Pain persists in mice lacking both Substance P and CGRPα signaling.

The neuropeptides Substance P and CGRPα have long been thought important for pain sensation. Both peptides and their receptors are expressed at high levels in pain-responsive neurons from the periphery to the brain making them attractive therapeutic targets. However, drugs targeting these pathways individually did not relieve pain in clinical trials. Since Substance P and CGRPα are extensively co-expressed we hypothesized that their simultaneous inhibition would be required for effective analgesia. We therefore generated Tac1 and Calca double knockout (DKO) mice and assessed their behavior using a wide range of pain-relevant assays. As expected, Substance P and CGRPα peptides were undetectable throughout the nervous system of DKO mice. To our surprise, these animals displayed largely intact responses to mechanical, thermal, chemical, and visceral pain stimuli, as well as itch. Moreover, chronic inflammatory pain and neurogenic inflammation were unaffected by loss of the two peptides. Finally, neuropathic pain evoked by nerve injury or chemotherapy treatment was also preserved in peptide-deficient mice. Thus, our results demonstrate that even in combination, Substance P and CGRPα are not required for the transmission of acute and chronic pain.

A unique case of Schistosoma-related ureteral stricture: Diagnosis and surgical reconstruction.

Background: Chronic infection with Schistosoma haematobium can lead to pathology of the upper and lower urinary tracts. While well known as a cause of squamous cell carcinoma of the bladder, relatively little research exists on ureteral involvement. Here, we present a unique case of bilateral ureteral obstruction from schistosomiasis with concomitant ureteral stone disease. Case presentation: A 43-year-old male Somalian immigrant was diagnosed with a right proximal ureteral stone and bilateral multifocal ureteral narrowing causing obstruction with preserved renal function. He underwent a staged repair with right robotic pyelolithotomy and non-transecting ureteroureterostomy, followed by left robotic ureteroureterostomy with stricture excision. Pathology revealed Schistosoma ova. Conclusion: Ureteral stricture from schistosomiasis represents a rare diagnosis for urologists in non-endemic countries. Bilateral ureteral narrowing and concomitant ureteral stone burden presented both diagnostic and reconstructive challenges, requiring a staged repair. Minimally invasive reconstruction was achieved using robotic assistance with good functional outcome.

Opportunistic Infections After Induction With Alemtuzumab or Basiliximab: A 3-Year Kidney Transplantation Experience.

BACKGROUND: Antibody induction immunosuppression is commonly used in kidney transplantation to decrease the risk of early acute rejection. However, infectious complications may arise in patients treated with higher intensity induction immunosuppression. In this study, we compared the rate of opportunistic infections during the 3 years after kidney transplantation in recipients who received either alemtuzumab or basiliximab for induction therapy. METHODS: All renal transplant recipients from our center who received induction with alemtuzumab between 2011 and 2016 were included and matched 1:2 (by age and date of transplant) to renal transplant recipients who received basiliximab. The primary outcome was the rate of opportunistic infections. RESULTS: Twenty-seven patients received alemtuzumab (mean age = 50.8 years; SD ±12), and 54 received basiliximab (mean age = 50.8 years; SD ±11.8). Infections within 3 years posttransplant were not different between groups: BK viremia (P = .99), BK nephritis (P = .48), cytomegalovirus infection (P = .13), varicella zoster virus (P = .22), and all infections (P = .87). Time to infection (P = .67), patient survival (P = .21), and time to rejection (P = .098) were similar in both groups. There were also no group differences in delayed graft function (P = .76), graft loss (P = .97), or rejection (P = .2). CONCLUSION: The rate of infection was not significantly increased in recipients receiving lymphocyte-depleting alemtuzumab compared to recipients receiving basiliximab induction therapy, despite receiving similar maintenance immunosuppression. Although the immunologic risks differed between the 2 groups, there was no observable difference in clinical outcomes.

Online Pricing in Urology: The Example of Male Fertility Care.

