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With an objective to extend the shelf life with improved microbial safety by natural means without compromising sensory attributes, lyophilised khejri tree leaf extract (LKE) was evaluated in vitro and in vivo. The LKE was subjected to preliminary scrutiny followed by FTIR mediated analysis and RP-HPLC, then its antioxidant activity was accessed. Five batches of minced mutton were prepared as C-1 (negative control with no LKE or BHA), C-2 (positive control with 200 ppm BHA), T-1 (0.05% LKE), T-2 (0.1% LKE), and T-3 (0.5% LKE). FTIR analysis showed C=O-CH3, C=C, C-O, and O-H peaks at 1028, 1609, and 1445 cm-1 wave numbers, respectively. The LKE was found to have six phenolic compounds namely p-hydroxybenzoic acid, gallic acid, syringic acid, p-coumaric acid, ferulic acid, and sinapic acid. In minced mutton, LKE was found to be significantly (p ≤ 0.05) more effective preservative than BHA at 0.5% level of incorporation in terms of TBARS value, tyrosine values, and microbial quality. During storage, the decrease in ERV and increase in total plate count were at a slower rate in treated samples and were devoid of coliform counts. However, sensory attributes at 0.5% LKE incorporation level were negatively affected, and imparted greenish tinge to minced mutton. The present work concluded that LKE might be incorporated without compromising sensory attributes. Further, preservative effects at 0.1% level were at par with BHA (200 ppm), and extended the minced mutton’s shelf life up to 9 d at refrigerated temperature.

Understanding the mechanisms responsible for the kidney's defense against ascending uropathogen is critical to devise novel treatment strategies against increasingly antibiotic resistant uropathogen. Growing body of evidence indicate Intercalated cells of the kidney as the key innate immune epithelial cells against uropathogen. The aim of this study was to find orthologous and differentially expressed bacterial defense genes in human versus murine intercalated cells. We simultaneously analyzed 84 antibacterial genes in intercalated cells enriched from mouse and human kidney samples. Intercalated cell "reporter mice" were exposed to saline versus uropathogenic Escherichia coli (UPEC) transurethrally for 1 h in vivo, and intercalated cells were flow sorted. Human kidney intercalated cells were enriched from kidney biopsy using magnetic-activated cell sorting and exposed to UPEC in vitro for 1 h. RT

Antimicrobial peptides (AMPs) are critical to the protection of the urinary tract of humans and other animals from pathogenic microbial invasion. AMPs rapidly destroy pathogens by disrupting microbial membranes and/or augmenting or inhibiting the host immune system through a variety of signaling pathways. We have previously demonstrated that alpha-defensins 1-3 (

Antimicrobial peptides (AMPs) are critical to the protection of the urinary tract of humans and other animals from pathogenic microbial invasion. AMPs rapidly destroy pathogens by disrupting microbial membranes and/or augmenting or inhibiting the host immune system through a variety of signaling pathways. We have previously demonstrated that alpha-defensins 1-3 ( DEFA1A3 ) are AMPs expressed in the epithelial cells of the human kidney collecting duct in response to uropathogens. We also demonstrated that DNA copy number variations in the DEFA1A3 locus are associated with UTI and pyelonephritis risk. Because DEFA1A3 is not expressed in mice, we utilized human DEFA1A3 gene transgenic mice ( DEFA 4/4 ) to further elucidate the biological relevance of this locus in the murine urinary tract. We demonstrate that the kidney transcriptional and translational expression pattern is similar in humans and the human gene transgenic mouse upon uropathogenic Escherichia coli (UPEC) stimulus in vitro and in vivo. We also demonstrate transgenic human DEFA 4/4 gene mice are protected from UTI and pyelonephritis under various UPEC challenges. This study serves as the foundation to start the exploration of manipulating the DEFA1A3 locus and alpha-defensins 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.

Urinary tract infections (UTI), associated with vesicoureteral reflux (VUR), can lead to chronic kidney disease. Genetic alterations in the innate immune defenses contribute to UTI risk. We investigated a novel gene, Dachsous Cadherin-Related 1 ( DCHS1), in children with UTI. We determined absolute DNA copy number (CN) of DCHS1 in children with UTI. In this case-control study, we utilized multiple complementary methods to determine the genomic CN of DCHS1. Children with ( n = 370) and without ( n = 71) VUR from two well-phenotyped clinical trials of UTI were copy-typed and compared to 491 healthy controls with no known history of VUR or UTI. Less than 1% of controls had a single copy of DCHS1, while 31% of children with UTI and no VUR and 7% of children with UTI and VUR had a single copy of the DCHS1 gene. Using immunostaining, we localized expression postnatally to the bladder and renal epithelia. Mice were also challenged with two uropathogenic Escherichia coli strains, and Dchs1 mRNA was quantified. This study represents the first report of DCHS1 in association with pediatric UTI. We hypothesize that its role in innate immunity is critical to lower urinary tract defense. Further investigation is required to determine the role of DCHS1 in innate immunity.

