Elongation of the cytoplasmic domain,due to a point deletion at exon 7, results in an HLA-C null allele,Cw*0409 N

The development of molecular techniques for HLA typing has allowed the identification of genes previously assigned as serologic blank alleles. Lack or poor cell surface expression has been found for molecules coded by HLA-A, -B, -DRB4, -DRB5, and -DPB1 genes. In this report we describe the first HLA-C gene encoding for a null cell surface molecule. HLA-Cw*0409 N shows a point deletion at position 1095 within exon 7. This mutation provokes a codon reading shift, generating a new translation stop codon 97 bp downstream to that described in alleles normally expressed. This new stop codon location implies the presence of 32 extra amino acid residues in the cytoplasmic domain. Transfection experiments suggest that elongation of the cytoplasmic domain in Cw*0409 N would be the cause of cell surface expression failure, although Cw*0409 N heavy chain is able to create stable complexes with beta2-microglobulin. HLA-C fragment length analysis in a small selected group of samples with B44-Cblk haplotypic associations allowed us to identify two additional subjects showing both a serologic silent Cw*04 allele and a point base deletion at the 3' end of the HLA-C gene. This finding indicates that the allele frequency of Cw*0409 N within serologic C blank alleles would be appreciable, although basically restricted to the (A23)-Cw*0409 N-B*4403-DR7-DQ2 haplotype.     

Aging and the human vestibular nuclei: morphometric analysis

The data concerning the effects of age on the brainstem are scarce and few works are devoted to the human vestibular nuclear complex. The study of the effects of aging in the vestibular nuclei could have clinical interest due to the high prevalence of balance control and gait problems in the elderly. We have used in this work eight human brainstems of different ages sectioned and stained by the formaldehyde-thionin technique. The neuron's profiles were drawn with a camera lucida and Abercrombie's method was used to estimate the total number of neurons. The test of Kolmogorov-Smirnov with the correction of Lilliefors was used to evaluate the fit of our data to a normal distribution and a regression analysis was done to determine if the variation of our data with age was statistically significant. Aging does not affect the volume or length of the vestibular nuclear complex. Our results clearly show that neuronal loss occurs with aging in the descending (DVN), medial (MVN), and lateral (LVN) vestibular nuclei, but not in the superior (SVN). There are changes in the proportions of neurons of different sizes but they are not statistically significant. The neuronal loss could be related with the problems that elderly people have to compensate unilateral vestibular lesions and the alterations of the vestibulospinal reflexes. The preservation of SVN neurons can explain why vestibulo-ocular reflexes are compensated after unilateral vestibular injuries.     

Prosthetic mitral valve endocarditis due to Ochrobactrum anthropi: case report

We describe a case of infective endocarditis in a prosthetic mitral valve due to Ochrobactrum anthropi. Although O. anthropi is an emerging pathogen in immunocompromised patients, infections with the bacterium have very rarely been documented in healthy hosts, and endocarditis is rare. To our knowledge, only two cases of O. anthropi endocarditis have been reported in the medical literature.     

Visualizing cytokine-secreting cells in situ in the rhesus macaque model of chronic gut inflammation

Cytokine-producing cells in gut-associated lymphoid tissues of rhesus macaques with chronic enterocolitis were studied. The confocal microscopy technique that we developed enables simultaneous in situ visualization of multiple extra- and/or intracellular antigens at a resolution higher than that allowed by light or epifluorescence microscopy. The presence of interleukin-6 (IL-6)-, tumor necrosis factor alpha (TNF-alpha)-, and IL-1-alpha-producing cells was focally intense in the colon lamina propria of the affected animals. The IL-1-alpha-producing cells were T lymphocytes (CD3+), while the TNF-alpha-producing cells were both macrophages (CD68+/HAM56+/LN5+) and T lymphocytes (CD3+). The IL-6-producing cells within the colon consisted of T lymphocytes and macrophages. The amount of IL-6-producing cells seen in macaques with enterocolitis was significantly higher (P<0.001) than that seen in the healthy control animal, while TNF-alpha- and IL-1-alpha-producing cells were seen only in macaques with enterocolitis. Most of the T lymphocytes that produced cytokines were detected in the lamina propria, while the macrophages were most prominent in highly inflamed regions of the lamina propria. Taken together, our findings indicate that there might be immunological similarity between chronic enterocolitis of rhesus macaques and humans, suggesting the potential use of the nonhuman primate model for the validation of novel therapies.     

Study of CYP2D6 gene in children with autoimmune hepatitis and P450 IID6 autoantibodies.

Cytochrome P450 IID6 is an autoantigen recognized by the sera of children affected with a subtype of autoimmune hepatitis. It was hypothesized that a mutation in the CYP2D6 gene could explain the autoimmune response in these patients. To examine this question, genomic DNA from peripheral lymphocytes (n = 9) and liver (n = 1) of 10 patients with anti-LKM-1 antibody was analysed by Southern blot for genetic association studies between a particular CYP2D6 haplotype and autoimmune hepatitis. In addition, a region of CYP2D6, from the same genomic DNA, was amplified by polymerase chain reaction (PCR) and digested by BstNI, in a search for the most prevalent 29B mutation, described in subjects who do not express the P450 IID6. Total RNA and proteins, prepared from the liver of an anti-LKM-1+ patient, were analysed by Northern and Western (immunoblot) blots respectively. Our results do not reveal any major structural change in the DNA of this patient at the CYP2D6 locus that could explain their autoimmune response. Corroborating this observation, no changes were noted either in P450 IID6 mRNA size or in the corresponding protein. However, these data do not exclude the possibility of subtle changes in the protein due to point mutations in critical regions that might trigger an autoimmune response.     