INTRODUCTION: Health care prices in the United States are often opaque to providers and patients while prices of nonhealth care services are displayed prominently across the Internet. Posting prices online will soon be a national requirement for hospitals, including for urological care. Male infertility care is rarely covered by insurance so many providers have developed cash prices to assist their patients. Expected success rate is also of interest to patients. The online availability of this information is unknown. METHODS: Membership databases of the Society for the Study of Male Reproduction and Society for Male Reproduction and Urology were searched to identify U.S. based clinical urologists. Websites were found with Google and analyzed for infertility services provided, prices and success rates. Specifically, websites were reviewed for information on any infertility care, vasectomy, vasectomy reversal, varicocele, sperm retrieval or microscopic testicular sperm extraction. RESULTS: A total of 222 individual providers were identified of which 204 had associated websites. Information about general male infertility services was readily available (85%), while specific procedures were described on 66% of websites or lower. Pricing information was available on 7.4% (15) of websites. Success rates were most frequently described for vasectomy reversal (23%). Pricing and success rates were more commonly found for private practice or personal websites. CONCLUSIONS: Pricing and success rates for male infertility are uncommonly listed on websites for U.S. male infertility providers. Several barriers may contribute including institutional policies and lack of physician input on website content. As pricing online becomes required, hospitals and likely urology practices will need to provide this information publicly.

Efficacy and risk of harms of repeat ivermectin mass drug administrations for control of malaria (RIMDAMAL): a cluster-randomised trial.

BACKGROUND: Ivermectin is widely used in mass drug administrations for controlling neglected parasitic diseases, and can be lethal to malaria vectors that bite treated humans. Therefore, it could be a new tool to reduce plasmodium transmission. We tested the hypothesis that frequently repeated mass administrations of ivermectin to village residents would reduce clinical malaria episodes in children and would be well tolerated with minimal harms. METHODS: We invited villages (clusters) in Burkina Faso to participate in a single-blind (outcomes assessor), parallel-assignment, two-arm, cluster-randomised trial over the 2015 rainy season. Villages were assigned (1:1) by random draw to either the intervention group or the control group. In both groups, all eligible participants who consented to the treatment and were at least 90 cm in height received single oral doses of ivermectin (150-200 µg/kg) and albendazole (400 mg), and those in the intervention group received five further doses of ivermectin alone at 3-week intervals thereafter over the 18-week treatment phase. The primary outcome was cumulative incidence of uncomplicated malaria episodes over 18 weeks (analysed on a cluster intention-to-treat basis) in an active case detection cohort of children aged 5 years or younger living in the study villages. This trial is registered with ClinicalTrials.gov, number NCT02509481. FINDINGS: Eight villages agreed to participate, and four were randomly assigned to each group. 2712 participants (1333 [49%] males and 1379 [51%] females; median age 15 years [IQR 6-34]), including 590 children aged 5 years or younger, provided consent and were enrolled between May 22 and July 20, 2015 (except for 77 participants enrolled after these dates because of unavailability before the first mass drug administration, travel into the village during the trial, or birth), with 1447 enrolled into the intervention group and 1265 into the control group. 330 (23%) participants in the intervention group and 233 (18%) in the control group met the exclusion criteria for mass drug administration. Most children in the active case detection cohort were not treated because of height restrictions. 14 (4%) children in the intervention group and 10 (4%) in the control group were lost to follow-up. Cumulative malaria incidence was reduced in the intervention group (648 episodes among 327 children; estimated mean 2·00 episodes per child) compared with the control group (647 episodes among 263 children; 2·49 episodes per child; risk difference -0·49 [95% CI -0·79 to -0·21], p=0·0009, adjusted for sex and clustering). The risk of adverse events among all participants did not differ between groups (45 events [3%] among 1447 participants in the intervention group vs 24 events [2%] among 1265 in the control group; risk ratio 1·63 [1·01 to 2·67]; risk difference 1·21 [0·04 to 2·38], p=0·060), and no adverse reactions were reported. INTERPRETATION: Frequently repeated mass administrations of ivermectin during the malaria transmission season can reduce malaria episodes among children without significantly increasing harms in the populace. FUNDING: Bill & Melinda Gates Foundation.

Engineering of a GLP-1 analogue peptide/anti-PCSK9 antibody fusion for type 2 diabetes treatment.