UropathogenicWe studied the effects of UPEC infection using single cell RNA sequencing (scRNAseq) in zebrafish. Bulk RNA sequencing has historically been used to evaluate gene expression patterns, but scRNAseq allows gene expression to be evaluated at the single cell level and is optimal for evaluating heterogeneity within cell types and rare cell types. Zebrafish cohorts were injected with either saline or UPEC, and scRNAseq and canonical pathway analyses were performed.Canonical pathway analysis of scRNAseq data provided key information regarding innate immune pathways in the cells determined to be thymus cells, ionocytes, macrophages/monocytes, and pronephros cells. Pathways activated in thymus cells included interleukin 6 (IL-6) signaling and production of reactive oxygen species. Fc receptor-mediated phagocytosis was a leading canonical pathway in the pronephros and macrophages. Genes that were downregulated in UPEC vs saline exposed embryos involved the cellular response to the Gram-negative endotoxin lipopolysaccharide (LPS) and included Forkhead Box O1aBecause 4-day post fertilization zebrafish embryos only have innate immune systems, the scRNAseq provides insights into pathways and genes that cell types utilize in the bacterial response. Based on our analysis, we have identified genes and pathways that might serve as genetic targets for treatment and further investigation in UPEC infections at the single cell level.

Carbonic anhydrase II knockout ( Car2−/−) mice have depleted numbers of renal intercalated cells, which are increasingly recognized to be innate immune effectors. We compared pyelonephritis susceptibility following reciprocal renal transplantations between Car2−/−and wild-type mice. We examined the effect of pharmacological CA suppression using acetazolamide in an experimental murine model of urinary tract infection. Car2−/−versus wild-type mice were compared for differences in renal innate immunity. In our transplant scheme, mice lacking CA-II in the kidney had increased pyelonephritis risk. Mice treated with acetazolamide had lower kidney bacterial burdens at 6 h postinfection, which appeared to be due to tubular flow from diuresis because comparable results were obtained when furosemide was substituted for acetazolamide. Isolated Car2−/−kidney cells enriched for intercalated cells demonstrated altered intercalated cell innate immune gene expression, notably increased calgizzarin and insulin receptor expression. Intercalated cell number and function along with renal tubular flow are determinants of pyelonephritis risk.

The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanism by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells that consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H+-ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cre+tdT+mice to fluorescently label ICs and AQP2-cre+tdT+mice to fluorescently label PCs, and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure antimicrobial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC) challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge, this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.

Background The mechanisms by which intestinal bacteria impact liver diseases remain poorly understood. The aim of this study was to develop a mouse model of small-bowel bacterial overgrowth and to determine its impact on hepatobiliary injury. Materials and methods A jejunal self-filling blind loop (SFBL) was created in C57BL/6 mice. Three weeks after surgery, the mice were euthanized, and bacterial cultures of luminal content of the loop and extraintestinal tissues were performed. Liver and jejunum were collected for histological grading of inflammation and injury. Serum liver biochemistry assays were performed. Hepatobiliary transporter mRNA expression in liver was measured by quantitative real-time polymerase chain reaction. Bile and blood were collected for measurement of total bile acids, phospholipid, and cholesterol. Mice undergoing jejunal transection and reanastomosis and laparotomy only served as control groups. Results SFBL induced a dramatic increase in intraluminal bacterial counts, mesenteric lymph node bacterial translocation, and evidence of jejunal and hepatobiliary injury. Significant reductions in hepatic expression of hepatobiliary transporters involved in biliary canalicular export and basolateral uptake were observed in SFBL mice. SFBL resulted in a significant increase in biliary total bile acid concentration, decreases in bile phospholipid and cholesterol output, and an increase in the bile acid/phospholipid ratio. Conclusions We have developed a reproducible mouse model of small-bowel bacterial overgrowth with evidence of liver inflammation, altered hepatobiliary transporter expression, and alterations in bile composition. This model may help to elucidate the mechanisms by which gut-derived bacterial factors impact the liver and contribute to the exacerbation of liver diseases and biliary injury.

Urinary stone disease (USD) is increasing in adult and pediatric populations. Adult and pediatric studies have demonstrated decreased bone mineral density and increased fracture rates. USD has also been independently linked to increased rates of myocardial infarction and cerebral vascular accidents. Although USD is a multisystem disorder involving the kidneys, bone, and vasculature, the molecular mechanisms linking these three organs remain unknown. Calcium oxalate nephropathy was induced in C57BL/6J mice with intra-peritoneal (ip) injection of sodium glyoxolate. Half of each kidney underwent Pizzalato staining and half was snap frozen for RNA extraction. RT2 Profiler Mouse Atherosclerosis, Osteoporosis, and Calcium Signaling PCR Arrays (Qiagen) were performed. Only results that passed quality checks in PCR array reproducibility and genomic DNA contamination were included. Genes had to show at least fourfold differential expression and P 10-fold increase. All 10 have P ≤ 0.003. The calcium signaling array showed significant fourfold upregulation of 10 genes, four of which were ≥10-fold. All 10 have P ≤ 0.03. We have demonstrated that calcium oxalate nephropathy can induce upregulation of atherosclerotic, metabolic bone, and calcium homeostasis genes in a murine model. This may be and initial step in identifying the molecular mechanisms linking stone, bone, and cardiovascular disease. J. Cell. Biochem. 118: 2744–2751, 2017. © 2017 Wiley Periodicals, Inc.

