Calretinin- and parvalbumin-immunoreactive neurons in the rat main olfactory bulb do not express NADPH-diaphorase activity

The presence of nitric oxide synthase (NOS) in neuronal elements expressing the calcium-binding proteins calretinin (CR) and parvalbumin (PV) was studied in the rat main olfactory bulb. CR and PV were detected by using immunocytochemistry and the nitric oxide (NO) -synthesizing cells were identified by means of the reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) direct histochemical method. The possible coexistence of NADPH-diaphorase and each calcium-binding protein marker was determined by sequential histochemical-immunohistochemical double-labeling of the same sections. Specific neuronal populations were positive for these three markers. A subpopulation of olfactory fibers and olfactory glomeruli were positive for either NADPH-diaphorase or CR. In the most superficial layers, groups of juxtaglomerular cells, superficial short-axon cells and Van Gehuchten cells demonstrated staining for all three markers. In the deep regions, abundant granule cells were NADPH-diaphorase- and CR-positive and a few were PV-immunoreactive. Scarce deep short-axon cells demonstrated either CR-, PV-, or NADPH-diaphorase staining. Among all these labeled elements, no neuron expressing CR or PV colocalized NADPH-diaphorase staining. The present data contribute to a more detailed classification of the chemically- and morphologically-defined neuronal types in the rodent olfactory bulb. The neurochemical differences support the existence of physiologically distinct groups within morphologically homogeneous populations. Each of these groups would be involved in different modulatory mechanisms of the olfactory information. In addition, the absence of CR and PV in neuronal groups displaying NADPH-diaphorase, which moreover are calmodulin-negative, indicate that the regulation of NOS activity in calmodulin-negative neurons of the rat olfactory bulb is not mediated by CR or PV.     

DQB1*0602 confers genetic susceptibility to multiple sclerosis in Afro-Brazilians

We studied the distribution of the HLA-DRB1, -DQA1 and -DQB1 alleles in 44 Afro-Brazilian patients with multiple sclerosis and 88 controls. Although no significant differences were found between the patients and controls for the DRB1 and DQA1 alleles, the HLA-DQB1*0602 allele was positively associated with multiple sclerosis (45.0% vs. 17.0%, Pc=0.024, RR=3.31). The positive extended haplotypes for DQB1*0602 were more frequent in patients than controls, although the differences were not statistically significant in any of them. These results in Afro-Brazilians are in line with other studies which have found DQB1*0602 to be associated with the disease in the absence of the DRB1*1501 allele. We therefore think that the association with the disease in this ethnic group is more allelic than haplotypic.     

Immunocytochemical localization of the sigma(1) receptor in the adult rat central nervous system

In order to characterize the localization of the sigma(1) receptor in the adult rat central nervous system, a polyclonal antibody was raised against a 20 amino acid peptide, corresponding to the fragment 143-162 of the cloned sigma(1) receptor protein. Throughout the rostrocaudal regions of the central nervous system extending from the olfactory bulb to the spinal cord, intense to moderate immunostaining was found to be associated with: (i) ependymocytes bordering the entire ventricular system, and (ii) neuron-like structures located within the parenchyma. Double fluorescence studies confirmed that, throughout the parenchyma, sigma(1) receptor-immunostaining was essentially associated with neuronal structures immunostained for the neuronal marker betaIII-tubulin. In all rats examined, high levels of immunostaining were always associated with neurons located within specific regions including the granular layer of the olfactory bulb, various hypothalamic nuclei, the septum, the central gray, motor nuclei of the hindbrain and the dorsal horn of the spinal cord. In contrast, only faint immunostaining was associated with neurons located in the caudate-putamen and the cerebellum. Electron microscope studies indicated that sigma(1) receptor immunostaining was mostly associated with neuronal perikarya and dendrites, where it was localized to the limiting plasma membrane, the membrane of mitochondria and of some cisternae of the endoplasmic reticulum. At the level of synaptic contacts, intense immunostaining was associated with postsynaptic structures including the postsynaptic thickening and some polymorphous vesicles, whereas the presynaptic axons were devoid of immunostaining.These data indicate that the sigma(1) receptor antibody prepared here, represents a promising tool for further investigating the role of sigma(1) receptors.     

The Leishmania infantum acidic ribosomal protein LiP2a induces a prominent humoral response in vivo and stimulates cell proliferation in vitro and interferon-gamma (IFN-gamma) production by murine splenocytes

The acidic ribosomal proteins of the protozoan parasite Leishmania infantum have been described as prominent antigens during both human and canine visceral leishmaniasis. In this study we present data showing that the intraperitoneal administration in BALB/c mice of the Leishmania LiP2a protein, in the absence of any added adjuvants, elicited a strong humoral response as an indication that the protein is a potent immunogen. Despite the evolutionary conservation of the acidic ribosomal proteins, the antibody response was found to be specific for the Leishmania protein. Another remarkable finding was the observation that the LiP2a protein stimulates the in vitro proliferation of splenocytes from either LiP2a-immunized or naive BALB/c mice. Since similar proliferative indices were observed in T cell-enriched cultures, it is likely that the LiP2a stimulating activity is due mainly to T lymphocyte expansion. Also, the stimulatory effect was demonstrated to be antigen-specific, since the proliferation was abrogated by the presence of anti-LiP2a antibodies. Interestingly, the LiP2a protein stimulated the production of substantial amounts of IFN-gamma in cultured splenocytes from LiP2a-immunized mice. Our data indicate therefore that the immunostimulatory properties shown by this antigen should be taken into account when developing therapeutic and prophylactic vaccines against leishmaniasis.     

