Murine Ribonuclease 6 Limits Bacterial Dissemination during Experimental Urinary Tract Infection.

INTRODUCTION: The ribonuclease (RNase) A superfamily encodes cationic antimicrobial proteins with potent microbicidal activity toward uropathogenic bacteria. Ribonuclease 6 (RNase6) is an evolutionarily conserved, leukocyte-derived antimicrobial peptide with potent microbicidal activity toward uropathogenic Escherichia coli (UPEC), the most common cause of bacterial urinary tract infections (UTIs). In this study, we generated Rnase6-deficient mice to investigate the hypothesis that endogenous RNase 6 limits host susceptibility to UTI. METHODS: We generated a Rnase6EGFP knock-in allele to identify cellular sources of Rnase6 and determine the consequences of homozygous Rnase6 deletion on antimicrobial activity and UTI susceptibility. RESULTS: We identified monocytes and macrophages as the primary cellular sources of Rnase6 in bladders and kidneys of Rnase6EGFP/+ mice. Rnase6 deficiency (i.e., Rnase6EGFP/EGFP) resulted in increased upper urinary tract UPEC burden during experimental UTI, compared to Rnase6+/+ controls. UPEC displayed increased intracellular survival in Rnase6-deficient macrophages. CONCLUSION: Our findings establish that RNase6 prevents pyelonephritis by promoting intracellular UPEC killing in monocytes and macrophages and reinforce the overarching contributions of endogenous antimicrobial RNase A proteins to host UTI defense.

Concentration of novel urinary tract infection biomarkers in neonates.

Urinary tract infections (UTIs) are a common comorbidity in hospitalized neonates. The current UTI diagnostics have several limitations including invasive collection of urinary samples to ensure sterility, risk of contamination and lack of consensus definitions of UTI based on urine culture. Antimicrobial peptides (AMPs) have been recently utilized as novel biomarkers that can efficiently and accurately diagnose pediatric UTI. However, the concentration of AMPs in neonatal urine is not well-defined. Urine from neonates admitted to a single level IV neonatal intensive care unit was obtained to determine baseline concentration of two AMPs, Ribonuclease 7 (RNase 7) and Beta Defensin-1 (BD-1) and to define the relationship between AMP concentration and gestational age (GA). AMP levels were normalized to urine creatinine. RNase 7 and BD-1 were expressed in neonatal urine (n = 66) regardless of GA and as early as 22 weeks gestation. Urinary concentrations of both AMPs decreased as GA and birthweight increased. The overall median urinary RNase 7/UCr and BD-1/UCr values were 271 ng/mg, and 116 ng/mg, respectively. Median urinary concentrations of RNase 7/UCr for infants born at < 27, 27-32, 33-35 and ≥ 36 weeks were 569, 308, 254, and 124 ng/mg respectively. Similarly, the concentrations of BD-1/UCr at these GA were 166, 115, 108, and 14 ng/mg, respectively. Baseline neonatal urinary concentration of two AMPs (RNase 7 and BD-1) and the variation by GA were identified. This is an essential first step toward the potential utilization of AMPs in improving neonatal UTI diagnostics.

Human Ribonuclease 6 Has a Protective Role during Experimental Urinary Tract Infection.

Mounting evidence suggests that antimicrobial peptides and proteins (AMPs) belonging to the RNase A superfamily have a critical role in defending the bladder and kidney from bacterial infection. RNase 6 has been identified as a potent, leukocyte-derived AMP, but its impact on urinary tract infection (UTI) in vivo has not been demonstrated. To test the functional role of human RNase 6, we generated RNASE6 transgenic mice and studied their susceptibility to experimental UTI. In addition, we generated bone marrow-derived macrophages to study the impact of RNase 6 on antimicrobial activity within a cellular context. When subjected to experimental UTI, RNASE6 transgenic mice developed reduced uropathogenic Escherichia coli (UPEC) burden, mucosal injury, and inflammation compared to non-transgenic controls. Monocytes and macrophages were the predominant cellular sources of RNase 6 during UTI, and RNASE6 transgenic macrophages were more proficient at intracellular UPEC killing than non-transgenic controls. Altogether, our findings indicate a protective role for human RNase 6 during experimental UTI.