Type 2 diabetes (T2D) is a complex and progressive disease requiring polypharmacy to manage hyperglycaemia and cardiovascular risk factors. However, most patients do not achieve combined treatment goals. To address this therapeutic gap, we have developed MEDI4166, a novel glucagon-like peptide-1 (GLP-1) receptor agonist peptide fused to a proprotein convertase subtilisin/kexin type 9 (PCSK9) neutralising antibody that allows for glycaemic control and low-density lipoprotein cholesterol (LDL-C) lowering in a single molecule. The fusion has been engineered to deliver sustained peptide activity in vivo in combination with reduced potency, to manage GLP-1 driven adverse effects at high dose, and a favourable manufacturability profile. MEDI4166 showed robust and sustained LDL-C lowering in cynomolgus monkeys and exhibited the anticipated GLP-1 effects in T2D mouse models. We believe MEDI4166 is a novel molecule combining long acting agonist peptide and neutralising antibody activities to deliver a unique pharmacology profile for the management of T2D.

Characterization and application of two RANK-specific antibodies with different biological activities.

Antibodies play an important role in therapy and investigative biomedical research. The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation and epithelial cell activation. However, the study of RANK biology has been hampered by missing or insufficient characterization of high affinity tools that recognize RANK. Here, we present a careful description and comparison of two antibodies, RANK-02 obtained by phage display (Newa, 2014 [1]) and R12-31 generated by immunization (Kamijo, 2006 [2]). We found that both antibodies recognized mouse RANK with high affinity, while RANK-02 and R12-31 recognized human RANK with high and lower affinities, respectively. Using a cell apoptosis assay based on stimulation of a RANK:Fas fusion protein, and a cellular NF-κB signaling assay, we showed that R12-31 was agonist for both species. R12-31 interfered little or not at all with the binding of RANKL to RANK, in contrast to RANK-02 that efficiently prevented this interaction. Depending on the assay and species, RANK-02 was either a weak agonist or a partial antagonist of RANK. Both antibodies recognized human Langerhans cells, previously shown to express RANK, while dermal dendritic cells were poorly labeled. In vivo R12-31 agonist activity was demonstrated by its ability to induce the formation of intestinal villous microfold cells in mice. This characterization of two monoclonal antibodies should now allow better evaluation of their application as therapeutic reagents and investigative tools.

Age and prior blood feeding of Anopheles gambiae influences their susceptibility and gene expression patterns to ivermectin-containing blood meals.

BACKGROUND: Ivermectin has been proposed as a novel malaria transmission control tool based on its insecticidal properties and unique route of acquisition through human blood. To maximize ivermectin's effect and identify potential resistance/tolerance mechanisms, it is important to understand its effect on mosquito physiology and potential to shift mosquito population age-structure. We therefore investigated ivermectin susceptibility and gene expression changes in several age groups of female Anopheles gambiae mosquitoes. METHODS: The effect of aging on ivermectin susceptibility was analyzed in three age groups (2, 6, and 14-days) of colonized female Anopheles gambiaemosquitoes using standard survivorship assays. Gene expression patterns were then analyzed by transcriptome sequencing on an Illumina HiSeq 2500 platform. RT-qPCR was used to validate transcriptional changes and also to examine expression in a different, colonized strain and in wild mosquitoes, both of which blood fed naturally on an ivermectin-treated person. RESULTS: Mosquitoes of different ages and blood meal history died at different frequencies after ingesting ivermectin. Mortality was lowest in 2-day old mosquitoes exposed on their first blood meal and highest in 6-day old mosquitoes exposed on their second blood meal. Twenty-four hours following ivermectin ingestion, 101 and 187 genes were differentially-expressed relative to control blood-fed, in 2 and 6-day groups, respectively. Transcription patterns of select genes were similar in membrane-fed, colonized, and naturally-fed wild vectors. Transcripts from several unexpected functional classes were highly up-regulated, including Niemann-Pick Type C (NPC) genes, peritrophic matrix-associated genes, and immune-response genes, and these exhibited different transcription patterns between age groups, which may explain the observed susceptibility differences. Niemann-Pick Type 2 genes were the most highly up-regulated transcripts after ivermectin ingestion (up to 160 fold) and comparing phylogeny to transcriptional patterns revealed that NPCs have rapidly evolved and separate members respond to either blood meals or to ivermectin. CONCLUSION: We present evidence of increased ivermectin susceptibility in older An. gambiae mosquitoes that had previously bloodfed. Differential expression analysis suggests complex midgut interactions resulting from ivermectin ingestion that likely involve blood meal digestion physiological responses, midgut microflora, and innate immune responses. Thus, the transcription of certain gene families is consistently affected by ivermectin ingestion, and may provide important clues to ivermectin's broad effects on malaria vectors. These findings contribute to the growing understanding of ivermectin's potential as a transmission control tool.

