The involvement of peritoneal GATA6+ macrophages in the pathogenesis of endometriosis.

Endometriosis is a chronic inflammatory disease that causes debilitating pelvic pain in women. Macrophages are considered to be key players in promoting disease progression, as abundant macrophages are present in ectopic lesions and elevated in the peritoneum. In the present study, we examined the role of GATA6+ peritoneal macrophages on endometriosis-associated hyperalgesia using mice with a specific myeloid deficiency of GATA6. Lesion induction induced the disappearance of TIM4hi MHCIIlo residential macrophages and the influx of increased Ly6C+ monocytes and TIM4lo MHCIIhi macrophages. The recruitment of MHCIIhi inflammatory macrophages was extensive in Mac Gata6 KO mice due to the severe disappearance of TIM4hi MHCIIlo residential macrophages. Ki67 expression confirmed GATA6-dependent proliferative ability, showing different proliferative phenotypes of TIM4+ residential macrophages in Gata6f/f and Mac Gata6 KO mice. Peritoneal proinflammatory cytokines were elevated after lesion induction. When cytokine levels were compared between Gata6f/f and Mac Gata6 KO mice, TNFα at day 21 in Gata6f/f mice was higher than in Mac Gata6 KO mice. Lesion induction increased both abdominal and hind paw sensitivities. Gata6f/f mice tended to show higher sensitivity in the abdomen after day 21. Elevated expression of TRPV1 and CGRP was observed in the dorsal root ganglia after ELL induction in Gata6f/f mice until days 21 and 42, respectively. These results support that peritoneal GATA6+ macrophages are involved in the recruitment and reprogramming of monocyte-derived macrophages. The extensive recruitment of monocyte-derived macrophages in Mac Gata6 KO mice might protect against inflammatory stimuli during the resolution phase, whereas GATA6 deficiency did not affect lesion initiation and establishment at the acute phase of inflammation. GATA6+ residential macrophages act to sustain local inflammation in the peritoneum and sensitivities in the neurons, reflecting endometriosis-associated hyperalgesia.

Normal Ovarian Function in Subfertile Mouse with Amhr2-Cre-Driven Ablation of Insr and Igf1r.

Insulin receptor signaling promotes cell differentiation, proliferation, and growth which are essential for oocyte maturation, embryo implantation, endometrial decidualization, and placentation. The dysregulation of insulin signaling in women with metabolic syndromes including diabetes exhibits poor pregnancy outcomes that are poorly understood. We utilized the Cre/LoxP system to target the tissue-specific conditional ablation of insulin receptor (Insr) and insulin-like growth factor-1 receptor (Igf1r) using an anti-Mullerian hormone receptor 2 (Amhr2) Cre-driver which is active in ovarian granulosa and uterine stromal cells. Our long-term goal is to examine insulin-dependent molecular mechanisms that underlie diabetic pregnancy complications, and our conditional knockout models allow for such investigation without confounding effects of ligand identity, source and cross-reactivity, or global metabolic status within dams. Puberty occurred with normal timing in all conditional knockout models. Estrous cycles progressed normally in Insrd/d females but were briefly stalled in diestrus in Igf1rd/d and double receptor (DKO) mice. The expression of vital ovulatory genes (Lhcgr, Pgr, Ptgs2) was not significantly different in 12 h post-hCG superovulated ovaries in knockout mice. Antral follicles exhibited an elevated apoptosis of granulosa cells in Igf1rd/d and DKO mice. However, the distribution of ovarian follicle subtypes and subsequent ovulations was normal in all insulin receptor mutants compared to littermate controls. While ovulation was normal, all knockout lines were subfertile suggesting that the loss of insulin receptor signaling in the uterine stroma elicits implantation and decidualization defects responsible for subfertility in Amhr2-Cre-derived insulin receptor mutants.

High-fat diets promote peritoneal inflammation and augment endometriosis-associated abdominal hyperalgesia.

