The IL-4-IL-4Rα axis modulates olfactory neuroimmune signaling to induce loss of smell.

IL-4 and IL-13 have non-redundant effects in olfaction, with loss of smell in mice evoked only by intranasal administration of IL-4, but not IL-13. IL-4-evoked pathophysiological effects on olfaction is independent of compromised structural integrity of the olfactory neuroepithelium. IL-4-IL-4Rα signaling modulates neuronal crosstalk with immune cells, suggesting a functional link between olfactory impairment and neuroinflammation. Abbreviations: IL, interleukin; KO, knock-out; wk, week; WT, wild-type.

BACKGROUND: Loss of smell is a part of the diagnostic criteria for CRSwNP. Although the mechanistic understanding is poor, inhibition of IL­4Rα and IL­4/IL­13 signaling improves loss of smell in CRSwNP patients. In the present study, we compare the effects of IL­4, IL­13, and IL­4Rα blockade on murine olfaction and identify the underlying pathophysiological mechanisms of loss of smell. METHODS: To evaluate the effects of IL­4 and IL­13 on olfactory function, we administered these cytokines intranasally to BALB/c mice for 5 consecutive days and assessed their latency to find hidden food. Calcium uptake assays were conducted to measure olfactory neuronal activity in vitro and ex vivo. We also performed histological analyses, proteomics, bulk RNA sequencing, and single­cell RNA sequencing to assess the impact of IL­4, IL­13, and IL­4Rα blockade on the olfactory epithelium and to identify potential molecular or cellular correlations with smell loss in human CRSwNP patients. RESULTS: Here, we provide evidence for non­redundant effects of IL­4 and IL­13 in olfaction, with loss of smell in mice evoked by intranasal administration of IL­4, not IL­13. We demonstrate that an IL­4­IL­4Rα axis has a direct functional impact on murine olfactory sensory neurons and evokes inflammatory cell infiltration and pathophysiologic modulation of unique molecular signatures in olfactory epithelium without compromising structural integrity. Furthermore, single­cell analysis of olfactory epithelium reveals that IL­4­IL­4Rα signaling modulates neuronal crosstalk with mast cells, macrophages, and NK cells, suggesting a functional link between olfactory impairment and neuroinflammation. CONCLUSION: Collectively, this study suggests that an IL­4­IL­4Rα signaling axis plays a unique pathophysiological role in olfactory dysfunction via direct effect on neurons and modulation of neuroimmune interactions.

Defining disease severity in atopic dermatitis and psoriasis for the application to biomarker research: an interdisciplinary perspective.

More severe atopic dermatitis and psoriasis are associated with a higher cumulative impact on quality of life, multimorbidity and healthcare costs. Proactive, early intervention in those most at risk of severe disease may reduce this cumulative burden and modify the disease trajectory to limit progression. The lack of reliable biomarkers for this at-risk group represents a barrier to such a paradigm shift in practice. To expedite discovery and validation, the BIOMarkers in Atopic Dermatitis and Psoriasis (BIOMAP) consortium (a large-scale European, interdisciplinary research initiative) has curated clinical and molecular data across diverse study designs and sources including cross-sectional and cohort studies (small-scale studies through to large multicentre registries), clinical trials, electronic health records and large-scale population-based biobanks. We map all dataset disease severity instruments and measures to three key domains (symptoms, inflammatory activity and disease course), and describe important codependencies and relationships across variables and domains. We prioritize definitions for more severe disease with reference to international consensus, reference standards and/or expert opinion. Key factors to consider when analysing datasets across these diverse study types include explicit early consideration of biomarker purpose and clinical context, candidate biomarkers associated with disease severity at a particular point in time and over time and how they are related, taking the stage of biomarker development into account when selecting disease severity measures for analyses, and validating biomarker associations with disease severity outcomes using both physician- and patient-reported measures and across domains. The outputs from this exercise will ensure coherence and focus across the BIOMAP consortium so that mechanistic insights and biomarkers are clinically relevant, patient-centric and more generalizable to current and future research efforts.