Role of Streptococcus pneumoniae extracellular glycosidases in immune evasion.

Streptococcus pneumoniae (pneumococcus) typically colonizes the human upper airway asymptomatically but upon reaching other sites of the host body can cause an array of diseases such as pneumonia, bacteremia, otitis media, and meningitis. Be it colonization or progression to disease state, pneumococcus faces multiple challenges posed by host immunity ranging from complement mediated killing to inflammation driven recruitment of bactericidal cells for the containment of the pathogen. Pneumococcus has evolved several mechanisms to evade the host inflicted immune attack. The major pneumococcal virulence factor, the polysaccharide capsule helps protect the bacteria from complement mediated opsonophagocytic killing. Another important group of pneumococcal proteins which help bacteria to establish and thrive in the host environment is surface associated glycosidases. These enzymes can hydrolyze host glycans on glycoproteins, glycolipids, and glycosaminoglycans and consequently help bacteria acquire carbohydrates for growth. Many of these glycosidases directly or indirectly facilitate bacterial adherence and are known to modulate the function of host defense/immune proteins likely by removing glycans and thereby affecting their stability and/or function. Furthermore, these enzymes are known to contribute the formation of biofilms, the bacterial communities inherently resilient to antimicrobials and host immune attack. In this review, we summarize the role of these enzymes in host immune evasion.

Laboratory diagnosis of COVID-19: current status and challenges.

The magnitude and pace of global affliction caused by Coronavirus Disease-19 (COVID-19) is unprecedented in the recent past. From starting in a busy seafood market in the Chinese city of Wuhan, the virus has spread across the globe in less than a year, infecting over 76 million people and causing death of close to 1.7 million individuals worldwide. As no specific antiviral treatment is currently available, the major strategy in containing the pandemic is focused on early diagnosis and prompt isolation of the infected individuals. Several diagnostic modalities have emerged within a relatively short period, which can be broadly classified into molecular and immunological assays. While the former category is centered around real-time PCR, which is currently considered the gold standard of diagnosis, the latter aims to detect viral antigens or antibodies specific to the viral antigens and is yet to be recommended as a stand-alone diagnostic tool. This review aims to provide an update on the different diagnostic modalities that are currently being used in diagnostic laboratories across the world as well as the upcoming methods and challenges associated with each of them. In a rapidly evolving diagnostic landscape with several testing platforms going through various phases of development and/or regulatory clearance, it is prudent that the clinical community familiarizes itself with the nuances of different testing modalities currently being employed for this condition.

Trans IL-6 signaling does not appear to play a role in renal scarring after urinary tract infection.

INTRODUCTION: While inflammation is an important innate defense mechanism against infection, it can also lead to local tissue damage. The trans signaling pathway of interleukin (IL)-6 is a known mediator of inflammation. We hypothesized that the trans IL-6 signaling pathway is associated with the development of post febrile urinary tract infection (UTI) renal scarring. OBJECTIVE: To compare soluble regulators of trans IL-6 signaling between patients with a history of febrile UTI who do or do not have renal scarring. STUDY DESIGN: After IRB-approval, we collected urine samples in pediatric patients with a history of febrile (≥38 °C) UTI (urine culture >50 K uropathogen) with documented presence or absence of renal scarring on imaging. Samples were collected at a time when patients were not actively infected. Enzyme-linked immunosorbent assays were performed on samples for markers of trans IL-6 signaling: IL-6, soluble (s) IL-6 receptor (R), and soluble (s)gp130, a buffer in trans IL-6 signaling. Values were normalized to urine creatinine. Results were analyzed by t-test or Mann-Whitney U. Spearman rank correlation was used. A p-value of <0.05 was considered significant. RESULTS: A total of 50 urines from patients with a history of febrile UTI were collected: 23 with and 27 without scarring. There was no difference between groups regarding age or gender. There was no significant difference in urine IL-6, sIL-6R, or sgp130 between those with and without scarring (Figure). While IL-6 values significantly correlated with sIL-6R and sgp130 in those without renal scarring, IL-6 did not correlate with sgp130 in those with scarring. Ratios of IL-6 to sgp130 and sIL-6R to sgp130 were not different between groups. DISCUSSION: The inflammatory response generated in response to infection is believed to be largely responsible for the development of renal scarring after UTI. IL-6 is a cytokine known to be induced during UTI with a pro-inflammatory pathway, known as trans signaling. This study investigated for differences in markers of trans IL-6 signaling between patients with a history of febrile UTI with and without renal scarring. There was no significant difference between the absolute values or ratio of these markers between groups. CONCLUSIONS: Markers of trans IL-6 signaling are not different between individuals with a history of febrile UTI with and without renal scarring in the non-acute setting.

