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Women with uncomplicated urinary tract infection (UTI) symptoms are commonly treated with empirical antibiotics, resulting in overuse of antibiotics, which promotes antimicrobial resistance. Available diagnostic tools are either not cost-effective or diagnostically sub-optimal. Here, we identified clinical and urinary immunological predictors for UTI diagnosis. We explored 17 clinical and 42 immunological potential predictors for bacterial culture among women with uncomplicated UTI symptoms using random forest or support vector machine coupled with recursive feature elimination. Urine cloudiness was the best performing clinical predictor to rule out (negative likelihood ratio [LR−] = 0.4) and rule in (LR+ = 2.6) UTI. Using a more discriminatory scale to assess cloudiness (turbidity) increased the accuracy of UTI prediction further (LR+ = 4.4). Urinary levels of MMP9, NGAL, CXCL8 and IL-1β together had a higher LR+ (6.1) and similar LR− (0.4), compared to cloudiness. Varying the bacterial count thresholds for urine culture positivity did not alter best clinical predictor selection, but did affect the number of immunological predictors required for reaching an optimal prediction. We conclude that urine cloudiness is particularly helpful in ruling out negative UTI cases. The identified urinary biomarkers could be used to develop a point of care test for UTI but require further validation.

Neutrophil infiltration is a hallmark of peritoneal inflammation, but mechanisms regulating neutrophil recruitment in patients with peritoneal dialysis (PD)-related peritonitis are not fully defined. We examined 104 samples of PD effluent collected during acute peritonitis for correspondence between a broad range of soluble parameters and neutrophil counts. We observed an association between peritoneal IL-17 and neutrophil levels. This relationship was evident in effluent samples with low but not high IFN-γ levels, suggesting a differential effect of IFN-γ concentration on neutrophil infiltration. Surprisingly, there was no association of neutrophil numbers with the level of CXCL1, a key IL-17-induced neutrophil chemoattractant. We investigated therefore the production of CXCL1 by human peritoneal mesothelial cells (HPMCs) under in vitro conditions mimicking clinical peritonitis. Stimulation of HPMCs with IL-17 increased CXCL1 production through induction of transcription factor SP1 and activation of the SP1-binding region of the CXCL1 promoter. These effects were amplified by TNFα. In contrast, IFN-γ dose-dependently suppressed IL-17-induced SP1 activation and CXCL1 production through a transcriptional mechanism involving STAT1. The SP1-mediated induction of CXCL1 was also observed in HPMCs exposed to PD effluent collected during peritonitis and containing IL-17 and TNFα, but not IFN-γ. Supplementation of the effluent with IFN-γ led to a dose-dependent activation of STAT1 and a resultant inhibition of SP1-induced CXCL1 expression. Transmesothelial migration of neutrophils in vitro increased upon stimulation of HPMCs with IL-17 and was reduced by IFN-γ. In addition, HPMCs were capable of binding CXCL1 at their apical cell surface. These observations indicate that changes in relative peritoneal concentrations of IL-17 and IFN-γ can differently engage SP1-STAT1, impacting on mesothelial cell transcription of CXCL1, whose release and binding to HPMC surface may determine optimal neutrophil recruitment and retention during peritonitis. © 2020 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Infection remains a major cause of morbidity, mortality and technique failure in patients with end stage kidney failure who receive peritoneal dialysis (PD). Recent research suggests that the early inflammatory response at the site of infection carries diagnostically relevant information, suggesting that organ and pathogen-specific “immune fingerprints” may guide targeted treatment decisions and allow patient stratification and risk prediction at the point of care. Here, we recorded microRNA profiles in the PD effluent of patients presenting with symptoms of acute peritonitis and show that elevated peritoneal miR-223 and reduced miR-31 levels were useful predictors of bacterial infection. Cell culture experiments indicated that miR-223 was predominantly produced by infiltrating immune cells (neutrophils, monocytes), while miR-31 was mainly derived from the local tissue (mesothelial cells, fibroblasts). miR-223 was found to be functionally stabilised in PD effluent from peritonitis patients, with a proportion likely to be incorporated into neutrophil-derived exosomes. Our study demonstrates that microRNAs are useful biomarkers of bacterial infection in PD-related peritonitis and have the potential to contribute to disease-specific immune fingerprints. Exosome-encapsulated microRNAs may have a functional role in intercellular communication between immune cells responding to the infection and the local tissue, to help clear the infection, resolve the inflammation and restore homeostasis.

