clinic assessment during which socio-demographics, anthropometrics, resting systolic blood pressure (SBP) and health behaviours were measured. Symptoms of depression and anxiety were measured using the Hospital Anxiety and Depression Scale. In 2008-2009, participants completed a questionnaire, which asked whether they ever had a physician diagnosing them as suffering from hypertension. In separate regression models that initially adjusted for age and then additionally for sex, socio-economic status, smoking, sports participation, alcohol consumption, resting SBP, antidepressive and anxiolytic medication, whether or not participants were exposed to the Dutch famine in utero, BMI and waist:hip ratio, both depression and anxiety were positively associated with hypertension status. Those who met the criterion for possible clinical depression and anxiety were also more likely to be hypertensive, and these associations remained statistically significant in the fully adjusted regression model. In conclusion, symptoms of depression and anxiety were associated with a diagnosis of hypertension assessed 5 years later, although the mechanisms underlying these associations remain to be determined.

White blood cell subsets are associated with carotid intima-media thickness and pulse wave velocity in an older Chinese population: the Guangzhou Biobank Cohort Study.

Cross-sectional associations between white blood cell (WBC) count, lymphocyte and granulocyte numbers, and carotid intima-media thickness (IMT) and brachial-ankle pulse wave velocity (PWV) were examined in a novel older Chinese community sample. A total of 817 men and 760 women from a sub-study of the Guangzhou Biobank Cohort Study had a full blood count measured by an automated hematology analyzer, carotid IMT by B-mode ultrasonography and brachial-ankle PWV by a non-invasive automatic waveform analyzer. Following adjustment for confounders, WBC count (ß=0.07, P<0.001) and granulocyte (ß=0.07, P<0.001) number were significantly positively related to PWV, but not lymphocyte number. Similarly, WBC count (ß=0.08, P=0.03), lymphocyte (ß=0.08, P=0.002) and granulocyte (ß=0.03, P=0.04) number were significantly positively associated with carotid IMT, but only the association with lymphocyte count survived correction for other cardiovascular risk factors. In conclusion, higher WBC, particularly lymphocyte and granulocyte, count could be used, respectively, as markers of cardiovascular disease risk, measured through indicators of atherosclerosis and arterial stiffness. The associations for WBC count previously observed by others were likely driven by higher granulocytes; an index of systemic inflammation.

Sex hormones and cause-specific mortality in the male veterans: the Vietnam Experience Study.

BACKGROUND: Testosterone levels have been linked to life expectancy in men, less is known about the sex hormones follicular stimulating hormone and luteinizing hormone. AIM: To examine the association of testosterone, follicular stimulating hormone, luteinizing hormone with mortality. DESIGN: Prospective cohort analysis. METHODS: Participants were 4255 Vietnam-era US army veterans with a mean age of 38.3 years. From military service files, telephone interviews and a medical examination, socio-demographic and health data were collected. Contemporary morning fasted hormone concentrations were determined. All-cause, cardiovascular, cancer, external and 'other' cause mortality was ascertained over the subsequent 15 years. Hazard ratios were calculated, first with adjustment for age and then, additionally, for a range of confounders. RESULTS: Individuals within the highest tertiles of follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels were at increased risk of all-cause mortality following adjustment for a range of risk factors. However, with mutual adjustment, neither FSH nor LH significantly predicted mortality. Testosterone levels did not show an association with all-cause mortality, and none of the hormones were significantly associated with CVD, cancer, 'other' or external-cause mortality in fully adjusted models. CONCLUSION: Greater FSH and LH levels are associated with all-cause mortality, but not independently of one another.

Cortisol, dehydroepiandrosterone sulphate, their ratio and hypertension: evidence of associations in male veterans from the Vietnam Experience Study.