Pre-test prediction models of BRCA1 or BRCA2 mutation in breast/ovarian families attending familial cancer clinics

Objective: To test whether statistical models developed to calculate pre-test probability of being a BRCA1/2 carrier can differentiate better between the breast/ovarian families to be referred to the DNA test laboratory.

Study design: A retrospective analysis was performed in 109 Spanish breast/ovarian families previously screened for germline mutations in both the BRCA1 and BRCA2 genes. Four easy to use logistic regression models originally developed in Spanish (HCSC model), Dutch (LUMC model), Finnish (HUCH model), and North American (U Penn model) families and one model based on empirical data of Frank 2002 were tested. A risk counsellor was asked to assign a subjective pre-test probability for each family. Sensitivity, specificity, negative and positive predictive values, and areas under receiver operator characteristics (ROC) curves were calculated in each case. Correlation between predicted probability and mutation prevalence was tested. All statistical tests were two sided.

Results: Overall, the models performed well, improving the performances of a genetic counsellor. The median ROC curve area was 0.80 (range 0.77-0.82). At 100% sensitivity, the median specificity was 30% (range 25-33%). At 92% sensitivity, the median specificity was 42% (range 33.3-54.2%) and the median negative predictive value was 93% (range 89.7-98%). BRCA1 families tended to score higher risk than BRCA2 families in all models tested.

Conclusions: All models increased the discrimination power of an experienced risk counsellor, suggesting that their use is valuable in the context of clinical counselling and genetic testing to optimise selection of patients for screening and allowing for more focused management. Models developed in different ethnic populations performed similarly well in a Spanish series of families, suggesting that models targeted to specific populations may not be necessary in all cases. Carrier probability as predicted by the models is consistent with actual prevalence, although in general models tend to underestimate it. Our study suggests that these models may perform differently in populations with a high prevalence of BRCA2 mutations.

     

Codon optimization of the human papillomavirus 11 (HPV 11) L1 gene leads to increased gene expression and formation of virus-like particles in mammalian epithelial cells

The 505 amino acid L1 protein of the human papillomavirus type 11 (HPV 11) is the major capsid polypeptide that has been shown to self-assemble into virus-like particles (VLPs) in vivo and in vitro. While L1 is essential for viral infection, expression studies in mammalian cells have been hampered by different codon preference between the virus and its host. To optimize L1 gene expression in mammalian cells, we converted wild-type HPV 11 L1 (11 L1wt) codons to those more common in human genes. The modified HPV 11 L1 gene (11 L1h) generated protein levels that were at least 100-fold higher than those of wild-type HPV 11 L1, while no obvious differences were seen in the level of mRNA. HPV 11 L1 protein was detected in mammalian epithelial and fibroblast cells, by immunoblotting and indirect immunofluorescence (IIF) techniques. Unlike the situation in situ, IIF revealed the presence of L1 mainly at perinuclear sites. Virus-like particles assembled intranuclearly only to a low extent, as indicated by transmission electron microscopy. DNA vaccination using the HPV 11 L1h gene yielded a drastic increase in L1-specific antibody production in mice as compared to immunization with the wild-type gene.     

Two new mutations and three novel polymorphisms in the<Emphasis Type="Italic"> RB1</Emphasis> gene in Ecuadorian patients

RB1 is the gene responsible for retinoblastoma, the most common malignant intraocular tumor of infancy and early childhood. There are no reports about this gene in Ecuadorian populations, and only a few studies have been published in Latin America about this subject. There is a spectrum of more than 370 mutations described in the RB1 gene mutation database (http://www.d-lohmann.de/Rb/mutations.html), and alterations have been found in 25 of the 27 exons. During the exon-by-exon analysis of 31 tumor and blood samples from Ecuadorian patients, we found two new mutations and three novel polymorphisms. One of the polymorphisms is located in intron 26 where no alterations of the gene have been described previously. The polymorphisms were found in all of the patients tumor samples, but not in normal population, suggesting there might be a relationship between these polymorphisms and the development of retinoblastoma in the Ecuadorian population.The nucleotide sequence data reported are available in the GenBank database under the accession numbers: AY243567, AY260472, AY260473, AY273783     

Sarcomatoid carcinoma (so-called pseudosarcoma) of the larynx simulating malignant giant cell tumor of soft parts. A case report

A tumor of the larynx composed of a small infiltrating and well-differentiated squamous cell carcinoma and a large polypoid sarcomatous mass showing the histologic features of the malignant giant cell tumor of soft parts (MGCT) is reported. Other types of sarcomatous components have been described with squamous cell carcinoma (SCC) of the larynx and names such as pseudosarcoma, carcinosarcoma, and spindle cell carcinoma have been used for these peculiar tumors. Because most data support that the sarcomatous component is an unusual carcinoma transformation due to unknown factors the authors prefer the term sarcomatoid carcinoma. Sarcomatoid carcinomas having the features of MGCT have been described in multiple epithelial organs. Therefore, an extensive search for a carcinoma component always should be carried out in tumors arising in any organ, including the larynx, and showing the typical histologic features of MGCT.