Introduction: Type 2 diabetes mellitus involves a variety of dysfunctions bringing about a few delicate tissue variations from the norm in the oral cavity. These incorporate gingivitis periodontitis, salivary dysfunction, taste dysfunction, oral fungal and bacterial infections, geographic tongue, benign migratory glossitis, fissured tongue, traumatic ulcer, lichen planus, angular cheilitis, delayed mucosal healing, dental root caries, tooth loss, mucosal neurosensory disorders, and oral mucosal lesions. Aim and objectives: This study aims to determine the frequency of different oral manifestations in patients of Type 2 Diabetes Mellitus of Bhopal city which will expand the consciousness of oral signs of diabetes mellitus subsequently helping in speeding up the examination regarding this matter. Methodology: This descriptive study was carried out at the Department of Medicine, People’s Hospital, hitech division, Bhopal from March 2016 to January 2017. 200 patients satisfying the inclusion criteria (diagnosed cases of type 2 diabetes mellitus of > 35 years of age) were included in the study. 84 (42%) were female and 116(58%) were male patients. Result: The frequency of different oral manifestations seen in female diabetic patients in descending order were; gingivitis (49%), periodontitis (36.7%), dental root caries (20%), taste dysfunction (8%), burning mouth syndrome (8%), xerostomia (31%), recurrent apthous stomatitis (11%) and oral candidiasis (2%) and in male diabetic patients in descending order were; gingivitis (41%), periodontitis (29%), dental root caries (18%), taste dysfunction (9%), burning mouth syndrome (3%), xerostomia (31%), recurrent apthous stomatitis (5%) and oral candidiasis (3%). Conclusion: It was inferred that diabetic patients are more inclined to get oral complications so health education, timely diagnosis and effective treatment should be carried out to prevent these complications.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ObjectiveCurrent urinary tract infection (UTI) diagnostic strategies that rely on leukocyte esterase have limited accuracy. We performed an aptamer-based proteomics pilot study to identify urine protein levels that could differentiate a culture proven UTI from culture negative samples, regardless of pyuria status.MethodsWe analyzed urine from 16 children with UTIs, 8 children with culture negative pyuria and 8 children with negative urine culture and no pyuria. The urine levels of 1,310 proteins were quantified using the Somascan™ platform and normalized to urine creatinine. Machine learning with support vector machine (SVM)-based feature selection was performed to determine the combination of urine biomarkers that optimized diagnostic accuracy.ResultsEight candidate urine protein biomarkers met filtering criteria. B-cell lymphoma protein, C-X-C motif chemokine 6, C-X-C motif chemokine 13, cathepsin S, heat shock 70kDA protein 1A, mitogen activated protein kinase, protein E7 HPV18 and transgelin. AUCs ranged from 0.91 to 0.95. The best prediction was achieved by the SVMs with radial basis function kernel.ConclusionsBiomarkers panel can be identified by the emerging technologies of aptamer-based proteomics and machine learning that offer the potential to increase UTI diagnostic accuracy, thereby limiting unneeded antibiotics.

BACKGROUND: Urinary stone are increasing in children, primarily during adolescence. Although urinary stones are often viewed in the context of intermittent stone events, increasing evidence indicates that stones are a metabolic process associated with chronic kidney disease and low bone mass. These aforementioned stone associated conditions may have pediatric origins. OBJECTIVE: To compare urine inflammatory markers in otherwise healthy stone forming children versus matched controls. METHODS: Urine samples were collected from 12 adolescents with urinary stones along with 15 controls. The levels of 30 urine cytokines were measured using a Mesoscale 30-Plex Human Cytokine panel and normalized to urine creatinine levels. RESULTS: Macrophage inflammatory protein 1β and Interleukin 13 levels were significantly elevated in the urine of the stone forming adolescents compared to controls. Interleukin 17A was elevated in the urine of controls. CONCLUSIONS: This study indicates that urine levels of cytokines involved in chronic inflammation and fibrosis are elevated urinary stone formers as early as adolescence. Because stone formers are at risk for chronic kidney disease, Macrophage inflammatory protein 1β and Interleukin 13 represent investigative targets.

Objectives: Low hydration has a deleterious effect on many conditions. In the absence of a urine concentrating defect, urine concentration is a marker of hydration status. However, markers to evaluate hydration status have not been well studied in children. The objectives of this paper are to compare measures of thirst and urine concentration in children and to develop a novel mobile technology application to measure urine concentration.Study Design: Children age 12–17 years were selected (n = 21) for this pilot study. Thirst perception, specific gravity (automated dipstick analysis and refractometer), and urine color scale results were correlated to urine osmolality. The technology department developed a mobile technology camera application to measure light penetrance into urine which was tested on 25 random anonymized urine samples.Results: The patients' thirst perception and color scale as well as two researchers color scale did not significantly correlate with osmolality. Correlation between osmolality and hydration markers resulted in the following Pearson coefficients: SG automated dipstick, 0.61 (P 0.003); SG refractometer, 0.98 (P < 0.0001); urine color scale (patient), 0.37 (P 0.10), and light penetrance, −0.77 (P < 0.0001). The correlation of light penetrance with osmolality was stronger than all measures except SG by refractometer and osmolality.Conclusion: The mobile technology application may be a more accurate tool for urine concentration measurement than specific gravity by automated dipstick, subjective thirst, and urine color scale, but lags behind specific gravity measured by refractometer. The mobile technology application is a step toward patient oriented hydration strategies.

