Skewed Distribution of Circulating Activated Natural Killer T (NKT) Cells in Patients with Common Variable Immunodeficiency Disorders (CVID)

Common variable immunodeficiency disorder (CVID) is the commonest cause of primary antibody failure in adults and children, and characterized clinically by recurrent bacterial infections and autoimmune manifestations. Several innate immune defects have been described in CVID, but no study has yet investigated the frequency, phenotype or function of the key regulatory cell population, natural killer T (NKT) cells. We measured the frequencies and subsets of NKT cells in patients with CVID and compared these to healthy controls. Our results show a skewing of NKT cell subsets, with CD4+ NKT cells at higher frequencies, and CD8+ NKT cells at lower frequencies. However, these cells were highly activated and expression CD161. The NKT cells had a higher expression of CCR5 and concomitantly expression of CCR5+CD69+CXCR6 suggesting a compensation of the remaining population of NKT cells for rapid effector action.

CYP2C19 and ABCB1 gene polymorphisms are differently distributed according to ethnicity in the Brazilian general population

Background: Recent studies have reported the clinical importance of CYP2C19 and ABCB1 polymorphisms in an individualized approach to clopidogrel treatment. The aims of this study were to evaluate the frequencies of CYP2C19 and ABCB1 polymorphisms and to identify the clopidogrel-predicted metabolic phenotypes according to ethnic groups in a sample of individuals representative of a highly admixtured population. Methods: One hundred and eighty-three Amerindians and 1,029 subjects of the general population of 4 regions of the country were included. Genotypes for the ABCB1c.C3435T (rs1045642), CYP2C19*2 (rs4244285), CYP2C19*3 (rs4986893), CYP2C19*4 (rs28399504), CYP2C19*5 (rs56337013), and CYP2C19*17 (rs12248560) polymorphisms were detected by polymerase chain reaction followed by high resolution melting analysis. The CYP2C19*3, CYP2C19*4 and CYP2C19*5 variants were genotyped in a subsample of subjects (300 samples randomly selected). Results: The CYP2C19*3 and CYP2C19*5 variant alleles were not detected and the CYP2C19*4 variant allele presented a frequency of 0.3%. The allelic frequencies for the ABCB1c.C3435T, CYP2C19*2 and CYP2C19*17 polymorphisms were differently distributed according to ethnicity: Amerindian (51.4%, 10.4%, 15.8%); Caucasian descent (43.2%, 16.9%, 18.0%); Mulatto (35.9%, 16.5%, 21.3%); and African descent (32.8%, 20.2%, 26.3%) individuals, respectively. As a result, self-referred ethnicity was able to predict significantly different clopidogrel-predicted metabolic phenotypes prevalence even for a highly admixtured population. Conclusion: Our findings indicate the existence of inter-ethnic differences in the ABCB1 and CYP2C19 variant allele frequencies in the Brazilian general population plus Amerindians. This information could help in stratifying individuals from this population regarding clopidogrel-predicted metabolic phenotypes and design more cost-effective programs towards individualization of clopidogrel therapy.

Frequency of LCT-13910C > T single nucleotide polymorphism associated with adult-type hypolactasia/lactase persistence among Brazilians of different ethnic groups

Background: Adult-type hypolactasia, the physiological decline of lactase some time after weaning, was previously associated with the LCT -13910C>T polymorphism worldwide except in Africa. Lactase non-persistence is the most common phenotype in humans, except in northwestern Europe with its long history of pastoralism and milking. We had previously shown association of LCT -13910C>T polymorphism with adult-type hypolactasia in Brazilians; thus, we assessed its frequency among different Brazilian ethnic groups. Methods: We investigated the ethnicity-related frequency of this polymorphism in 567 Brazilians [mean age, 42.1 +/- 16.8 years; 157 (27.7%) men]; 399 (70.4%) White, 50 (8.8%) Black, 65 (11.5%) Brown, and 53 (9.3%) Japanese-Brazilian. DNA was extracted from leukocytes; LCT -13910C>T polymorphism was analyzed by PCR-restriction fragment length polymorphism. Results: Prevalence of the CC genotype associated with hypolactasia was similar (57%) among White and Brown groups; however, prevalence was higher among Blacks (80%) and those of Japanese descent (100%). Only 2 (4%) Blacks had TT genotype, and 8 (16%) had the CT genotype. Assuming an association between CC genotype and hypolactasia, and CT and TT genotypes with lactase persistence, 356 (62.8%) individuals had hypolactasia and 211 (37.2%) had lactase persistence. The White and Brown groups had the same hypolactasia prevalence (similar to 57%); nevertheless, was 80% among Black individuals and 100% among Japanese-Brazilians (P < 0.01). Conclusion: The lactase persistence allele, LCT -13910T, was found in about 43% of both White and Brown and 20% of the Black Brazilians, but was absent among all Japanese Brazilians studied.

