The PTPN22 1858T allele but not variants in the proximal promoter region of IL-21 gene is associated with the susceptibility to type 1 diabetes and the presence of autoantibodies in a Brazilian cohort.

Interleukin (IL)-21 and protein tyrosine phosphatase non-receptor 22 (PTPN22) regulate lymphocyte function and have been implicated in the pathogenesis of autoimmune diabetes. We sequenced the proximal promoter of the IL-21 gene for the first time and analysed the PTPN22 1858T polymorphism in type 1A diabetes (T1AD) patients and healthy controls (HC). We correlated the frequencies of islet and extra-pancreatic autoantibodies with genotypes from both loci. The case series comprised 612 T1AD patients and 792 HC. Genotyping of PTPN22 C1858T was performed on 434 T1AD patients and 689 HC. The -448 to +83 base pairs (bp) region of the IL-21 gene was sequenced in 309 Brazilian T1AD and 189 HC subjects. We also evaluated human leucocyte antigen (HLA) DR3/DR4 alleles. The frequencies of glutamic acid decarboxylase (GAD65), tyrosine phosphatase-like protein (IA)-2, anti-nuclear antibody (ANA), thyroid peroxidase (TPO), thyroglobulin (TG), thyrotrophin receptor autoantibody (TRAb), anti-smooth muscle (ASM) and 21-hydroxylase (21-OH) autoantibodies were higher in T1AD patients than in HC. The PTPN22 1858T allele was associated with an increased risk for developing T1AD [odds ratio (OR) = 1·94; P < 0·001], particularly in patients of European ancestry, and with a higher frequency of GAD65 and TG autoantibodies. HLA-DR3/DR4 alleles predominated in T1AD patients. A heterozygous allelic IL-21 gene variant (g.-241 T > A) was found in only one patient. In conclusion, only PTPN22 C1858T polymorphism and HLA-DR3 and/or DR4 alleles, but not allelic variants in the 5'-proximal region of the IL-21 gene were associated with T1AD risk. Patients with T1AD had increased frequencies of anti-islet-cell, anti-thyroid, anti-nuclear, anti-smooth muscle and anti-21-OH autoantibodies. The C1858T PTPN22 polymorphism was also associated with a higher frequency of GAD65 and TG autoantibodies.

Binding kinetics of calbindin-D(28k) determined by flash photolysis of caged Ca(2+)

We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca(2+) binding to calbindin-D(28K). The experiments used saturated DM-nitrophen at a [Ca(2+)] of 1.5 microM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca(2+)] in the absence of calbindin-D(28K), and in its presence the decay of the flash-induced fluorescence was due solely to the Ca(2+) buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca(2+) buffer EGTA to confirm its validity. We provide evidence that calbindin-D(28K) binds Ca(2+) in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca(2+) with a k(on) comparable to that of EGTA (i.e., approximately 1 x 10(7) M(-1) s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca(2+) binding kinetics of Ca(2+) buffers, this method promises to be highly valuable for studying the Ca(2+) binding properties of other biologically important Ca(2+) binding proteins.

Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus.

Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus and Micrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC(2)(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC(2)(3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 microg/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 microg/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of <1 microg/ml did not change permeability, while a tetracycline concentration of 4 microg/ml permeabilized 50% of the bacteria; 4 microg of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.

Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique.

BACKGROUND: Membrane potential (MP) plays a critical role in bacterial physiology. Existing methods for MP estimation by flow cytometry are neither accurate nor precise, due in part to the heterogeneity of size of the particles analyzed. The ratio of a size- and MP-sensitive measurement, and an MP-independent, size-sensitive measurement, should provide a better estimate of MP. METHODS: Flow cytometry and spectrofluorometry were used to detect red (488 --> 600 nm) fluorescence associated with aggregates of diethyloxacarbocyanine (DiOC2(3)), which, in the monomeric state, is normally green (488 --> 530 nm) fluorescent. RESULTS: In bacteria incubated with 30 microM dye, aggregate formation increases with the magnitude of the interior-negative membrane potential. Green fluorescence from stained bacteria predominantly reflects particle size, and is relatively independent of MP, whereas red fluorescence is highly dependent on both MP and size. The ratio of red to green fluorescence provides a measure of MP that is largely independent of cell size, with a low coefficient of variation (CV). Calibration with valinomycin and potassium demonstrates that the method is accurate over the range from -50 mV through -120 mV; it also accurately tracks reversible reductions in MP produced by incubation at 4 degrees C and washing in glucose-free medium. CONCLUSIONS: The ratiometric technique for MP estimation using DiOC2(3) is substantially more accurate and precise than those previously available, and may be useful in studies of bacterial physiology and in investigations of the effects of antibiotics and other agents on microorganisms.

Absolute linkage of celiac disease and dermatitis herpetiformis to HLA-DQ.

This report shows the absolute genetic linkage of celiac disease (CD) to the HLA-DQ region, and supports the fact that dermatitis herpetiformis (DH) follows the same pattern of HLA-mediated susceptibility in extensive series of Caucasian Spanish patients. Ninety-five percent of CD (201 of 212) and 100% of DH (55) patients could produce DQ alpha 1*0501-DQ beta 1*02 heterodimers. Negative CD patients for this combination were mostly DR4-DQ8 (DQA1*03-DQB1*0302) (9 OF 11), along with a restricted number of complementary chromosomes. Comparison of observed and expected DQA1-DQB1 genotype distributions (Hardy-Weinberg equilibrium) showed that the excess of patients with DQB1*02 in double doses would be the consequence for which this allele should be complemented by DQA1*0501. Homozygosity for DQA1*0501 would restrain susceptibility to CD and DH.

[Alpha-1 antitrypsin deficiency in infancy and childhood]. / Déficit de alfa 1 antitripsina en la infancia.

We present our experience with 5 pediatric patients, 3 males and 2 females, with alpha 1 antitrypsin deficiency. These patients were between the ages of 15 months and 8 years and 4 were of the PI ZZ phenotype and 1 of the PI SZ phenotype. All cases presented with liver disease (neonatal cholestasis, cirrhosis, hepatitis). We comment on the different clinical forms of this entity during childhood, most of which are liver diseases, whereas in the adult it is generally manifest as lung disease.

Aspectos psicologicos da gestante sob o ponto de vista da teoria do Nucleo do Eu / Psychological aspects of the pregnant woman under the point of view of the theory of the Ego Center

Foi apresentada, sucintamente, a teoria do Nucleo do Eu de Rojas-Bermudez. Fez-se analogia de alguns aspectos psicologicos que ocorrem nos tres trimestres da gestacao com essa teoria, nos tempos de determinacao dos papeis psicossomaticos de ingeridor, defecador e urinador, que se marcam respectivamente, ate o terceiro mes de vida, do terceiro ao oitavo e do oitavo aos 2 anos de vida. Sugeriu-se a necessidade de implantacao do metodo psicoprofilatico em maior numero de servicos de assistencia a mulher gravida, adaptando-o aos recursos da area atendida e as possibilidades da gestante