Study was aimed to quantify plasma level of total, short and long fragmented cell-free DNA (cfDNA) along with DNA integrity in patients with oral cancer, oral precancer and tobacco users without lesions and normal controls. In addition, study evaluated the correlation of cfDNA with clinicopathologic parameters of oral cancer.Plasma samples were collected preoperatively from 44 patients with oral cancer, 40 patients with oral precancer, 40 tobacco users without any oral lesion and 40 healthy controls without any tobacco habit. cfDNA extraction was carried out from the plasma followed by quantitative and qualitative assessment of extracted DNA. Quantity of short and long fragmented DNA was assessed by using PCR with two different primer sets for the beta-actin gene, amplifying short (102 bp) and long (253 bp) products. The DNA integrity index was measured by calculating the ratio of quantity of long fragmented to short fragmented DNA. All quantitative cfDNA parameters were statistically analyzed to verify their correlation with clinicopathologic parameters.Results showed that total cfDNA level, short and long fragmented cfDNA concentration and DNA integrity was significantly higher in oral cancer group as compare to other (p=0.0001). Study demonstrated that there is no correlation total, short and long cfDNA and DNA integrity with tumor size and histologic type or grading. But positive correlation of total cfDNA was found with nodal metastasis (p=0.001) and clinical stages (p=0.006).Quantitative analysis of total cfDNA may be applied as a screening marker for early detection of precancer and cancer as well as for prognostication of oral cancer. Additionally, plasma levels of short and long fragmented cfDNA and DNA integrity index can be applied for early detection of oral cancer.

The renal collecting duct consists of intercalated cells (ICs) and principal cells (PCs). We have previously demonstrated that collecting ducts have a role in the innate immune defense of the kidney. Transcriptomics is an important tool used to enhance systems-level understanding of cell biology. However, transcriptomics performed on whole kidneys provides limited insight of collecting duct cell gene expression, because these cells comprise a small fraction of total kidney cells. Recently we generated reporter mouse models to enrich collecting duct specific PC and ICs and reported targeted gene expression of anti-microbial peptide genes. Here we report transcriptomics on enriched ICs and PCs and performed a pilot study sequencing four single ICs. We identified 3,645 genes with increased relative expression in ICs compared to non-ICs. In comparison to non-PCs, 2,088 genes had higher relative expression in PCs. IC associated genes included the innate interleukin 1 receptor, type 1 and the antimicrobial peptide (AMP) adrenomedullin. The top predicted canonical pathway for enriched ICs was lipopolysaccharide/Interleukin 1 mediated inhibition of Retinoid X Receptor alpha function and decreased Retinoid X Receptor expression was confirmed to occur 1-hour post experimental murine UTI in ICs but not in non-ICs.

Objectives Urinary tract infection (UTI) often represents a diagnostic challenge in the emergency department (ED) where urine culture results are generally not available and other tests demonstrate limited sensitivity and specificity. Antimicrobial peptides (AMPs) are components of the innate immune system that have demonstrated increased urinary levels in response to infection both in children and in adults with chronic UTI. The objective of this study was to determine the relationship between urinary AMP levels and positive urine cultures in adult ED patients with suspected UTI. Methods This was a prospective, observational study of adult ED patients with suspected UTI. Enzyme-linked immunosorbent assays were performed to measure urine levels of AMPs: human neutrophil peptides 1–3 (HNP1–3), human α-defensin 5 (HD5), human beta defensin 2 (hBD-2), and cathelicidin (LL-37). Comparisons between positive and negative cultures were performed using Wilcoxon rank sum tests and receiver operating characteristic curves, with calculation of area under the curve (AUC). Data were also analyzed for the older adult subgroup. Results Of 40 patients enrolled, 23 (58%) were ≥ 65 years, 25 were female (64%), and seven (17%) were nonwhite. Cultures were positive in 13 (32%), including seven in those ≥ 65 years old. HNP1–3, HD5, and hBD-2 levels were significantly higher in those with positive than negative urine cultures. Median HNP1–3 was 5.39 ng/mg (interquartile range [IQR] = 2.74 to 11.09) in positive vs. 0.81 ng/mg (IQR = 0.06 to 3.87) in negative cultures. Median HD5 was 4.75 pg/mg (IQR = 1.6 to 22.7) in positive versus 0.00 pg/mg (IQR = 0 to 2.60) in negative cultures, and median hBD-2 was 0.13 pg/mg (IQR = 0.08 to 0.17) in positive versus 0.02 pg/mg (IQR = 0 to 0.04) in negative cultures (p < 0.05 for all). Findings were similar for adults ≥ 65 years. The AUC was ≥ 0.75 for all three AMPs, both overall and in the older adult subgroup. LL-37 was not significantly higher in patients with positive urine culture. However, LL-37 expression is vitamin D dependent, and inadequate serum levels (< 30 ng/mL) were present in 72% of those tested. Conclusions Urinary levels of HNP1–3, HD5, and hBD-2 are significantly greater in the presence of positive urine cultures in ED patients with suspected UTI. These findings are maintained in the high-risk subgroup of older adults.

Urinary stone disease (USD) is increasing in adult and pediatric populations. Adult and pediatric studies have demonstrated decreased bone mineral density and increased fracture rates. USD has also been independently linked to increased rates of myocardial infarction and cerebral vascular accidents. Although USD is a multisystem disorder involving the kidneys, bone, and vasculature, the molecular mechanisms linking these three organs remain unknown. Calcium oxalate nephropathy was induced in C57BL/6J mice with intra-peritoneal (ip) injection of sodium glyoxolate. Half of each kidney underwent Pizzalato staining and half was snap frozen for RNA extraction. RT

Summary In order to examine the association between HIV/AIDS knowledge and perceptions, and risk intentions and behaviours among adolescents in Goa, India, cross-sectional data from 942 youth were collected and assessed. The prevalence rates in the past six months for fighting, smoking, drinking and drug use were 16.5%, 3.8%, 17.8% and 1.1%, respectively; 5.2% acknowledged ever having engaged in sex. Prior risk involvement was significantly correlated with future risk intention (odds ratio [OR]: 9.7–19.7), and those involved in one risk behaviour were more likely to engage in other risk behaviours (OR: 1.3–23.5). The findings suggest the importance of targeted interventions for youth engaging or intending to engage in risk behaviours and universal interventions regarding basic facts and skills for all youth in Goa.

