RNA-seq gene expression profiling of the bladder in a mouse model of urogenital schistosomiasis.

Background: Parasitic flatworms of the Schistosoma genus cause schistosomiasis, which affects over 230 million people. Schistosoma haematobium causes the urogenital form of schistosomiasis (UGS), which can lead to hematuria, fibrosis, and increased risk of secondary infections by bacteria or viruses. UGS is also linked to bladder cancer. To understand the bladder pathology during S. haematobium infection, our group previously developed a mouse model that involves the injection of S. haematobium eggs into the bladder wall. Using this model, we studied changes in epigenetics profile, as well as changes in gene and protein expression in the host bladder tissues. In the current study, we expand upon this work by examining the expression level of both host and parasite genes using RNA sequencing (RNA-seq) in the mouse bladder wall injection model of S. haematobium infection. Methods: We used a mouse model of S. haematobium infection in which parasite eggs or vehicle control were injected into the bladder walls of female BALB/c mice. RNA-seq was performed on the RNA isolated from the bladders four days after bladder wall injection. Results/Conclusions: RNA-seq analysis of egg- and vehicle control-injected bladders revealed the differential expression of 1025 mouse genes in the egg-injected bladders, including genes associated with cellular infiltration, immune cell chemotaxis, cytokine signaling, and inflammation We also observed the upregulation of immune checkpoint-related genes, which suggests that while the infection causes an inflammatory response, it also dampens the response to avoid excessive inflammation-related damage to the host. Identifying these changes in host signaling and immune responses improves our understanding of the infection and how it may contribute to the development of bladder cancer. Analysis of the differential gene expression of the parasite eggs between bladder-injected versus uninjected eggs revealed 119 S. haematobium genes associated with transcription, intracellular signaling, and metabolism. The analysis of the parasite genes also revealed fewer transcript reads compared to that found in the analysis of mouse genes, highlighting the challenges of studying parasite egg biology in the mouse model of S. haematobium infection. Author summary: More than 230 million people worldwide are estimated to carry infection with parasites belonging to the Schistosoma genus, which cause morbidity associated with parasite egg deposition. Praziquantel, the drug of choice to treat the infection, does not prevent reinfection, and its decades-long history as the main treatment raises concerns for drug resistance. Of the schistosome species, Schistosoma haematobium causes urogenital disease and has a strong association with bladder cancer. The possibility for drug resistance and the gap in knowledge with respect to the mechanisms driving S. haematobium -related bladder cancer highlight the need to better understand the biology of the infection to aid in the development of new therapeutic strategies. In this study, we used a mouse model of S. haematobium infection that delivers parasite eggs directly to the host mouse bladder wall, and we examined the changes in the gene expression profile of the host and the parasite by RNA-sequencing. The results corroborated previous findings with respect to the host's inflammatory responses against the parasite eggs, as well as revealed alterations in other immune response genes that deepen our understanding of the mechanisms involved in urogenital schistosomiasis pathogenesis.

A Camelid-Derived STAT-Specific Nanobody Inhibits Neuroinflammation and Ameliorates Experimental Autoimmune Encephalomyelitis (EAE).

Proinflammatory T-lymphocytes recruited into the brain and spinal cord mediate multiple sclerosis (MS) and currently there is no cure for MS. IFN-γ-producing Th1 cells induce ascending paralysis in the spinal cord while IL-17-producing Th17 cells mediate cerebellar ataxia. STAT1 and STAT3 are required for Th1 and Th17 development, respectively, and the simultaneous targeting of STAT1 and STAT3 pathways is therefore a potential therapeutic strategy for suppressing disease in the spinal cord and brain. However, the pharmacological targeting of STAT1 and STAT3 presents significant challenges because of their intracellular localization. We have developed a STAT-specific single-domain nanobody (SBT-100) derived from camelids that targets conserved residues in Src homolog 2 (SH2) domains of STAT1 and STAT3. This study investigated whether SBT-100 could suppress experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We show that SBT-100 ameliorates encephalomyelitis through suppressing the expansion of Th17 and Th1 cells in the brain and spinal cord. Adoptive transfer experiments revealed that lymphocytes from SBT-100-treated EAE mice have reduced capacity to induce EAE, indicating that the immunosuppressive effects derived from the direct suppression of encephalitogenic T-cells. The small size of SBT-100 makes this STAT-specific nanobody a promising immunotherapy for CNS autoimmune diseases, including multiple sclerosis.