Impact of successful pediatric ureteropelvic junction obstruction surgery on urinary HIP/PAP and BD-1 levels.

INTRODUCTION: In the pediatric patient whose ureteropelvic junction obstruction (UPJO) is not always symptomatic, imaging is the most common means of detecting surgical success. There is interest, however, in other means of post-operative monitoring. A panel of antimicrobial peptides (AMPs) has been previously found to be elevated in UPJO, but the impact of surgical correction on these AMPs is unknown. OBJECTIVE: To determine if elevated levels of candidate urinary AMP biomarkers of urinary tract obstruction decrease following UPJO repair. STUDY DESIGN: Pediatric patients undergoing surgical correction of an UPJO were recruited for participation. Bladder urine from uninfected consenting/assenting patients was collected immediately prior to surgery and then at least 6 months afterward. Based on prior studies demonstrating significant elevation of beta defensin 1 (BD-1), hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), cathelicidin (LL-37), and neutrophil gelatinase-associated lipocalin (NGAL) in patients with UPJO versus control patients, we performed enzyme-linked immunosorbent assays on these four AMPs to compare their expression before and after surgical intervention. If found to significantly decrease, AMP levels were compared to healthy controls. AMP levels were normalized to urine creatinine. Results were analyzed with paired t test or Wilcoxon test using Graphpad software. Correlation was calculated using Pearson or Spearman correlation. A p-value of <0.05 was considered significant. RESULTS: 13 UPJO patients were included in this study; 9 were male (69%). Age at surgery was a median of 4.3 years (average 6.1, range 0.4-18.4 years). Follow-up urine samples were collected a median of 27.4 months after surgery (average 27.4; range 7.8-45.3 months). All 13 patients had clinical improvement and/or signs of improved hydronephrosis on post-operative imaging. HIP/PAP and BD-1 significantly decreased in post-surgical samples compared to pre-surgical samples (p = 0.02 and 0.01, respectively); NGAL and LL-37 did not significantly change. Overall, HIP/PAP decreased in 12 patients (92%) and BD-1 decreased in 11 patients (85%). BD-1 levels after successful repair were not different from healthy controls (p = 0.06). DISCUSSION: Urinary biomarkers of obstruction should detect significant obstructive pathology as well as reflect its resolution. This would enable their use in post-operative monitoring and augment current methods of determining successful surgical outcome through imaging. CONCLUSIONS: The AMPs HIP/PAP and BD-1 are significantly elevated in UPJO but then significantly decrease after pyeloplasty, with BD-1 returning to healthy control levels. As a result, these AMPs could serve as markers of successful surgical intervention.

Differentiating Asymptomatic Bacteriuria From Urinary Tract Infection in the Pediatric Neurogenic Bladder Population: NGAL As a Promising Biomarker.