Although clinical observations implicate cortisol in hypertension, the epidemiological evidence is less compelling. Little is known about the relationship between dehydroepiandrosterone sulphate (DHEAS) and hypertension, and nothing about the association with the cortisol:DHEAS ratio. The present analyses of data obtained from Vietnam-era US veterans examined the associations between cortisol, DHEAS, their ratio and hypertension. Participants were 4180 male veterans. From military files, telephone interviews and a medical examination, sociodemographic and health data were collected. At medical examination, a fasted morning blood sample was collected to assay serum cortisol and DHEAS, blood pressure measured and body mass index (BMI) determined. Hypertension was defined by having one of the following: a reported physician diagnosis, taking antihypertensive medication, an average systolic blood pressure ≥ 140 mm Hg and an average diastolic blood pressure ≥ 90 mm Hg. Cortisol and the cortisol:DHEAS ratio were positively associated with hypertension (P < 0.001), whereas DHEAS was negatively associated; the latter relationship was attenuated to non-significance (P = 0.06) in models that adjusted for age, sociodemographics, place of service, health behaviours and BMI. The present analyses provide confirmation of a positive association between cortisol and the cortisol:DHEAS ratio and population hypertension.

Lymphocyte cell counts in middle age are positively associated with subsequent all-cause and cardiovascular mortality.

BACKGROUND: There is an association between higher white blood cell counts and all-cause and cardiovascular disease (CVD) mortality. However, little is known about the prognostic significance of circulating lymphocyte and lymphocyte subset numbers. AIMS: The present study examined the association between T-, CD4-, CD8- and B-cell numbers, and the CD4:CD8 ratio, and all-cause and CVD mortality. METHODS: Lymphocyte and lymphocyte subset numbers were measured by flow cytometry in a cohort of 4256 male middle-aged Vietnam-era US veterans. Mortality was tracked for 15 years and cause of death was determined from death certificates. RESULTS: In fully adjusted survival analyses, high circulating T-cells numbers were associated with increased risk of both all-cause [hazard ratio (HR)=1.75, 95% confidence interval (CI) 1.15-2.66] and cardiovascular (HR=3.57, 95% CI 1.53-8.33) mortality. The former association appeared to reflect an effect for high CD8-cells numbers, the latter an effect for high CD4-cell numbers. For all-cause mortality, a high CD4:CD8 ratio was protective (HR=0.58, 95% CI 0.41-0.81). Cardiovascular mortality was also predicted by high B-cells numbers (HR=1.87, 95% CI 1.10-3.17). CONCLUSION: Circulating lymphocyte and lymphocyte subset numbers may have substantial prognostic significance for both all-cause and CVD mortality.

Adenoviral-mediated Rybp expression promotes tumor cell-specific apoptosis.

Previous work demonstrated that exogenous expression of Rybp (Ring 1 YY1-binding protein or DEDAF) kills tumor but not non-transformed cells. This tumor-preferential killing activity could be exploited in a gene therapy approach to treat cancer. To test the potential of viral-mediated delivery of Rybp as an anticancer treatment, we generated an adenovirus expressing Rybp (Ad-Rybp). Infection with Ad-Rybp inhibits the proliferation of a range of tumor cell lines, but has no effect on normal cell types. This inhibition of proliferation is the result of the induction of apoptosis, consistent with reports that Rybp regulates apoptosis. Combined Ad-Rybp infection and etoposide treatment resulted in an additive cytotoxic effect in the osteosarcoma cell line U20S. Furthermore, Ad-Rybp infection synergistically cooperates with the death receptor ligand, tumor necrosis factor-alpha, in the induction of apoptosis. These results suggest that Ad-Rybp may have clinical applicability, either alone or in combination with other agents for the treatment of cancer.

E2F-1 induced apoptosis.

Members of the E2F family of transcription factors play an important role in regulating the cell cycle, and their activity is often perturbed during the development of human malignancies. More recent work has shown that E2F-1 regulates apoptosis as well as proliferation, in part by stabilizing the p53 tumor suppressor, an important mediator of apoptosis. This has led to the suggestion that E2F-1 may function as a tumor surveillance mechanism, detecting aberrant proliferation and engaging apoptotic pathways to protect the organism from developing tumors.