Background & Aims Although recent studies have identified important roles for T and NK cells in the pathogenesis of biliary atresia (BA), the mechanisms by which susceptibility to bile duct injury is restricted to the neonatal period are unknown. Methods We characterised hepatic regulatory T cells (Tregs) by flow cytometry in two groups of neonatal mice challenged with rhesus rotavirus (RRV) at day 7 (no ductal injury) or day 1 of life (resulting in BA), determined the functional interaction with effector cells in co-culture assays, and examined the effect of adoptive transfer of CD4+ cells on the BA phenotype. Results While day 7 RRV infection increased hepatic Tregs (Foxp3+ CD4+ CD25+) by 10-fold within 3days, no increase in Tregs occurred at this time point following infection on day 1. In vitro , Tregs effectively suppressed NK cell activation by hepatic dendritic cells and decreased the production of pro-inflammatory cytokines, including TNFα and IL-15, following RRV infection. In vivo , adoptive transfer of CD4+ cells prior to RRV inoculation led to increased survival, improved weight gain, decreased population of hepatic NK cells, and persistence of donor Tregs in the liver. Conclusions (1) The liver is devoid of Tregs early after perinatal RRV infection; (2) Tregs suppress DC-dependent activation of naive NK cells in vitro , and Treg-containing CD4+ cells inhibit hepatic NK cell expansion in vivo . Thus, the post-natal absence of Tregs may be a key factor that allows hepatic DCs to act unopposed in NK cell activation during the initiation of neonatal bile duct injury.

Ample evidence suggests a role of TGF-β in preventing autoimmunity. Multiorgan inflammatory disease, spontaneous activation of self-reactive T cells, and autoantibody production are hallmarks of autoimmune diseases, such as lupus. These features are reminiscent of the immunopathology manifest in TGF-β1-deficient mice. In this study, we show that lupus-prone (New Zealand Black and White)F1 mice have reduced expression of TGF-β1 in lymphoid tissues, and TGF-β1 or TGF-β1-producing T cells suppress autoantibody production. In contrast, the expression of TGF-β1 protein and mRNA and TGF-β signaling proteins (TGF-β receptor type II and phosphorylated SMAD3) increases in the target organs, i.e., kidneys, of these mice as they age and develop progressive organ damage. In fact, the levels of TGF-β1 in kidney tissue and urine correlate with the extent of chronic lesions that represent local tissue fibrosis. In vivo TGF-β blockade by treatment of these mice with an anti-TGF-β Ab selectively inhibits chronic fibrotic lesions without affecting autoantibody production and the inflammatory component of tissue injury. Thus, TGF-β plays a dual, seemingly paradoxical, role in the development of organ damage in multiorgan autoimmune diseases. According to our working model, reduced TGF-β in immune cells predisposes to immune dysregulation and autoantibody production, which causes tissue inflammation that triggers the production of anti-inflammatory cytokines such as TGF-β in target organs to counter inflammation. Enhanced TGF-β in target organs, in turn, can lead to dysregulated tissue repair, progressive fibrogenesis, and eventual end-organ damage.

Islet Ag-specific CD4+ T cells receive antigenic stimulation from MHC class II-expressing APCs. Herein, we delineate the direct in vivo necessity for distinct subsets of macrophages and dendritic cells (DC) in type 1 diabetes mellitus of the NOD mouse by using diphtheria toxin-mediated cell ablation. The ablation of macrophages had no impact on islet Ag presentation or on the induction of insulitis or diabetes in either transfer or spontaneous models. However, the ablation of CD11b+CD11c+ DC led to the loss of T cell activation, insulitis, and diabetes mediated by CD4+ T cells. When the specific myeloid DC subset was “added-back” to mice lacking total DC, insulitis and diabetes were restored. Interestingly, when NOD mice were allowed to progress to the insulitis phase, the ablation of DC led to accelerated insulitis. This accelerated insulitis was mediated by the loss of plasmacytoid DC (pDC). When pDC were returned to depleted mice, the localized regulation of insulitis was restored. The loss of pDC in the pancreas itself was accompanied by the localized loss of IDO and the acceleration of insulitis. Thus, CD11c+CD11b+ DC and pDC have countervailing actions in NOD diabetes, with myeloid DC providing critical antigenic stimulation to naive CD4+ T cells and pDC providing regulatory control of CD4+ T cell function in the target tissue.

