Host genetic background affects regulatory T-cell activity that influences the magnitude of cellular immune response against Mycobacterium tuberculosis

Using two mouse strains with different abilities to generate interferon (IFN)-gamma production after Mycobacterium tuberculosis infection, we tested the hypothesis that the frequency and activity of regulatory T (Treg) cells are influenced by genetic background. Our results demonstrated that the suppressive activity of spleen Treg cells from infected or uninfected BALB/c mice was enhanced, inhibiting IFN-gamma and interleukin (IL)-2 production. Infected C57BL/6 mice exhibited a decrease in the frequency of lung Treg cells and an increased ratio CD4(+):CD4(+)Foxp3(+) cells compared with infected BALB/c mice and uninfected C57BL/6 mice. Moreover, infected C57BL/6 mice also had a decrease in the immunosuppressive capacity of spleen Treg cells, higher lung IFN-gamma and IL-17 production, and restricted the infection better than BALB/c mice. Adoptive transfer of BALB/c Treg cells into BALB/c mice induced an increase in bacterial colony-forming unit (CFU) counts. Furthermore, BALB/c mice treated with anti-CD25 antibody exhibited lung CFU counts significantly lower than mice treated with irrelevant antibody. Our results show that in BALB/c mice, the Treg cells have a stronger influence than that in C57BL/6 mice. These data suggest that BALB/c and C57BL/6 mice may use some different mechanisms to control M. tuberculosis infection. Therefore, the role of Treg cells should be explored during the development of immune modulators, both from the perspective of the pathogen and the host. Immunology and Cell Biology (2011) 89, 526-534; doi:10.1038/icb.2010.116; published online 19 October 2010

Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function

The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine-phosphorylated ITAM subunits of immunoreceptors, such as Fc epsilon RI on mast cells, results in a conformational change, with an increase of enzymatic activity of Syk. This conformational change exposes the COOH-terminal tail of Syk, which has three conserved Tyr residues (Tyr-623, Tyr-624, and Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased Fc epsilon RI-induced degranulation, nuclear factor for T cell activation and NF kappa B activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity toward an exogenous substrate, whereas its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog ZAP-70, which depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Tyr-624 but especially Tyr-625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk.

A transposon toolkit for gene transfer and mutagenesis in protozoan parasites

Protozoan parasites affect millions of people around the world. Treatment and control of these diseases are complicated partly due to the intricate biology of these organisms. The interactions of species of Plasmodium, Leishmania and trypanosomes with their hosts are mediated by an unusual control of gene expression that is not fully understood. The availability of the genome sequence of these protozoa sets the stage for using more comprehensive, genome-wide strategies to study gene function. Transposons are effective tools for the systematic introduction of genetic alterations and different transposition systems have been adapted to study gene function in these human pathogens. A mariner transposon toolkit for use in vivo or in vitro in Leishmania parasites has been developed and can be used in a variety of applications. These modified mariner elements not only permit the inactivation of genes, but also mediate the rescue of translational gene fusions, bringing a major contribution to the investigation of Leishmania gene function. The piggyBac and Tn5 transposons have also been shown to mobilize across Plasmodium spp. genomes circumventing the current limitations in the genetic manipulation of these organisms.

Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma

We used high-resolution SNP genotyping to identify regions of genomic gain and loss in the genomes of 212 medulloblastomas, malignant pediatric brain tumors. We found focal amplifications of 15 known oncogenes and focal deletions of 20 known tumor suppressor genes (TSG), most not previously implicated in medulloblastoma. Notably, we identified previously unknown amplifications and homozygous deletions, including recurrent, mutually exclusive, highly focal genetic events in genes targeting histone lysine methylation, particularly that of histone 3, lysine 9 (H3K9). Post-translational modification of histone proteins is critical for regulation of gene expression, can participate in determination of stem cell fates and has been implicated in carcinogenesis. Consistent with our genetic data, restoration of expression of genes controlling H3K9 methylation greatly diminishes proliferation of medulloblastoma in vitro. Copy number aberrations of genes with critical roles in writing, reading, removing and blocking the state of histone lysine methylation, particularly at H3K9, suggest that defective control of the histone code contributes to the pathogenesis of medulloblastoma.