Regulation and function of the p53 tumor suppressor protein.

Loss of the p53 tumor suppressor pathway contributes to the development of most human cancers. p53 is a nuclear protein that functions as a regulator of transcription. Significant advances have been made recently in our understanding of how p53 function is regulated and the mechanisms by which p53 mediates its effects.

Characterization of type XI collagen-glycosaminoglycan interactions.

Using competitive binding experiments, it was found that native type XI collagen binds heparin, heparan sulfate, and dermatan sulfate. However, interactions were not evident with hyaluronic acid, keratan sulfate, or chondroitin sulfate chains over the concentration range studied. Chondrocyte-matrix interactions were investigated using cell attachment to solid phase type XI collagen. Pretreatment of chondrocytes with either heparin or heparinase significantly reduced attachment to type XI collagen. Incubation of denatured and cyanogen bromide-cleaved type XI collagen with radiolabeled heparin identified sites of interaction on the alpha1(XI) and alpha2(XI) chains. NH(2)-terminal sequence data confirmed that the predominant heparin-binding peptide contained the sequence GKPGPRGQRGPTGPRGSRGAR from the alpha1(XI) chain. Using rotary shadowing electron microscopy of native type XI collagen molecules and heparin-bovine serum albumin conjugate, an additional binding site was identified at one end of the triple helical region of the collagen molecule. This coincides with consensus heparin binding motifs present at the amino-terminal ends of both the alpha1(XI) and the alpha2(XI) chains. The contribution of glycosaminoglycan-type XI collagen interactions to cartilage matrix stabilization is discussed.

Role for the p53 homologue p73 in E2F-1-induced apoptosis.

The transcription factor E2F-1 induces both cell-cycle progression and, in certain settings, apoptosis. E2F-1 uses both p53-dependent and p53-independent pathways to kill cells. The p53-dependent pathway involves the induction by E2F-1 of the human tumour-suppressor protein p14ARF, which neutralizes HDM2 (human homologue of MDM2) and thereby stabilizes the p53 protein. Here we show that E2F-1 induces the transcription of the p53 homologue p73. Disruption of p73 function inhibited E2F-1-induced apoptosis in p53-defective tumour cells and in p53-/- mouse embryo fibroblasts. We conclude that activation of p73 provides a means for E2F-1 to induce death in the absence of p53.

Acetyltransferases and tumour suppression.

The acetyltransferase p300 was first identified associated with the adenoviral transforming protein E1A, suggesting a potential role for p300 in the regulation of cell proliferation. Direct evidence demonstrating a role for p300 in human tumours was lacking until the recently publication by Gayther et al, which strongly supports a role for p300 as a tumour suppressor. The authors identify truncating mutations associated with the loss or mutation of the second allele in both tumour samples and cell lines, suggesting that loss of p300 may play a role in the development of a subset of human cancers.

E2F-1 potentiates cell death by blocking antiapoptotic signaling pathways.

The E2F family of transcription factors plays an essential role in promoting cell cycle progression, and one member of the family, E2F-1, is also capable of inducing apoptosis. We show here that E2F-1 can induce apoptosis by a death receptor-dependent mechanism, by downregulating TRAF2 protein levels and inhibiting activation of antiapoptotic signals including NF-kappa B. In this way, E2F-1 expression can lead to the sensitization of cells to apoptosis by a number of agents independently of p53. Deregulation of E2F-1 activity occurs in the majority of human tumors, and the ability of E2F-1 to inhibit antiapoptotic signaling may contribute to the enhanced sensitivity of transformed cells to chemotherapeutic agents.