Genetic polymorphism of N-acetyltransferase-2, glutathione S-transferase-M1, and cytochromes P450IIE1 and P450IID6 in the susceptibility to head and neck cancer.

AIMS: To analyse the allele frequencies of DNA polymorphisms at the genes for cytochromes P450IIE1 and P450IID6, N-acetyltransferase-2, and glutathione S-transferase-M1 in patients with head and neck squamous cell carcinoma, in an attempt to define genetic factors involved in the susceptibility to this cancer, which is strongly associated with tobacco consumption. METHODS: Determination of restriction fragment length polymorphism (RFLP) at cytochromes P450IIE1/P450IID6 and NAT2 genes, and the presence of homozygous deletion of the GSTM1 gene, in 200 controls and 75 head and neck cancer patients. Allelic frequencies between the two groups were compared using a chi 2 test, and odds ratio with 95% confidence intervals were calculated. RESULTS: There was no evidence of an association between alleles of CYP2D6 and CYP2E1 and head and neck cancer in our population. Similarly, frequencies of individuals lacking the GSTM1 gene did not differ between controls and patients. However, individuals with the NAT2-SA phenotype were at higher risk of developing head and neck cancer. The frequencies of the most common SA genotype (homozygous for the NAT2*5 allele) were higher in patients than in controls (27% v 15%, respectively). Slow acetylators homozygous for the NAT2*6 allele, the second most common SA allele, were also more common in patients than in controls (11% v 5%, respectively). CONCLUSIONS: Slow NAT2 activity is a risk factor possibly leading to the development of head and neck cancer in response to tobacco carcinogens.     

Discrimination of Strains of Salmonella enteritidis with Differing Levels of Virulence by an In Vitro Glass Adherence Test

The objective of this study was the in vitro differentiation of isolates of Salmonella enteritidis whose virulences differed in a chick model. A total of 14 strains of S. enteritidis were isolated from either the environment, dairy products, or infected patients. The isolates could be divided into two groups on the basis of their virulence (50% lethal dose) in chickens infected intraperitoneally. When the strains were incubated in adherence test medium (Spanish patent 9700408), only the virulent strains produced aggregates and formed visible filaments attached to the glass tube. These results suggest, although for a limited number of strains, that aggregation in such a medium could be used as a diagnostic tool to discriminate virulent strains of S. enteritidis.     

Antigen-induced inhibition of human in vitro spontaneous IgE synthesis. I. Factors and kinetic studies.

Peripheral blood mononuclear cells from patients with hypersensitivity to L. perenne and D. pteronyssinus were incubated with specific antigen. They were then cultured for 7 days in the absence of antigen and the IgE contained in the supernatants was determined using an enzyme immunoassay (ELISA). Pre-incubation of the cells with antigen produced an inhibitory effect on the spontaneous IgE synthesis in most of the cases (101 of the 134 individual studied). This inhibition was more pronounced and more frequent in those cultures with an elevated spontaneous production of IgE (3,000-10,000 pg/ml). This effect depended on the dose of antigen and the duration of cell exposure. Both spontaneous IgE production kinetics and antigen-mediated inhibition were studied. A study of the IgE content of the cell pellets indicated that the antigen did not induce inhibition of the IgE release. We therefore believe that the inhibition observed must be due to some kind of IgE synthesis suppression.     

NK cells mediate increase of phagocytic activity but not of proinflammatory cytokine (interleukin-6 [IL-6], tumor necrosis factor alpha,and IL-12) production elicited in splenic macrophages by tilorone treatment of mice during acute systemic candidiasis

The participation of NK cells in the activation of splenic macrophages or in resistance to systemic candidiasis is still a matter of debate. We had previously reported that there is a correlation between natural killer cell activation and resistance to systemic candidiasis. In those experiments we had used tilorone to boost NK cell activity in mice. Here we show a mechanism elicited by tilorone in splenic macrophages which could explain their effect on mouse survival during acute disseminated Candida albicans infection. The results demonstrate that tilorone treatment elicits, by a direct effect, the production of proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-alpha], and IL-12) by splenic macrophages. In addition, it increases the capacity of splenic macrophages to phagocytize C. albicans through activation of NK cells. We also demonstrate that the presence of NK cells is essential for maintaining a basal level of phagocytic activity, which characterizes splenic macrophages of na?ve control mice. The results demonstrate that it is possible to identify two phenotypically and functionally peculiar cell populations among splenic macrophages: (i). cells of the "stimulator/secretor phenotype," which show high levels of major histocompatibility complex (MHC) class II surface expression, are poorly phagocytic, and synthesize the proinflammatory cytokines IL-6, TNF-alpha, and IL-12, and (ii). cells of the "phagocytic phenotype," which express low levels of MHC class II molecules, are highly phagocytic, and do not secrete proinflammatory cytokines.