Atopic dermatitis (AD), and psoriasis are long-term skin conditions that can significantly affect people's lives, especially when symptoms are severe. Approximately 10% of adults and 20% of children are affected by AD, while psoriasis affects around 5% of people in the UK. Both conditions are associated with debilitating physical symptoms (such as itch) and have been linked to depression and anxiety. Biomarkers are naturally occurring chemicals in the human body and have potential to enhance the longer-term management of AD and psoriasis. Currently, there are no routinely used biomarkers that can identify people who experience or will go on to develop severe AD and psoriasis. For this reason, research is under way to understand which biomarkers are linked to severity. In this study, a multidisciplinary team of skin researchers from across Europe, along with patient groups, discussed the complexities of studying severity-related biomarkers. We identified a number of severity measurement approaches and there were recommendations for future biomarker research, including (i) considering multiple measures as no single measure can encompass all aspects of severity, (ii) exploring severity measures recorded by both healthcare professionals and patients, as each may capture different aspects, and (iii) accounting for influencing factors, such as different treatment approaches, that may impact AD and psoriasis severity, which make it challenging to compare findings across studies. Overall, we anticipate that the insights gained from these discussions will increase the likelihood of biomarkers being effectively applied in real-world settings, to ultimately improve outcomes for people with AD and psoriasis.

A cancer personalised activity and lifestyle tool (CAN-PAL): A codesign study with patients and healthcare professionals.

AIMS: To codesign a cancer personalised activity and lifestyle tool (CAN-PAL) based on an existing tool. To help cancer care workers support people affected by cancer to plan and integrate physical activity into lifestyles. DESIGN: Mixed-methods codesign study. METHODS: Phase 1: Focus groups with people affected by cancer (n = 10) or interviews (n = 2) to discuss suitable physical activities and adaptation of the existing tool. Data were recorded, transcribed and analysed thematically. Themes informed the design of the prototype CAN-PAL and user guide. Phase 2: Healthcare professionals considered the potential use of the CAN-PAL prototype and completed an online survey including the system usability scale and free text responses. RESULTS: Phase 1: Identified suitable physical activities and four themes were identified including: Capability, benefits, barriers and resources which informed the prototype CAN-PAL and user guide. Phase 2: The user survey was completed by 12 healthcare professionals. Median (range) system usability scale was 80 (50-95) (best score 100), scores >68 indicate good or better usability. Themes from the free text comments included strengths, amendments, considerations and limitations. Results were used to finalise CAN-PAL and the user guide. CONCLUSION: The codesigned CAN-PAL tool had good usability. Further work is needed to evaluate the impact of CAN-PAL on activity levels and behaviour in people affected by cancer. RELEVANCE TO CLINICAL PRACTICE: People affected by cancer need support to undertake physical activity. The purpose of CAN-PAL is to assist cancer care workers to support people affected by cancer to plan and integrate physical activity into lifestyles. PATIENT OR PUBLIC CONTRIBUTION: Public partners considered the findings from Phase 1 and 2 and informed the design of the prototype, final CAN-PAL and user guide and coauthored the paper. REPORTING METHOD: The study adhered to relevant EQUATOR guidelines; the study was reported according to the COREQ checklist.

Potent, non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-a]pyrazine core featuring 3-position bicyclic ring substitutes.

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition, and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine we recently reported. The X-ray crystal structures of BTK with inhibitors were also published, which provided great help for the SAR design. Here we report our SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine. This modification improved the potency in BTK inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model.

Discovery of novel BTK inhibitors with carboxylic acids.

We report the design and synthesis of a series of novel Bruton's Tyrosine Kinase (BTK) inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has demonstrated much improved off-target selectivities including adenosine uptake (AdU) inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4 based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat and dog.

Discovery of 3-morpholino-imidazole[1,5-a]pyrazine BTK inhibitors for rheumatoid arthritis.