Evaluation of muscle strength and motor abilities in children with type II and III spinal muscle atrophy treated with valproic acid

Background: Spinal muscular atrophy (SMA) is an autosomal recessive disorder that affects the motoneurons of the spinal anterior horn, resulting in hypotonia and muscle weakness. The disease is caused by deletion or mutation in the telomeric copy of SMN gene (SMN1) and clinical severity is in part determined by the copy number of the centromeric copy of the SMN gene (SMN2). The SMN2 mRNA lacks exon 7, resulting in a production of lower amounts of the full-length SMN protein. Knowledge of the molecular mechanism of diseases has led to the discovery of drugs capable of increasing SMN protein level through activation of SMN2 gene. One of these drugs is the valproic acid (VPA), a histone deacetylase inhibitor. Methods: Twenty-two patients with type II and III SMA, aged between 2 and 18 years, were treated with VPA and were evaluated five times during a one-year period using the Manual Muscle Test (Medical Research Council scale-MRC), the Hammersmith Functional Motor Scale (HFMS), and the Barthel Index. Results: After 12 months of therapy, the patients did not gain muscle strength. The group of children with SMA type II presented a significant gain in HFMS scores during the treatment. This improvement was not observed in the group of type III patients. The analysis of the HFMS scores during the treatment period in the groups of patients younger and older than 6 years of age did not show any significant result. There was an improvement of the daily activities at the end of the VPA treatment period. Conclusion: Treatment of SMA patients with VPA may be a potential alternative to alleviate the progression of the disease.

Expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in medullary thyroid carcinoma: Prognostic implications

Background: Metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) participate in the degeneration of the extracellular matrix and are associated with carcinogenesis. MMP-2 is one of the main metalloproteinases active in neoplasia and is a marker of the malignant phenotype. Since the biological behavior of medullary thyroid carcinoma (MTC) varies widely, the present study was undertaken to determine if there is a correlation between the clinical evolution of MTC and the immunohistochemically detected expression of these enzymes in thyroid surgical specimens containing MTC. If so, their expression would be a novel indicator of the prognosis of MTC. Methods: Thirty-seven patients with MTC who had undergone thyroid surgery were followed for an average of 73 months. Immunohistochemical staining for metalloproteinase-related enzymes was performed in surgical paraffin blocks. The clinical status of the patients after surgery and at the end of the study period was characterized to determine correlations between these and the immunohistochemical markers. A value of p < 0.05 was considered statistically significant. Results: At the end of the study period, 15 patients (40.5%) were alive and without evidence of MTC, 17 (45.9%) had persistent MTC, and 5 (13.5%) had a relapse of their neoplasia. Four patients (10.8%) died during the course of the study. There was a significant correlation (p = 0.0005) between the immunohistochemical staining for MMP-2 and the clinical condition of the patients at the end of the study period, and a correlation between the state of apparent cure compared to persistence of MTC after thyroid surgery (p = 0.0207). No significant correlations were observed between either TIMP-2 expression or immune marking of metastatic lymph nodes and the clinical variables studied. Conclusion: Immunohistochemical expression of MMP-2 in thyroid surgical specimens from patients with MTC is a novel indicator of the prognosis of this cancer.

