Haploinsufficiency of Dmxl2, Encoding a Synaptic Protein,Causes Infertility Associated with a Loss of GnRH Neurons in Mouse

Characterization of the genetic defects causing gonadotropic deficiency has made a major contribution to elucidation of the fundamental role of Kisspeptins and Neurokinin B in puberty onset and reproduction. The absence of puberty may also reveal neurodevelopmental disorders caused by molecular defects in various cellular pathways. Investigations of these neurodevelopmental disorders may provide information about the neuronal processes controlling puberty onset and reproductive capacity. We describe here a new syndrome observed in three brothers, which involves gonadotropic axis deficiency, central hypothyroidism, peripheral demyelinating sensorimotor polyneuropathy, mental retardation, and profound hypoglycemia, progressing to nonautoimmune insulin-dependent diabetes mellitus. High-throughput sequencing revealed a homozygous in-frame deletion of 15 nucleotides in DMXL2 in all three affected patients. This homozygous deletion was associated with lower DMXL2 mRNA levels in the blood lymphocytes of the patients. DMXL2 encodes the synaptic protein rabconnectin-3α, which has been identified as a putative scaffold protein for Rab3-GAP and Rab3-GEP, two regulators of the GTPase Rab3a. We found that rabconnectin-3α was expressed in exocytosis vesicles in gonadotropin-releasing hormone (GnRH) axonal extremities in the median eminence of the hypothalamus. It was also specifically expressed in cells expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) within the pituitary. The conditional heterozygous deletion of Dmxl2 from mouse neurons delayed puberty and resulted in very low fertility. This reproductive phenotype was associated with a lower number of GnRH neurons in the hypothalamus of adult mice. Finally, Dmxl2 knockdown in an insulin-secreting cell line showed that rabconnectin-3α controlled the constitutive and glucose-induced secretion of insulin. In conclusion, this study shows that low levels of DMXL2 expression cause a complex neurological phenotype, with abnormal glucose metabolism and gonadotropic axis deficiency due to a loss of GnRH neurons. Our findings identify rabconectin-3α as a key controller of neuronal and endocrine homeostatic processes.     

Intravaginal practices, bacterial vaginosis, and HIV infection in women: individual participant data meta-analysis

Background

Identifying modifiable factors that increase women''s vulnerability to HIV is a critical step in developing effective female-initiated prevention interventions. The primary objective of this study was to pool individual participant data from prospective longitudinal studies to investigate the association between intravaginal practices and acquisition of HIV infection among women in sub-Saharan Africa. Secondary objectives were to investigate associations between intravaginal practices and disrupted vaginal flora; and between disrupted vaginal flora and HIV acquisition.

Methods and Findings

We conducted a meta-analysis of individual participant data from 13 prospective cohort studies involving 14,874 women, of whom 791 acquired HIV infection during 21,218 woman years of follow-up. Data were pooled using random-effects meta-analysis. The level of between-study heterogeneity was low in all analyses (I ² values 0.0%–16.1%). Intravaginal use of cloth or paper (pooled adjusted hazard ratio [aHR] 1.47, 95% confidence interval [CI] 1.18–1.83), insertion of products to dry or tighten the vagina (aHR 1.31, 95% CI 1.00–1.71), and intravaginal cleaning with soap (aHR 1.24, 95% CI 1.01–1.53) remained associated with HIV acquisition after controlling for age, marital status, and number of sex partners in the past 3 months. Intravaginal cleaning with soap was also associated with the development of intermediate vaginal flora and bacterial vaginosis in women with normal vaginal flora at baseline (pooled adjusted odds ratio [OR] 1.24, 95% CI 1.04–1.47). Use of cloth or paper was not associated with the development of disrupted vaginal flora. Intermediate vaginal flora and bacterial vaginosis were each associated with HIV acquisition in multivariable models when measured at baseline (aHR 1.54 and 1.69, p<0.001) or at the visit before the estimated date of HIV infection (aHR 1.41 and 1.53, p<0.001), respectively.