MAFB in macrophages regulates cold-induced neuronal density in brown adipose tissue.

Transcription factor MAFB regulates various homeostatic functions of macrophages. This study explores the role of MAFB in brown adipose tissue (BAT) thermogenesis using macrophage-specific Mafb-deficient (Mafbf/f::LysM-Cre) mice. We find that Mafb deficiency in macrophages reduces thermogenesis, energy expenditure, and sympathetic neuron (SN) density in BAT under cold conditions. This phenotype features a proinflammatory environment that is characterized by macrophage/granulocyte accumulation, increases in interleukin-6 (IL-6) production, and IL-6 trans-signaling, which lead to decreases in nerve growth factor (NGF) expression and reduction in SN density in BAT. We confirm MAFB regulation of IL-6 expression using luciferase readout driven by IL-6 promoter in RAW-264.7 macrophage cell lines. Immunohistochemistry shows clustered organization of NGF-producing cells in BAT, which are primarily TRPV1+ vascular smooth muscle cells, as additionally shown using single-cell RNA sequencing and RT-qPCR of the stromal vascular fraction. Treating Mafbf/f::LysM-Cre mice with anti-IL-6 receptor antibody rescues SN density, body temperature, and energy expenditure.

IL-27-containing exosomes secreted by innate B-1a cells suppress and ameliorate uveitis.

Introduction: IL-27 is a heterodimeric cytokine composed of Ebi3 and IL-27p28 and can exert proinflammatory or immune suppressive effects depending on the physiological context. Ebi3 does not contain membrane-anchoring motifs, suggesting that it is a secreted protein while IL-27p28 is poorly secreted. How IL-27p28 and Ebi3 dimerize in-vivo to form biologically active IL-27 is unknown. Major impediment to clinical use of IL-27 derives from difficulty of determining exact amount of bioavailable heterodimeric IL-27 needed for therapy. Methods: To understand how IL-27 mediates immune suppression, we characterized an innate IL-27-producing B-1a regulatory B cell population (i27-Breg) and mechanisms i27-Bregs utilize to suppress neuroinflammation in mouse model of uveitis. We also investigated biosynthesis of IL-27 and i27-Breg immunobiology by FACS, immunohistochemical and confocal microscopy. Results: Contrary to prevailing view that IL-27 is a soluble cytokine, we show that i27-Bregs express membrane-bound IL-27. Immunohistochemical and confocal analyses co-localized expression of IL-27p28 at the plasma membrane in association with CD81 tetraspanin, a BCR-coreceptor protein and revealed that IL-27p28 is a transmembrane protein in B cells. Most surprising, we found that i27-Bregs secrete IL-27-containing exosomes (i27-exosomes) and adoptive transfer of i27-exosomes suppressed uveitis by antagonizing Th1/Th17 cells, up-regulating inhibitory-receptors associated with T-cell exhaustion while inducing Treg expansion. Discussion: Use of i27-exosomes thus obviates the IL-27 dosing problem, making it possible to determine bioavailable heterodimeric IL-27 needed for therapy. Moreover, as exosomes readily cross the blood-retina-barrier and no adverse effects were observed in mice treated with i27-exosome, results of this study suggest that i27-exosomes might be a promising therapeutic approach for CNS autoimmune diseases.

STAT3-Specific Single Domain Nanobody Inhibits Expansion of Pathogenic Th17 Responses and Suppresses Uveitis in Mice.