Arabinose and protocatechuate catabolism genes are important for growth of Rhizobium leguminosarum biovar viciae in the pea rhizosphere.

BACKGROUND AND AIMS: To form nitrogen-fixing nodules on pea roots, Rhizobium leguminosarum biovar viciae must be competitive in the rhizosphere. Our aim was to identify genes important for rhizosphere fitness. METHODS: Signature-tagged mutants were screened using microarrays to identify mutants reduced for growth in pea rhizospheres. Candidate mutants were assessed relative to controls for growth in minimal medium, growth in pea rhizospheres and for infection of peas in mixed inoculants. Mutated genes were identified by DNA sequencing and confirmed by transduction. RESULTS: Of 5508 signature-tagged mutants, microarrays implicated 50 as having decreased rhizosphere fitness. Growth tests identified six mutants with rhizosphere-specific phenotypes. The mutation in one of the genes (araE) was in an arabinose catabolism operon and blocked growth on arabinose. The mutation in another gene (pcaM), encoding a predicted solute binding protein for protocatechuate and hydroxybenzoate uptake, decreased growth on protocatechuate. Both mutants were decreased for nodule infection competitiveness with mixed inoculants, but nodulated peas normally when inoculated alone. Other mutants with similar phenotypes had mutations predicted to affect secondary metabolism. CONCLUSIONS: Catabolism of arabinose and protocatechuate in the pea rhizosphere is important for competitiveness of R.l. viciae. Other genes predicted to be involved in secondary metabolism are also important.

Sampling host-seeking anthropophilic mosquito vectors in west Africa: comparisons of an active human-baited tent-trap against gold standard methods.

In this study, we characterize the ability of the previously described Infoscitex tent (IST) to capture mosquitoes in comparison to either the Centers for Disease Control Light Trap hung next to individuals under a bed net (LTC) or to human landing catches (HLC). In Senegal, the IST caught 6.14 times the number of Anopheles gambiae sensu lato (s.l.), and 8.78 times the Culex group V mosquitoes as LTC. In one of two locations in Burkina Faso, the IST caught An. gambiae at a rate not significantly different than HLC. Of importance, 9.1-36.1% of HLC caught An. gambiae were blood fed, mostly with fresh blood, suggesting they fed upon the collector, whereas only 0.5-5.0% from the IST had partial or old blood. The IST also caught outdoor biting species in proportions comparable to HLC. The results show this tent provides a safer and effective alternative to the skill-dependent, risky, and laborious HLC method.

Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1.

Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding VH CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long VH CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long VH CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.

Design and testing of a novel, protective human-baited tent trap for the collection of anthropophilic disease vectors.

Currently, there exists a deficit of safe, active trapping methods for the collection of host-seeking Anopheles and other disease-causing arthropod vectors. The gold-standard approach for mosquito collection is that of human landing catch (HLC), in which an individual exposes bare skin to possibly infected vectors. Here, we present the development of a new method for mosquito collection, the Infoscitex tent, which uses modern tent materials coupled with a novel trap design. This provides an efficacious, a non-labor-intensive, and a safe method for vector collection. In these initial studies, we found it collected an average of 27.7 Anopheles gambiae s.l. per trap per night in rural villages in southeastern Senegal, and 43.8 Culex group Vper trap per night in the semiurban town of Kedougou, Senegal. In direct comparisons with HLC, the tent was not statistically different for collection of Culex quinquefasciatus in crepuscular sampling, but was significantly less efficacious at trapping the highly motile dusk-biter Aedes aegypti. These studies suggest that the Infoscitex tent is a viable and safe alternative to HLC for Anopheles and Culex sampling in areas of high vector-borne disease infection risk.