DSD Due to 5 alpha-Reductase 2 Deficiency - from Diagnosis to Long Term Outcome

Most of the patients with 5 alpha-RD 2 deficiency are reared in the female social sex due to their severely undervirilized external genitalia but similar to 60% who have not been submitted to orchiectomy in childhood undergo male social sex change at puberty. In our cohort of 30 cases from 18 families, all subjects were registered in the female social sex except for two children-one who had an affected uncle and the other who was diagnosed before being registered. The majority of the patients were satisfied with the long-term results of their treatment and surprisingly, penile length was not associated with satisfactory or unsatisfactory sexual activity. Steroid 5 alpha-RD2 deficiency should be included in the differential diagnosis of all newborns with 46,XY DSD with normal testosterone production before gender assignment or any surgical intervention because these patients should be considered males at birth.

Synthesis, spectroscopic characterization, photochemical and photophysical properties and biological activities of ruthenium complexes with mono- and bi-dentate histamine ligand

The monodentate cis-[Ru(phen)(2)(hist)(2)](2+) 1R and the bidentate cis-[Ru(phen)(2)(hist)](2+) 2A complexes were prepared and characterized using spectroscopic (H-1, (H-1-H-1) COSY and (H-1-C-13) HSQC NMR, UV-vis, luminescence) techniques. The complexes presented absorption and emission in the visible region, as well as a tri-exponential emission decay. The complexes are soluble in aqueous and non-aqueous solution with solubility in a buffer solution of pH 7.4 of 1.14 x 10(-3) mol L-1 for (1R + 2A) and 6.43 x 10(-4) mol L-1 for 2A and lipophilicity measured in an aqueous-octanol solution of -1.14 and -0.96, respectively. Photolysis in the visible region in CH3CN converted the starting complexes into cis-[Ru(phen)(2)(CH3CN)(2)](2+). Histamine photorelease was also observed in pure water and in the presence of BSA (1.0 x 10(-6) mol L-1). The bidentate coordination of the histamine to the ruthenium center in relation to the monodentate coordination increased the photosubstitution quantum yield by a factor of 3. Pharmacological studies showed that the complexes present a moderate inhibition of AChE with an IC50 of 21 mu mol L-1 (referred to risvagtini, IC50 181 mu mol L-1 and galantamine IC50 0.006 mu mol L-1) with no appreciable cytotoxicity toward to the HeLa cells (50% cell viability at 925 mu mol L-1). Cell uptake of the complexes into HeLa cells was detected by fluorescence confocal microscopy. Overall, the observation of a luminescent complex that penetrates the cell wall and has low cytotoxicity, but is reactive photochemically, releasing histamine when irradiated with visible light, are interesting features for application of these complexes as phototherapeutic agents.

Anti-IL-2 Treatment Impairs the Expansion of T-reg Cell Population during Acute Malaria and Enhances the Th1 Cell Response at the Chronic Disease

Plasmodium chabaudi infection induces a rapid and intense splenic CD4(+) T cell response that contributes to both disease pathogenesis and the control of acute parasitemia. The subsequent development of clinical immunity to disease occurs concomitantly with the persistence of low levels of chronic parasitemia. The suppressive activity of regulatory T (T-reg) cells has been implicated in both development of clinical immunity and parasite persistence. To evaluate whether IL-2 is required to induce and to sustain the suppressive activity of T-reg cells in malaria, we examined in detail the effects of anti-IL-2 treatment with JES6-1 monoclonal antibody (mAb) on the splenic CD4(+) T cell response during acute and chronic P. chabaudi AS infection in C57BL/6 mice. JES6-1 treatment on days 0, 2 and 4 of infection partially inhibits the expansion of the CD4(+)CD25(+)Foxp3(+) cell population during acute malaria. Despite the concomitant secretion of IL-2 and expression of high affinity IL-2 receptor by large CD4(+) T cells, JES6-1 treatment does not impair effector CD4+ T cell activation and IFN-gamma production. However, at the chronic phase of the disease, an enhancement of cellular and humoral responses occurs in JES6-1-treated mice, with increased production of TNF-alpha and parasite-specific IgG2a antibodies. Furthermore, JES6-1 mAb completely blocked the in vitro proliferation of CD4(+) T cells from non-treated chronic mice, while it further increased the response of CD4(+) T cells from JES6-1-treated chronic mice. We conclude that JES6-1 treatment impairs the expansion of T-reg cell population during early P. chabaudi malaria and enhances the Th1 cell response in the late phase of the disease.

