Deficiency of thrombospondin-1 reduces Th17 differentiation and attenuates experimental autoimmune encephalomyelitis

Transforming growth factor beta (TGF-beta) plays a role both in the induction of Treg and in the differentiation of the IL-17-secreting T cells (Th17) which drive inflammation in experimental autoimmune encephalomyelitis (EAE). We investigated the role that thrombospondin-1 (TSP-1) dependent activation of TGF-beta played in the generation of an encephalitic Th17 response in EAE. Upon immunization with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)), TSP-1 deficient (TSP-1(null)) mice and MOG(35-55) TCR transgenic mice that lack of TSP-1 (2D2.TSP-1(null)) exhibited an attenuated form of EAE, and secreted lower levels of IL-17. Adoptive transfer of in vitro-activated 2D2.TSP-1(null) T cells induced a milder form of EAE, independent of TSP-1 expression in the recipient mice. Furthermore, in vitro studies demonstrated that anti-CD3/anti-CD28 pre-activated CD4+ T cells transiently upregulated latent TGF-beta in a TSP-1 dependent way, and such activation of latent TGF-beta was required for the differentiation of Th17 cells. These results demonstrate that TSP-1 participates in the differentiation of Th17 cells through its ability to activate latent TGF-beta, and enhances the inflammatory response in EAE. (C) 2009 Elsevier Ltd. All rights reserved.

Complement 4 phenotypes and genotypes in Brazilian patients with classical 21-hydroxylase deficiency

The aim of this work was to analyse C4 genotypes, C4 protein levels, phenotypes and genotypes in patients with the classical form of 21-hydroxylase deficiency. Fifty-four patients from 46 families (36 female, 18 male; mean age 10.8 years) with different clinical manifestations (31 salt-wasting; 23 simple-virilizing) were studied. Taq I Southern blotting was used to perform molecular analysis of the C4/CYP21 gene cluster and the genotypes were defined according to gene organization within RCCX modules. Serum C4 isotypes were assayed by enzyme-linked immunosorbent assay. The results revealed 12 different haplotypes of the C4/CYP21 gene cluster. Total functional activity of the classical pathway (CH50) was reduced in individuals carrying different genotypes because of low C4 concentrations (43% of all patients) to complete or partial C4 allotype deficiency. Thirteen of 54 patients presented recurrent infections affecting the respiratory and/or the urinary tracts, none of them with severe infections. Low C4A or C4B correlated well with RCCX monomodular gene organization, but no association between C4 haplotypes and recurrent infections or autoimmunity was observed. Considering this redundant gene cluster, C4 seems to be a well-protected gene segment along the evolutionary process.

ADCK3, an ancestral kinase, is mutated in a form of recessive ataxia associated with coenzyme Q(10) deficiency

Muscle coenzyme Q(10) (CoQ(10) or ubiquinone) deficiency has been identified in more than 20 patients with presumed autosomal-recessive ataxia. However, mutations in genes required for CoQ(10) biosynthetic pathway have been identified only in patients with infantile-onset multisystemic diseases or isolated nephropathy. Our SNP-based genome-wide scan in a large consanguineous family revealed a locus for autosomal-recessive ataxia at chromosome 1q41. The causative mutation is a homozygous splice-site mutation in the aarF-domain-containing kinase 3 gene (ADCK3). Five additional mutations in ADCK3 were found in three patients with sporadic ataxia, including one known to have CoQ(10) deficiency in muscle. All of the patients have childhood-onset cerebellar ataxia with slow progression, and three of six have mildly elevated lactate levels. ADCK3 is a mitochondrial protein homologous to the yeast COQ8 and the bacterial UbiB proteins, which are required for CoQ biosynthesis. Three out of four patients tested showed a low endogenous pool of CoQ(10) in their fibroblasts or lymphoblasts, and two out of three patients showed impaired ubiquinone synthesis, strongly suggesting that ADCK3 is also involved in CoQ(10) biosynthesis. The deleterious nature of the three identified missense changes was confirmed by the introduction of them at the corresponding positions of the yeast COQ8 gene. Finally, a phylogenetic analysis shows that ADCK3 belongs to the family of atypical kinases, which includes phosphomositide and choline kinases, suggesting that ADCK3 plays an indirect regulatory role in ubiquinone biosynthesis possibly as part of a feedback loop that regulates ATP production.