STAT3 activates transcription of genes that regulate cell growth, differentiation, and survival of mammalian cells. Genetic deletion of Stat3 in T cells has been shown to abrogate Th17 differentiation, suggesting that STAT3 is a potential therapeutic target for Th17-mediated diseases. However, a major impediment to therapeutic targeting of intracellular proteins such as STAT3 is the lack of efficient methods for delivering STAT3 inhibitors into cells. In this study, we developed a novel antibody (SBT-100) comprised of the variable (V) region of a STAT3-specific heavy chain molecule and demonstrate that this 15 kDa STAT3-specific nanobody enters human and mouse cells, and induced suppression of STAT3 activation and lymphocyte proliferation in a concentration-dependent manner. To investigate whether SBT-100 would be effective in suppressing inflammation in vivo, we induced experimental autoimmune uveitis (EAU) in C57BL/6J mice by active immunization with peptide from the ocular autoantigen, interphotoreceptor retinoid binding protein (IRBP651-670). Analysis of the retina by fundoscopy, histological examination, or optical coherence tomography showed that treatment of the mice with SBT-100 suppressed uveitis by inhibiting expansion of pathogenic Th17 cells that mediate EAU. Electroretinographic (ERG) recordings of dark and light adapted a- and b-waves showed that SBT-100 treatment rescued mice from developing significant visual impairment observed in untreated EAU mice. Adoptive transfer of activated IRBP-specific T cells from untreated EAU mice induced EAU, while EAU was significantly attenuated in mice that received IRBP-specific T cells from SBT-100 treated mice. Taken together, these results demonstrate efficacy of SBT-100 in mice and suggests its therapeutic potential for human autoimmune diseases.

Varenicline limits ischemia reperfusion injury following testicular torsion in mice.

BACKGROUND: Torsion of the spermatic cord and the resulting testicular ischemia leads to the production of inflammatory cytokines and cell death due to impaired aerobic metabolism. Following reperfusion of the testis, a robust innate inflammatory response furthers tissue injury due to the production of reactive oxygen species and disruption of normal capillary function. Blunting the innate immune response with antioxidants, anti-inflammatory medications and targeted genetic interventions reduces long term testicular injury in animal models of torsion, however these approaches have limited clinical applicability. Mediated via α7 nACh receptors, the cholinergic anti-inflammatory pathway limits NFKB signaling and prevents renal fibrosis following warm renal ischemia. We identified varenicline as an FDA approved α7 nAChR agonist and hypothesized that varenicline administration would decrease long-term testicular atrophy and fibrosis in a murine model of testicular torsion. METHODS: Using an established model, unilateral testicular torsion was induced in mature male CD1 mice by rotating the right testicle 720° for 2 h. In the treatment group, 4 doses of varenicline (1mg/grm) were administered via intraperitoneal injection every 12 h, with the first dose given 1 h after the creation of testicular torsion. The acute inflammatory response was evaluated 48 h following reperfusion of the testis. Long term outcomes were evaluated 30 days following testicular perfusion. RESULTS: 48 h following reperfusion, the testis of animals treated with varenicline demonstrated a significant reduction in the inflammatory response as measured by the acute immune cell infiltrate, myeloperoxidase activity, concentration of reduced glutathione and expression of downstream NF-KB targets. 30 days following reperfusion, animals treated with varenicline, demonstrated decreased testicular atrophy (Summary Figure), fibrosis and expression of pro-fibrotic genes. CONCLUSION: Activation of a central immunosuppressive cascade with varenicline after the onset of testicular torsion reduces ischemia reperfusion injury and prevents long term testicular atrophy and fibrosis. Further studies are needed to define the optimum dose and varenicline administration regimen. Our results suggest that varenicline offers a novel, FDA approved, adjunct to the current management of testicular torsion.

Uveitis: Molecular Pathogenesis and Emerging Therapies.

The profound impact that vision loss has on human activities and quality of life necessitates understanding the etiology of potentially blinding diseases and their clinical management. The unique anatomic features of the eye and its sequestration from peripheral immune system also provides a framework for studying other diseases in immune privileged sites and validating basic immunological principles. Thus, early studies of intraocular inflammatory diseases (uveitis) were at the forefront of research on organ transplantation. These studies laid the groundwork for foundational discoveries on how immune system distinguishes self from non-self and established current concepts of acquired immune tolerance and autoimmunity. Our charge in this review is to examine how advances in molecular cell biology and immunology over the past 3 decades have contributed to the understanding of mechanisms that underlie immunopathogenesis of uveitis. Particular emphasis is on how advances in biotechnology have been leveraged in developing biologics and cell-based immunotherapies for uveitis and other neuroinflammatory diseases.