Objective: To evaluate whether urinary antimicrobial peptides (AMPs) can discriminate between asymptomatic bacteriuria (ASB) and urinary tract infection (UTI) in pediatric patients with neurogenic bladder (NGB). Design/Methods: Bladder urine was collected from pediatric patients (≤18 years old) with NGB without augmentation cystoplasty. Patients were divided into the following groups based on symptomatology and results of urinalysis/urine culture: (a) UTI, (b) ASB, and (c) sterile. Urine AMPs ß defense 1 (BD-1), neutrophil gelatinase-associated lipocalin (NGAL), cathelicidin (LL-37), hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), and human α defensin 5 (HD-5) were compared between groups by enzyme-linked immunosorbent assays. In addition, urines from pediatric controls without NGB or UTI were also analyzed. Significance was determined using Student's t test for parametric or Mann-Whitney U test for nonparametric data. A p value of <.05 was considered significant. Results: Thirty-six patients with NGB from a spinal dysraphism were evaluated: UTI, n = 6; ASB, n = 18; sterile, n = 12. These groups did not differ significantly by age but did significantly differ by gender (p = .0139). NGAL significantly differed between UTI and ASB groups (median 38.5 ng/mg vs 15.5 ng/mg, respectively; p = .0197) with a sensitivity and specificity of 82.4% and 83.3%, respectively. HIP/PAP, BD-1, HD-5, LL-37, and NGAL levels were all significantly higher in sterile NGB urines compared to 17 non-NGB pediatric controls (p < .0001, p = .0020, p = .0035, p = .0006, and p = .0339, respectively). Conclusion: All five urinary AMPs evaluated were significantly elevated in NGB patients compared to controls. NGAL levels may help differentiate between UTI and ASB in pediatric NGB patients.

Implications of Bacteriuria in Myelomeningocele Patients at Time of Urodynamic Testing.

Objective: To identify those myelomeningocele (MMC) patients at risk for post-urodynamic study (UDS) complications. We hypothesized that patients who manage their bladder with clean intermittent catheterization (CIC) would have a greater risk of post-instrumentation complications due to higher rates of bacteriuria compared to those who freely void (FV). Design/Methods: Urine was collected from patients with MMC without augmentation cystoplasty undergoing routine renal ultrasound or urodynamic study (UDS). Samples were divided into those with bacteriuria (urine culture ≥10,000 colony-forming units) and those without. Post-UDS complications were evaluated and compared between CIC and FV patients. Results: A total of 91 urine samples from 82 total MMC patients were included for evaluation. Significantly more patients on CIC than those who FV had bacteriuria (67% vs 33%, p = .0457). From these urine samples, 54 were obtained at time of UDS of which 45 were from patients on CIC and 9 from FV patients. More patients on CIC had bacteriuria at the time of UDS than those who FV (60% vs 33%, respectively), but this did not reach significance (p = .1416). No patient with bacteriuria on CIC had a complication after UDS while one FV patient with bacteriuria developed post-UDS pyelonephritis. Conclusion: MMC patients with bacteriuria on CIC did not have post-UDS complications. Patients with bacteriuria who FV may be at particular risk for post-instrumentation UTI, providing guidance as to which MMC patients should undergo urine testing prior to UDS in order to prevent post-instrumentation pyelonephritis.

Ribonuclease 7 Shields the Kidney and Bladder from Invasive Uropathogenic Escherichia coli Infection.

BACKGROUND: Evidence suggests that antimicrobial peptides, components of the innate immune response, protect the kidneys and bladder from bacterial challenge. We previously identified ribonuclease 7 (RNase 7) as a human antimicrobial peptide that has bactericidal activity against uropathogenic Escherichia coli (UPEC). Functional studies assessing RNase 7's contributions to urinary tract defense are limited. METHODS: To investigate RNase 7's role in preventing urinary tract infection (UTI), we quantified urinary RNase 7 concentrations in 29 girls and adolescents with a UTI history and 29 healthy female human controls. To assess RNase 7's antimicrobial activity in vitro in human urothelial cells, we used siRNA to silence urothelial RNase 7 production and retroviral constructs to stably overexpress RNase 7; we then evaluated UPEC's ability to bind and invade these cells. For RNase 7 in vivo studies, we developed humanized RNase 7 transgenic mice, subjected them to experimental UTI, and enumerated UPEC burden in the urine, bladder, and kidneys. RESULTS: Compared with controls, study participants with a UTI history had 1.5-fold lower urinary RNase 7 concentrations. When RNase 7 was silenced in vitro, the percentage of UPEC binding or invading human urothelial cells increased; when cells overexpressed RNase 7, UPEC attachment and invasion decreased. In the transgenic mice, we detected RNase 7 expression in the kidney's intercalated cells and bladder urothelium. RNase 7 humanized mice exhibited marked protection from UPEC. CONCLUSIONS: These findings provide evidence that RNase 7 has a role in kidney and bladder host defense against UPEC and establish a foundation for investigating RNase 7 as a UTI prognostic marker or nonantibiotic-based therapy.

