Repurposing HDAC inhibitors to enhance ribonuclease 4 and 7 expression and reduce urinary tract infection.

With the emergence of antibiotic-resistant bacteria, innovative approaches are needed for the treatment of urinary tract infections. Boosting antimicrobial peptide expression may provide an alternative to antibiotics. Here, we developed reporter cell lines and performed a high-throughput screen of clinically used drugs to identify compounds that boost ribonuclease 4 and 7 expression (RNase 4 and 7), peptides that have antimicrobial activity against antibiotic-resistant uropathogens. This screen identified histone deacetylase (HDAC) inhibitors as effective RNase 4 and RNase 7 inducers. Validation studies in primary human kidney and bladder cells confirmed pan-HDAC inhibitors as well as the HDAC class I inhibitor, MS-275, induce RNase 4 and RNase 7 to protect human kidney and bladder cells from uropathogenic Escherichia coli. When we administered MS-275 to mice, RNase 4 and 7 expression increased and mice were protected from acute transurethral E. coli challenge. In support of this mechanism, MS-275 treatment increased acetylated histone H3 binding to the RNASE4 and RNASE7 promoters. Overexpression and knockdown of HDAC class I proteins identified HDAC3 as a primary regulator of RNase 4 and 7. These results demonstrate the protective effects of enhancing RNase 4 and RNase 7, opening the door to repurposing medications as antibiotic conserving therapeutics for urinary tract infection.

Human neutrophil peptides 1-3 protect the murine urinary tract from uropathogenic Escherichia coli challenge.

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 (DEFA4/4) 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 DEFA4/4 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.

p38 MAPK and MKP-1 control the glycolytic program via the bifunctional glycolysis regulator PFKFB3 during sepsis.

Hyperlactatemia often occurs in critically ill patients during severe sepsis/septic shock and is a powerful predictor of mortality. Lactate is the end product of glycolysis. While hypoxia due to inadequate oxygen delivery may result in anaerobic glycolysis, sepsis also enhances glycolysis under hyperdynamic circulation with adequate oxygen delivery. However, the molecular mechanisms involved are not fully understood. Mitogen-activated protein kinase (MAPK) families regulate many aspects of the immune response during microbial infections. MAPK phosphatase (MKP)-1 serves as a feedback control mechanism for p38 and JNK MAPK activities via dephosphorylation. Here, we found that mice deficient in Mkp-1 exhibited substantially enhanced expression and phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) 3, a key enzyme that regulates glycolysis following systemic Escherichia coli infection. Enhanced PFKFB3 expression was observed in a variety of tissues and cell types, including hepatocytes, macrophages, and epithelial cells. In bone marrow-derived macrophages, Pfkfb3 was robustly induced by both E. coli and lipopolysaccharide, and Mkp-1 deficiency enhanced PFKFB3 expression with no effect on Pfkfb3 mRNA stability. PFKFB3 induction was correlated with lactate production in both WT and Mkp-1-/- bone marrow-derived macrophage following lipopolysaccharide stimulation. Furthermore, we determined that a PFKFB3 inhibitor markedly attenuated lactate production, highlighting the critical role of PFKFB3 in the glycolysis program. Finally, pharmacological inhibition of p38 MAPK, but not JNK, substantially attenuated PFKFB3 expression and lactate production. Taken together, our studies suggest a critical role of p38 MAPK and MKP-1 in the regulation of glycolysis during sepsis.

Mitogen-activated protein kinase phosphatase-1 controls PD-L1 expression by regulating type I interferon during systemic Escherichia coli infection.

Mitogen-activated protein kinase phosphatase 1 (Mkp-1) KO mice produce elevated cytokines and exhibit increased mortality and bacterial burden following systemic Escherichia coli infection. To understand how Mkp-1 affects immune defense, we analyzed the RNA-Seq datasets previously generated from control and E. coli-infected Mkp-1+/+ and Mkp-1-/- mice. We found that E. coli infection markedly induced programmed death-ligand 1 (PD-L1) expression and that Mkp-1 deficiency further amplified PD-L1 expression. Administration of a PD-L1-neutralizing monoclonal antibody (mAb) to Mkp-1-/- mice increased the mortality of the animals following E. coli infection, although bacterial burden was decreased. In addition, the PD-L1-neutralizing mAb increased serum interferon (IFN)-γ and tumor necrosis factor alpha, as well as lung- and liver-inducible nitric oxide synthase levels, suggesting an enhanced inflammatory response. Interestingly, neutralization of IFN-α/ß receptor 1 blocked PD-L1 induction in Mkp-1-/- mice following E. coli infection. PD-L1 was potently induced in macrophages by E. coli and lipopolysaccharide in vitro, and Mkp-1 deficiency exacerbated PD-L1 induction with little effect on the half-life of PD-L1 mRNA. In contrast, inhibitors of Janus kinase 1/2 and tyrosine kinase 2, as well as the IFN-α/ß receptor 1-neutralizing mAb, markedly attenuated PD-L1 induction. These results suggest that the beneficial effect of type I IFNs in E. coli-infected Mkp-1-/- mice is, at least in part, mediated by Janus kinase/signal transducer and activator of transcription-driven PD-L1 induction. Our studies also support the notion that enhanced PD-L1 expression contributes to the bactericidal defect of Mkp-1-/- mice.