Background The role of calcium oxalate crystals and deposits in UTI pathogenesis has not been established. The objectives of this study were to identify bacteria present in pediatric urolithiasis and, using in vitro and in vivo models, to determine the relevance of calcium oxalate deposits during experimental pyelonephritis. Methods Pediatric kidney stones and urine were collected and both cultured and sequenced for bacteria. Bacterial adhesion to calcium oxalate was compared. Murine kidney calcium oxalate deposits were induced by intraperitoneal glyoxalate injection and kidneys were transurethrally inoculated with uropathogenic Escherichia coli to induce pyelonephritis Results E. coli of the family Enterobacteriaceae was identified in patients by calcium oxalate stone culture. Additionally Enterobacteriaceae DNA was sequenced from multiple calcium oxalate kidney stones. E. coli selectively aggregated on and around calcium oxalate monohydrate crystals. Mice inoculated with glyoxalate and uropathogenic E. coli had higher bacterial burdens, increased kidney calcium oxalate deposits and an increased kidney innate immune response compared to mice with only calcium oxalate deposits or only pyelonephritis. Conclusions In a murine model, the presence of calcium oxalate deposits increases pyelonephritis risk, likely due to preferential aggregation of bacteria on and around calcium oxalate crystals. When both calcium oxalate deposits and uropathogenic bacteria were present, calcium oxalate deposit number increased along with renal gene transcription of inner stone core matrix proteins increased. Therefore renal calcium oxalate deposits may be a modifiable risk factor for infections of the kidney and urinary tract. Furthermore, bacteria may be present in calcium oxalate deposits and potentially contribute to calcium oxalate renal disease.

An ACE inhibitor reduces Th2 cytokines and TGF-β1 and TGF-β2 isoforms in murine lupus nephritis. Background Angiotensin-converting enzyme (ACE) inhibitors, such as captopril, are used to control hypertension. In patients and animals with primary nephropathies, these agents improve renal function more than that would be expected from their control of hypertension. Here, we examine the effects of treatment with captopril on lupus nephritis and discuss the potential mechanism(s) by which this agent exerts its renoprotective effects. Methods Lupus-prone, NZB/NZW F1 and MRL- lpr/lpr , mice were treated with captopril or with a control antihypertensive agent, verapamil. Mice were monitored for nephritis, and their sera and tissues analyzed for cytokine and transforming growth factor-β (TGF-β) expression. Results Captopril treatment delayed the onset of proteinuria when administered to prenephritic mice, whereas verapamil did not. Captopril treatment also retarded disease progression when given to lupus mice that had early disease, and even reversed severe proteinuria in at least some older animals with advanced disease. It reduced chronic renal lesions, but had no effect on autoantibody production. The improvement in renal disease correlated with reduced TGF-β expression, particularly of the TGF-β1 and TGF-β2 isoforms, in the kidneys. Interestingly, in vivo or in vitro exposure to captopril reduced splenic levels of type 2 cytokines, interleukin (IL)-4 and IL-10, suggesting a possible role of the immune system in captopril-mediated disease modulation. Conclusion Since type 2 cytokines are known to promote lupus glomerulosclerosis, decreased IL-4 and IL-10 production in captopril-treated mice may be related to this agent's renoprotective effects. We argue here that ACE inhibitors not only act as selective TGF-β inhibitors, but also as selective immunomodulators, to improve lupus nephritis.

Mechanisms that initiate lupus nephritis and cause progression to end-stage renal disease remain poorly understood. In this study, we show that lupus-prone New Zealand Mixed 2410 mice that develop a severe glomerulosclerosis and rapidly progressive renal disease overexpress IL-4 in vivo. In these mice, STAT6 deficiency or anti-IL-4 Ab treatment decreases type 2 cytokine responses and ameliorates kidney disease, particularly glomerulosclerosis, despite the presence of high levels of IgG anti-dsDNA Abs. STAT4 deficiency, however, decreases type 1 and increases type 2 cytokine responses, and accelerates nephritis, in the absence of high levels of IgG anti-dsDNA Abs. Thus, STAT6 and IL-4 may selectively contribute to the development of glomerulosclerosis, whereas STAT4 may play a role in autoantibody production.

Many individuals develop a single or a few brief episodes of autoimmunity from which they recover. Mechanisms that quell pathologic autoimmunity following such a breakdown of self-tolerance are not clearly understood. In this study, we show that in nonautoimmune mice, dsDNA-specific autoreactive B cells exist but remain inactive. This state of inactivation in dsDNA-specific B cells could be disrupted by autoreactive Th cells; in this case T cells that react with peptides from the VH region of anti-DNA Abs (hereafter called anti-VH T cells). Immunization with anti-DNA mAb, its γ-chain or peptides derived from its VH region induced anti-VH Th cells, IgG anti-dsDNA Ab, and proteinuria. The breakdown of B cell tolerance in nonautoimmune mice, however, was short-lived: anti-DNA Ab and nephritis subsided despite subsequent immunizations. The recovery from autoimmunity temporally correlated with the appearance of T cells that inhibited anti-DNA Ab production. Such inhibitory T cells secreted TGFβ; the inhibition of anti-DNA Ab production by these cells was partly abolished by anti-TGFβ Ab. Even without immunization, nonautoimmune mice possess T cells that can inhibit autoantibody production. Thus, inhibitory T cells in nonautoimmune mice may normally inhibit T-dependent activation of autoreactive B cells and/or reverse such activation following stimulation by Th cells. The induction of such inhibitory T cells may play a role in protecting nonautoimmune mice from developing chronic autoimmunity.