The Aurora A and B kinases are up-regulated in bone marrow-derived chronic lymphocytic leukemia cells and represent potential therapeutic targets

Background The malignant B cells in chronic lymphocytic leukemia receive signals from the bone marrow and lymph node microenvironments which regulate their survival and proliferation. Characterization of these signals and the pathways that propagate them to the interior of the cell is important for the identification of novel potential targets for therapeutic intervention. Design and Methods We compared the gene expression profiles of chronic lymphocytic leukemia B cells purified from bone marrow and peripheral blood to identify genes that are induced by the bone marrow microenvironment. Two of the differentially expressed genes were further studied in cell culture experiments and in an animal model to determine whether they could represent appropriate therapeutic targets in chronic lymphocytic leukemia. Results Functional classification analysis revealed that the majority of differentially expressed genes belong to gene ontology categories related to cell cycle and mitosis. Significantly up-regulated genes in bone marrow-derived tumor cells included important cell cycle regulators, such as Aurora A and B, survivin and CDK6. Down-regulation of Aurora A and B by RNA interference inhibited proliferation of chronic lymphocytic leukemia-derived cell lines and induced low levels of apoptosis. A similar effect was observed with the Aurora kinase inhibitor VX-680 in primary chronic lymphocytic leukemia cells that were induced to proliferate by CpG-oligonucleotides and interleukin-2. Moreover, VX-680 significantly blocked leukemia growth in a mouse model of chronic lymphocytic leukemia. Conclusions Aurora A and B are up-regulated in proliferating chronic lymphocytic leukemia cells and represent potential therapeutic targets in this disease.

Advice on the Management of Ambiguous Genitalia to a Young Endocrinologist from Experienced Clinicians

The birth of a child with ambiguous genitalia is a challenging and distressing event for the family and physician and one with life-long consequences. Most disorders of sexual differentiation (DSD) associated with ambiguous genitalia are the result either of inappropriate virilization of girls or incomplete virilization of boys. It is important to establish a diagnosis as soon as possible, for psychological, social, and medical reasons, particularly for recognizing accompanying life-threatening disorders such as the salt-losing form of congenital adrenal hyperplasia. In most instances, there is sufficient follow-up data so that making the diagnosis also establishes the appropriate gender assignment (infants with congenital adrenal hyperplasia, those with androgen resistance syndromes), but some causes of DSD such as steroid 5 alpha-reductase 2 deficiency and 17 beta-hydroxysteroid dehydrogenase deficiency are associated with frequent change in social sex later in life. In these instances, guidelines for sex assignment are less well established.

Purinergic and glutamatergic interactions in the hypothalamic paraventricular nucleus modulate sympathetic outflow

P2X receptors are expressed on ventrolateral medulla projecting paraventricular nucleus (PVN) neurons. Here, we investigate the role of adenosine 5′-triphosphate (ATP) in modulating sympathetic nerve activity (SNA) at the level of the PVN. We used an in situ arterially perfused rat preparation to determine the effect of P2 receptor activation and the putative interaction between purinergic and glutamatergic neurotransmitter systems within the PVN on lumbar SNA (LSNA). Unilateral microinjection of ATP into the PVN induced a dose-related increase in the LSNA (1 nmol: 38 ± 6 %, 2.5 nmol: 72 ± 7 %, 5 nmol: 96 ± 13 %). This increase was significantly attenuated by blockade of P2 receptors (pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid, PPADS) and glutamate receptors (kynurenic acid, KYN) or a combination of both. The increase in LSNA elicited by L-glutamate microinjection into the PVN was not affected by a previous injection of PPADS. Selective blockade of non-N-methyl-D-aspartate receptors (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, CNQX), but not N-methyl-D-aspartate receptors (NMDA) receptors (DL-2-amino-5-phosphonopentanoic acid, AP5), attenuated the ATP-induced sympathoexcitatory effects at the PVN level. Taken together, our data show that purinergic neurotransmission within the PVN is involved in the control of SNA via P2 receptor activation. Moreover, we show an interaction between P2 receptors and non-NMDA glutamate receptors in the PVN suggesting that these functional interactions might be important in the regulation of sympathetic outflow