8-Amino-imidazo[1,5-a]pyrazine-based Bruton's tyrosine kinase (BTK) inhibitors, such as 6, exhibited potent inhibition of BTK but required improvements in both kinase and hERG selectivity (Liu et al., 2016; Gao et al., 2017). In an effort to maintain the inhibitory activity of these analogs and improve their selectivity profiles, we carried out SAR exploration of groups at the 3-position of pyrazine compound 6. This effort led to the discovery of the morpholine group as an optimized pharmacophore. Compounds 13, 23 and 38 displayed excellent BTK potencies, kinase and hERG selectivities, and pharmacokinetic profiles.

Implementation of a multidisciplinary approach to care for people with HIV aged 80 years and over.

BACKGROUND: Frailty adversely affects health and quality of life of people with HIV (PWH). A multidisciplinary-team (MDT) was established to perform a Comprehensive Geriatric Assessment (CGA) and to address modifiable risk factors in elderly PWH. METHODS: PWH aged over 80 years were reviewed by a dedicated HIV/geriatric clinic at Chelsea and Westminster Hospital. Descriptive statistics were used to report the variables of interest. RESULTS: Sixty-three PWH were reviewed, with a median age of 82 (IQR 80-86). Fifty-seven (88%) were men. The median number of co-morbidities was five (IQR 4-7) and polypharmacy was seen in 55 (85%), with a median of six (IQR 5-8) co-medications reported. The median Rockwood Clinical Frailty Score was two (IQR 2-5). Antiretrovirals were modernised in 18 (29%) individuals; co-medication changes and de-prescribing was recommended in 39 (62%). CONCLUSION: A MDT approach helped reducing polypharmacy and frailty, suggesting that specialised tools and MDT input are key to identify medical and social concerns in elderly PWH.

The interleukin-4/interleukin-13 pathway in type 2 inflammation in chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is predominantly a type 2 inflammatory disease associated with type 2 (T2) cell responses and epithelial barrier, mucociliary, and olfactory dysfunction. The inflammatory cytokines interleukin (IL)-4, IL-13, and IL-5 are key mediators driving and perpetuating type 2 inflammation. The inflammatory responses driven by these cytokines include the recruitment and activation of eosinophils, basophils, mast cells, goblet cells, M2 macrophages, and B cells. The activation of these immune cells results in a range of pathologic effects including immunoglobulin E production, an increase in the number of smooth muscle cells within the nasal mucosa and a reduction in their contractility, increased deposition of fibrinogen, mucus hyperproduction, and local edema. The cytokine-driven structural changes include nasal polyp formation and nasal epithelial tissue remodeling, which perpetuate barrier dysfunction. Type 2 inflammation may also alter the availability or function of olfactory sensory neurons contributing to loss of sense of smell. Targeting these key cytokine pathways has emerged as an effective approach for the treatment of type 2 inflammatory airway diseases, and a number of biologic agents are now available or in development for CRSwNP. In this review, we provide an overview of the inflammatory pathways involved in CRSwNP and describe how targeting key drivers of type 2 inflammation is an effective therapeutic option for patients.

SARS-CoV-2 burden on the floor was associated with COVID-19 cases and outbreaks in two acute care hospitals: a prospective cohort study.