Interleukin-10 production by tumor infiltrating macrophages plays a role in Human Papillomavirus 16 tumor growth

Background: Human Papillomavirus, HPV, is the main etiological factor for cervical cancer. Different studies show that in women infected with HPV there is a positive correlation between lesion grade and number of infiltrating macrophages, as well as with IL-10 higher expression. Using a HPV16 associated tumor model in mice, TC-1, our laboratory has demonstrated that tumor infiltrating macrophages are M2-like, induce T cell regulatory phenotype and play an important role in tumor growth. M2 macrophages secrete several cytokines, among them IL-10, which has been shown to play a role in T cell suppression by tumor macrophages in other tumor models. In this work, we sought to establish if IL-10 is part of the mechanism by which HPV tumor associated macrophages induce T cell regulatory phenotype, inhibiting anti-tumor activity and facilitating tumor growth. Results: TC-1 tumor cells do not express or respond to IL-10, but recruit leukocytes which, within the tumor environment, produce this cytokine. Using IL-10 deficient mice or blocking IL-10 signaling with neutralizing antibodies, we observed a significant reduction in tumor growth, an increase in tumor infiltration by HPV16 E7 specific CD8 lymphocytes, including a population positive for Granzyme B and Perforin expression, and a decrease in the percentage of HPV specific regulatory T cells in the lymph nodes. Conclusions: Our data shows that in the HPV16 TC-1 tumor mouse model, IL-10 produced by tumor macrophages induce regulatory phenotype on T cells, an immune escape mechanism that facilitates tumor growth. Our results point to a possible mechanism behind the epidemiologic data that correlates higher IL-10 expression with risk of cervical cancer development in HPV infected women.

rst Transcriptional Activity Influences kirre mRNA Concentration in the Drosophila Pupal Retina during the Final Steps of Ommatidial Patterning

Background: Drosophila retinal architecture is laid down between 24-48 hours after puparium formation, when some of the still uncommitted interommatidial cells (IOCs) are recruited to become secondary and tertiary pigment cells while the remaining ones undergo apoptosis. This choice between survival and death requires the product of the roughest (rst) gene, an immunoglobulin superfamily transmembrane glycoprotein involved in a wide range of developmental processes. Both temporal misexpression of Rst and truncation of the protein intracytoplasmic domain, lead to severe defects in which IOCs either remain mostly undifferentiated and die late and erratically or, instead, differentiate into extra pigment cells. Intriguingly, mutants not expressing wild type protein often have normal or very mild rough eyes. Methodology/Principal Findings: By using quantitative real time PCR to examine rst transcriptional dynamics in the pupal retina, both in wild type and mutant alleles we showed that tightly regulated temporal changes in rst transcriptional rate underlie its proper function during the final steps of eye patterning. Furthermore we demonstrated that the unexpected wild type eye phenotype of mutants with low or no rst expression correlates with an upregulation in the mRNA levels of the rst paralogue kin-of-irre (kirre), which seems able to substitute for rst function in this process, similarly to their role in myoblast fusion. This compensatory upregulation of kirre mRNA levels could be directly induced in wild type pupa upon RNAi-mediated silencing of rst, indicating that expression of both genes is also coordinately regulated in physiological conditions. Conclusions/Significance: These findings suggest a general mechanism by which rst and kirre expression could be fine tuned to optimize their redundant roles during development and provide a clearer picture of how the specification of survival and apoptotic fates by differential cell adhesion during the final steps of retinal morphogenesis in insects are controlled at the transcriptional level.

Genotypic Characteristics of HIV Type 1 Based on gp120 Hypervariable Region 3 of Isolates from Southern Brazil

The aim of this study was to investigate HIV-1 molecular diversity and the epidemiological profile of HIV-1-infected patients from Ribeirao Preto, Brazil. A nested PCR followed by sequencing of a 302-base pair fragment of the env gene (C2-V3 region) was performed in samples from HIV-1-positive patients. A total of 45 sequences were aligned with final manual adjustments. The phylogenetic analyses showed a higher prevalence of HIV-1 subtype B in the studied population (97.8%) with only one sample yielding an F1 subtype. The viral genotyping prediction showed that CCR5 tropism was the most prevalent in the studied cohort. Geno2pheno analysis showed that R5 and CXCR4 prediction were 69% and 31%, respectively. There was no statistical significance, either in viral load or in CD4(+) T cell count when R5 and X4 prediction groups were compared. Moreover, the GPGR tetramer was the most common V3 loop core motif identified in the HIV-1 strains studied (34.1%) followed by GWGR, identified in 18.1% of the samples. The high level of B subtype in this Brazilian population reinforces the nature of the HIV epidemic in Brazil, and corroborates previous data obtained in the Brazilian HIV-infected population.