Loss of Interleukin-10 Signaling and Infantile Inflammatory Bowel Disease: Implications for Diagnosis and Therapy

BACKGROUND & AIMS: Homozygous loss of function mutations in interleukin-10 (IL10) and interleukin-10 receptors (IL10R) cause severe infantile (very early onset) inflammatory bowel disease (IBD). Allogeneic hematopoietic stem cell transplantation (HSCT) was reported to induce sustained remission in 1 patient with IL-10R deficiency. We investigated heterogeneity among patients with very early onset IBD, its mechanisms, and the use of allogeneic HSCT to treat this disorder. METHODS: We analyzed 66 patients with early onset IBD (younger than 5 years of age) for mutations in the genes encoding IL-10, IL-10R1, and IL-10R2. IL-10R deficiency was confirmed by functional assays on patients' peripheral blood mononuclear cells (immunoblot and enzyme-linked immunosorbent assay analyses). We assessed the therapeutic effects of standardized allogeneic HSCT. RESULTS: Using a candidate gene sequencing approach, we identified 16 patients with IL-10 or IL-10R deficiency: 3 patients had mutations in IL-10, 5 had mutations in IL-10R1, and 8 had mutations in IL-10R2. Refractory colitis became manifest in all patients within the first 3 months of life and was associated with perianal disease (16 of 16 patients). Extraintestinal symptoms included folliculitis (11 of 16) and arthritis (4 of 16). Allogeneic HSCT was performed in 5 patients and induced sustained clinical remission with a median follow-up time of 2 years. In vitro experiments confirmed reconstitution of IL-10R-mediated signaling in all patients who received the transplant. CONCLUSIONS: We identified loss of function mutations in IL-10 and IL-10R in patients with very early onset IBD. These findings indicate that infantile IBD patients with perianal disease should be screened for IL-10 and IL-10R deficiency and that allogeneic HSCT can induce remission in those with IL-10R deficiency.

MyD88 Signaling Pathway Is Involved in Renal Fibrosis by Favoring a T(H)2 Immune Response and Activating Alternative M2 Macrophages

Inflammation contributes to the pathogenesis of chronic kidney disease (CKD). Molecules released by the inflamed injured tissue can activate toll-like receptors (TLRs), thereby modulating macrophage and CD4+ T-cell activity. We propose that in renal fibrogenesis, M2 macrophages are recruited and activated in a T helper subset 2 cell (TH2)-prone inflammatory milieu in a MyD88- dependent manner. Mice submitted to unilateral ureteral ligation (UUO) demonstrated an increase in macrophage infiltration with collagen deposition after 7 d. Conversely, TLR2, TLR4 and MyD88 knockout (KO) mice had an improved renal function together with diminished TH2 cytokine production and decreased fibrosis formation. Moreover, TLR2, TLR4 and MyD88 KO animals exhibited less M2 macrophage infiltration, namely interleukin (IL)-10+ and CD206+ CD11bhigh cells, at 7 d after surgery. We evaluated the role of a TH2 cytokine in this context, and observed that the absence of IL-4 was associated with better renal function, decreased IL-13 and TGF- β levels, reduced arginase activity and a decrease in fibrosis formation when compared with IL-12 KO and wild-type (WT) animals. Indeed, the better renal outcomes and the decreased fibrosis formation were restricted to the deficiency of IL-4 in the hematopoietic compartment. Finally, macrophage depletion, rather than the absence of T cells, led to reduced lesions of the glomerular filtration barrier and decreased collagen deposition. These results provide evidence that future therapeutic strategies against renal fibrosis should be accompanied by the modulation of the M1:M2 and TH1:TH2 balance, as TH2 and M2 cells are predictive of fibrosis toward mechanisms that are sensed by innate immune response and triggered in a MyD88-dependent pathway.

Toll-like receptor 2 knockdown modulates Interleukin (IL)-6 and IL-8 but not Stromal Derived Factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Cocaine effects on mouse incentive-learning and human addiction are linked to alpha 2 subunit-containing GABA(A) receptors

Because GABA(A) receptors containing alpha 2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with alpha 2 gene deletion showed reduced synaptic GABA(A) receptor-mediated responses. Behaviorally, the deletion abolished cocaine`s ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of alpha 2-GABA(A) receptors (alpha 2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In alpha 2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of alpha 2-GABA(A) receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.