Multilevel modelling of the risk of malaria among children aged under five years in Nigeria.

BACKGROUND: Malaria is still a major cause of morbidity and mortality among children aged <5 y (U5s). This study assessed individual, household and community risk factors for malaria in Nigerian U5s. METHODS: Data from the Nigerian Malaria Health Indicator Survey 2015 were pooled for analyses. This comprised a national survey of 329 clusters. Children aged 6-59 mo who were tested for malaria using microscopy were retained. Multilevel logit model accounting for sampling design was used to assess individual, household and community factors associated with malaria parasitaemia. RESULTS: A total of 5742 children were assessed for malaria parasitaemia with an overall prevalence of 27% (95% CI 26 to 28%). Plasmodium falciparum constituted 98% of the Plasmodium species. There was no significant difference in parasitaemia between older children and those aged ≤12 mo. In adjusted analyses, rural living, northwest region, a household size of >7, dependence on river and rainwater as primary water source were associated with higher odds of parasitaemia, while higher wealth index, all U5s who slept under a bed net and dependence on packaged water were associated with lower odds of parasitaemia. CONCLUSION: Despite sustained investment in malaria control and prevention, a quarter of the overall study population of U5s have malaria. Across the six geopolitical zones, the highest burden was in children living in the poorest rural households.

IPSE, a parasite-derived, host immunomodulatory infiltrin protein, alleviates resiniferatoxin-induced bladder pain.

The transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor is an important mediator of nociception and its expression is enriched in nociceptive neurons. TRPV1 signaling has been implicated in bladder pain and is a potential analgesic target. Resiniferatoxin is the most potent known agonist of TRPV1. Acute exposure of the rat bladder to resiniferatoxin has been demonstrated to result in pain-related freezing and licking behaviors that are alleviated by virally encoded IL-4. The interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE) is a powerful inducer of IL-4 secretion, and is also known to alter host cell transcription through a nuclear localization sequence-based mechanism. We previously reported that IPSE ameliorates ifosfamide-induced bladder pain in an IL-4- and nuclear localization sequence-dependent manner. We hypothesized that pre-administration of IPSE to resiniferatoxin-challenged mice would dampen pain-related behaviors. IPSE indeed lessened resiniferatoxin-triggered freezing behaviors in mice. This was a nuclear localization sequence-dependent phenomenon, since administration of a nuclear localization sequence mutant version of IPSE abrogated IPSE's analgesic effect. In contrast, IPSE's analgesic effect did not seem IL-4-dependent, since use of anti-IL-4 antibody in mice given both IPSE and resiniferatoxin did not significantly affect freezing behaviors. RNA-Seq analysis of resiniferatoxin- and IPSE-exposed bladders revealed differential expression of TNF/NF-κb-related signaling pathway genes. In vitro testing of IPSE uptake by urothelial cells and TRPV1-expressing neuronal cells showed uptake by both cell types. Thus, IPSE's nuclear localization sequence-dependent therapeutic effects on TRPV1-mediated bladder pain may act on TRPV1-expressing neurons and/or may rely upon urothelial mechanisms.

IPSE, a urogenital parasite-derived immunomodulatory molecule, suppresses bladder pathogenesis and anti-microbial peptide gene expression in bacterial urinary tract infection.