Immune dysfunction is one of the central components in the development and progression of endometriosis by establishing a chronic inflammatory environment. Western-style high-fat diets (HFD) have been linked to greater systemic inflammation to cause metabolic and chronic inflammatory diseases, and are also considered an environmental risk factor for gynecologic diseases. Here, we aimed to examine how HFD cause an inflammatory environment in endometriosis and discern their contribution to endometriotic-associated hyperalgesia. Our results showed that HFD-induced obesity enhanced abdominal hyperalgesia that was induced by endometriotic lesions. Peritoneal inflammatory macrophages and cytokine levels increased by lesion induction were elevated by chronic exposure to HFD. Increased expression of pain-related mediators in the dorsal root ganglia was observed after lesion induction under the HFD condition. Although HFD did not affect inflammatory macrophages in the peritoneal cavity without lesion induction, the diversity and composition of the gut microbiota were clearly altered by HFD as a sign of low-grade systemic inflammation. Thus, HFD alone might not establish a local inflammatory environment in the pelvic cavity, but it can contribute to further enhancing chronic inflammation, leading to the exacerbation of endometriosis-associated abdominal hyperalgesia following the establishment and progression of the disease.

Who performs neonatal hip assessment: is there a cause for concern?

OBJECTIVE: The UK falls behind other European countries in the early detection of developmental dysplasia of the hip (DDH) and screening strategies differ for early detection. Clinical detection of DDH is challenging and recognised to be dependent on examiner experience. No studies exist assessing the number of personnel currently involved in such assessments.Our objective was to review the current screening procedure by studying a cohort of newborn babies in one teaching hospital and assess the number of health professionals involved in neonatal hip assessment and the number of examinations undertaken during one period by each individual. METHODS: This was a retrospective observational study assessing all babies born consecutively over a 14-week period in 2020. Record of each initial baby check was obtained from BadgerNet. Follow-up data on ultrasound or orthopaedic outpatient referrals were obtained from clinical records. RESULTS: 1037 babies were examined by 65 individual examiners representing 9 different healthcare professional groups. The range of examinations conducted per examiner was 1-97 with a median of 5.5 examinations per person. 49% of individuals examined 5 or less babies across the 14 weeks, with 18% only performing 1 examination. Of the six babies (0.48%) treated for DDH, one was picked up on neonatal assessment. CONCLUSION: In a system where so many examiners are involved in neonatal hip assessment, the experience is limited for most examiners. Currently high rates of late presentation of DDH are observed locally, which are in accordance with published national experience. The potential association merits further investigation.

High-fat diets promote peritoneal inflammation and augment endometriosis-associated abdominal hyperalgesia.

Immune dysfunction is one of the central components in the development and progression of endometriosis by establishing a chronic inflammatory environment. Western-style high-fat diets (HFD) have been linked to greater systemic inflammation to cause metabolic and chronic inflammatory diseases, and are also considered an environmental risk factor for gynecologic diseases. Here, we aimed to examine how HFD alter an inflammatory environment in endometriosis and discern their contribution to endometriotic-associated hyperalgesia. Our results showed that HFD-induced obesity enhanced abdominal mechanical allodynia that was induced by endometriotic lesions. Peritoneal inflammatory macrophages and cytokine levels increased by lesion induction were elevated by chronic exposure to HFD. Pain-related mediators in the dorsal root ganglia were further stimulated after lesion induction under the HFD condition. Although HFD did not affect inflammatory macrophages in the peritoneal cavity without lesion induction, the diversity and composition of the gut microbiota were clearly altered by HFD as a sign of low-grade systemic inflammation. Thus, HFD alone might not establish a local inflammatory environment in the pelvic cavity, but it can contribute to further enhancing chronic inflammation, leading to the exacerbation of endometriosis-associated abdominal hyperalgesia following the establishment and progression of the disease.

Investigating the Heaviest Halogen: Lessons Learned from Modeling the Electronic Structure of Astatine's Small Molecules.

We present a systematic study of electron-correlation and relativistic effects in diatomic molecular species of the heaviest halogen astatine (At) within relativistic single- and multireference coupled-cluster approaches and relativistic density functional theory. We establish revised reference ab initio data for the ground states of At2, HAt, AtAu, and AtO+ using a highly accurate relativistic effective core potential model and in-house basis sets developed for accurate modeling of molecules with large spin-orbit effects. Spin-dependent relativistic effects on chemical bonding in the ground state are comparable to the binding energy or even exceed it in At2. Electron-correlation effects near the equilibrium internuclear separation are mostly dynamical and can be adequately captured using single-reference CCSD(T). However, bond elongation in At2 and, especially, AtO+ results in rapid manifestation of its multireference character. While useful for evaluating the spin-orbit effects on the ground-state bonding and properties, the two-component density functional theory lacks predictive power, especially in combination with popular empirically adjusted exchange-correlation functionals. This drawback supports the necessity to develop new functionals for reliable quantum-chemical models of heavy-element compounds with strong relativistic effects.