Conclusions

This study provides evidence to suggest that some intravaginal practices increase the risk of HIV acquisition but a direct causal pathway linking intravaginal cleaning with soap, disruption of vaginal flora, and HIV acquisition has not yet been demonstrated. More consistency in the definition and measurement of specific intravaginal practices is warranted so that the effects of specific intravaginal practices and products can be further elucidated. Please see later in the article for the Editors'' Summary     

Indoleamine 2,3-Dioxygenase 1 (Ido1) Is Involved in the Control of Mouse Caput Epididymis Immune Environment

The epididymis maintains a state of immune tolerance towards spermatozoa while also protecting them and itself against infection and acute inflammation. The immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (Ido1) participates in this delicate local equilibrium. Using the mouse Ido1^−/− model, we show here that the absence of IDO1 expression leads in the epididymis but not in serum to (1) an increase in the inflammatory state as evidenced by changes in the content of cytokines and chemokines, (2) the engagement of a Th1-driven inflammatory response as evidenced by changes in the Th17/Treg as well as Th1/Th2 equilibria, as well as (3) differences in the content of lipid intermediates classically involved in inflammation. Despite this more pronounced inflammatory state, Ido1^−/− animals succeed in preserving the local epididymal immune situation due to the activation of compensatory mechanisms that are discussed.     

Symptom Burden of Patients with Dry Eye Disease: A Four Domain Analysis

Purpose

To determine which sensory (symptom persistence and intensity) and reactive (activity and affective interference) domains of symptom analysis are essential for assessing symptom burden in dry eye disease (DED) patients.

Methods

A symptom domain tool was developed to investigate all four symptom domains in DED. In a cross-sectional pilot study, we administered the symptom burden tool and the Ocular Surface Disease Index (OSDI) questionnaire to 48 DED patients. Total and domain scores from the symptom burden tool and the OSDI were normalized to achieve comparability. Spearman correlation coefficients were calculated to measure the relationship between domains and subscales. Agreement between the symptom burden tool and OSDI was assessed by Bland-Altman plot. Assigned treatments were compared by symptom burden to determine whether treatment aggressiveness is linked to symptom intensity.

Results

There was high agreement between the symptom burden tool and the OSDI. Symptom persistence had a stronger correlation with affective interference (r  =  0.62 for the symptom burden tool and r = 0.73 for the OSDI) than activity interference (r = 0.58 for the symptom burden tool and r = 0.60 for the OSDI). Symptom intensity correlated weakly with affective interference (r = 0.38) and activity interference (r = 0.37) in the symptom burden tool (OSDI does not have a subscale for intensity). In patients with equal persistence of symptoms, those having high symptom intensity were receiving more aggressive treatment (66.7%) than those with lower symptom intensity (33.3%).

Conclusions

Persistence of symptoms correlates better with affective interference than activity interference. Intensity of symptoms may be important for treatment decisions.     

Relationship of IgG and IgM autoantibodies and immune complexes to oxidized LDL with markers of oxidation and inflammation and cardiovascular events: results from the EPIC-Norfolk Study

Levels of IgG and IgM autoantibodies (AA) to malondialdehyde (MDA)-LDL and apoB-immune complexes (ICs) were measured in 748 cases and 1,723 controls in the EPIC-Norfolk cohort and their association to coronary artery disease (CAD) events determined. We evaluated whether AA and IC modify CAD risk associated with secretory phospholipase A(2) (sPLA(2)) type IIA mass and activity, lipoprotein-associated PLA(2) activity, lipoprotein (a) [Lp(a)], oxidized phospholipids on apoB-100 (OxPL/apoB), myeloperoxidase, and high sensitivity C-reactive protein. IgG ICs were higher in cases versus controls (P = 0.02). Elevated levels of IgM AA and IC were inversely associated with Framingham Risk Score and number of metabolic syndrome criteria (p range 0.02-0.001). In regression analyses adjusted for age, smoking, diabetes, LDL-cholesterol, HDL-cholesterol, and systolic blood pressure, the highest tertiles of IgG and IgM AA and IC were not associated with higher risk of CAD events compared with the lowest tertiles. However, elevated levels of IgM IC reduced the risk of Lp(a) (P = 0.006) and elevated IgG MDA-LDL potentiated the risk of sPLA(2) mass (P = 0.018). This epidemiological cohort of initially healthy subjects shows that IgG and IgM AA and IC are not independent predictors of CAD events but may modify CAD risk associated with elevated levels of oxidative biomarkers.     