Evidence of the Importance of the First Intracellular Loop of Prokineticin Receptor 2 in Receptor Function

Prokineticin receptors (PROKR) are G protein-coupled receptors (GPCR) that regulate diverse biological processes, including olfactory bulb neurogenesis and GnRH neuronal migration. Mutations in PROKR2 have been described in patients with varying degrees of GnRH deficiency and are located in diverse functional domains of the receptor. Our goal was to determine whether variants in the first intracellular loop (ICL1) of PROKR2 (R80C, R85C, and R85H) identified in patients with hypogonadotropic hypogonadism interfere with receptor function and to elucidate the mechanisms of these effects. Because of structural homology among GPCR, clarification of the role of ICL1 in PROKR2 activity may contribute to a better understanding of this domain across other GPCR. The effects of the ICL1 PROKR2 mutations on activation of signal transduction pathways, ligand binding, and receptor expression were evaluated. Our results indicated that the R85C and R85H PROKR2 mutations interfere only modestly with receptor function, whereas the R80C PROKR2 mutation leads to a marked reduction in receptor activity. Cotransfection of wild-type (WT) and R80C PROKR2 showed that the R80C mutant could exert a dominant negative effect on WT PROKR2 in vitro by interfering with WT receptor expression. In summary, we have shown the importance of Arg80 in ICL1 for PROKR2 expression and demonstrate that R80C PROKR2 exerts a dominant negative effect on WT PROKR2. (Molecular Endocrinology 26: 1417-1427, 2012)

The pattern recognition receptors expressed on neutrophils and the associated cytokine profile from different aged patients with Candida-related denture stomatitis

This study evaluated the expression of pattern recognition receptors (PRRs) and activation factors associated with salivary and blood neutrophils from different aged patients diagnosed with Candida-related denture stomatitis (DS). Expression of neutrophil PRRs was determined by flow cytometry and immunofluorescence, and the levels of selected cytokines that influence immune activation were determined by ELISA. The salivary (but not the serum derived) neutrophils of individuals with DS were found to have an increased expression of CD69 regardless of the age of the patient compared to patients without DS. However, these salivary neutrophils had a lower expression of CD66b and CD64. Expression of TLR2 was lower on the salivary-and serum-derived neutrophils from elderly individuals compared to the neutrophils of younger subjects, regardless of whether the individual had DS. Salivary interleukin (IL)-4 was elevated in both of the elderly subject groups (with or without DS). Only elderly DS patients were observed to have increased serum IL-4 levels and reduced salivary IL-12 levels. Younger DS patients showed an increase in salivary IL-10 levels, and both the saliva and the serum levels of IFN-gamma were increased in all of the younger subjects. Our data demonstrated that changes in both the oral immune cells and the protein components could be associated with DS. Furthermore, changes in the blood-derived factors were more associated with age than DS status. (C) 2012 Elsevier Inc. All rights reserved.

Nitric oxide modulates metalloproteinase-2, collagen deposition and adhesion rate after polypropylene mesh implantation in the intra-abdominal wall

Prosthetic meshes are commonly used to correct abdominal wall defects. However, the inflammatory reaction induced by these devices in the peritoneum is not completely understood. We hypothesized that nitric oxide (NO), produced by nitric oxide synthase 2 (NOS2) may modulate the response induced by mesh implants in the abdominal wall and, consequently, affect the outcome of the surgical procedure. Polypropylene meshes were implanted in the peritoneal side of the abdominal wall in wild-type and NOS2-deficient (NOS2(-/-)) mice. After 15 days tissues around the mesh implant were collected, and inflammatory markers (the cytokine interleukin 1 beta (IL-1 beta) and NO) and tissue remodeling (collagen and metalloproteinases (MMP) 2 and 9) were analyzed. The lack of NOS2-derived NO induced a higher incidence of visceral adhesions at the mesh implantation site compared with wild-type mice that underwent the same procedure (P < 0.05). Additionally, higher levels of IL-1 beta were present in the mesh-implanted NOS2(-/-) animals compared with control and wild-type mice. Mesh implantation induced collagen I and III deposition, but in smaller amounts in NOS2(-/-) mice. MMP-9 activity after the surgical procedure was similarly increased in both groups. Conversely, MMP-2 activity was unchanged in mesh-implanted wild-type mice, but was significantly increased in NOS2(-/-) mice (P < 0.01), due to decreased S-nitrosylation of the enzyme in these animals. We conclude that NOS2-derived NO is crucial for an adequate response to and integration of polypropylene mesh implants in the peritoneum. NO deficiency results in a prolonged inflammatory reaction to the mesh implant, and reduced collagen deposition may contribute to an increased incidence of visceral adhesions. (C) 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Growth hormone response to growth hormone-releasing peptide-2 in growth hormone-deficient Little mice

