Identification of genes associated with environmental persistence in Campylobacter jejuni and Campylobacter coli isolates from processing in a broiler abattoir.

The aim of this study was to determine the prevalence of the htrA, htrB and ppk1 genes -all of which are related to environmental persistence- in C. jejuni and C. coli isolates obtained from abattoir samples at the arrival of broilers (initial stage) and in meat products after processing (final stage). A total of 119 DNA extracts (55 C. jejuni and 64 C. coli) were included in the study. Identification of genes was performed by conventional PCR (one for each gene). The overall prevalence was 40.3%, 93.3% and 68.9% for the htrA, htrB and ppk1 genes, respectively. Statistically significant differences were found (p < 0.05) between prevalence of C. jejuni and C. coli for all three genes. In C. coli the prevalence was significantly higher for the htrA (p = 0.007) and htrB (p = 0.015) genes, while ppk1 gene prevalence was significantly higher in C. jejuni (p < 0.001). In addition, statistically significant increase in the frequency of htrA (p = 0.007) and htrB (p = 0.013) genes in the final product compared to broilers on arrival at the abattoir was observed in C. jejuni, but not in C. coli. These results suggest that htrA and htrB genes are involved in environmental persistence of Campylobacter jejuni.

Combined genetic approaches yield a 48% diagnostic rate in a large cohort of French hearing-impaired patients.

Hearing loss is the most common sensory disorder and because of its high genetic heterogeneity, implementation of Massively Parallel Sequencing (MPS) in diagnostic laboratories is greatly improving the possibilities of offering optimal care to patients. We present the results of a two-year period of molecular diagnosis that included 207 French families referred for non-syndromic hearing loss. Our multi-step strategy involved (i) DFNB1 locus analysis, (ii) MPS of 74 genes, and (iii) additional approaches including Copy Number Variations, in silico analyses, minigene studies coupled when appropriate with complete gene sequencing, and a specific assay for STRC. This comprehensive screening yielded an overall diagnostic rate of 48%, equally distributed between DFNB1 (24%) and the other genes (24%). Pathogenic genotypes were identified in 19 different genes, with a high prevalence of GJB2, STRC, MYO15A, OTOF, TMC1, MYO7A and USH2A. Involvement of an Usher gene was reported in 16% of the genotyped cohort. Four de novo variants were identified. This study highlights the need to develop several molecular approaches for efficient molecular diagnosis of hearing loss, as this is crucial for genetic counselling, audiological rehabilitation and the detection of syndromic forms.

Mutations in SDCCAG8/NPHP10 Cause Bardet-Biedl Syndrome and Are Associated with Penetrant Renal Disease and Absent Polydactyly.

The ciliopathies are an expanding group of disorders caused by mutations in genes implicated in the biogenesis and function of primary cilia. Bardet-Biedl syndrome (BBS) is a model ciliopathy characterized by progressive retinal degeneration, obesity, polydactyly, cognitive impairment, kidney anomalies and hypogonadism. Mutations in SDCCAG8(NPHP10) were described recently in patients with nephronophthisis and retinal degeneration (Senior-Loken syndrome; SLS). Given the phenotypic and genetic overlap between known ciliopathy genes, we hypothesized that mutations in SDCCAG8 might also contribute alleles to more severe, multisystemic ciliopathies. We performed genetic and phenotypic analyses of 2 independent BBS cohorts. Subsequent to mutation screening, we made a detailed phenotypic analysis of 5 families mutated for SDCCAG8 (3 homozygous and 2 compound heterozygous mutations) and conducted statistical analyses across both cohorts to examine possible phenotype-genotype correlations with mutations at this locus. All patients with mutations in SDCCAG8 fulfilled the diagnostic criteria for BBS (retinal degeneration, obesity, cognitive defects, renal failure, hypogonadism). Interestingly, none of the patients with primary SDCCAG8 mutations had polydactyly, a frequent but not obligatory BBS feature. In contrast, the same patients displayed early-onset renal failure, obesity, as well as recurrent pulmonary and ENT infections. Comparison of the phenotypes of these families with our entire BBS cohort indicated that renal impairment and absent polydactyly correlated significantly with causal SDCCAG8 mutations. Thus, SDCCAG8 mutations are sufficient to cause BBS in 1-2% of our combined cohorts, and define this gene as the sixteenth BBS locus (BBS16). The absence of polydactyly and the concomitant, apparently fully penetrant association with early kidney failure represents the first significant genotype-phenotype correlation in BBS that potentially represents an indicator for phenotype-driven priority screening and informs specific patient management.