Primary Hyperparathyroidism as the First Clinical Manifestation of Multiple Endocrine Neoplasia Type 2A in a 5-Year-Old Child

Background: Primary hyperparathyroidism occurs in only 10%-30% of patients with multiple endocrine neoplasia type 2A (MEN2A), rarely as the sole clinical manifestation, and is usually diagnosed after the third decade of life. Summary: A5-year-old girl was referred for prophylactic thyroidectomy as she carried the p.C634R RET mutation. She was clinically asymptomatic, with a normally palpable thyroid and with the cervical region free of lymphadenopathy or other nodules. Preoperative tests revealed hypercalcemia associated with elevation of parathyroid hormone (PTH) (calcium = 11.2mg/dL, calcium ion = 1.48mmol/L, phosphorus = 4.0 mg/dL, alkaline phosphatase = 625U/L, parathyroid hormone (PTH) PTH = 998 pg/mL). A thyroid ultrasound was normal and parathyroid scintigraphy with (99m)Tc-Sestamibi revealed an area of radioconcentration in the upper half of the left thyroid lobe suggesting hyperfunctioning parathyroid tissue. She underwent total thyroidectomy and parathyroidectomy and developed hypocalcemia. The anatomopathological examination showed no histopathological changes in the thyroid tissue and an adenoma of the parathyroid gland, confirming the diagnosis of hyperparathyroidism. Conclusions: Primary hyperparathyroidism can be a precocious manifestation of MEN2A. This case report highlights that asymptomatic hypercalcemia should be scrutinized in children related to patients with MEN2A who carry a mutation in the RET proto-oncogene, especially mutations in the codon 634, before the currently recommended age of 8 years.

Differential expression of HIF-1 alpha in CD44(+)CD24(-/) (low) breast ductal carcinomas

Background: Cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of tumor. Stem cells renewal and differentiation can be directly influenced by the oxygen levels of determined tissues, probably by the reduction of oxidative DNA damage in hypoxic regions, thus leading to a friendlier microenvironment, regarding to clonal expansion and for resistance to chemotherapeutic regimens. Furthermore, there have been strong data indicating a pivotal role of hypoxic niche in cancer stem cells development. There are evidence that hypoxia could drive the maintenance of CSC, via HIF-1 alpha expression, but it still to be determined whether hypoxia markers are expressed in breast tumors presenting CD44(+)CD24(-/low) immunophenotype. Methods: Immunohistochemical analysis of CD44(+)CD24(-/low) expression and its relationship with hypoxia markers and clinical outcome were evaluated in 253 samples of breast ductal carcinomas. Double-immunolabeling was performed using EnVision Doublestain System (Dako, Carpinteria, CA, USA). Slides were then scanned into high-resolution images using Aperio ScanScope XT and then, visualized in the software Image Scope (Aperio, Vista, CA, USA). Results: In univariate analysis, CD44(+)CD24(-/low) expression showed association with death due to breast cancer (p = 0.035). Breast tumors expressing CD44(+)CD24(-/low) immunophenotype showed relationship with HIF-1 alpha (p = 0.039) and negativity for HER-2 (p = 0.013). Conclusion: Considering that there are strong evidences that the fraction of a tumour considered to be cancer stem cells is plastic depending upon microenvironmental signals, our findings provide further evidence that hypoxia might be related to the worse prognosis found in CD44(+)CD24(-/low) positive breast tumors.

Cross talk Initiated by Endothelial Cells Enhances Migration and Inhibits Anoikis of Squamous Cell Carcinoma Cells through STAT3/Akt/ERK Signaling

It is well known that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effect of endothelial cell-secreted factors on the phenotype and behavior of tumor cells. The hypothesis underlying this study is that endothelial cells initiate signaling pathways that enhance tumor cell survival and migration. Here, we observed that soluble mediators from primary human dermal microvascular endothelial cells induce phosphorylation of signal transducer and activator of transcription 3 (STAT3), Akt, and extracellular signal-regulated kinase (ERK) in a panel of head and neck squamous cell carcinoma (HNSCC) cells (OSCC-3, UM-SCC-1, UM-SCC-17B, UM-SCC-74A). Gene expression analysis demonstrated that interleukin-6 (IL-6), interleukin-8 (CXCL8), and epidermal growth factor (EGF) are upregulated in endothelial cells cocultured with HNSCC. Blockade of endothelial cell-derived IL-6, CXCL8, or EGF by gene silencing or neutralizing antibodies inhibited phosphorylation of STAT3, Akt, and ERK in tumor cells, respectively. Notably, activation of STAT3, Akt, and ERK by endothelial cells enhanced migration and inhibited anoikis of tumor cells. We have previously demonstrated that Bcl-2 is upregulated in tumor microvessels in patients with HNSCC. Here, we observed that Bcl-2 signaling induces expression of IL-6, CXCL8, and EGF, providing a mechanism for the upregulation of these cytokines in tumor-associated endothelial cells. This study expands the contribution of endothelial cells to the pathobiology of tumor cells. It unveils a new mechanism in which endothelial cells function as initiators of molecular crosstalks that enhance survival and migration of tumor cells.