BACKGROUND: Parasitic infections can increase susceptibility to bacterial co-infections. This may be true for urogenital schistosomiasis and bacterial urinary tract co-infections (UTI). We previously reported that this co-infection is facilitated by S. haematobium eggs triggering interleukin-4 (IL-4) production and sought to dissect the underlying mechanisms. The interleukin-4-inducing principle from Schistosoma mansoni eggs (IPSE) is one of the most abundant schistosome egg-secreted proteins and binds to IgE on the surface of basophils and mast cells to trigger IL-4 release. IPSE can also translocate into host nuclei using a nuclear localization sequence (NLS) to modulate host transcription. We hypothesized that IPSE is the factor responsible for the ability of S. haematobium eggs to worsen UTI pathogenesis. METHODS: Mice were intravenously administered a single 25 µg dose of recombinant S. haematobium-derived IPSE, an NLS mutant of IPSE or PBS. Following IPSE exposure, mice were serially weighed and organs analyzed by histology to assess for toxicity. Twenty-four hours after IPSE administration, mice were challenged with the uropathogenic E. coli strain UTI89 by urethral catheterization. Bacterial CFU were measured using urine. Bladders were examined histologically for UTI-triggered pathogenesis and by PCR for antimicrobial peptide and pattern recognition receptor expression. RESULTS: Unexpectedly, IPSE administration did not result in significant differences in urine bacterial CFU. However, IPSE administration did lead to a significant reduction in UTI-induced bladder pathogenesis and the expression of anti-microbial peptides in the bladder. Despite the profound effect of IPSE on UTI-triggered bladder pathogenesis and anti-microbial peptide production, mice did not demonstrate systemic ill effects from IPSE exposure. CONCLUSIONS: Our data show that IPSE may play a major role in S. haematobium-associated urinary tract co-infection, albeit in an unexpected fashion. These findings also indicate that IPSE either works in concert with other IL-4-inducing factors to increase susceptibility of S. haematobium-infected hosts to bacterial co-infection or does not contribute to enhancing vulnerability to this co-infection.

IPSE, an abundant egg-secreted protein of the carcinogenic helminth Schistosoma haematobium, promotes proliferation of bladder cancer cells and angiogenesis.

BACKGROUND: Schistosoma haematobium, the helminth causing urogenital schistosomiasis, is a known bladder carcinogen. Despite the causal link between S. haematobium and bladder cancer, the underlying mechanisms are poorly understood. S. haematobium oviposition in the bladder is associated with angiogenesis and urothelial hyperplasia. These changes may be pre-carcinogenic events in the bladder. We hypothesized that the Interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE), an S. haematobium egg-secreted "infiltrin" protein that enters host cell nuclei to alter cellular activity, is sufficient to induce angiogenesis and urothelial hyperplasia. Methods: Mouse bladders injected with S. haematobium eggs were analyzed via microscopy for angiogenesis and urothelial hyperplasia. Endothelial and urothelial cell lines were incubated with recombinant IPSE protein or an IPSE mutant protein that lacks the native nuclear localization sequence (NLS-) and proliferation measured using CFSE staining and real-time monitoring of cell growth. IPSE's effects on urothelial cell cycle status was assayed through propidium iodide staining. Endothelial and urothelial cell uptake of fluorophore-labeled IPSE was measured. Findings: Injection of S. haematobium eggs into the bladder triggers angiogenesis, enhances leakiness of bladder blood vessels, and drives urothelial hyperplasia. Wild type IPSE, but not NLS-, increases proliferation of endothelial and urothelial cells and skews urothelial cells towards S phase. Finally, IPSE is internalized by both endothelial and urothelial cells. Interpretation: IPSE drives endothelial and urothelial proliferation, which may depend on internalization of the molecule. The urothelial effects of IPSE depend upon its NLS. Thus, IPSE is a candidate pro-carcinogenic molecule of S. haematobium. SUMMARY: Schistosoma haematobium acts as a bladder carcinogen through unclear mechanisms. The S. haematobium homolog of IPSE, a secreted schistosome egg immunomodulatory molecule, enhances angiogenesis and urothelial proliferation, hallmarks of pre-carcinogenesis, suggesting IPSE is a key pro-oncogenic molecule of S. haematobium.

Interleukin-4 Signaling Plays a Major Role in Urogenital Schistosomiasis-Associated Bladder Pathogenesis.