Effects of vascular endothelial growth factor on the lymphocyte-endothelium interactions: identification of caveolin-1 and nitric oxide as control points of endothelial cell anergy

Tumors may evade immune responses at multiple levels, including through a defect in the lymphocyte-vessel wall interactions. The angiogenic nature of endothelial cells (EC) lining tumor blood vessels may account for such anergy. In this study, we examined whether mechanisms other than down-regulation of adhesion molecules could be involved, particularly signaling pathways dependent on the caveolae platforms. To mimic the influence of the tumor microenvironment, EC were exposed to TNF-alpha and the proangiogenic vascular endothelial growth factor (VEGF). We identified a dramatic inhibition of lymphocyte adhesion on activated EC following either short or long VEGF pretreatments. We further documented that VEGF did not influence the abundance of major adhesion molecules, but was associated with a defect in ICAM-1 and VCAM-1 clustering at the EC surface. We also found that overexpression of the caveolar structural protein, caveolin-1, overcame the VEGF-mediated inhibition of adhesion and restored ICAM-1 clustering. Conversely, EC transduction with a caveolin-1 small interfering RNA reduced the TNF-alpha-dependent increase in adhesion. Finally, we identified VEGF-induced NO production by the endothelial NO synthase as the main target of the changes in caveolin-1 abundance. We found that the NO synthase inhibitor N-nitro-l-arginine methyl ester could reverse the inhibitory effects of VEGF on lymphocyte adhesion and EC cytoskeleton rearrangement. Symmetrically, a NO donor was shown to prevent the ICAM clustering-mediated lymphocyte adhesion, thereby recapitulating the effects of VEGF. In conclusion, this study provides new insights on the mechanisms leading to the tumor EC anergy vs immune cells and opens new perspectives for the use of antiangiogenic strategies as adjuvant approaches to cancer immunotherapy.     

Increasing methotrexate resistance by combination of active-site mutations in human dihydrofolate reductase

Methotrexate-resistant forms of human dihydrofolate reductase have the potential to protect healthy cells from the toxicity of methotrexate (MTX), to improve prognosis during cancer therapy. It has been shown that synergistic MTX-resistance can be obtained by combining two active-site mutations that independently confer weak MTX-resistance. In order to obtain more highly MTX-resistant human dihydrofolate reductase (hDHFR) variants for this application, we used a semi-rational approach to obtain combinatorial active-site mutants of hDHFR that are highly resistant towards MTX. We created a combinatorial mutant library encoding various amino acids at residues Phe31, Phe34 and Gln35. In vivo library selection was achieved in a bacterial system on medium containing high concentrations of MTX. We characterized ten novel MTX-resistant mutants with different amino acid combinations at residues 31, 34 and 35. Kinetic and inhibition parameters of the purified mutants revealed that higher MTX-resistance roughly correlated with a greater number of mutations, the most highly-resistant mutants containing three active site mutations (Ki(MTX)=59-180 nM; wild-type Ki(MTX)<0.03 nM). An inverse correlation was observed between resistance and catalytic efficiency, which decreased mostly as a result of increased KM toward the substrate dihydrofolate. We verified that the MTX-resistant hDHFRs can protect eukaryotic cells from MTX toxicity by transfecting the most resistant mutants into DHFR-knock-out CHO cells. The transfected variants conferred survival at concentrations of MTX between 100-fold and >4000-fold higher than the wild-type enzyme, the most resistant triple mutant offering protection beyond the maximal concentration of MTX that could be included in the medium. These highly resistant variants of hDHFR offer potential for myeloprotection during administration of MTX in cancer treatment.