Identification of three distinguishable phenotypes in golden retriever muscular dystrophy

Duchenne muscular dystrophy (DMD) is a human disease characterized by progressive and irreversible skeletal muscle degeneration caused by mutations in genes coding for important muscle proteins. Unfortunately, there is no efficient treatment for this disease; it causes progressive loss of motor and muscular ability until death. The canine model (golden retriever muscular dystrophy) is similar to DMD, showing similar clinical signs. Fifteen dogs were followed from birth and closely observed for clinical signs. Dogs had their disease status confirmed by polymerase chain reaction analysis and genotyping. Clinical observations of musculoskeletal, morphological, gastrointestinal, respiratory, cardiovascular, and renal features allowed us to identify three distinguishable phenotypes in dystrophic dogs: mild (grade I), moderate (grade II) and severe (grade III). These three groups showed no difference in dystrophic alterations of muscle morphology and creatine kinase levels. This information will be useful for therapeutic trials, because DMD also shows significant, inter- and intra-familiar clinical variability. Additionally, being aware of phenotypic differences in this animal model is essential for correct interpretation and understanding of results obtained in pre-clinical trials.

Long-Lasting Priming of Endothelial Cells by Plasma Melatonin Levels

Background: Endothelial cells are of great interest for cell therapy and tissue engineering. Understanding the heterogeneity among cell lines originating from different sources and culture protocols may allow more standardized material to be obtained. In a recent paper, we showed that adrenalectomy interferes with the expression of membrane adhesion molecules on endothelial cells maintained in culture for 16 to 18 days. In addition, the pineal hormone, melatonin, reduces the adhesion of neutrophils to post-capillary veins in rats. Here, we evaluated whether the reactivity of cultured endothelial cells maintained for more than two weeks in culture is inversely correlated to plasma melatonin concentration. Methodology/Principal Findings: The nocturnal levels of melatonin were manipulated by treating rats with LPS. Nocturnal plasma melatonin, significantly reduced two hours after LPS treatment, returned to control levels after six hours. Endothelial cells obtained from animals that had lower nocturnal melatonin levels significantly express enhanced adhesion molecules and iNOS, and have more leukocytes adhered than cells from animals that had normal nocturnal levels of melatonin (naive or injected with vehicle). Endothelial cells from animals sacrificed two hours after a simultaneous injection of LPS and melatonin present similar phenotype and function than those obtained fromcontrol animals. Analyzing together all the data, taking into account the plasma melatonin concentration versus the expression of adhesion molecules or iNOS we detected a significant inverse correlation. Conclusions/Significance: Our data strongly suggest that the plasma melatonin level primes endothelial cells ""in vivo,"" indicating that the state of the donor animal is translated to cells in culture and therefore, should be considered for establishing cell banks in ideal conditions.

A novel COL1A1 gene-splicing mutation (c. 1875+1G > C) in a Brazilian patient with osteogenesis imperfecta

Osteogenesis imperfecta is a heterogeneous genetic disorder characterized by bone fragility and deformity, recurrent fractures, blue sclera, short stature, and dentinogenesis imperfecta. Most cases are caused by mutations in COL1A1 and COL1A2 genes. We present a novel splicing mutation in the COL1A1 gene (c. 1875+ 1G>C) in a 16-year-old Brazilian boy diagnosed as a type III osteogenesis imperfecta patient. This splicing mutation and its association with clinical phenotypes will be submitted to the reference database of COL1A1 mutations, which has no other description of this mutation.

Deletion of the Basement Membrane Heparan Sulfate Proteoglycan Type XVIII Collagen Causes Hypertriglyceridemia in Mice and Humans

Background: Lipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism. Methods and Findings: We examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia. Conclusions: This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.