The role of wild rodents in the transmission of Trypanosoma evansi infection in an endemic area of the Canary Islands (Spain).

Trypanosoma evansi was diagnosed for the first time in camels in the Canary Islands in 1997. Several sanitary measures including treatment of infected animals were taken; however, nowadays a little area is still infected. In order to determine possible reservoirs 138 wild rodents were trapped, 64 of them in the infected farms and the remaining 74 in other areas. The captured species were Rattus rattus (24), Rattus norvegicus (69) and Mus musculus domesticus (45). Serological (CATT/T. evansi), parasitological (micro-Hematocrit Centrifugation technique and stained smears) and molecular (PCR) methods for T. evansi and T. lewisi were used as diagnostic methods. None of the examined rodents was positive for T. evansi; 18, however, showed motile trypanosomes at micro-Hematocrit Centrifugation technique and resulted positive for T. lewisi by PCR. The results would suggest that the studied rodent species would not play a relevant role in the epidemiology of T. evansi infection in Canaries.

Confirmation of ADAMTSL4 mutations for autosomal recessive isolated bilateral ectopia lentis.

Ectopia lentis (EL) is a zonular disease where alteration of the zonular fibers leads progressively to lens dislocation. It is most often associated with systemic diseases such as Marfan syndrome, Weill-Marchesani syndrome or homocystinuria. Isolated non syndromic ectopia lentis (IEL) is reported in families with autosomal inheritance, with dominant forms being more common than recessive. LTBP2 truncating mutations have been described as a cause of autosomal recessive ectopia lentis as a primary or secondary feature in patients showing ocular (eg, glaucoma) or extraocular manifestations (eg, Marfanoid habitus). Recently, ADAMTSL4 has been shown to be responsible for isolated autosomal recessive ectopia lentis in an inbred family. Herein we show a consanguineous family that carries a novel homozygous splice mutation IVS4-1G>A/IVS4-1G>A in ADAMTSL4 responsible for isolated autosomal recessive EL, thus confirming the involvement of this gene in this condition and underlining the major role of ADAMTS proteases in zonular fibers homeostasis.

Insight into the role of CYBA A640G and C242T gene variants and coronary heart disease risk. A case-control study.

The CYBA gene variants have been inconsistently associated with coronary heart disease (CHD) risk. A case-control study was conducted genotyping 619 subjects to explore the contribution of C242T and A640G to CHD risk in the population. A significant risk was found associated with GG homozygosity (odds ratio (OR) 2.132, 95% confidence interval, 1.113-4.085). The C242T variant was associated with CHD risk in women. Bias due to population stratification was analysed. Phenotype changes linked to these polymorphisms were evaluated. Superoxide measurements revealed higher production as indicated by the presence of the G and T alleles. Differences in mRNA concentration in heterozygous A640G samples were analysed. Higher levels of G allele mRNA compared with A allele mRNA were found. NAD(P)H oxidase p22phox sub-unit expression was evaluated with Western blot. Experiments revealed a gradual relationship in p22phox protein expression according to genotypes of the analysed variants. Those GG TT double homozygous showed increased p22phox protein expressions regarding AA CC double homozygous. This study has demonstrated increased expression and activity of the NAD(P)H system components during atherogenesis and the results could help explain the relevance of the A640G variant as a CHD marker.

Rosiglitazone but not losartan prevents Nrf-2 dependent CD36 gene expression up-regulation in an in vivo atherosclerosis model.

BACKGROUND: Thiazolidinediones exert anti-inflammatory and anti-oxidative roles and attenuate atherosclerosis by mechanisms partially independent of their metabolizing actions. High doses of angiotensin type 1 receptor (AT1R) blocker losartan (LST) seem to promote fat cell formation by preserving PPARgamma activity. METHODS: C57BL/6J diet-induced atherosclerotic susceptible mice randomly received a normal or a high-fat high-cholesterol (HFHC) diet and were treated with rosiglitazone (RG), LST or a vehicle for 12 weeks. RESULTS: HFHC was associated with increased PPARgamma gene expression without an over regulation of PPARgamma responsive genes, whereas RG and LST treatments were found to maintain PPARgamma activity without resulting in increased PPARgamma gene expression. A better anti-inflammatory and antioxidant profile in mice treated with RG regarding LST was observed in spite of a similar PPARgamma preserved activity. Chromatin immunoprecipitation (ChIP) assays revealed that animals under HFHC diet treated with RG showed a significant nuclear factor erythroid 2-like 2 (Nrf2)-dependent down-regulation of the expression of the CD36 gene. CONCLUSION: The PPARgamma agonist RG exerts antioxidant properties that significantly reduced Nrf-2-dependent CD-36 up-regulation in mice under HFHC diet. Because LST treatment was also associated with a preserved PPARgamma activity, our data suggests that these RG antioxidant effects are partially independent of its PPARgamma metabolizing properties.