Peritoneal dialysis (PD) remains limited by dialysis failure due to peritoneal membrane fibrosis driven by inflammation caused by infections or sterile cellular stress. Given the fundamental role of Toll-like receptors (TLRs) and complement in inflammation, we assessed the potential of peritoneal TLR2, TLR4 and C5a receptors, C5aR and C5L2, as therapeutic targets in PD-associated fibrosis. We detected TLR2–, TLR4–, and C5aR–mediated proinflammatory and fibrotic responses to bacteria that were consistent with the expression of these receptors in peritoneal macrophages (TLR2/4, C5aR) and mesothelial cells (TLR2, C5aR). Experiments in knockout mice revealed a major role for TLR2, a lesser role for TLR4, a supplementary role for C5aR, and no apparent activity of C5L2 in infection–induced peritoneal fibrosis. Similarly, antibody blockade of TLR2, TLR4, or C5aR differentially inhibited bacteria–induced profibrotic and inflammatory mediator production by peritoneal leukocytes isolated from the peritoneal dialysis effluent (PDE) of noninfected uremic patients. Additionally, antibodies against TLR2, TLR4, or the coreceptor CD14 reduced the profibrotic responses of uremic leukocytes to endogenous components present in the PDE of noninfected patients. Enhancing TLR2-mediated inflammation increased fibrosis in vivo. Furthermore, soluble TLR2 (sTLR2), a negative modulator of TLRs that we detected in PDE, inhibited PDE–induced, TLR2– or TLR4–mediated profibrotic responses. Notably, sTLR2 treatment markedly reduced Gram–positive and –negative bacteria–induced fibrosis in vivo, inhibiting proinflammatory and fibrotic genes without affecting infection clearance. These findings reveal the influence of peritoneal TLR2 and TLR4 on PD-associated fibrosis and describe a therapeutic strategy against fibrosis.

Accurate and timely diagnosis of bacterial infection is crucial for effective and targeted treatment, yet routine microbiological identification is inefficient and often delayed to an extent that makes it clinically unhelpful. The immune system is capable of a rapid, sensitive and specific detection of a broad spectrum of microbes, which has been optimized over millions of years of evolution. A patient's early immune response is therefore likely to provide far better insight into the true nature and severity of microbial infections than conventional tests. To assess the diagnostic potential of pathogen-specific immune responses, we characterized the local responses of 52 adult patients during episodes of acute peritoneal dialysis (PD)-associated peritonitis by multicolor flow cytometry and multiplex ELISA, and defined the immunologic signatures in relation to standard microbiological culture results and to clinical outcomes. We provide evidence that unique local "immune fingerprints" characteristic of individual organisms are evident in PD patients on the day of presentation with acute peritonitis and discriminate between culture-negative, Gram-positive, and Gram-negative episodes of infection. Those humoral and cellular parameters with the most promise for defining disease-specific immune fingerprints include the local levels of IL-1β, IL-10, IL-22, TNF-α, and CXCL10, as well as the frequency of local γδ T cells and the relative proportion of neutrophils and monocytes/macrophages among total peritoneal cells. Our data provide proof of concept for the feasibility of using immune fingerprints to inform the design of point-of-care tests that will allow rapid and accurate infection identification and facilitate targeted antibiotic prescription and improved patient management.