Carbonic anhydrase 2 regulates acid-base homeostasis, and recent findings have indicated a correlation between cellular control of acid-base status and the innate defense of the kidney. Mice deficient in carbonic anhydrase 2 ( Car2−/−mice) have metabolic acidosis, impaired urine acidification, and are deficient in normal intercalated cells. The objective of the present study was to evaluate the biological consequences of carbonic anhydrase 2 deficiency in a murine model of pyelonephritis. Infection susceptibility and transcription of bacterial response components in Car2−/−mice were compared with wild-type littermate controls. Car2−/−mice had increased kidney bacterial burdens along with decreased renal bacterial clearance after inoculation compared with wild-type mice. Standardization of the urine pH and serum HCO3−levels did not substantially alter kidney infection susceptibility between wild-type and Car2−/−mice; thus, factors other than acid-base status are responsible. Car2−/−mice had significantly increased neutrophil-gelatinase-associated lipocalin mRNA and protein and expression at baseline and a marked decreased ability to upregulate key bacterial response genes during pyelonephritis. Our findings provide in vivo evidence that supports a role for carbonic anhydrase 2 and intercalated cells in promoting renal bacterial clearance. Decreased carbonic anhydrase expression results in increased antimicrobial peptide production by cells other than renal intercalated cells, which is not sufficient to prevent infection after a bacterial challenge.

Biliary atresia is the most common cholangiopathy of childhood. During infancy, an idiopathic activation of the neonatal immune system targets the biliary epithelium, obstructs bile ducts, and disrupts the anatomic continuity between the liver and the intestine. Here, we use a model of virus-induced biliary atresia in newborn mice to trace the initiating pathogenic disease mechanisms to resident plasmacytoid (pDCs) and conventional (cDCs) dendritic cells. We found pDCs to be the most abundant DC population in the livers of newborn mice, and we observed pDCs in the livers of infants at the time of diagnosis. In the livers of newborn mice, cDCs spontaneously overexpressed the costimulatory molecule CD80 soon after birth, and pDCs produced the cytokine interleukin-15 (IL-15) in response to a virus insult. Both subtypes of primed DCs were required for the proliferation of T lymphocytes and the activation of natural killer cells. Disruption of this cellular network by depletion of pDCs or blockade of IL-15 signaling in mice in vivo prevented epithelial injury, maintained anatomic continuity of the bile duct, and promoted long-term survival. These findings identify cellular triggers of biliary injury and have implications for future therapies to block the progression of biliary atresia and liver disease.

Diets high in saturated fat and fructose have been implicated in the development of obesity and nonalcoholic steatohepatitis (NASH) in humans. We hypothesized that mice exposed to a similar diet would develop NASH with fibrosis associated with increased hepatic oxidative stress that would be further reflected by increased plasma levels of the respiratory chain component, oxidized coenzyme Q9 (oxCoQ9). Adult male C57Bl/6 mice were randomly assigned to chow, high-fat (HF), or high-fat high-carbohydrate (HFHC) diets for 16 weeks. The chow and HF mice had free access to pure water, whereas the HFHC group received water with 55% fructose and 45% sucrose (wt/vol). The HFHC and HF groups had increased body weight, body fat mass, fasting glucose, and were insulin-resistant compared with chow mice. HF and HFHC consumed similar calories. Hepatic triglyceride content, plasma alanine aminotransferase, and liver weight were significantly increased in HF and HFHC mice compared with chow mice. Plasma cholesterol (P < 0.001), histological hepatic fibrosis, liver hydroxyproline content (P = 0.006), collagen 1 messenger RNA (P = 0.003), CD11b-F4/80+Gr1+ monocytes (P < 0.0001), transforming growth factor β1 mRNA (P = 0.04), and α-smooth muscle actin messenger RNA (P = 0.001) levels were significantly increased in HFHC mice. Hepatic oxidative stress, as indicated by liver superoxide expression (P = 0.002), 4-hydroxynonenal, and plasma oxCoQ9 (P < 0.001) levels, was highest in HFHC mice. Conclusion: These findings demonstrate that nongenetically modified mice maintained on an HFHC diet in addition to developing obesity have increased hepatic ROS and a NASH-like phenotype with significant fibrosis. Plasma oxCoQ9 correlated with fibrosis progression. The mechanism of fibrosis may involve fructose inducing increased ROS associated with CD11b+F4/80+Gr1+ hepatic macrophage aggregation, resulting in transforming growth factor β1–signaled collagen deposition and histologically visible hepatic fibrosis. (HEPATOLOGY 2010)

TGF-β1 plays an important role in the maintenance of immune homeostasis and self-tolerance. To determine the mechanism by which TGF-β1 prevents autoimmunity we have analyzed T cell activation in splenic lymphocytes from TGF-β1-deficient mice. Here we demonstrate that unlike wild-type splenic lymphocytes, those from Tgfb1−/− mice are hyporesponsive to receptor-mediated mitogenic stimulation, as evidenced by diminished proliferation and reduced IL-2 production. However, they have elevated levels of IFN-γ and eventually undergo apoptosis. Receptor-independent stimulation of Tgfb1−/− T cells by PMA plus ionomycin induces IL-2 production and mitogenic response, and it rescues them from anergy. Tgfb1−/− T cells display decreased CD3 expression; increased expression of the activation markers LFA-1, CD69, and CD122; and increased cell size, all of which indicate prior activation. Consistently, mutant CD4+ T cells have elevated intracellular Ca2+ levels. However, upon subsequent stimulation in vitro, increases in Ca2+ levels are less than those in wild-type cells. This is also consistent with the anergic phenotype. Together, these results demonstrate that the ex vivo proliferative hyporesponsiveness of Tgfb1−/− splenic lymphocytes is due to prior in vivo activation of T cells resulting from deregulated intracellular Ca2+ levels.