Diagnostic accuracy of optical coherence tomography for assessing surgical margins of canine soft tissue sarcomas in observers of different specialties.

OBJECTIVE: To determine the diagnostic accuracy of optical coherence tomography (OCT) to assess surgical margins of canine soft tissue sarcoma (STS) and determine the influence of observer specialty and training. STUDY DESIGN: Blinded clinical prospective study. ANIMALS: Twenty-five dogs undergoing surgical excision of STS. METHODS: In vivo and ex vivo surgical margins were imaged with OCT after tumor resection. Representative images and videos were used to generate a training presentation and data sets. These were completed by 16 observers of four specialties (surgery, radiology, pathology, and OCT researchers). Images and videos from data sets were classified as cancerous or noncancerous. RESULTS: The overall sensitivity and specificity were 88.2% and 92.8%, respectively, for in vivo tissues and 82.5% and 93.3%, respectively, for ex vivo specimens. The overall accurate classification for all specimens was 91.4% in vivo and 89.5% ex vivo. There was no difference in accuracy of interpretation of OCT imaging by observers of different specialties or experience levels. CONCLUSION: Use of OCT to accurately assess surgical margins after STS excision was associated with a high sensitivity and specificity among various specialties. Personnel of all specialties and experience levels could effectively be trained to interpret OCT imaging. CLINICAL SIGNIFICANCE: Optical coherence tomography can be used by personnel of different specialty experience levels and from various specialties to accurately identify canine STS in vivo and ex vivo after a short training session. These encouraging results provide evidence to justify further research to assess the ability of OCT to provide real-time assessments of surgical margins and its applicability to other neoplasms.

Nail abnormalities identified in an ageing study of 30 inbred mouse strains.

In a large-scale ageing study, 30 inbred mouse strains were systematically screened for histologic evidence of lesions in all organ systems. Ten strains were diagnosed with similar nail abnormalities. The highest frequency was noted in NON/ShiLtJ mice. Lesions identified fell into two main categories: acute to chronic penetration of the third phalangeal bone through the hyponychium with associated inflammation and bone remodelling or metaplasia of the nail matrix and nail bed associated with severe orthokeratotic hyperkeratosis replacing the nail plate. Penetration of the distal phalanx through the hyponychium appeared to be the initiating feature resulting in nail abnormalities. The accompanying acute to subacute inflammatory response was associated with osteolysis of the distal phalanx. Evaluation of young NON/ShiLtJ mice revealed that these lesions were not often found, or affected only one digit. The only other nail unit abnormality identified was sporadic subungual epidermoid inclusion cysts which closely resembled similar lesions in human patients. These abnormalities, being age-related developments, may have contributed to weight loss due to impacts upon feeding and should be a consideration for future research due to the potential to interact with other experimental factors in ageing studies using the affected strains of mice.

Optical coherence tomography for surgical margin evaluation of excised canine cutaneous and subcutaneous tumours.

Currently, intraoperative tumour margin imaging is not routinely utilized in veterinary medicine. Optical coherence tomography (OCT) allows for real-time assessment of tissue morphology of 1-2 mm depth. The aims of this study were (1) to compare the histologic and OCT features of excised canine skin and subcutaneous specimens, and (2) to determine the diagnostic accuracy of OCT for surgical margin evaluation. The authors hypothesized that OCT imaging would correlate well with histopathology and that OCT would be sensitive for detection of incomplete margins. Eighty dogs were prospectively enrolled. Tumours were excised, and the surgical margins were imaged using a spectral domain OCT system. The tumour type and completeness of excision were determined by histopathology. Nine blinded observers received training in OCT image interpretation and were then given a set of OCT images and videos. The observers assigned each image/video a grade from 1 (no tumour) to 4 (tumour) and the results were compared to histopathology. The overall median sensitivity and specificity of OCT imaging for detection of incomplete margins were 86.7% and 84.6%, respectively. A potential limitation is that observers had varied experience with OCT image interpretation, ranging from no prior experience to participating in a previous OCT project. OCT is sensitive for detection of incomplete margins and could be a promising real-time surgical margin imaging modality. Further study is needed to evaluate intraoperative applications of OCT and its impact on tumour recurrence and long-term outcome.