Cell cycle arrest and DNA endoreduplication following p21Waf1/Cip1 expression.

p21Waf1/Cip1 is a major transcriptional target of p53 and has been shown to be one of the principal mediators of the p53 induced G1 cell cycle arrest. We show that in addition to the G1 block, p21Waf1/Cip1 can also contribute to a delay in G2 and expression of p21Waf1/Cip1 gives rise to cell cycle profiles essentially indistinguishable from those obtained following p53 expression. Arrest of cells in G2 likely reflects an inability to induce cyclin B1/cdc2 kinase activity in the presence of p21Waf1/Cip1, although the inefficient association of p21Waf1/Cip1 and cyclin B1 suggests that the mechanism of inhibition is indirect. Cells released from an S-phase block were not retarded in their ability to progress through S-phase by the presence of p21Waf1/Cip1, despite efficient inhibition of cyclin E, A and B1 dependent kinase activity, suggesting that p21Waf1/Cip1 is inefficient at inhibiting replicative DNA synthesis in vivo. Interestingly, significant numbers of cells released from the p21Waf1/Cip1 activated G2 block undergo endoreduplication, passing through another S-phase before undergoing mitosis. This supports a function of the mitotic kinases in both entry into mitosis, and also in preventing re-replication of DNA following S-phase and suggests a role for p21Waf1/Cip1 in coupling DNA synthesis and mitosis. Unlike p53, which induces apoptosis in these cells, extended expression of p21Waf1/Cip1 resulted in the expression of a senescent-like phenotype in these p53 null, pRB null tumor cells.

Oral iloprost treatment in patients with Raynaud's phenomenon secondary to systemic sclerosis: a multicenter, placebo-controlled, double-blind study.

OBJECTIVE: To evaluate the efficacy and tolerability of an oral preparation of iloprost, a prostacyclin analog, in patients with Raynaud's phenomenon (RP) secondary to systemic sclerosis (scleroderma). METHODS: A multicenter, randomized, parallel-group, placebo-controlled double-blind study was performed at university and community-based medical centers. Patients were randomly assigned to receive either 50 microg of iloprost orally twice daily or an identical gelatin-coated capsule containing placebo for 6 weeks. Outcome measures included average total daily duration of RP attacks, average number of RP attacks, and RP condition scored via a standardized daily diary. RESULTS: Three hundred eight patients with scleroderma (272 women, 36 men, mean age 49 years [range 18-80]) were enrolled. One hundred fifty seven were assigned to receive iloprost and 151 to receive placebo. One hundred forty-three patients in the iloprost group (91.1%) and 144 in the placebo group (95.4%) completed the 6-week treatment phase. Fifteen of these treated patients (8 iloprost, 7 placebo) failed to complete all of the followup visits. The mean reduction in the average duration of attacks from baseline to week 5-6 was 24.32 minutes in the iloprost group and 34.34 minutes in the placebo group (P = 0.569). Likewise, the mean reduction from baseline to week 5-6 in the daily frequency of attacks was 1.02 in the iloprost group and 0.83 in the placebo group (P = 0.459). The Raynaud's condition score, a patient-completed assessment of the severity of RP attacks, was reduced by 1.32 in the iloprost group and 1.00 in the placebo group (P = 0.323). The lack of significant difference between treatment groups did not change when a variety of factors, including use of other vasodilators, duration of disease, classification of scleroderma (limited versus diffuse), or number of baseline digital ulcers were taken into account. Premature withdrawal from the study due to adverse events occurred in 10 patients (6.4%) in the iloprost group and 3 (2.0%) in the placebo group (P = 0.058). CONCLUSION: Oral iloprost at a dosage of 50 microg twice daily is no better than placebo for management of RP secondary to scleroderma, either during 6 weeks of treatment or during 6 weeks of posttreatment followup.

Induction of DNA synthesis and apoptosis are separable functions of E2F-1.

The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E2F-1, suggesting that it may be mediated through alleviation of E2F-dependent transcriptional repression. These results indicate that E2F-1 can show independent cell cycle progression and apoptotic functions, consistent with its putative role as a tumor suppressor.