[Arterial blood pressure variations: homocysteine and nitric oxide synthase gene polymorphisms (NOS3)]. / Presión arterial, homocisteína y variantes polimórficas del gen de la óxido nítrico sintasa (NOS3).

BACKGROUND: The nitric oxide synthase (NOS3) G894T gene polymorphism seems as a genetic determinant of total homocysteine (tHcy) concentrations through an effect on folate catabolism. We tested for a significant contribution to blood pressure values for the NOS3 G894T and 4a/b gene polymorphisms and whether those changes could explain the modulating effect on tHcy concentrations. PATIENTS AND METHODS: We analyzed 158 healthy men. The NOS3 gene polymorphisms were determined by polymerase chain reaction (PCR) and restriction fragment analysis (G894T) and by PCR (4a/b). Total homocysteine concentrations were evaluated by the fluorescence polarization immunoassay method. RESULTS: In our population we did not obtain a significant contribution of the G894T to blood pressure variations. However, tHcy mean concentration values differed according G894T genotypes (P = 0.01). Interestingly, we did not obtain a significant modulating effect on tHcy concentrations according to 4a/b genotypes although the 4a/b genotype distribution was statistically associated with blood pressure variations. CONCLUSION: Our results showed a modulating effect of the NOS3 4a/b gene variant on tHcy concentrations that is at least partially provoked by discrete blood pressure increments. Nevertheless, our multivariate analysis did not show a statistical significant role for the NOS3 G894T gene polymorphism on tHcy concentrations.

Presión arterial, homocisteína y variantes polimórficas del gen de la óxido nítrico sintasa (NOS3) / Arterial blood pressure variations: homocysteine and nitric oxide synthase gene polymorphisms (NOS3)

Fundamento y objetivo: El polimorfismo G894T en el gen humano NOS3 se ha sugerido como un determinante genético que contribuye a modular las variaciones de las concentraciones de homocisteína (tHcy) a través de un efecto indirecto sobre el catabolismo del folato. Nos planteamos estudiar la contribución de las variantes NOS3 G894T y 4a/b a las variaciones de presión arterial y la contribución de estas variaciones a la modulación de concentraciones de tHcy. Pacientes y métodos: Analizamos 158 hombres sanos. Los genotipos de los polimorfismos NOS3 se determinaron mediante amplificación por reacción en cadena de la polimerasa (PCR) y posterior digestión enzimática (G894T) y por PCR (4a/b). Las concentraciones de tHcy mediante inmunofluorescencia polarizada. Resultados: En nuestra población la variante G894T no parece contribuir de forma significativa a las variaciones de presión arterial, pero obtuvimos una gradación de concentraciones de tHcy en función de los genotipos G894T (P = 0,01). No obtuvimos un efecto modulador significativo sobre los niveles de tHcy para la variante 4a/b, si bien su contribución a la variación de cifras de presión arterial resultó estadísticamente significativa. Conclusiones: Nuestros resultados muestran un efecto modulador de la variante 4a/b en el gen NOS3 sobre la variación de concentraciones de tHcy que se produce, parcialmente y a expensas de incrementos de valores de presión arterial. En análisis multivariante, el papel del polimorfismo G894T del gen NOS3 sobre la modulación de las concentraciones de tHcy, no resultó sin embargo estadísticamente relevante

Background: The nitric oxide synthase (NOS3) G894T gene polymorphism seems as a genetic determinant of total homocysteine (tHcy) concentrations through an effect on folate catabolism. We tested for a significant contribution to blood pressure values for the NOS3 G894T and 4a/b gene polymorphisms and whether those changes could explain the modulating effect on tHcy concentrations. Patients and methods: We analyzed 158 healthy men. The NOS3 gene polymorphisms were determined by polymerase chain reaction (PCR) and restriction fragment analysis (G894T) and by PCR (4a/b). Total homocysteine concentrations were evaluated by the fluorescence polarization immunoassay method. Results: In our population we did not obtain a significant contribution of the G894T to blood pressure variations. However, tHcy mean concentration values differed according G894T genotypes (P = 0.01). Interestingly, we did not obtain a significant modulating effect on tHcy concentrations according to 4a/b genotypes although the 4a/b genotype distribution was statistically associated with blood pressure variations. Conclusion: Our results showed a modulating effect of the NOS3 4a/b gene variant on tHcy concentrations that is at least partially provoked by discrete blood pressure increments. Nevertheless, our multivariate analysis did not show a statistical significant role for the NOS3 G894T gene polymorphism on tHcy concentrations