BACKGROUND: Recent work demonstrated that detection of SARS-CoV-2 on the floor of long-term care facilities is associated with impending COVID-19 outbreaks. It is unknown if similar results will be observed in hospitals. METHODS: Floor swabs were prospectively collected weekly from healthcare worker-only areas (eg, staff locker rooms) at two hospitals in Ontario, Canada for 39 weeks. Floor swabs were processed for SARS-CoV-2 using quantitative reverse-transcriptase polymerase chain reaction. Results were reported as percentage of positive floor swabs and viral copy number. Grouped fivefold cross-validation was used to evaluate model outbreak discrimination. RESULTS: SARS-CoV-2 RNA was detected on 537 of 760 floor swabs (71%). At Hospital A, overall positivity was 90% (95% CI: 85%-93%; N = 280); at Hospital B, overall positivity was 60% (95% CI: 55%-64%; N = 480). There were four COVID-19 outbreaks at Hospital A and seven at Hospital B during the study period. The outbreaks consisted of primarily patient cases (ie, 140 patient cases and 4 staff cases). For every 10-fold increase in viral copies, there was a 22-fold higher odds of a COVID-19 outbreak (OR = 22.0, 95% CI 7.3, 91.8). The cross-validated area under the receiver operating curve for SARS-CoV-2 viral copies for predicting a contemporaneous outbreak was 0.86 (95% CI 0.82-0.90). CONCLUSION: Viral burden of SARS-CoV-2 on floors, even in healthcare worker-only areas, was strongly associated with COVID-19 outbreaks in those hospital wards. Built environment sampling may support hospital COVID-19 outbreak identification, fill gaps in traditional surveillance, and guide infection prevention and control measures.

Zinc finger nuclease-mediated gene editing in hematopoietic stem cells results in reactivation of fetal hemoglobin in sickle cell disease.

BIVV003 is a gene-edited autologous cell therapy in clinical development for the potential treatment of sickle cell disease (SCD). Hematopoietic stem cells (HSC) are genetically modified with mRNA encoding zinc finger nucleases (ZFN) that target and disrupt a specific regulatory GATAA motif in the BCL11A erythroid enhancer to reactivate fetal hemoglobin (HbF). We characterized ZFN-edited HSC from healthy donors and donors with SCD. Results of preclinical studies show that ZFN-mediated editing is highly efficient, with enriched biallelic editing and high frequency of on-target indels, producing HSC capable of long-term multilineage engraftment in vivo, and express HbF in erythroid progeny. Interim results from the Phase 1/2 PRECIZN-1 study demonstrated that BIVV003 was well-tolerated in seven participants with SCD, of whom five of the six with more than 3 months of follow-up displayed increased total hemoglobin and HbF, and no severe vaso-occlusive crises. Our data suggest BIVV003 represents a compelling and novel cell therapy for the potential treatment of SCD.

Selective Btk inhibition by PRN1008/PRN473 blocks human CLEC-2 & PRN473 reduces venous thrombosis formation in mice.

Platelet CLEC-2 is a hemITAM-containing receptor which has a critical role in venous thrombosis, but minimal involvement in haemostasis. CLEC-2 can be blocked by Btk inhibitors. Treatment with ibrutinib is associated with increased bleeding due to off-target inhibition of Src family kinases (SFKs). Patients with X-linked agammaglobulinemia (XLA) who lack Btk however do not bleed, suggesting selective Btk inhibition is a viable antithrombotic strategy. We assessed the effects of selective Btk inhibitors PRN1008 (rilzabrutinib) and PRN473 on platelet signalling and function mediated by CLEC-2 and GPVI. We used healthy donor and XLA platelets to determine off-target inhibitor effects. Inferior vena cava (IVC) stenosis and Salmonella infection mouse models were used to assess antithrombotic effects of PRN473 in vivo. PRN1008 and PRN473 potently inhibited CLEC-2-mediated platelet activation to rhodocytin. No off-target inhibition of SFKs was seen. PRN1008 treatment of Btk-deficient platelets resulted in minor additional inhibition of aggregation and tyrosine phosphorylation, likely reflecting inhibition of Tec. No effect on GPCR-mediated platelet function was observed. PRN473 significantly reduced the number of thrombi in podoplanin positive vessels following Salmonella infection and the presence of IVC thrombosis following vein stenosis. The potent inhibition of human platelet CLEC-2, and reduced thrombosis in in vivo models, together with the lack of off-target SFK inhibition and absence of bleeding reported in rilzabrutinib treated immune thrombocytopenia patients, suggest Btk inhibition as a promising antithrombotic strategy.

Cellular mechanisms and effects of IL-4 receptor blockade in experimental conjunctivitis evoked by skin inflammation.