OBJECTIVE: To investigate a possible direct, growth hormone-releasing, hormone-independent action of a growth hormone secretagogue, GHRP-2, in pituitary somatotroph cells in the presence of inactive growth hormone-releasing hormone receptors. MATERIALS AND METHODS: The responses of serum growth hormone to acutely injected growth hormone-releasing P-2 in lit/litmice, which represent a model of GH deficiency arising frommutated growth hormone-releasing hormone-receptors, were compared to those observed in the heterozygous (lit/+) littermates and wild-type (+/+) C57BL/6J mice. RESULTS: After the administration of 10 mcg of growth hormone-releasing P-2 to lit/lit mice, a growth hormone release of 9.3 +/- 1.5 ng/ml was observed compared with 1.04 +/- 1.15 ng/ml in controls (p<0.001). In comparison, an intermediate growth hormone release of 34.5 +/- 9.7 ng/ml and a higher growth hormone release of 163 +/- 46 ng/ml were induced in the lit/+ mice and wild-type mice, respectively. Thus, GHRP-2 stimulated growth hormone in the lit/lit mice, and the release of growth hormone in vivo may be only partially dependent on growth hormone-releasing hormone. Additionally, the plasma leptin and ghrelin levels were evaluated in the lit/lit mice under basal and stimulated conditions. CONCLUSIONS: Here, we have demonstrated that lit/lit mice, which harbor a germline mutation in the Growth hormone-releasing hormone gene, maintain a limited but statistically significant growth hormone elevation after exogenous stimulation with GHRP-2. The present data probably reflect a direct, growth hormone-independent effect on Growth hormone S (ghrelin) stimulation in the remaining pituitary somatotrophs of little mice that is mediated by growth hormone S-R 1a.

Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT(1)/AT(2) receptors to activate SERCA and to promote Ca2+ mobilization

Ferrao FM, Lara LS, Axelband F, Dias J, Carmona AK, Reis RI, Costa-Neto CM, Vieyra A, Lowe J. Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT(1)/AT(2) receptors to activate SERCA and to promote Ca2+ mobilization. Am J Physiol Renal Physiol 302: F875-F883, 2012. First published January 4, 2012; doi:10.1152/ajprenal.00381.2011.-ANG II is secreted into the lumens of proximal tubules where it is also synthesized, thus increasing the local concentration of the peptide to levels of potential physiological relevance. In the present work, we studied the effect of ANG II via the luminal membranes of LLC-PK1 cells on Ca2+-ATPase of the sarco(endo) plasmic reticulum (SERCA) and plasma membrane (PMCA). ANG II (at concentrations found in the lumen) stimulated rapid (30 s) and persistent (30 min) SERCA activity by more than 100% and increased Ca2+ mobilization. Pretreatment with ANG II for 30 min enhanced the ANG II-induced Ca2+ spark, demonstrating a positively self-sustained stimulus of Ca2+ mobilization by ANG II. ANG II in the medium facing the luminal side of the cells decreased with time with no formation of metabolites, indicating peptide internalization. ANG II increased heterodimerization of AT(1) and AT(2) receptors by 140%, and either losartan or PD123319 completely blocked the stimulation of SERCA by ANG II. Using the PLC inhibitor U73122, PMA, and calphostin C, it was possible to demonstrate the involvement of a PLC -> DAG(PMA)-> PKC pathway in the stimulation of SERCA by ANG II with no effect on PMCA. We conclude that ANG II triggers SERCA activation via the luminal membrane, increasing the Ca2+ stock in the reticulum to ensure a more efficient subsequent mobilization of Ca2+. This first report on the regulation of SERCA activity by ANG II shows a new mechanism for Ca2+ homeostasis in renal cells and also for regulation of Ca2+-modulated fluid reabsorption in proximal tubules.