Background It is known that during normal pregnancy and after immunotherapy blocking antibodies are developed, these antibodies inhibit mixed lymphocyte reaction and are also anti-mitogenic in nature. Mixed lymphocyte reaction blocking antibodies are specific to the husband's lymphocytes. In the present study an attempt has been made to characterize the mixed lymphocyte reaction blocking antibodies in normal pregnancy and in women with recurrent spontaneous abortion after immunotherapy. Methods Serum was obtained from women of different gestational windows of pregnancy (Ist, IInd, IIIrd trimesters and post delivery period of normal pregnancy), recurrent spontaneous aborters from pre and post immunization. Healthy (male and females) controls were screened for the presence of mixed lymphocyte reaction blocking antibodies. The standard mixed lymphocyte reaction technique was used to evaluate the inhibitory effect of serum in the mixed lymphocyte reaction. Each serum was tested for cytotoxic antibodies. Immunoglobulin G and its isotypes were isolated according to the standard protocol. Results In the present study we have observed that there was significant inhibition of proliferation response when immunoglobulin G from different trimesters of pregnancy were added to one way mixed lymphocyte reaction or to phytohemagglutinin activated lymphocyte proliferation assay. Similar pattern was seen when immunoglobulin G isolated from adequately immunized women with recurrent spontaneous abortion was used. It was further confirmed that amongst all the isotypes of immunoglobulin G, only immunoglobulin G-3 was found to be positive for the inhibitory effect. Conclusions Present study indicates that mixed lymphocyte reaction blocking antibodies are immunoglobulin G-3 in nature. It is developed during pregnancy and also after immunotherapy in women with recurrent spontaneous abortion who subsequently have the successful pregnancy.

Islets specific CD4+ T Cells receive instructional signals from antigen bearing MHC class II-expressing antigen presenting cells (APC), yet the influence of APC on autoimmune diabetes remains elusive. Herein we delineate the direct in vivo necessity for macrophages and dendritic cells in Type 1 Diabetes of the non-obese diabetic (NOD) mouse by using diphtheria toxin-mediated conditional abaltion. We find that specific loss of macrophages had no impact on diabetes or insulitis in both spontaneous and transfer model. Loss of both myeloid (mDC) and lymphoid DC (ly DC) led to protection of NOD mice from diabetes and accompanying loss of T cell activation, while specific loss of plasmacytoid DC (pDC) and Indoleamine 2,3,dioxygenase (IDO) led to breakdown in regulatory control and accelerated insultis. These data imply that autoimmune diabetes is regulated by imbalance of mDC and pDC function in vivo.

To investigate the metabolic relationship between urea and guanidinosuccinic acid (GSA), we determined the levels of the guanidino compounds, including GSA, and urea in serum and urine of cirrhotic patients. Linear correlation studies between serum urea and GSA levels were performed. Good positive linear correlation coefficients were found in the Child-Turcotte C subgroup (r = .847, P < .001) and in the total subgroup including B and C patients (r = .848; P < .0001). Serum guanidinoacetic acid levels were significantly increased in the Child-Turcotte C subgroup (P < .0001 for men and P < .001 for women). In contrast, GSA levels were significantly (P < .0001) decreased in the three studied subgroups. Similar results were found for urinary GSA excretion levels. Within each subgroup, serum and urinary GSA levels were significantly lower in patients with alcohol-induced cirrhosis than in nonalcoholic cirrhotic patients. Similar results were obtained for urea. The findings in cirrhotic patients clearly demonstrate a metabolic relationship between urea and GSA. They also show that urea and GSA biosynthesis is significantly lower in cirrhotic patients with an alcoholic origin than in cirrhotic patients with a nonalcoholic origin.

Biliary atresia results from an NK cell-mediated inflammatory injury of the bile duct epithelium with onset in the early postnatal period. Based on key immunoregulatory roles of dendritic cells (DC), we hypothesized that early neonatal DC activation triggers an effector immune response that results in biliary atresia. We tested this hypothesis in a mouse model of rotavirus-induced biliary atresia. Following rotavirus challenge on day 1 of life, viral immunogenic protein NSP3 was detected in hepatic PDCA-1+ plasmacytoid DC (pDC) but not CD11c+ conventional DC (cDC) within 3 days of infection. Hepatic pDC increased 3 fold, while non-pDC (CD11b+CD11c+ cells) decreased 2.5 fold. Rotavirus-naïve perinatal hepatic pDC and cDC spontaneously expressed B7.1/2 (2-3 fold higher than adults), with a much higher increased expression in B7.1/2, IFNγ, and IL-12p40 by pDC after rotavirus challenge. IL-15 was spontaneously expressed by naïve perinatal pDC and increased dramatically after rotavirus infection, but in vitro activation of NK cells required co-culture with both pDC and cDC. Most notably, ablation of pDC decreased the liver population of NK cells and prevented the mucosa injury and obstruction of neonatal bile ducts, leading to improved symptoms and long-term survival. Thus early activation of pDC due upon viral exposure regulates neonatal duct injury and obstruction in biliary atresia.

Air velocities at several points near the spark plug location were measured using a laser Doppler anemometer (LDA) in a motoring internal combustion engine. This engine was equipped with three intake configurations: a standard valve in a standard port, a standard valve in a helical (swirl) port, and a shrouded valve in a standard port. Both ensemble-averaged and cycle-resolved mean velocities were determined; this allows comparison of rms velocity fluctuation, cycle-resolved turbulence, and cycle-by-cycle variations in mean velocity. Frequency domain analysis of these components was also carried out. The main conclusion of this experimental study was that starting from a very low swirl situation (standard valve case), addition of a moderate amount of swirl (swirl port case) goes a long way towards improving cycle repeatability.