Ocular surface diseases, including conjunctivitis, are recognized as common comorbidities in atopic dermatitis (AD) and occur at an increased frequency in patients with AD treated with biologics targeting IL-4 receptor α (IL-4Rα) or IL-13. However, the inflammatory mechanisms underlying this pathology are unknown. Here, we developed a potentially novel mouse model of skin inflammation-evoked conjunctivitis and showed that it is dependent on CD4+ T cells and basophils. Blockade of IL-4Rα partially attenuated conjunctivitis development, downregulated basophil activation, and led to a reduction in expression of genes related to type 2 cytokine responses. Together, these data suggest that an IL-4Rα/basophil axis plays a role in the development of murine allergic conjunctivitis. Interestingly, we found a significant augmentation of a number of genes that encode tear proteins and enzymes in anti-IL-4Rα-treated mice, and it may underlie the partial efficacy in this model and may represent candidate mediators of the increased frequency of conjunctivitis following dupilumab in patients with AD.

The mitochondrial genomes of two Gryllus crickets (Grylloidea: Gryllidae) via RNA-seq.

Here, we used RNA-seq reads to assemble the complete mitochondrial genomes of the spring field cricket, Gryllus veletis, and the variable field cricket, Gryllus lineaticeps. The mitochondrial genomes of G. veletis (15,686 bp, MW322713) and G. lineaticeps (15,607 bp, MW315773) each contain the expected 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a large control (D-loop) region. The arrangements of these features were similar for both species and consistent with other closely related Orthoptera. A phylogenetic analysis of the mitochondrial genome sequences revealed that G. veletis and G. lineaticeps cluster with the other Gryllus species and all reside in a clade with the Gryllidae.

cAMP-induced decrease in cell-surface laminin receptor and cellular prion protein attenuates amyloid-ß uptake and amyloid-ß-induced neuronal cell death.

Previous studies have shown that amyloid-ß oligomers (AßO) bind with high affinity to cellular prion protein (PrPC ). The AßO-PrPC complex binds to cell-surface co-receptors, including the laminin receptor (67LR). Our current studies revealed that in Neuroscreen-1 cells, 67LR is the major co-receptor involved in the cellular uptake of AßO and AßΟ-induced cell death. Both pharmacological (dibutyryl-cAMP, forskolin and rolipram) and physiological (pituitary adenylate cyclase-activating polypeptide) cAMP-elevating agents decreased cell-surface PrPC and 67LR, thereby attenuating the uptake of AßO and the resultant neuronal cell death. These cAMP protective effects are dependent on protein kinase A, but not dependent on the exchange protein directly activated by cAMP. Conceivably, cAMP protects neuronal cells from AßO-induced cytotoxicity by decreasing cell-surface-associated PrPC and 67LR.

MetAP2 inhibition modifies hemoglobin S to delay polymerization and improves blood flow in sickle cell disease.

Sickle cell disease (SCD) is associated with hemolysis, vascular inflammation, and organ damage. Affected patients experience chronic painful vaso-occlusive events requiring hospitalization. Hypoxia-induced polymerization of sickle hemoglobin S (HbS) contributes to sickling of red blood cells (RBCs) and disease pathophysiology. Dilution of HbS with nonsickling hemoglobin or hemoglobin with increased oxygen affinity, such as fetal hemoglobin or HbS bound to aromatic aldehydes, is clinically beneficial in decreasing polymerization. We investigated a novel alternate approach to modify HbS and decrease polymerization by inhibiting methionine aminopeptidase 2 (MetAP2), which cleaves the initiator methionine (iMet) from Val1 of α-globin and ßS-globin. Kinetic studies with MetAP2 show that ßS-globin is a fivefold better substrate than α-globin. Knockdown of MetAP2 in human umbilical cord blood-derived erythroid progenitor 2 cells shows more extensive modification of α-globin than ß-globin, consistent with kinetic data. Treatment of human erythroid cells in vitro or Townes SCD mice in vivo with selective MetAP2 inhibitors extensively modifies both globins with N-terminal iMet and acetylated iMet. HbS modification by MetAP2 inhibition increases oxygen affinity, as measured by decreased oxygen tension at which hemoglobin is 50% saturated. Acetyl-iMet modification on ßS-globin delays HbS polymerization under hypoxia. MetAP2 inhibitor-treated Townes mice reach 50% total HbS modification, significantly increasing the affinity of RBCs for oxygen, increasing whole blood single-cell RBC oxygen saturation, and decreasing fractional flow velocity losses in blood rheology under decreased oxygen pressures. Crystal structures of modified HbS variants show stabilization of the nonpolymerizing high O2-affinity R2 state, explaining modified HbS antisickling activity. Further study of MetAP2 inhibition as a potential therapeutic target for SCD is warranted.