HLA-DRB1*allele-associated genetic susceptibility and protection against multiple sclerosis in Brazilian patients

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system that causes neurological disorders in young adults. Previous studies in various populations highlighted an association between the HLA-DRB1*1.5 allele and MS. This study investigated the association between HLA-DRB1*15 and other HLA-DRB1 alleles and MS in a Brazilian Caucasian population sample from Londrina, Southern Brazil. HLA-DRB1 alleles were analyzed by polymerase chain reaction with specific sequence oligonucleotide primers in 119 MS patients and in 305 healthy blood donors as a control. Among the MS patients, 89 (75.0%) presented with relapsing remitting MS, 24 (20.0%) with secondary progressive MS and 6 (5.0%) with primary progressive MS. The frequency of the HLA-DRB1*15 allele observed in the MS Brazilian patients was similar to findings reported in previous studies carried out in populations worldwide. However, the results showed a higher frequency of the HLA-DRB1*15 allele in the MS patients compared to the controls, with a relative frequency of 0.1050 (10.50%) and 0.0443 (4.4%), respectively (OR=2.53; 95% CI 1.43-4.46; p=0.0009). A protector allele was also detected. The frequency of the HLA-DRB1*11 allele was reduced in the MS patients compared to the controls, with a relative frequency of 0.1345 (13.4%) and 0.1869 (18.7%), respectively (OR=0.67; 95% CI 0.44-1.03; p=0.0692). The results demonstrated that the HLA-DRB1*15 allele in heterozygosity is positively associated with MS (p=0.0079), and may be considered a genetic marker of susceptibility to the disease. A negative association between the HLA-DRB1*11 allele in homozygosity and MS was also verified (p=0.0418); this allele may be considered a genetic marker of resistance to MS in the Brazilian population.

Changes in Myocardial Perfusion Correlate With Deterioration of Left Ventricular Systolic Function in Chronic Chagas` Cardiomyopathy

OBJECTIVES This study aimed at analyzing the association between myocardial perfusion changes and the progression of left ventricular systolic dysfunction in patients with chronic Chagas` cardiomyopathy (CCC). BACKGROUND Pathological and experimental studies have suggested that coronary microvascular derangement, and consequent myocardial perfusion disturbance, may cause myocardial damage in CCC. METHODS Patients with CCC (n = 36, ages 57 +/- 10 years, 17 males), previously having undergone myocardial perfusion single-positron emission computed tomography and 2-dimensional echocardiography, prospectively underwent a new evaluation after an interval of 5.6 +/- 1.5 years. Stress and rest myocardial perfusion defects were quantified using polar maps and normal database comparison. RESULTS Between the first and final evaluations, a significant reduction of left ventricular ejection fraction was observed (55 +/- 11% and 50 +/- 13%, respectively; p = 0.0001), as well as an increase in the area of the perfusion defect at rest (18.8 +/- 14.1% and 26.5 +/- 19.1%, respectively; p = 0.0075). The individual increase in the perfusion defect area at rest was significantly correlated with the reduction in left ventricular ejection fraction (R = 0.4211, p = 0.0105). Twenty patients with normal coronary arteries (56%) showed reversible perfusion defects involving 10.2 +/- 9.7% of the left ventricle. A significant topographic correlation was found between reversible defects and the appearance of new rest perfusion defects at the final evaluation. Of the 47 segments presenting reversible perfusion defects in the initial study, 32 (68%) progressed to perfusion defects at rest, and of the 469 segments not showing reversibility in the initial study, only 41 (8.7%) had the same progression (p < 0.0001, Fisher exact test). CONCLUSIONS In CCC patients, the progression of left ventricular systolic dysfunction was associated with both the presence of reversible perfusion defects and the increase in perfusion defects at rest. These results support the notion that myocardial perfusion disturbances participate in the pathogenesis of myocardial injury in CCC. (J Am Coll Cardiol Img 2009;2:164-72) (c) 2009 by the American College of Cardiology Foundation

Val/Leu(247) Polymorphism of beta 2-glycoprotein I in Brazilian Patients with Antiphospholipid Syndrome-A Genetic Risk Factor?