Analysis of the Ribonuclease A Superfamily of Antimicrobial Peptides in Patients Undergoing Chronic Peritoneal Dialysis.

Infectious peritonitis is a common complication in patients undergoing chronic peritoneal dialysis (PD), limiting the duration of PD as a modality for renal replacement therapy and increasing patient morbidity and mortality. Antimicrobial peptides (AMPs) serve critical roles in mucosal defense, but their expression and activity during peritonitis are poorly understood. We hypothesized that AMPs belonging to the Ribonuclease (RNase) A Superfamily are present in peritoneal fluid and increase during peritonitis in patients undergoing chronic PD. In the absence of peritonitis, we detected RNase 3, RNase 6, and RNase 7 in cell-free supernatants and viable cells obtained from peritoneal fluid of chronic PD patients. The cellular sources of these RNases were eosinophils (RNase 3), macrophages (RNase 6), and mesothelial cells (RNase 7). During peritonitis, RNase 3 increased 55-fold and RNase 7 levels increased 3-fold on average, whereas RNase 6 levels were unchanged. The areas under the receiver-operating characteristic curves for RNase 3 and RNase 7 were 0.99 (95% confidence interval (CI): 0.96-1.0) and 0.79 (95% CI: 0.64-0.93), respectively, indicating their potential as biomarkers of peritonitis. Discrete omental reservoirs of these RNases were evident in patients with end stage kidney disease prior to PD initiation, and omental RNase 3 reactive cells increased in patients undergoing PD with a history of peritonitis. We propose that constitutive and inducible pools of antimicrobial RNases form a network to shield the peritoneal cavity from microbial invasion in patients undergoing chronic PD.

Interleukin-6/Stat3 signaling has an essential role in the host antimicrobial response to urinary tract infection.

The signaling networks regulating antimicrobial activity during urinary tract infection (UTI) are incompletely understood. Interleukin-6 (IL-6) levels increase with UTI severity, but the specific contributions of IL-6 to host immunity against bacterial uropathogens are unknown. To clarify this we tested whether IL-6 activates the Stat3 transcription factor, to drive a program of antimicrobial peptide gene expression in infected urothelium during UTI. Transurethral inoculation of uropathogenic Escherichia coli led to IL-6 secretion, urothelial Stat3 phosphorylation, and activation of antimicrobial peptide transcription, in a Toll-like receptor 4-dependent manner in a murine model of cystitis. Recombinant IL-6 elicited Stat3 phosphorylation in primary urothelial cells in vitro, and systemic IL-6 administration promoted urothelial Stat3 phosphorylation and antimicrobial peptide expression in vivo. IL-6 deficiency led to decreased urothelial Stat3 phosphorylation and antimicrobial peptide mRNA expression following UTI, a finding mirrored by conditional Stat3 deletion. Deficiency in IL-6 or Stat3 was associated with increased formation of intracellular bacterial communities, and exogenous IL-6 reversed this phenotype in IL-6 knockout mice. Moreover, chronic IL-6 depletion led to increased renal bacterial burden and severe pyelonephritis in C3H/HeOuJ mice. Thus, IL-6/Stat3 signaling drives a transcriptional program of antimicrobial gene expression in infected urothelium, with key roles in limiting epithelial invasion and ascending infection.

Proteomic profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.

Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1) through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01) in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1), VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61) and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein expression in T cells by HIV-1 Nef variants.