Niclosamide targets the dynamic progression of macrophages for the resolution of endometriosis in a mouse model.

Due to the vital roles of macrophages in the pathogenesis of endometriosis, targeting macrophages could be a promising therapeutic direction. Here, we investigated the efficacy of niclosamide for the resolution of a perturbed microenvironment caused by dysregulated macrophages in a mouse model of endometriosis. Single-cell transcriptomic analysis revealed the heterogeneity of macrophages including three intermediate subtypes with sharing characteristics of traditional "small" or "large" peritoneal macrophages (SPMs and LPMs) in the peritoneal cavity. Endometriosis-like lesions (ELL) enhanced the differentiation of recruited macrophages, promoted the replenishment of resident LPMs, and increased the ablation of embryo-derived LPMs, which were stepwise suppressed by niclosamide. In addition, niclosamide restored intercellular communications between macrophages and B cells. Therefore, niclosamide rescued the perturbed microenvironment in endometriosis through its fine regulations on the dynamic progression of macrophages. Validation of similar macrophage pathogenesis in patients will further promote the clinical usage of niclosamide for endometriosis treatment.

An optimized Factor H-Fc fusion protein against multidrug-resistant Neisseria gonorrhoeae.

Novel therapeutics against the global threat of multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococci evade killing by complement by binding factor H (FH), a key inhibitor of the alternative pathway. FH comprises 20 short consensus repeat (SCR) domains organized as a single chain. Gonococci bind FH through domains 6 and 7, and C-terminal domains 18 through 20. Previously, we showed that a chimeric protein comprising (from the N- to C-terminus) FH domains 18-20 (containing a point mutation in domain 19 to prevent lysis of host cells) fused to human IgG1 Fc (called FH*/Fc1) killed gonococci in a complement-dependent manner and reduced the duration and bacterial burden in the mouse vaginal colonization model of gonorrhea. Considering the N. gonorrhoeae-binding FH domains 18-20 are C-terminal in native FH, we reasoned that positioning Fc N-terminal to FH* (Fc1/FH*) would improve binding and bactericidal activity. Although both molecules bound gonococci similarly, Fc1/FH* displayed a 5-fold lower IC50 (the concentration required for 50% killing in complement-dependent bactericidal assays) than FH*/Fc1. To further increase complement activation, we replaced human IgG1 Fc in Fc1/FH* with Fc from human IgG3, the most potent complement-activating IgG subclass, to obtain Fc3/FH*. Bactericidal activity was further increased ~2.3-fold in Fc3/FH* compared to Fc1/FH*. Fc3/FH* killed (defined by <50% survival) 45/45 (100%) diverse PorB1B-expessing gonococci, but only 2/15 PorB1A-expressing isolates, in a complement-dependent manner. Decreased Fc3/FH* binding accounted for the limited activity against PorB1A strains. Fc3/FH* was efficacious against all four tested PorB1B gonococcal strains in the mouse vaginal colonization model when administered at a dose of 5 µg intravaginally, daily. Furthermore, Fc3/FH* retained bactericidal activity when reconstituted following lyophilization or spray-drying, suggesting feasibility for formulation into intravaginal rings. In conclusion, Fc3/FH* represents a promising prophylactic immunotherapeutic against multidrug-resistant gonococci.

Mirels' score for upper limb metastatic lesions: do we need a different cutoff for recommending prophylactic fixation?

