LtpD Is a Novel Legionella pneumophila Effector That Binds Phosphatidylinositol 3-Phosphate and Inositol Monophosphatase IMPA1

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of the Legionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD_471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD_471-626 bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitro and colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-binding L. pneumophila effector that has a role in intracellular bacterial replication.     

Chronic pulsatile infusion of growth hormone to growth-restricted fetal sheep increases circulating fetal insulin-like growth factor-I levels but not fetal growth

Intra-uterine growth restriction (IUGR) is a major cause of perinatal mortality and morbidity. Postnatally, growth hormone (GH) increases growth, increases circulating insulin-like growth factor (IGF)-I levels, and alters metabolism. Our aim was to determine if GH infusion to IUGR fetal sheep would alter fetal growth and metabolism, and thus provide a potential intra-uterine treatment for the IUGR fetus. We studied three groups of fetuses: control, IUGR+ vehicle and IUGR+GH (n=5 all groups). IUGR was induced by repeated embolisation of the placental vascular bed between 110 and 116 days of gestation (term=145 days). GH (3.5 mg/kg/day) or vehicle was infused in a pulsatile manner from 117 to 127 days of gestation. Embolisation reduced fetal growth rate by 25% (P<0.01) and reduced the weight of the fetal liver (20%), kidney (23%) and thymus (31%; all P<0.05). GH treatment further reduced the weight of the fetal kidneys (32%) and small intestine (35%; both P<0.04), but restored the relative weight of the fetal thymus and liver (P<0.05). Embolisation decreased fetal plasma IGF-I concentrations (48%, P<0.001) and increased IGF binding protein 1 (IGFBP-1) concentrations (737%, P<0.002). GH treatment restored fetal plasma IGF-I concentrations to control levels, while levels in IUGR+vehicle fetuses stayed low (P<0.05 vs control). IGFBP-1 and IGFBP-2 concentrations were about sevenfold lower in amniotic fluid than in fetal plasma, but amniotic and plasma concentrations were closely correlated (r=0.75, P<0.0001 and r=0.55 P<0.0001 respectively). Embolisation transiently decreased fetal blood oxygen content (40%, P<0.002), and increased blood lactate concentrations (213%, P<0.04). Both returned to pre-embolisation levels after embolisation stopped, but blood glucose concentrations declined steadily in IUGR+vehicle fetuses. GH treatment maintained fetal blood glucose concentrations at control levels. Our study shows that GH infusion to the IUGR fetal sheep restores fetal IGF-I levels but does not improve fetal growth, and further reduces the fetal kidney and intestine weights. Thus, fetal GH therapy does not seem a promising treatment stratagem for the IUGR fetus.     

Effects of sex, litter size and periconceptional ewe nutrition on offspring behavioural and physiological response to isolation

Maternal periconceptional undernutrition alters fetal hypothalamic-pituitary-adrenal (HPA) axis development. However, the effects of this early nutritional insult on postnatal HPA axis function and stress-related behaviours are unknown. We investigated in sheep the effects of different periods of undernutrition, and of sex and litter size, on offspring behavioural and cortisol responses to isolation stress. We studied four nutritional groups: controls well nourished throughout pregnancy (n = 39), or ewes undernourished (UN, 10-15% body weight reduction) before mating (−60 to 0 d, n = 26), after mating (−2 to + 30 d, n = 20) or both (−60 to + 30 d, n = 36). At 4 and 18 months of age, offspring were isolated for 5 min, their behaviour video recorded, and plasma cortisol concentrations measured.Offspring of all undernourished groups demonstrated 50% fewer escape attempts than controls at 4 months of age, and offspring of UN−60 + 30 ewes had 20% lower plasma cortisol area under the curve in response to isolation at 18 months. Females had higher cortisol concentrations and vocalised more than males at 4 and 18 months, and were more active at 18 months. After isolation, UN−2 + 30 males had higher cortisol concentrations than UN−2 + 30 females whereas in all other groups males had lower concentrations than females. Singleton males made more escape attempts than females, whereas in twins females made more escape attempts than males.These findings suggest that maternal periconceptional undernutrition in sheep can suppress behavioural reactions and cortisol secretion in response to isolation stress in the offspring into adulthood, and that these effects differ between males and females.     

Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studies

Linkage studies indicate close associations of certain HLA alleles with autoimmune diseases. To better understand how specific HLA alleles are related to disease pathogenesis, we have generated an HLA DR3/DQ2 transgenic mouse utilizing a 550-kb yeast artificial chromosome (YAC) construct containing the complete DRalpha, DRbeta1, DRbeta3, DQalpha, and DQbeta regions. The transgenic mouse (4D1/C2D) in an I-Abeta(o) background appears healthy with no signs of autoimmune diseases. Lymphoid tissues as well as CD4(+) T cells develop normally. Characterization of the transgene expression demonstrates that approximately 90% of B cells express high levels of DR3 and 50-70% of B cells express DQ2. CD11c(+) dendritic cells express high levels of DR and DQ. Approximately 12-18% of resting T cells are positive for DR expression, and further up-regulation to 40-50% expression is seen upon activation with anti-CD3/anti-CD28 mAb. These results suggest that the transgenic construct confers a high fidelity to the normal human temporal and spatial expression profile. Analysis of T cell receptor repertoire in transgenic mice confirms that DR3/DQ2 are able to mediate thymic selection. Furthermore, transgenic mice respond to a DR3-restricted antigen, demonstrating antigen processing and presentation by antigen-presenting cells (APC). Purified T cells from ovalbumin (OVA)-immunized 4D1 mice respond to human APC co-cultured with OVA, suggesting appropriate antigen/DR3 or DQ2 recognition by murine T cells. Immunoglobulin isotype switching is also observed, indicating functional T-B cognate interactions. Thus, the DR3/DQ2 transgenic mouse has normal lymphoid development and functionality that are mediated by HLA transgenes and can be used to investigate HLA-associated immunological questions.     

Does the Female Sexual Distress Scale‐Revised Cover the Feelings of Women with HSDD?

IntroductionDistress associated with low sexual desire is a key feature of hypoactive sexual desire disorder (HSDD). Accurate, reliable, and easy‐to‐use diagnostic tools to measure such distress are required. The Female Sexual Distress Scale‐Revised (FSDS‐R) has been shown to have good discriminant validity, test–retest reliability, and internal consistency in measuring sex‐related personal distress in women with HSDD. However, the content validity (relevance, clarity, comprehensiveness) of the scale must also be established.AimThe aim of this study was to assess the content validity of the FSDS‐R and to examine the potential of Item 13 as a stand‐alone measure of distress associated with decreased sexual desire.MethodsA single‐visit content validation study was conducted in three centers in the United States. Women were screened for HSDD; those with HSDD completed the FSDS‐R and then underwent debriefing to capture information on their perceptions of the instrument. Participants also rated the relevancy of every FSDS‐R item, from 0 (“not at all relevant”) to 4 (“extremely relevant”).Main Outcome MeasuresFemale HSDD patients' ratings of the relevance and ease of understanding of the 13 items of the FSDS‐R.ResultsTwenty‐five women with HSDD were interviewed. Mean relevancy ratings ranged from 1.96 (Item 9) to 3.33 (Item 13). Most participants (76–100%) found every item clear and easy to understand. Item 13 alone demonstrated good content validity, and 56% of participants felt that it covered all of their feelings about their low sexual desire.ConclusionsThis study established the content validity of the FSDS‐R and demonstrated that the FSDS‐R total score is a relevant endpoint for women with HSDD. The tool's one item specific to low sexual desire (Item 13) was given the highest score and highest relevancy of all items, and over half the sample felt that it covered all of their feelings about their low sexual desire. DeRogatis L, Pyke R, McCormack J, Hunter A, and Harding G. Does the female sexual distress scale‐revised© (FSDS‐R©) cover the feelings of women with HSDD? J Sex Med 2011;8:2810–2815.