Interleukin-4 (IL-4) is crucial in many helminth infections, but its role in urogenital schistosomiasis, infection with Schistosoma haematobium worms, remains poorly understood due to a historical lack of animal models. The bladder pathology of urogenital schistosomiasis is caused by immune responses to eggs deposited in the bladder wall. A range of pathology occurs, including urothelial hyperplasia and cancer, but associated mechanisms and links to IL-4 are largely unknown. We modeled urogenital schistosomiasis by injecting the bladder walls of IL-4 receptor-alpha knockout (Il4ra-/- ) and wild-type mice with S. haematobium eggs. Readouts included bladder histology and ex vivo assessments of urothelial proliferation, cell cycle, and ploidy status. We also quantified the effects of exogenous IL-4 on urothelial cell proliferation in vitro, including cell cycle status and phosphorylation patterns of major downstream regulators in the IL-4 signaling pathway. There was a significant decrease in the intensity of granulomatous responses to bladder-wall-injected S. haematobium eggs in Il4ra-/- versus wild-type mice. S. haematobium egg injection triggered significant urothelial proliferation, including evidence of urothelial hyper-diploidy and cell cycle skewing in wild-type but not Il4ra-/- mice. Urothelial exposure to IL-4 in vitro led to cell cycle polarization and increased phosphorylation of AKT. Our results show that IL-4 signaling is required for key pathogenic features of urogenital schistosomiasis and that particular aspects of this signaling pathway may exert these effects directly on the urothelium. These findings point to potential mechanisms by which urogenital schistosomiasis promotes bladder carcinogenesis.

IPSE, a parasite-derived host immunomodulatory protein, is a potential therapeutic for hemorrhagic cystitis.

Chemotherapy-induced hemorrhagic cystitis is characterized by bladder pain and voiding dysfunction caused by hemorrhage and inflammation. Novel therapeutic options to treat hemorrhagic cystitis are needed. We previously reported that systemic administration of the Schistosomiasis hematobium-derived protein H-IPSEH06 (IL-4-inducing principle from Schistosoma mansoni eggs) is superior to three doses of MESNA in alleviating hemorrhagic cystitis (Mbanefo EC, Le L, Pennington LF, Odegaard JI, Jardetzky TS, Alouffi A, Falcone FH, Hsieh MH. FASEB J 32: 4408-4419, 2018). Based on prior reports by others on S. mansoni IPSE (M-IPSE) and additional work by our group, we reasoned that H-IPSE mediates its effects on hemorrhagic cystitis by binding IgE on basophils and inducing IL-4 expression, promoting urothelial proliferation, and translocating to the nucleus to modulate expression of genes implicated in relieving bladder dysfunction. We speculated that local bladder injection of the S. hematobium IPSE ortholog IPSEH03, hereafter called H-IPSEH03, might be more efficacious in preventing hemorrhagic cystitis compared with systemic administration of IPSEH06. We report that H-IPSEH03, like M-IPSE and H-IPSEH06, activates IgE-bearing basophils in a nuclear factor of activated T-cells reporter assay, indicating activation of the cytokine pathway. Furthermore, H-IPSEH03 attenuates ifosfamide-induced increases in bladder wet weight in an IL-4-dependent fashion. H-IPSEH03 relieves hemorrhagic cystitis-associated allodynia and modulates voiding patterns in mice. Finally, H-IPSEH03 drives increased urothelial cell proliferation, suggesting that IPSE induces bladder repair mechanisms. Taken together, H-IPSEH03 may be a potential novel therapeutic to treat hemorrhagic cystitis by basophil activation, attenuation of allodynia, and promotion of urothelial cell proliferation.

IPSE, a urogenital parasite-derived immunomodulatory protein, ameliorates ifosfamide-induced hemorrhagic cystitis through downregulation of pro-inflammatory pathways.

Ifosfamide and other oxazaphosphorines can result in hemorrhagic cystitis, a constellation of complications caused by acrolein metabolites. We previously showed that a single dose of IPSE (Interleukin-4-inducing principle from Schistosoma eggs), a schistosome-derived host modulatory protein, can ameliorate ifosfamide-related cystitis; however, the mechanisms underlying this urotoxicity and its prevention are not fully understood. To provide insights into IPSE's protective mechanism, we undertook transcriptional profiling of bladders from ifosfamide-treated mice, with or without pretreatment with IPSE or IPSE-NLS (a mutant of IPSE lacking nuclear localization sequence). Ifosfamide treatment upregulated a range of proinflammatory genes. The IL-1ß-TNFα-IL-6 proinflammatory cascade via NFκB and STAT3 pathways was identified as the key driver of inflammation. The NRF2-mediated oxidative stress response pathway, which regulates heme homoeostasis and expression of antioxidant enzymes, was highly activated. Anti-inflammatory cascades, namely Wnt, Hedgehog and PPAR pathways, were downregulated. IPSE drove significant downregulation of major proinflammatory pathways including the IL-1ß-TNFα-IL-6 pathways, interferon signaling, and reduction in oxidative stress. IPSE-NLS reduced inflammation but not oxidative stress. Taken together, we have identified signatures of acute-phase inflammation and oxidative stress in ifosfamide-injured bladder, which are reversed by pretreatment with IPSE. This work revealed several pathways that could be therapeutically targeted to prevent ifosfamide-induced hemorrhagic cystitis.