Hypothesis: The aim of this study was to investigate the reproducibility, reliability, and accuracy of Mirels' score in upper limb bony metastatic disease and validate its use in predicting pathologic fractures. Methods: Forty-five patients with upper limb bony metastases met the inclusion criteria (62% male 28/45). The mean age was 69 years (SD 9.5), and the most common primaries were lung (29%, 13/45), followed by prostate and hematological (each 20%, 9/45). The most commonly affected bone was the humerus (76%, 35/45), followed by the ulna (6.5%, 3/45). Mirels' score was calculated in 32 patients; with plain radiographs at index presentation scored using Mirels' system by 6 raters. The radiological aspects (lesion size and appearance) were scored twice by each rater (2 weeks apart). Intraobserver and interobserver reliability were calculated using Fleiss' kappa test. Bland-Altman plots compared the variances of both individual components and the total Mirels' score. Results: The overall fracture rate of upper limb metastatic lesions was 76% (35/46) with a mean follow-up of 3.6 years (range 11 months-6.8 years). Where time from diagnosis to fracture was known (n = 20), fractures occurred at a median 19 days (interquartile range 60-10), and 80% (16/20) occurred within 3 months of diagnosis.Mirels' score of ≥9 did not accurately predict lesions that fractured (fracture rate 11%, 5/46, for Mirels' ≥ 9 vs. 65%, 30/46, for Mirels' ≤ 8, P < .001). Sensitivity was 14%, and specificity was 73%. When Mirels' cutoff was lowered to ≥7, patients were more likely to fracture than not (48%, 22/46, vs. 28%, 13/46, P = .045); sensitivity rose to 63%, but specificity fell to 55%.Kappa values for interobserver variability were κ = 0.358 (fair, 95% confidence interval [CI] 0.288-0.429) for lesion size, κ = 0.107 (poor, 95% CI 0.02-0.193) for radiological appearance, and κ = 0.274 (fair, 95% CI 0.229-0.318) for total Mirels' score. Values for intraobserver variability were κ = 0.716 (good, 95% CI 0.432-0.999) for lesion size, κ = 0.427 (moderate, 95% CI 0.195-0.768) for radiological appearance, and κ = 0.580 (moderate, 95% CI 0.395-0.765) for total Mirels' score. Conclusions: This study demonstrates moderate to substantial agreement between and within raters using Mirels' score on upper limb radiographs. However, Mirels' score had a poor sensitivity and specificity in predicting upper extremity fractures. Until a more valid scoring system has been developed, based on our study, we recommend a Mirels' threshold of ≥7/12 for considering prophylactic fixation of impending upper limb pathologic fractures. This contrasts with the current ≥9/12 cutoff, which is recommended for lower limb pathologic fractures.

Response to Severe Acute Respiratory Syndrome Coronavirus 2 Initial Series and Additional Dose Vaccine in Patients With Predominant Antibody Deficiency.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in patients with predominant antibody deficiency (PAD) is associated with high morbidity, yet data regarding the response to SARS-CoV-2 immunization in PAD patients, including additional dose vaccine, are limited. OBJECTIVE: To characterize antibody response to SARS-CoV-2 vaccine in PAD patients and define correlates of vaccine response. METHODS: We assessed the levels and function of anti-SARS-CoV-2 antibodies in 62 PAD patients compared with matched healthy controls at baseline, at 4 to 6 weeks after the initial series of immunization (a single dose of Ad26.COV2.S [Janssen] or two doses of BNT162b2 [Pfizer-BioNTech] or mRNA-1273 [Moderna]), and at 4 to 6 weeks after an additional dose immunization, if received. RESULTS: After the initial series of SARS-CoV-2 vaccination, PAD patients had lower mean anti-spike antibody levels compared with matched healthy controls (140.1 vs 547.3 U/mL; P = .02). Patients with secondary PAD (eg, B-cell depletion therapy was used) and those with severe primary PAD (eg, common variable immunodeficiency with autoinflammatory complications) had the lowest mean anti-spike antibody levels. Immune correlates of a low anti-spike antibody response included low CD4+ T helper cells, low CD19+ total B cells, and low class-switched memory (CD27+IgD/M-) B cells. In addition, a low (<100 U/mL) anti-spike antibody response was associated with prior exposure to B-cell depletion therapy, both at any time in the past (odds ratio = 5.5; confidence interval, 1.5-20.4; P = .01) and proximal to vaccination (odds ratio = 36.4; confidence interval, 1.7-791.9; P = .02). Additional dose immunization with an mRNA vaccine in a subset of 31 PAD patients increased mean anti-spike antibody levels (76.3 U/mL before to 1065 U/mL after the additional dose; P < .0001). CONCLUSIONS: Patients with secondary and severe primary PAD, characterized by low T helper cells, low B cells, and/or low class-switched memory B cells, were at risk for low antibody response to SARS-CoV-2 immunization, which improved after an additional dose vaccination in most patients.

Progesterone Actions and Resistance in Gynecological Disorders.