BACKGROUND: Human γδ T cells reactive to the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) contribute to acute inflammatory responses. We have previously shown that peritoneal dialysis (PD)-associated infections with HMB-PP producing bacteria are characterized by locally elevated γδ T-cell frequencies and poorer clinical outcome compared with HMB-PP negative infections, implying that γδ T cells may be of diagnostic, prognostic and therapeutic value in acute disease. The regulation by local tissue cells of these potentially detrimental γδ T-cell responses remains to be investigated. METHODS: Freshly isolated γδ or αβ T cells were cultured with primary mesothelial cells derived from omental tissue, or with mesothelial cell-conditioned medium. Stimulation of cytokine production and proliferation by peripheral T cells in response to HMB-PP or CD3/CD28 beads was assessed by flow cytometry. RESULTS: Resting mesothelial cells were potent suppressors of pro-inflammatory γδ T cells as well as CD4+ and CD8+ αβ T cells. The suppression of γδ T-cell responses was mediated through soluble factors released by primary mesothelial cells and could be counteracted by SB-431542, a selective inhibitor of TGF-β and activin signalling. Recombinant TGF-β1 but not activin-A mimicked the mesothelial cell-mediated suppression of γδ T-cell responses to HMB-PP. CONCLUSIONS: The present findings indicate an important regulatory function of mesothelial cells in the peritoneal cavity by dampening pro-inflammatory T-cell responses, which may help preserve the tissue integrity of the peritoneal membrane in the steady state and possibly during the resolution of acute inflammation.

The antimicrobial responsiveness and function of unconventional human T cells are poorly understood, with only limited access to relevant specimens from sites of infection. Peritonitis is a common and serious complication in individuals with end-stage kidney disease receiving peritoneal dialysis. By analyzing local and systemic immune responses in peritoneal dialysis patients presenting with acute bacterial peritonitis and monitoring individuals before and during defined infectious episodes, our data show that Vγ9/Vδ2(+) γδ T cells and mucosal-associated invariant T cells accumulate at the site of infection with organisms producing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and vitamin B2, respectively. Such unconventional human T cells are major producers of IFN-γ and TNF-α in response to these ligands that are shared by many microbial pathogens and affect the cells lining the peritoneal cavity by triggering local inflammation and inducing tissue remodeling with consequences for peritoneal membrane integrity. Our data uncover a crucial role for Vγ9/Vδ2 T cells and mucosal-associated invariant T cells in bacterial infection and suggest that they represent a useful predictive marker for important clinical outcomes, which may inform future stratification and patient management. These findings are likely to be applicable to other acute infections where local activation of unconventional T cells contributes to the antimicrobial inflammatory response.

5-Lipoxygenase (5-LOX) plays key roles in infection and allergic responses. Herein, four 5-LOX–derived lipids comprising 5-hydroxyeicosatetraenoic acid (HETE) attached to phospholipids (PLs), either phosphatidylethanolamine (PE) or phosphatidylcholine (18:0p/5-HETE-PE, 18:1p/5-HETE-PE, 16:0p/5-HETE-PE, and 16:0a/5-HETE-PC), were identified in primary human neutrophils. They formed within 2 minutes in response to serum-opsonized Staphylococcus epidermidis or f-methionine-leucine-phenylalanine, with priming by lipopolysaccharide, granulocyte macrophage colony-stimulating factor, or cytochalasin D. Levels generated were similar to free 5-HETE (0.37 ± 0.14 ng vs 0.55 ± 0.18 ng/106 cells, esterified vs free 5-HETE, respectively). They remained cell associated, localizing to nuclear and extranuclear membrane, and were formed by fast esterification of newly synthesized free 5-HETE. Generation also required Ca2+, phospholipase C, cytosolic and secretory phospholipase A2, 5-LOX activating protein, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1. 5-HETE-PLs were detected in murine S epidermidis peritonitis, paralleling neutrophil influx, and in effluent from Gram-positive human bacterial peritonitis. Formation of neutrophil extracellular traps was significantly enhanced by 5-LOX inhibition but attenuated by HETE-PE, whereas 5-HETE-PE enhanced superoxide and interleukin-8 generation. Thus, new molecular species of oxidized PL formed by human neutrophils during bacterial infection are identified and characterized.

