Comparative evaluation of the new version of the INNO-LiPA Mycobacteria and genotype Mycobacterium assays for identification of Mycobacterium species from MB/BacT liquid cultures artificially inoculated with Mycobacterial strains

The performance of two DNA line probe assays, a new version of INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium) and the GenoType Mycobacterium (Hain Diagnostika, Nehren, Germany), were evaluated for identification of mycobacterial species isolated from liquid cultures. Both tests are based on a PCR technique and designed for simultaneous identification of different mycobacterial species by reverse hybridization and line probe technology. The INNO-LiPA Mycobacteria v2 targeting the 16S-23S rRNA gene spacer region was developed for the simultaneous identification of 16 different mycobacterial species. The GenoType Mycobacterium, which targets the 23S rRNA gene, allows simultaneous identification of 13 mycobacterial species. Both tests were evaluated on 110 mycobacterial strains belonging to 22 different mycobacterial species (20 reference strains, 83 clinical strains, and 4 Mycobacterium kansasii strains isolated from tap water) that were previously inoculated into MB/BacT bottles. The sensitivity of both methods, defined as the number of positive results obtained with the Mycobacterium genus probe together with an interpretable result on the number of samples tested was 110 of 110 (100%) for INNO-LiPA and 102 of 110 (92.7%) for GenoType. For samples with interpretable results, INNO-LiPA was able to correctly identify 109 of 110 samples (99.1%), whereas the GenoType correctly identified 100 of 102 samples (98.0%). Both tests were easy to perform, rapid, and reliable when applied to mycobacterial identification directly from MB/BacT bottles.     

A subset of CD8 memory T cells from old mice have high levels of CD28 and produce IFN-gamma

Using carboxyfluorescein diacetate succinimidyl ester (CFSE)-tagged cells to measure proliferation in vivo, we found that only memory CD8(+) cells from mice older than 18 months gave measurable levels of proliferation and that the proportion of memory CD8(+) T cells able to proliferate in a nonirradiated recipient increased with age. CD8 cells that had proliferated in vivo contained higher levels of CD28 when compared to CD8 cells that had not divided. Cells with high levels of CD28 were preferentially able to divide in nonirradiated recipients. Using ex vivo intracellular staining analysis, we determined that most of the CD8(+) T cells that were capable of dividing in vivo produced IFN-gamma after isolation from recipient mice or their original host. These studies thus document the presence in aged mice of a population of CD28(hi) CD8(+) cells whose ability to proliferate in vivo without antigenic stimulation and to produce IFN-gamma may be involved in immune regulation.     

Retroposed new genes out of the X in Drosophila

New genes that originated by various molecular mechanisms are an essential component in understanding the evolution of genetic systems. We investigated the pattern of origin of the genes created by retroposition in Drosophila. We surveyed the whole Drosophila melanogaster genome for such new retrogenes and experimentally analyzed their functionality and evolutionary process. These retrogenes, functional as revealed by the analysis of expression, substitution, and population genetics, show a surprisingly asymmetric pattern in their origin. There is a significant excess of retrogenes that originate from the X chromosome and retropose to autosomes; new genes retroposed from autosomes are scarce. Further, we found that most of these X-derived autosomal retrogenes had evolved a testis expression pattern. These observations may be explained by natural selection favoring those new retrogenes that moved to autosomes and avoided the spermatogenesis X inactivation, and suggest the important role of genome position for the origin of new genes.     

Flutamide-metformin plus an oral contraceptive (OC) for young women with polycystic ovary syndrome: switch from third- to fourth-generation OC reduces body adiposity

BACKGROUND: Low-dose flutamide-metformin has been developed as a background therapy for non-obese adolescents and young women with hyperinsulinaemic hyperandrogenism, a variant of polycystic ovary syndrome (PCOS). We verified whether the lipolytic efficacy of flutamide-metformin in women with PCOS is enhanced by giving an oral contraceptive (OC) co-therapy that contains drospirenone, instead of gestodene, as progestin. METHODS: An open-labelled study was carried out in which non-obese women with PCOS (n = 29; age approximately 20 years), who had been on a combination of flutamide (62.5 mg/day), metformin (850 mg/day) and ethinylestradiol-gestodene for 8-15 months, were randomized for replacement of the gestodene OC by a drospirenone OC. Assessments of endocrine-metabolic state and body composition (by dual-energy X-ray absorptiometry) were performed at randomization and after 6 months. RESULTS: The switch to drospirenone OC was accompanied by a reduction of total and abdominal fat (mean -0.8 and -0.5 kg) and by an increment of lean body mass (+0.6 kg; all P < 0.01), so that body adiposity was strikingly reduced without changing body weight. CONCLUSION: In non-obese women with PCOS, low-dose flutamide-metformin reduces total and abdominal fat excess more effectively if contraceptive co-therapy contains drospirenone, instead of gestodene, as progestin.     

Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine

Alkylating agents are a structurally diverse group of compounds that cause a wide range of biological effects, including cell death, mutation and cancer. DNA damaged by these agents contains widely different amounts of 12 alkylated purines/pyrimidines and two phosphotriester isomers. The biological effects appear to be mediated predominantly by attack at the O(6) position of guanine. DNA extracted from various normal human tissues contains detectable levels of O(6)-alkylguanine, the source of which has not been defined. Given that, following DNA replication, this lesion cannot only generate point mutations but can also initiate mismatch repair-mediated DNA recombination and cell death, it seems worthwhile to consider the possible contribution of these events and cell killing to the aetiology of human cancer. There is increasing evidence that point mutations are not the only mechanism involved in malignant transformation by alkylating agents. Some cancer chemotherapeutic agents exploit the cytotoxic effects of O(6)-alkylguanine and an understanding of the processing of this lesion has allowed strategies to be developed that should increase the effectiveness of such agents.     