Estrogen and progesterone and their signaling mechanisms are tightly regulated to maintain a normal menstrual cycle and to support a successful pregnancy. The imbalance of estrogen and progesterone disrupts their complex regulatory mechanisms, leading to estrogen dominance and progesterone resistance. Gynecological diseases are heavily associated with dysregulated steroid hormones and can induce chronic pelvic pain, dysmenorrhea, dyspareunia, heavy bleeding, and infertility, which substantially impact the quality of women's lives. Because the menstrual cycle repeatably occurs during reproductive ages with dynamic changes and remodeling of reproductive-related tissues, these alterations can accumulate and induce chronic and recurrent conditions. This review focuses on faulty progesterone signaling mechanisms and cellular responses to progesterone in endometriosis, adenomyosis, leiomyoma (uterine fibroids), polycystic ovary syndrome (PCOS), and endometrial hyperplasia. We also summarize the association with gene mutations and steroid hormone regulation in disease progression as well as current hormonal therapies and the clinical consequences of progesterone resistance.

CD97 is associated with mitogenic pathway activation, metabolic reprogramming, and immune microenvironment changes in glioblastoma.

Glioblastoma (GBM) is the most common primary brain tumor with a median survival under two years. Using in silico and in vitro techniques, we demonstrate heterogeneous expression of CD97, a leukocyte adhesion marker, in human GBM. Beyond its previous demonstrated role in tumor invasion, we show that CD97 is also associated with upregulation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways in GBM. While CD97 knockout decreased Akt activation, CD97 targeting did not alter MAPK/Erk activation, did not slow GBM cell proliferation in culture, and increased levels of glycolytic and oxidative phosphorylation metabolites. Treatment with a soluble CD97 inhibitor did not alter activation of the MAPK/Erk and PI3K/Akt pathways. Tumors with high CD97 expression were associated with immune microenvironment changes including increased naïve macrophages, regulatory T cells, and resting natural killer (NK) cells. These data suggest that, while CD97 expression is associated with conflicting effects on tumor cell proliferative and metabolic pathways that overall do not affect tumor cell proliferation, CD97 exerts pro-tumoral effects on the tumor immune microenvironment, which along with the pro-invasive effects of CD97 we previously demonstrated, provides impetus to continue exploring CD97 as a therapeutic target in GBM.

Vapor Cannabis Exposure Generationally Affects Male Reproductive Functions in Mice.

This study was performed to examine whether vapor exposure to cannabis plant matter negatively impacts male reproductive functions and testis development in mice. Adult CD-1 male mice (F0) were exposed to air (control) or 200 mg of vaporized cannabis plant matter 3×/day over a 10-day period. Subsequently, F0 males were bred with drug-naïve CD-1 females to generate F1 males, and F1 offspring were used to generate F2 males. Cannabis vapor exposure decreased sperm count and/or motility in F0 and F1 males and disrupted the progression of germ cell development, as morphometric analyses exhibited an abnormal distribution of the stages of spermatogenesis in F0 males. Although plasma levels of testosterone were not affected by cannabis exposure in any ages or generations of males, dysregulated steroidogenic enzymes, Cyp11a1 and Cyp19a1, were observed in F0 testis. In the neonatal testis from F1 males, although apoptosis was not altered, DNA damage and DNMT1, but not DNMT3A and DNMT3B, were increased in germ cells following cannabis exposure. In contrast, the alterations of DNA damage and DNMT1 expression were not observed in F2 neonatal males. These results suggest that cannabis vapor exposure generationally affects male reproductive functions, probably due to disruption of spermatogenesis in the developing testis.

Effects of Dietary Soy Protein Isolate Versus Isoflavones Alone on Poststroke Skilled Ladder Rung Walking and Cortical mRNA Expression Differ in Adult Male Rats.

Dietary soy protein isolate (SPI) and the isoflavones daidzein and genistein have been shown to provide neuroprotection from stroke. However, the mechanisms remain uncertain. We sought to determine whether the addition of isoflavones to a diet containing caseinate (CAS) as the protein source would induce behavioral neuroprotection similar to that seen previously in rats fed SPI. Furthermore, we aimed to characterize the baseline and poststroke expression of mRNAs involved in pathways previously published as perhaps mediating soy-based neuroprotection from stroke and other markers of neuronal plasticity, oxidative stress, and inflammation. Adult male rats were fed a semipurified diet containing (1) sodium caseinate (CAS), (2) CAS plus daidzein and genistein (CAS+ISO), or (3) SPI for 2 weeks. A subset of rats was euthanized, and tissue was collected for quantitative real-time PCR (qPCR). Remaining rats underwent a middle cerebral artery occlusion to induce a stroke. Samples for qPCR were collected on day 3 poststroke. Rats fed SPI made fewer errors on the skilled ladder rung walking task after stroke compared to rats fed CAS (P < .05). Rats fed CAS+ISO were not different from rats fed CAS or SPI. Significant effects of diet were found at day 0 for Syp, Pparg, and Ywhae and at day 3 for Rtn4 expression. We concluded that the benefits of SPI are not solely attributable to daidzein and genistein.