Schistosoma haematobium cercarial infection alters subsequent systemic immune responses to eggs but has minimal impact on immune responses to egg injection of the bladder.

AIMS: Mouse bladder wall injection with Schistosoma haematobium eggs has been used to overcome limitations in animal models of urogenital schistosomiasis. However, the effect of the absence of cercarial infection on immune responses to eggs in this model is unknown. We hypothesized that cercarial infection would alter local bladder and systemic immune responses to eggs in this model. METHODS AND RESULTS: Mice were infected or not infected with S haematobium cercariae, and then, their bladder walls injected with S haematobium eggs or vehicle 5 weeks following cercarial infection. Three weeks later, mice were bled, sacrificed, perfused and their bladders harvested. Parasitological parameters and gross bladder pathology were not changed in egg-injected bladders by cercarial exposure. Figure S1 shows no changes in either granulomas or fibrosis. The only bladder cytokine upregulated in egg-injected bladders by cercarial exposure (vs no exposure) was leptin. Cercarial exposure, compared to no exposure, resulted in increased serum, IL-1α, IL-13 and TGF-ß in bladder egg-injected mice. CONCLUSION: Cercarial exposure altered systemic responses of several cytokines in bladder egg-injected mice, but surprisingly, only modified leptin expression in bladder tissue. This suggests that depending on the specific application, cercarial exposure may not be strictly necessary to model local immune responses in the bladder wall egg injection mouse model of urogenital schistosomiasis.

Therapeutic exploitation of IPSE, a urogenital parasite-derived host modulatory protein, for chemotherapy-induced hemorrhagic cystitis.

Chemotherapy-induced hemorrhagic cystitis (CHC) can be difficult to manage. Prior work suggests that IL-4 alleviates ifosfamide-induced hemorrhagic cystitis (IHC), but systemically administered IL-4 causes significant side effects. We hypothesized that the Schistosoma hematobium homolog of IL-4-inducing principle from Schistosoma mansoni eggs (H-IPSE), would reduce IHC and associated bladder pathology. IPSE binds IgE on basophils and mast cells, triggering IL-4 secretion by these cells. IPSE is also an "infiltrin," translocating into the host nucleus to modulate gene transcription. Mice were administered IL-4, H-IPSE protein or its nuclear localization sequence (NLS) mutant, with or without neutralizing anti-IL-4 antibody, or 2-mercaptoethane sulfonate sodium (MESNA; a drug used to prevent IHC), followed by ifosfamide. Bladder tissue damage and hemoglobin content were measured. Spontaneous and evoked pain, urinary frequency, and bladdergene expression analysis were assessed. Pain behaviors were interpreted in a blinded fashion. One dose of H-IPSE was superior to MESNA and IL-4 in suppressing bladder hemorrhage in an IL-4-dependent fashion and comparable with MESNA in dampening ifosfamide-triggered pain behaviors in an NLS-dependent manner. H-IPSE also accelerated urothelial repair following IHC. Our work represents the first therapeutic exploitation of a uropathogen-derived host modulatory molecule in a clinically relevant bladder disease model and indicates that IPSE may be an alternative to MESNA for mitigating CHC.-Mbanefo, E. C., Le, L., Pennington, L. F., Odegaard, J. I., Jardetzky, T. S., Alouffi, A., Falcone, F. H., Hsieh, M. H. Therapeutic exploitation of IPSE, a urogenital parasite-derived host modulatory protein, for chemotherapy-induced hemorrhagic cystitis.

Defining the Pathways of Urogenital Schistosomiasis-Associated Urothelial Carcinogenesis through Transgenic and Bladder Wall Egg Injection Models.