Pinealectomy increases ouabain high-affinity binding sites and dissociation constant in rat cerebral cortex.

The effect of the pineal gland on the ouabain high-affinity binding sites (Kd = 3.1 +/- 0.4 nM, Bmax = 246.4 +/- 18.4 fmol/mg protein) in rat cerebral cortex was studied. Pinealectomy increased Bmax (940.7 +/- 42.8 fmol/mg protein) and Kd (7.6 +/- 1.5 nM) while melatonin injection (100 micrograms/kg b.wt.) counteracted these effects, restoring kinetic parameters (Kd = 1.9 +/- 0.05 nM; Bmax = 262.2 +/- 29.6 fmol/mg prot) to control values. Melatonin activity on ouabain binding in vitro did not depend upon a direct effect on the binding sites themselves. However, in competition experiments, melatonin increased binding affinity of ouabain as shown by the decreased IC50 values.     

Recurrence of bronchioloalveolar carcinoma in donor lung after lung transplantation: microsatellite analysis demonstrates a recipient origin

Bronchioloalveolar carcinoma is a distinctive subtype of pulmonary adenocarcinoma, without effective therapy, although there have recently been some attempts to use lung transplantation. However, a high post-transplantation local recurrence rate is described with some controversy regarding the possible involved mechanisms, the main possibilities being the lymphatic spread and aerosolization. Presented herein is a case of a bilateral lung transplantation for a bilateral and pneumonic form of non-mucinous bronchioloalveolar carcinoma in a 43-year-old woman. The histological analysis of mediastinal lymph nodes during surgery did not show neoplastic cells. Thirty-five months after transplantation several nodular opacities in donor lungs were detected. Three pulmonary wedge resections were performed showing a non-mucinous bronchioloalveolar carcinoma with the same histological characteristics as the primary. Again, the mediastinal lymph nodes were tumor free. A complete microsatellites molecular analysis was performed to compare the primary and recurrent carcinoma using capillary electrophoresis, showing that the recurrent tumor was generated in a recipient cellular clone. The absence of lymph node metastasis and the molecular evidence of the recipient origin of the neoplasm supports the contamination of the new lungs at the time of implantation as being the reason for the high incidence of recurrence after lung transplantation in this kind of disease.     

Two CD14 promoter polymorphisms and atopic phenotypes in Czech patients with IgE-mediated allergy

BACKGROUND: Immunoglobulin E (IgE)-mediated allergy belongs to common chronic disorders resulting from an interaction between both genetic and environmental factors. The gene encoding CD14 is a positional candidate gene for allergic diseases as it is localized on chromosome 5q31.1, a region that is linked to asthma and bronchial hyperresponsiveness. Recently, several polymorphisms in the promoter region of this gene have been associated with atopic phenotypes in various populations. METHODS: We investigated relationship among atopic phenotypes and two polymorphisms [C(-159)T and G(-1359)T] in the promoter of the CD14 gene in the Czech population. Polymerase chain reaction with restriction fragment length polymorphism analyses was used to determine the CD14 genotypes in subjects with IgE-mediated allergic diseases (n = 562) and random controls (n = 320). RESULTS: The CD14 allele or genotype distributions were similar in patients and control group. However, the frequency of the C allele of the C(-159)T polymorphism was higher in patients with positive skin prick tests for moulds than in patients without reactivity to this antigen (P < 0.002, Pcorr<0.01). In addition, we found that patients with homozygous genotype (GG) of the G(-1359)T polymorphism had marginally lower percentage of positive skin prick tests compared with the other genotypes (P < 0.029, Pcorr > 0.05). CONCLUSIONS: Our study supports the idea that CD14 gene variants may act as disease modifiers of IgE-mediated allergic diseases.     

Pathogenesis of primary respiratory disease induced by isolates from a new genetic cluster of bovine viral diarrhea virus type I

The pathogenesis of infection induced by cytopathogenic isolates from the newly identified genetic cluster Id of bovine viral diarrhea virus (BVDV) type I was studied in two experimental infections of previously seronegative, immunocompetent calves. Experiment 1 focused on the evaluation of clinical patterns, viremia, and serological responses. All infected calves in this experiment developed respiratory symptoms and seroconverted to BVDV positivity. Contact calves also contracted a respiratory tract infection following exposure to infected animals. Viremia was demonstrated between postinfection days 2 and 17, and the virus was detected in organ specimens of all but one each of the infected and contact calves. In experiment 2, the distribution of BVDV in various tissues of calves euthanized at defined days postinfection was studied. In two of these calves recurrent shedding of BVDV in nasal secretions was shown. BVDV was detected in various tissues of all infected calves throughout the experiment and also following seroconversion and the clearance of BVDV from the circulatory system. Despite the widespread distribution of the virus in various organs, significant tissue damage was found mainly in respiratory tract and lymphoid tissues. These experiments revealed that viruses from cluster Id of BVDV are able to induce primary respiratory disease in previously seronegative, immunocompetent calves. Contact transmission and virus recurrence, contrary to observations from acute experimental infections with noncytopathogenic BVDV, are likely to reflect differences in biological features of these cytopathogenic isolates. Virus shedding and its presence in tissues following peripheral clearance and in the presence of antibodies may have implications in the diagnosis, pathogenesis, and epidemiology of BVDV-induced syndromes in cattle.