Anterior hip ultrasound: A useful technique in developmental dysplasia of the hips.

Introduction: Infant developmental dysplasia of the hips arises when there is an interruption to normal joint development, usually present at birth but may be a later development. It has a wide spectrum of severity with potentially disastrous long-term complications if left untreated. Incidence is highly variable, often being attributed to differences of opinion in definition and diagnosis; however, there is consensus that ultrasound be used in screening and management. Topic Description: The anterior hip ultrasound technique is underutilised but is of value due to the additional confidence it provides regarding joint stability. Discussion: The ability to relate the acquired ultrasound image to hip anatomy and a technical appreciation of the technique are crucial to successful use. The method is described and its use within previous literature briefly outlined. Using pictorial ultrasound imaging examples to demonstrate the technique, we aim to highlight the anterior approach as a useful addition to ultrasound assessment of infant developmental dysplasia of the hips. Conclusion: Knowledge and understanding of different techniques is essential for practitioners involved in the diagnosis and management of this multifaceted and vigorously debated condition.

Niclosamide's potential direct targets in ovarian cancer†.

Recent evidence indicates that niclosamide is an anti-cancer compound that is able to inhibit several signaling pathways. Although niclosamide has previously been identified by high-throughput screening platforms as a potential effective compound against several cancer types, no direct binding interactions with distinct biological molecule(s) has been established. The present study identifies key signal transduction mechanisms altered by niclosamide in ovarian cancer. Using affinity purification with a biotin-modified niclosamide derivative and mass spectrometry analysis, several RNA-binding proteins (RBPs) were identified. We chose the two RBPs, FXR1 and IGF2BP2, for further analysis. A significant correlation exists in which high-expression of FXR1 or IGF2BP2 is associated with reduced survival of ovarian cancer patients. Knockdown of FXR1 or IGF2BP2 in ovarian cancer cells resulted in significantly reduced cell viability, adhesion, and migration. Furthermore, FXR1 or IGF2BP2 deficient ovarian cancer cells exhibited reduced response to most doses of niclosamide showing greater cell viability than those with intact RBPs. These results suggest that FXR1 and IGF2BP2 are direct targets of niclosamide and could have critical activities that drive multiple oncogenic pathways in ovarian cancer.

Efficacy of niclosamide on the intra-abdominal inflammatory environment in endometriosis.

Endometriosis, a common gynecological disease, causes chronic pelvic pain and infertility in women of reproductive age. Due to the limited efficacy of current therapies, a critical need exists to develop new treatments for endometriosis. Inflammatory dysfunction, instigated by abnormal macrophage (MΦ) function, contributes to disease development and progression. However, the fundamental role of the heterogeneous population of peritoneal MΦ and their potential druggable functions is uncertain. Here we report that GATA6-expressing large peritoneal MΦ (LPM) were increased in the peritoneal cavity following lesion induction. This was associated with increased cytokine and chemokine secretion in the peritoneal fluid (PF), as well as MΦ infiltration, vascularization and innervation in endometriosis-like lesions (ELL). Niclosamide, an FDA-approved anti-helminthic drug, was effective in reducing LPM number, but not small peritoneal MΦ (SPM), in the PF. Niclosamide also inhibits aberrant inflammation in the PF, ELL, pelvic organs (uterus and vagina) and dorsal root ganglion (DRG), as well as MΦ infiltration, vascularization and innervation in the ELL. PF from ELL mice stimulated DRG outgrowth in vitro, whereas the PF from niclosamide-treated ELL mice lacked the strong stimulatory nerve growth response. These results suggest LPM induce aberrant inflammation in endometriosis promoting lesion progression and establishment of the inflammatory environment that sensitizes peripheral nociceptors in the lesions and other pelvic organs, leading to increased hyperalgesia. Our findings provide the rationale for targeting LPM and their functions with niclosamide and its efficacy in endometriosis as a new non-hormonal therapy to reduce aberrant inflammation which may ultimately diminish associated pain.

Insulin signaling is an essential regulator of endometrial proliferation and implantation in mice.