Urogenital schistosomiasis (infection with Schistosoma haematobium) is a major cause of bladder carcinogenesis. However, the exact mechanisms of the sequelae leading up to the development of bladder cancer are poorly understood, mainly because of a dearth of tractable mouse models. We developed a mouse model of urogenital schistosomiasis through intramural injection of parasite eggs into the bladder wall to mimic the trapping of parasite eggs in the bladder. This approach recapitulates many of the sequelae observed in infected humans. Here, we describe procedures for utilizing this surgical technique in combination with well-established transgenic mouse strains to dissect the role of cancer-related genes in the initiation and establishment of bladder carcinogenesis. The described method utilizes CRE-mediated flox activity to render mice p53 haploinsufficient before challenging them with bladder wall egg injection. These techniques are potentially amenable to studying the role of other pro-carcinogenic and cancer suppressor gene(s) in urogenital schistosomiasis-associated urothelial carcinogenesis.

H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage.

Urogenital schistosomiasis, caused by the parasitic trematode Schistosoma haematobium, affects over 112 million people worldwide. As with Schistosoma mansoni infections, the pathology of urogenital schistosomiasis is related mainly to the egg stage, which induces granulomatous inflammation of affected tissues. Schistosoma eggs and their secretions have been studied extensively for the related organism S. mansoni, which is more amenable to laboratory studies. Indeed, we have shown that IPSE/alpha-1 (here M-IPSE), a major protein secreted from S. mansoni eggs, can infiltrate host cells. Although the function of M-IPSE is unknown, its ability to translocate to the nuclei of host cells and bind DNA suggests a possible role in immune modulation of host cell tissues. Whether IPSE homologs are expressed in other schistosome species has not been investigated. Here, we describe the cloning of two paralog genes, H03-IPSE and H06-IPSE, which are orthologs of M-IPSE, from egg cDNA of S. haematobium Using PCR and immunodetection, we confirmed that the expression of these genes is restricted to the egg stage and female adult worms, while the H-IPSE protein is detectable only in mature eggs and not adults. We show that both H03-IPSE and H06-IPSE proteins can infiltrate HTB-9 bladder cells when added exogenously to culture medium. Monopartite C-terminal nuclear localization sequence (NLS) motifs conserved in H03-IPSE, SKRRRKY, and H06-IPSE SKRGRKY, are responsible for targeting the proteins to the nucleus of HTB-9 cells, as demonstrated by site-directed mutagenesis and green fluorescent protein (GFP) tagging. Thus, S. haematobium eggs express IPSE homologs that appear to perform similar functions in infiltrating host cells.

Onchocerciasis in Anambra State, Southeast Nigeria: clinical and psychological aspects and sustainability of community directed treatment with ivermectin (CDTI).

BACKGROUND: A cross-sectional study was performed to determine the psychological impact of onchocerciasis, and assess sustainability of the decade-old community directed treatment with ivermectin (CDTI) in Ayamelum Local Council, Anambra State, Southeast Nigeria. METHODS: Skin manifestations assessed using the rapid assessment method (RAM) in 894 subjects from 13 communities selected by multi-stage sampling were classified based on the anatomical sites affected. Focus group discussions and in-depth interviews were used to obtain information on the psychological impacts and sustainability of the CDTI programme. Qualitative data were summarised while quantitative data generated were analysed using charts and tables. RESULTS: Anatomical distribution showed a preponderance of onchodermatitis on the limbs (the most exposed parts of the body) and buttocks (an area considered 'private'), thus revealing some reasons for the psychological impacts of the skin disease and the psychosocial inclination of the victims. Itching (40%) and onchocercal skin manifestations (OSDs) (34.3%) were identified as the most troublesome signs and symptoms, while the most worrisome consequence of onchocerciasis was social seclusion (or stigmatisation) (34.3%). Focus group responses revealed the persistence of psychological impacts on the victims, affecting almost all facets of their lives. The CDTI programme has performed creditably well when assessed using the sustainability indicators, yet there are still challenges in the areas of coverage, monitoring, resources, and participation. A 'quick-win' was identified whereby the CDTI chain could be utilised to deliver other health interventions. CONCLUSION: It is recommended that onchocerciasis control programmes should include aspects that would address its psychosocial impacts and threats to the sustainability of the CDTI programme.