Case Report: Use of PET/CT to Guide Treatment in a Cat With Presentation Consistent With Hodgkin's-Like Lymphoma.

An 8-year-old male neutered Domestic Long Hair cat was presented for a cervical swelling that was suspected to be an enlarged left retropharyngeal lymph node. In the absence of other lymphadenopathy, this was initially suspected to be Hodgkin's-like lymphoma. A positron emission tomography-computed tomography (PET/CT) scan was performed using 2-deoxy-2-[18F]-fluorodeoxyglucose (18F-FDG) to assess for evidence of disease in other locations to guide treatment. Multifocal increased radiopharmaceutical uptake was identified, indicating disease in multiple organs. High-grade lymphoma was confirmed on tissue biopsy. As such, systemic cytotoxic chemotherapy was recommended instead of lymph node extirpation surgery. The cat received a modified CHOP chemotherapy protocol and attained a temporary partial remission. After 2 months of treatment, the cat stopped responding to chemotherapy and was eventually euthanized due to a relapse of disease and decreased quality of life. This case describes the utility of PET/CT to guide treatment in a cat with a presentation consistent with Hodgkin's-like lymphoma.

Does Supply Equal Demand? The Workforce of Direct Patient Care Genetic Counselors in Wisconsin.

BACKGROUND: Recent studies documented a shortage of direct patient care (DPC) genetic counselors in the United States. We aimed to survey genetic counselor members of the Wisconsin Genetic Counselors Association (WIGCA) to determine if the supply and demand was met within the state and where access to services can improve. METHODS: An email invitation was sent to all genetic counselor members of the WIGCA with a link to a confidential online survey. Survey questions addressed the workforce composition, elements that impact services, and professional satisfaction of practicing genetic counselors. RESULTS: The Wisconsin workforce currently has half of the projected need for full-time DPC genetic counselors. One-third of genetic counselors reported changing from direct to non-direct patient care positions. In-person services are concentrated within Milwaukee and Madison. Appointment wait times are decreased when patients meet with a genetic counselor only, and half of the genetic counselors reported moderate to high stress levels. DISCUSSION/CONCLUSION: A shortage of DPC genetic counselors in Wisconsin is confirmed due to the total full-time effort in direct patient care. Data provided here can be used to identify targets for increasing the number of DPC genetic counselors, maximizing time spent on patient care, and improving access.

Role of sex hormones in the development of osteoarthritis.

Women older than 50 years have a considerably higher prevalence of osteoarthritis than men of the same age group. Although several factors have been proposed, there is some evidence that sex hormones influence the development of osteoarthritis. This article will focus on the basic science and clinical evidence that describe the current state of knowledge regarding the relationship between sex hormones and the development of osteoarthritis.

Calcaneal quantitative ultrasound compared with hip and femoral neck dual-energy x-ray absorptiometry in people with a spinal cord injury.

OBJECTIVE: To evaluate the sensitivity and specificity of calcaneal quantitative ultrasound (QUS) measurements for identifying osteoporosis determined by dual-energy x-ray absorptiometry (DXA) at the hip in a spinal cord injury (SCI) population. DESIGN: Cross-sectional retrospective review of data collected in the bone health registry of persons with a disability. SETTING: Inpatients and outpatients at a single acute rehabilitation hospital. PARTICIPANTS: A convenience sample of 66 participants, both inpatients and outpatients, with a spinal cord injury. METHODS: Calcaneal T scores were determined by ultrasound, and bone density of the lumbar spine, total hip, and femoral neck were determined by DXA. MAIN OUTCOME MEASUREMENTS: Right and left calcaneal QUS T scores and right and left hip and femoral neck DXA T scores. RESULTS: Right and left hip DXA T scores were strongly associated with corresponding right and left calcaneal QUS T scores (right: r = .72, P < .001; left: r = .70, P < .001). Similar associations were found when we evaluated femoral neck T scores and calcaneal QUS T scores. Receiver operating characteristic analysis for evaluating QUS to identify DXA-defined osteoporosis demonstrated an area under the curve of 0.81 for all participants (acute and chronic injury) and 0.68 for those with a chronic SCI. CONCLUSIONS: A strong association exists between calcaneal QUS T scores and bone density T scores at the hip measured by DXA. QUS may have a place in the screening of people with SCI 1 year or more after their injury to evaluate their bone status.