Insulin signaling is critical for the development of preovulatory follicles and progression through the antral stage. Using a conditional knockout model that escapes this blockage, we recently described the role of insulin signaling in granulosa cells during the periovulatory window in mice lacking Insr and Igf1r driven by Pgr-Cre. These mice were infertile, exhibiting defects in ovulation, luteinization, steroidogenesis, and early embryo development. Herein, we demonstrate that while these mice exhibit normal uterine receptivity, uterine cell proliferation and decidualization are compromised resulting in complete absence of embryo implantation in uteri lacking both receptors. While the histological organization of double knockout mice appeared normal, the thickness of their endometrium was significantly reduced. This was supported by the reduced proliferation of both epithelial and stromal cells during the preimplantation stages of pregnancy. Expression and localization of the main drivers of uterine proliferation, ESR1 and PGR, was normal in knockouts, suggesting that insulin signaling acts downstream of these two receptors. While AKT/PI3K signaling was unaffected by insulin receptor ablation, activation of p44/42 MAPK was significantly reduced in both single and double knockout uteri at 3.5 dpc. Overall, we conclude that both INSR and IGF1R are necessary for optimal endometrial proliferation and implantation.

Development of Complement Factor H-Based Immunotherapeutic Molecules in Tobacco Plants Against Multidrug-Resistant Neisseria gonorrhoeae.

Novel therapeutics against the global threat of multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococci possess several mechanisms to evade killing by human complement, including binding of factor H (FH), a key inhibitor of the alternative pathway. FH comprises 20 short consensus repeat (SCR) domains organized in a head-to-tail manner as a single chain. N. gonorrhoeae binds two regions in FH; domains 6 and 7 and domains 18 through 20. We designed a novel anti-infective immunotherapeutic molecule that fuses domains 18-20 of FH containing a D-to-G mutation in domain 19 at position 1119 (called FH*) with human IgG1 Fc. FH*/Fc retained binding to gonococci but did not lyse human erythrocytes. Expression of FH*/Fc in tobacco plants was undertaken as an alternative, economical production platform. FH*/Fc was expressed in high yields in tobacco plants (300-600 mg/kg biomass). The activities of plant- and CHO-cell produced FH*/Fc against gonococci were similar in vitro and in the mouse vaginal colonization model of gonorrhea. The addition of flexible linkers [e.g., (GGGGS)2 or (GGGGS)3] between FH* and Fc improved the bactericidal efficacy of FH*/Fc 2.7-fold. The linkers also improved PMN-mediated opsonophagocytosis about 11-fold. FH*/Fc with linker also effectively reduced the duration and burden of colonization of two gonococcal strains tested in mice. FH*/Fc lost efficacy: i) in C6-/- mice (no terminal complement) and ii) when Fc was mutated to abrogate complement activation, suggesting that an intact complement was necessary for FH*/Fc function in vivo. In summary, plant-produced FH*/Fc represent promising prophylactic or adjunctive immunotherapeutics against multidrug-resistant gonococci.

Niclosamide suppresses macrophage-induced inflammation in endometriosis†.

Endometriosis is a common gynecological disease, which causes chronic pelvic pain and infertility in women of reproductive age. Due to limited efficacy of current treatment options, a critical need exists to develop new and effective treatments for endometriosis. Niclosamide is an efficacious and FDA-approved drug for the treatment of helminthosis in humans that has been used for decades. We have reported that niclosamide reduces growth and progression of endometriosis-like lesions via targeting STAT3 and NFĸB signaling in a mouse model of endometriosis. To examine the effects of niclosamide on macrophage-induced inflammation in endometriosis, a total of 29 stage III-IV endometrioma samples were used to isolate human endometriotic stromal cells (hESCs). M1 or M2 macrophages were isolated and differentiated from fresh human peripheral blood samples. Then, hESCs were cultured in conditioned media (CM) from macrophages with/without niclosamide. Niclosamide dose dependently reduced cell viability and the activity of STAT3 and NFκB signaling in hESCs. While macrophage CM stimulated cell viability in hESCs, niclosamide inhibited this stimulation. Macrophage CM stimulated the secretion of proinflammatory cytokines and chemokines from hESCs. Most of these secreted factors were inhibited by niclosamide. These results indicate that niclosamide is able to reduce macrophage-induced cell viability and cytokine/chemokine secretion in hESCs by inhibiting inflammatory mechanisms via STAT3 and/or NFκB signaling.