A genetic polymorphism of the beta 2-glycoprotein I (beta 2-GPI) is recognized by antiphospholipid antibodies (aPL) and may even play a role in the development of antiphospholipid syndrome (APS). The objectives of this study were to determine a Val/Leu SNP at position 247 of the beta 2-GPI gene in Brazilian patients with APS and to compare these data with clinical and laboratory manifestations. Polymorphism assignment was performed by PCR followed by Rsa I restriction endonuclease. The titration of anti-beta 2-GPI antibodies was detected by ELISA. The results showed significantly higher frequencies of the V-encoding allele and the homozygous VV genotype in patients with APS than in control subjects (OR = 1.781, P = 0.0068; and OR = 6.413, P < 0.0001, respectively). The frequency of this genotype was also significantly higher in patients with arterial and venous thrombosis than in the control group (52% and 44%, respectively, versus 13%). Anti-beta 2-GPI-positive patients had significantly higher frequencies of the VV genotype than the controls subjects (OR = 8.179, P < 0.0001). These results suggest that the V-encoding allele and the homozygous VV genotype at position 247 of the beta 2-GPI gene may play a role in the generation of anomalous beta 2-GPI, with consequent auto-antibody production, and in phenotype expression of arterial and venous thrombosis in APS patients.

Genetic Susceptibility Loci in Rheumatoid Arthritis Establish Transcriptional Regulatory Networks with Other Genes

Linkage studies have identified the human leukocyte antigen (HLA)-DRB1 as a putative rheumatoid arthritis (RA) susceptibility locus (SL). Nevertheless, it was estimated that its contribution was partial, suggesting that other non-HLA genes may play a role in RA susceptibility. To test this hypothesis, we conducted microarray transcription profiling of peripheral blood mononuclear cells in 15 RA patients and analyzed the data, using bioinformatics programs (significance analysis of microarrays method and GeneNetwork), which allowed us to determine the differentially expressed genes and to reconstruct transcriptional networks. The patients were grouped according to disease features or treatment with tumor necrosis factor blocker. Transcriptional networks that were reconstructed allowed us to identify the interactions occurring between RA SL and other genes, for example, HLA-DRB1 interacting with FNDC3A (fibronectin type III domain containing 3A). Given that fibronectin fragments can stimulate mediators of matrix and cartilage destruction in RA, this interaction is of special interest and may contribute to a clearer understanding of the functional role of HLA-DRB1 in RA pathogenesis.

Loss-of-function mutations in the genes encoding prokineticin-2 or prokineticin receptor-2 cause autosomal recessive Kallmann syndrome

Context: Physiological activation of the prokineticin pathway has a critical role in olfactory bulb morphogenesis and GnRH secretion in mice. Objective: To investigate PROK2 and PROKR2 mutations in patients with hypogonadotropic hypogonadism (HH) associated or not with olfactory abnormalities. Design: We studied 107 Brazilian patients with HH (63 with Kallmann syndrome and 44 with normosmic HH) and 100 control individuals. The coding regions of PROK2 and PROKR2 were amplified by PCR followed by direct automatic sequencing. Results: In PROK2, two known frameshift mutations were identified. Two brothers with Kallmann syndrome harbored the homozygous p. G100fsX121 mutation, whereas one male with normosmic HH harbored the heterozygous p. I55fsX56 mutation. In PROKR2, four distinct mutations (p. R80C, p. Y140X, p. L173R, and p. R268C) were identified in five patients with Kallmann syndrome and in one patient with normosmic HH. These mutations were not found in the control group. The p. R80C, p. L173R, and p. R268C missense mutations were identified in the heterozygous state in the HH patients and in their asymptomatic first-degree relatives. In addition, nomutations of FGFR1, KAL1, GnRHR, KiSS-1, or GPR54 were identified in these patients. Notably, the new nonsense mutation (p. Y140X) was identified in the homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism, and high-arched palate. His asymptomatic parents were heterozygous for this severe defect. Conclusion: We expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH. In addition, we show that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROKR2 or PROK2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models.

Effects of acute magnesium loading on pulmonary function of stable COPD patients

Background: Magnesium (Mg) use has the potential to promote bronchodilatation and to improve lung function in obstructive diseases. IV administration of Mg during exacerbations of chronic obstructive pulmonary disease (COPD) has led to improved peak flow. This study aimed to investigate the effects of acute IV Mg loading on respiratory parameters of stable COPD patients. Material/Methods: This was a randomized, double-blind, placebo-controlled crossover study. Twenty-two male COPD patients (64 +/- 6 years old, FEV1: 49 +/- 20%) received an IV infusion of 2 g of magnesium sulfate or placebo on two distinct occasions. Spirometry and mouth maximal respiratory pressures were obtained before and 45 minutes after the infusions. Results: Mg use led to significant changes in functional respiratory capacity (-0.48 1,95% CI: -0.96, -0.01), inspiratory capacity (0.21 1,95% CI: 0.04, 0.37). The treatment was also associated with a marginally significant decrease in residual volume (-0.47 1,95% CI: -0.96, 0.02, p=0.06). Conclusions: Acute IV Mg loading in stable COPD patients was associated with a reduction in lung hyperinflation and improvement of respiratory muscle strength. The clinical potential for chronic magnesium supplementation in COPD deserves further investigation.