Effects of LTB4 receptor antagonism on pulmonary inflammation in rodents and non-human primates.

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.

Arming oHSV with ULBP3 drives abscopal immunity in lymphocyte-depleted glioblastoma.

Oncolytic viruses induce local tumor destruction and inflammation. Whether virotherapy can also overcome immunosuppression in noninfected tumor areas is under debate. To address this question, we have explored immunologic effects of oncolytic herpes simplex viruses (oHSVs) in a genetically engineered mouse model of isocitrate dehydrogenase (IDH) wild-type glioblastoma, the most common and most malignant primary brain tumor in adults. Our model recapitulates the genomics, the diffuse infiltrative growth pattern, and the extensive macrophage-dominant immunosuppression of human glioblastoma. Infection with an oHSV that was armed with a UL16-binding protein 3 (ULBP3) expression cassette inhibited distant tumor growth in the absence of viral spreading (abscopal effect) and yielded accumulation of activated macrophages and T cells. There was also abscopal synergism of oHSVULBP3 with anti-programmed cell death 1 (anti-PD-1) against distant, uninfected tumor areas; albeit consistent with clinical trials in patients with glioblastoma, monotherapy with anti-PD-1 was ineffective in our model. Arming oHSV with ULBP3 led to upregulation of antigen processing and presentation gene sets in myeloid cells. The cognate ULBP3 receptor NKG2D, however, is not present on myeloid cells, suggesting a noncanonical mechanism of action of ULBP3. Overall, the myeloid-dominant, anti-PD-1-sensitive abscopal effect of oHSVULBP3 warrants further investigation in patients with IDH wild-type glioblastoma.

Influence of genetic background and gender on bladder function in the mouse.

Genetically targeted animals are used throughout research to investigate the role genes play in biological function, including the lower urinary tract. Generation of transgenic mice involves backcrossing for successive generations. Parental strain background genes can interact with the mutated gene potentially affecting interpretation of the mutant phenotype. Differences in physiological phenotypes may also be influenced by gender. The present study evaluated bladder function in five strains of male and female mice, 129S6/SvEvTac, A/J, B6129F1/Tac, BALB/cAnNCrL and C57BL/6NTacFBr. Urodynamic parameters were analyzed during infusion of saline (threshold and void volume, non-voiding contractions, pressure threshold and bladder contraction amplitude) in conscious mice and using voluntary urination in freely moving mice placed on filter paper (number of small and large diameter urine spots), which represent commonly used techniques in preclinical characterization of bladder function. Female BALB/c mice exhibited a significantly larger number of non-voiding contractions and urine dripping (increased number of small urine spots) compared to other female mice. Male BALB/c mice did not share this phenotype. Significant differences in threshold and void volumes were also noted amongst strains and genders. The numbers of large diameter urine spots differed amongst female, and not male, mouse strains. Gender differences were observed between sexes of the same strain in both large and small urine spots. These data demonstrate that genetic background and gender can influence bladder function in the mouse. These differences have a significant impact on the choice of strain and gender when investigating the effects of genetic manipulation on the micturition reflex.