CCR2 Deficiency Results in Increased Osteolysis in Experimental Periapical Lesions in Mice

Introduction: Periapical lesions are chronic inflammatory disorders of periradicular tissues caused by etiologic agents of endodontic origin. The inflammatory chemokines are thought to be involved in the latter observed osteolysis. With a murine model of experimental periapical lesion, the objective of this study was to evaluate the role of the chemokine receptor CCR2 in the lesion progression, osteoclast differentiation and activation, and expression of inflammatory osteolysis-related mediators. Methods: For lesion induction, right mandibular first molars were opened surgically with a (1)/(4) carbine bur, and 4 bacterial strains were inoculated in the exposed dental pulp; left mandibular first molars were used as controls. Animals were killed at 3, 7, 14, and 21 days after surgeries to evaluate the kinetics of lesion development. Results: CCR2 KO mice showed wider lesions than WT mice. CCR2 KO mice also expressed higher levels of the osteoclastogenic and osteolytic factors, receptor activator of nuclear factor kappa B ligand (RANKL) and cathepsin K, of the proinflammatory cytokine tumor necrosis factor alpha, and of the neutrophil migration related chemokine, KC. Conclusions: These results suggest that CCR2 is important in host protection to periapical osteolysis. (J Endod 2010;36:244-250)

Deficiency of the human complement regulatory protein factor H associated with low levels of component C9

We identified a 4-year-old Brazilian boy from a family of Japanese descent and history of consanguinity, who suffered from severe recurrent pneumonia. He carries factor H (FH) deficiency associated with reduced levels of component C9 and low serum levels of C3 and factor B. His mother also presented low levels of these proteins and factor I, while his father and sister had only lower levels of FH. Western blot assays confirmed the complete absence of FH and FHL-1 polypeptides in this patient. Sequencing of the proband`s FH cDNA revealed a homozygous G453A substitution, encoding an Arg(127)His change. His mother, father and sister are heterozygous for this substitution. Despite the absence of FH in the plasma, this protein was detected in the patient`s fibroblasts, suggesting that Arg(127) may be important for FH secretion. Low concentrations of C9 were detected in the proband serum but no mutations in the patient`s C9 gene or promoter have been identified, suggesting that this is a consequence of uncontrolled complement activation and high C9 consumption.

Helicobacter pylori and Iron-deficiency Anemia in Adolescents in Brazil

Aim: The aim of the study was to evaluate the association between Helicobacter pylori infection and iron deficiency (ID) in adolescents attending a public school. Patients and Methods: From March to June 2001, a cross-sectional study was conducted among adolescents (10-16 years) enrolled in a single public school in Sao Paulo, Brazil. Of 400 eligible students, 195 agreed to participate, but 1 was excluded due to sickle cell disease. A blood sample was collected from each subject to measure hemoglobin and ferritin. H pylori status was investigated with the 13 C-urea breath test. All of the subjects with either anemia or ID were given iron therapy. Results: H pylori prevalence was 40.7% (79/194), being higher in male subjects (45/90 vs 34/104, P = 0.014). There was no relation between infection and nutritional status. Abnormally low serum ferritin was observed in 12 subjects, half of whom were positive for H pylori (odds ratio [OR] 1.49, 95% confidence interval [CI] 0.38-5.81). The median serum ferritin was 33.6 ng/mL (interquartile range 23.9-50.9) in infected subjects and 35.1 ng/mL (interquartile range 23.7-53.9) in uninfected subjects. Anemia was detected in 2% (4/194) of the students, half of whom were infected (OR 1.47, 95% CI 0.1-20.6). The mean hemoglobin value in infected subjects was 13.83 g/dL +/- 1.02 versus 14 g/dL +/- 1.06 in uninfected subjects. Conclusions: The study was not able to find a relation between H pylori infection and ID or anemia.

Skipping of exon 30 in C5 gene results in complete human C5 deficiency and demonstrates the importance of C5d and CUB domains for stability

The deficiency of complement C5 is rare and frequently associated with severe and recurrent infections, especially caused by Neisseria spp. We observed the absence of component C5 in the serum of 3 siblings from a Brazilian family with history of consanguinity. The patients had suffered from recurrent episodes of meningitis and other less severe infections. Sera from these patients were unable to mediate hemolytic activity either by the classical or alternative pathways and presented extremely low levels of C5 protein (13, 0.9 and 1.0 mu g/ml-normal range: 45-190 mu g/ml). Hemolytic activity could be restored by the addition of purified C5 to deficient serum. Sequencing of sibling C5 cDNA revealed a homozygous 153 bp deletion that corresponds precisely to exon 30. The parents carried the same deletion but only in one allele. Sequencing of the corresponding region in the genomic DNA revealed a C to C substitution within intron 30 and, most significantly, the substitution of GAG(4028) for GAA(4028) at the 3` end of exon 30 which is most likely responsible for skipping of exon 30. The resulting in-frame deletion in the C5 mRNA codes for a mutant C5 protein lacking residues 1289-1339. These residues map to the CUB and C5d domains of the C5 alpha chain. This deletion is expected to produce a non-functional and unstable C5 protein which is more susceptible to degradation. (C) 2009 Published by Elsevier Ltd.