Estrategia del gen candidato en la hipertensión arterial: Estudios de ligamiento y asociación / Strategy of the candidate gene in arterial hypertension: linkage and association study

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Long-term survival and DNA ploidy in advanced epithelial ovarian cancer.

BACKGROUND AND OBJECTIVES: The relationship of the tumor DNA content to survival of patients with advanced epithelial cancer has not yet been clarified. A large amount of contradictory data exists in the literature. This study analyzes the putative relationship between ploidy and advanced ovarian carcinoma. METHODS: A retrospective analysis of tumor ploidy, DNA index, and the S-phase fraction from 35 patients with nonborderline epithelial ovarian carcinomas was determined by flow cytometry of paraffin-embedded tissue. All patients had FIGO stage III or IV disease. Those patients who survived > 5 years were assigned to Group A (10 patients). Group B consisted of 25 age-matched subjects who succumbed to their disease within 5 years of diagnosis. RESULTS: Group A had not reached a median overall survival with a median follow-up of 114 months (range 67-226), whereas Group B had a median overall survival of 17 months (range 1-48). Two of the patients in Group A and all of the patients in group B had died of the disease. The two groups were similar in age, histologic type, and treatment. In Group A, three patients had grade 1 tumors, in contrast to group B where all the patients had either grade 2 or 3 disease (P = 0.018). However, the distribution of aneuploidy was similar in both groups. Also, the DNA indices were similar: 1.40 +/- 0.42 in Group A, and 1.36 +/- 0.44 in Group B. The median S-phase fraction was 14% (range 3-23%) in Group A, and 15% (range 2-23%) in Group B. The grade and type of tumor were not related to the ploidy or the DNA index. There was no significant correlation between ploidy or the DNA index and survival. CONCLUSION: This study suggests that the DNA content of tumor as measured by flow cytometry is not a predictor of long-term survival in ovarian cancer patients with advanced disease.

Her2/neu overexpression is associated with treatment failure in women with high-risk stage II and stage IIIA breast cancer (>10 involved lymph nodes) treated with high-dose chemotherapy and autologous hematopoietic progenitor cell support following standard-dose adjuvant chemotherapy.

Twenty-five patients with high-risk stage II and IIIA breast cancer (>10 or more involved lymph nodes) were treated with six cycles of standard-dose chemotherapy (5-fluorouracil, doxorubicin, and cyclophosphamide) followed by high-dose chemotherapy (2.5 g/m2 cyclophosphamide for 3 days and 225 mg/m2 thiotepa for 3 days) with autologous hematopoietic progenitor cell support. The actuarial relapse free survival at 3 years is 80%; the actuarial survival at 3 years is 96%. Four patients relapsed systemically between 6 and 18 months; all four patients who relapsed had breast cancers that overexpressed Her2/neu. In contrast, none of the 21 patients who had no or borderline overexpression of Her2/neu relapsed (P = 0.00004, Fisher's exact test). Patients with high-risk stage II and IIIA breast cancer who have overexpression of Her2/neu appear to be at a high risk for relapse, even when treated with high-dose chemotherapy and autologous hematopoietic progenitor cell support.

Breast cancer metastatic to the uterus. Clinical manifestations of a rare event.

Adenocarcinoma of the breast rarely metastasizes to the mucosal surfaces of the uterus. We present two patients with endometrial involvement, in one of whom it was the initial manifestation of her breast cancer. Two additional patients with cervical involvement had abnormal Papanicolaou smears and grossly normal cervices. One of these patients underwent a biopsy, the results of which confirmed metastatic adenocarcinoma. Three of the four patients had previously well-established metastatic disease. The presence of genital, especially mucosal, metastases is indicative of widespread disease and imminent demise. This occurred in one of the patients described here; however, another patient survived 30 months. Breast cancer is a chronic disease for which the metastatic behavior is exceptionally unconventional. Tissue acquired by endometrial curettage or colposcopy may require an awareness on the part of the pathologist to such a clinical circumstance.

Granulocytic sarcoma of the ileum treated by bone marrow transplantation.