The linear glycosaminoglycan hyaluronan (HA) is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2, and -3 and performs multiple functions as part of the vertebrate extracellular matrix. Up-regulation of HA synthesis in the renal corticointerstitium, and the resultant extracellular matrix expansion, is a common feature of renal fibrosis. However, the regulation of expression of these HAS isoforms at transcriptional and translational levels is poorly understood. We have recently described the genomic structures of the human HAS genes, thereby identifying putative promoter regions for each isoform. Further analysis of the HAS2 gene identified the transcription initiation site and showed that region F3, comprising the proximal 121 bp of promoter sequence, mediated full constitutive transcription. In the present study, we have analyzed this region in the human renal proximal tubular epithelial cell line HK-2. Electrophoretic mobility shift and promoter assay data demonstrated that transcription factors Sp1 and Sp3 bound to three sites immediately upstream of the HAS2 transcription initiation site and that mutation of the consensus recognition sequences within these sites ablated their transcriptional response. Furthermore, subsequent knockdown of Sp1 or Sp3 using small interfering RNAs decreased constitutive HAS2 mRNA synthesis. In contrast, significant binding of HK-2 nuclear proteins by putative upstream NF-Y, CCAAT, and NF-kappaB recognition sites was not observed. The identification of Sp1 and Sp3 as principal mediators of HAS2 constitutive transcription augments recent findings identifying upstream promoter elements and provides further insights into the mechanism of HAS2 transcriptional activation.

Interaction between tumour cells and endothelial cells is an essential step during the systemic step of cancer metastasis in the body and has been an active area pursued for the past decades. However, methods to investigate tumour-endothelial interactions mostly are tedious and lack of high throughput capability. Similarly, tumour-mesothelial interactions are also important part of the transcoelomic spreading in the body cavities. However, methods to determine tumour-mesothelial interaction face similar challenges to that of tumour-endothelial interactions. This chapter discusses the application of electric cell-substrate impedance sensing (ECIS) in the investigation of tumour-endothelial/mesothelial interactions.

Introduction. Peritoneal metastasis is a frequently occurring complication patients with gastrointestinal, pancreatic, ovarian cancers and is a condition difficult to manage, clinically, due largely to the lack of effective treatment. In the present study, we report an accidental and novel discovery that the extract of Yangzheng Xiaoji (YZXJ), a traditional medical formula used in cancer treatment had an anti-peritoneal metastasis function. Materials and methods. Human primary cultured peritoneal mesothelial cells were used as an in vitro cellular model. Human gastric, colorectal, pancreatic, ovarian and prostate cancer cells were used in the tests. Tumour-mesothelial interaction, invasion and migration were assessed with YZXJ. Athymic nude mice peritoneal metastasis model was used to evaluate the effects in vivo. Results. Soluble YZXJ was firstly extracted in a highly reproducible manner. YZXJ extract (DME25) had a negletable toxicity to cancer cells and mesothelial cells. However, it profoundly inhibited the tumour-mesothelial adhesions in the type of tumour cells tested with pancreatic and colorectal cancer cells amongst the most sensitive ones to YZXJ (p Conclusion. Yangzheng Xiaoji has an potent inhibitory effect on tumour-mesothelial interactions of a variety of tumour types and also markedly reduces the peritoneal metastasis in vivo. This effect is likely to be achieved by its effect on the SRC signalling pathway. Yangzheng Xiaoji presents a new way of treating peritoneal metastasis. Citation Format: Wen G. Jiang, Yongning Jia, Lin Ye, Ke Ji, Yong Gao, Cong Wei, Yiling Wu, Nick Topley, Anna Kift-Morgan, Jiafu Ji. The anti-peritoneal metastasis properties of Yangzheng Xiaoji, the potential role of hyaluronan and CD44. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4034. doi:10.1158/1538-7445.AM2014-4034