The expression of Delta NTP73, TATP73 and TP53 genes in acute myeloid leukaemia is associated with recurrent cytogenetic abnormalities and in vitro susceptibility to cytarabine cytotoxicity

TP73 encodes for two proteins: full-length TAp73 and Delta Np73, which have little transcriptional activity and exert dominant-negative function towards TP53 and TAp73. We compared TATP73 and Delta NTP73 expression in acute myeloid leukaemia (AML) samples and normal CD34(+) progenitors. Both forms were more highly expressed in leukaemic cells. Amongst AML blasts, TATP73 was more expressed in AML harbouring the recurrent genetic abnormalities (RGA): PML-RARA, RUNX1-RUNX1T1 and CBFB-MYH11, whereas higher Delta NTP73 expression was detected in non-RGA cases. TP53 expression did not vary according to Delta NTP73/TATP73 expression ratio. Leukaemic cells with higher Delta NTP73/TATP73 ratios were significantly more resistant to cytarabine-induced apoptosis.

Liver mitochondrial function in familial amyloidotic polyneuropathy

The objective of the present study was to analyze hepatic mitochondrial function in patients with familial amyloidotic polyneuropathy (FAP) undergoing cadaveric donor orthotopic liver transplantation. From February `2005 to May 2007, eight patients with FAP, ranging in age from 34 to 41 years and with Model for End-Stage Liver Disease scores ranging from 24 to 29. Underwent orthotopic transplantation using a liver from a deceased donor by the piggyback method. Immediately before beginning the recipient hepatectomy in a patient with FAP, a biopsy was obtained for analysis of mitochondrial function (FAP group). The control group consisted of 15 patients undergoing hepatic surgery to treat small tumors of the liver. Mitochondrial respiration was determined on the basis of oxygen consumption by energized mitochondria using a polarographic method. The membrane potential of the mitochondria was determined spectrofluorometrically. Data were analyzed statistically by the Mann-Whitney test, with the level of significance set at 5%. State 3 and 4 values, respiratory control ratio, and membrane potential were 47 +/- 8 versus 28 +/- 10 natoms O/min/mg protein (P <.05); 14 +/- 3 vs 17 +/- 7 nat.O/min/ mg.prot.mit. (P >.05); 3.6+/- .5 vs 1.7 +/- 0.7 (P <.05); and 135 +/- 5.2 vs 135 +/- 6 mV (P >.05) for control versus FAP patients, respectively, demonstrating a decreased energy status of the liver in FAP.

Natural Host Relationships and Genetic Diversity of Rodent-Associated Hantaviruses in Southeastern Brazil

Objective: Hantaviruses are rodent-borne RNA viruses that have caused hantavirus cardiopulmonary syndrome in several Brazilian regions. In the present study, geographical distribution, seroprevalence, natural host range, and phylogenetic relations of rodent-associated hantaviruses collected from seven counties of Southeastern Brazil were evaluated. Methods: ELISA, RT-PCR and phylogenetic analysis were used in this study. Results: Antibodies to hantavirus were detected in Bolomys lasiurus, Akodon sp. and Oligoryzomys sp., performing an overall seroprevalence of 5.17%. All seropositive rodents were associated with grasslands or woods surrounded by sugar cane fields. Phylogenetic analysis of partial S- and M-segment sequences showed that viral sequences isolated from B. lasiurus specimens clustered with Araraquara virus. However, a sequence from Akodon sp. shared 100% similarity with Argentinian/Chilean viruses based on the partial S- segment amino acid sequence. Conclusion: These results indicate that there are associations between rodent reservoirs and hantaviruses in some regions of Southeastern Brazil, and suggest the existence of additional hantavirus genetic diversity and host ecology in these areas. Copyright (C) 2008 S. Karger AG, Basel