An 8-year-old boy with a granulocytic sarcoma of the proximal ileum metastatic to mesenteric lymph nodes was placed into complete remission with surgical excision of the primary tumor and conventional induction chemotherapy with daunorubicin and cytosine arabinoside. He was then treated with high dose cytosine arabinoside, fractionated total body irradiation, and allogeneic marrow transplantation from his 22-month-old brother who was completely matched at the major histocompatibility complex. Methotrexate was given following the transplant to prevent graft-versus-host disease (GVHD). His post-transplantation course was complicated by a transient autoimmune hemolytic anemia related to an ABO blood group incompatibility and hepatic fungal microabscesses which responded to Amphotericin therapy. Four years following the transplant the patient remains in complete remission. The prognosis for patients with granulocytic sarcoma has been poor although, perhaps, improved over the past decade. This is the first published case report of successful treatment of a granulocytic sarcoma of the ileum by allogeneic marrow transplantation.

Flow and image cytometric DNA analysis in rhabdomyosarcoma.

Rhabdomyosarcoma is the most common malignant soft-tissue tumor in childhood, with an overall 3-year disease-free survival of 73%. DNA content is known to correlate with prognosis and therapy response in many cancers. To determine the role of DNA content in rhabdomyosarcoma, 23 tumor samples were studied retrospectively: 18 primary tumors and 5 post-chemotherapy recurrences or specimens obtained at second-look surgeries. The DNA analysis was performed on disaggregated paraffin-embedded tissue nuclei by flow and image cytometry and correlated with the histology and clinical history. Of the primary tumors 4 were diploid, 4 polyploid, and 10 aneuploid (9 with a single aneuploid G0G1 peak and 1 multiploid) by flow cytometry. The concordance rate between flow and image cytometry was 19 of 23 (83%); one case did not have flow cytometry available. Most embryonal rhabdomyosarcomas were aneuploid (10 of 12; 83%), and they had a high incidence of recurrence in Stages III and IV (4 of 12; 33%). Although aneuploidy in pediatric cancers may predict a therapeutic response and good prognosis, this was not supported by our findings in rhabdomyosarcoma. The tumor DNA content correlated with the clinical stage but not with the patient's clinical course or tumor histopathological type. DNA content did not appear to be as important a prognostic tool as tumor stage.

Myxoglobulosis of the appendix.

Myxoglobulosis is a rare morphologic variant of appendiceal mucocele characterized by intraluminal mucinous globules of the appendix. Most reported cases have presented clinically as an acute abdomen or as an incidental laparotomy or autopsy finding. We report a case of myxoglobulosis in a 32-year-old man who presented with an extra-appendiceal mass following a 10-year symptomatic course. Laparotomy disclosed a pericecal collection of opaque, white globules originating from a perforated appendix walled off by fibrous adhesions. The globules exhibited some histologic and staining properties at variance with those described in previous reports. These findings suggest that myxoglobulosis may be more heterogeneous pathogenetically than the distinctive gross appearance of the lesion would indicate.

Association of dengue hemorrhagic fever with the HLA system.

The frequency of HLA antigens was determined in 82 unrelated patients who had been hospitalized with the most severe form of dengue hemorrhagic fever: shock, dehydration and severe hemorrhages (DHF/DSS). The HLA-A1, HLA-B plank, HLA Cw1 and HLA-A29 antigens showed a significant difference when their values were compared with the normal control group.

Utility of specimen angiography in angiodysplasia of the colon.

This study reviews the utility of specimen angiography in identifying colonic lesions clinically thought to represent angiodysplasia. Four elderly patients presented with acute rectal bleeding. The diagnosis of angiodysplasia was made preoperatively in 3 patients by colonoscopy or angiography, or both. These specimens were injected with silicon rubber compound, xerographed, cleared with methyl salicylate, and examined with transillumination before histologic sampling. Although this technique was not used in the fourth case, fortuitous random sampling of the ascending colon revealed vascular changes indicative of angiodysplasia that correlated with a preoperative bleeding scan. Associated lesions in 1 patient were carcinoma of the colon and primary amyloidosis, the concurrence of which has not been described previously. With the injection technique the lesions of angiodysplasia appear grossly as spiderlike, dilated blood vessels. Microscopically, dilated veins, venules, and capillaries are found in the submucosa only or in the mucosa and submucosa. The importance of the postoperative injection is that it prevents the collapse of the blood vessels and enables the pathologist to identify the lesions grossly. As these lesions are usually small, this is important for proper sampling and histologic documentation.