Duration of Time CD4/CD8 Ratio is Below 0.5 is Associated with Progression to Anal Cancer in Patients with HIV and High-Grade Dysplasia.

BACKGROUND: A CD4/CD8 ratio < 0.5 is associated with increased risk of advanced anal disease (AAD) but it is unknown if duration below 0.5 matters. The purpose of this study was to determine if duration of a CD4/CD8 ratio < 0.5 is associated with increased risk of invasive anal cancer (IC) in people living with HIV and high-grade dysplasia (HSIL). METHODS: This single institution, retrospective study used the University of Wisconsin Hospital and Clinics Anal Dysplasia and Anal Cancer Database. Patients with IC versus HSIL alone were compared. Independent variables were mean and percentage of time the CD4/CD8 ratio was < 0.5. Multivariate logistic regression was performed to estimate the adjusted odds of anal cancer. RESULTS: We identified 107 patients with HIV infection and AAD (87 with HSIL, 20 with IC). A history of smoking was significantly associated with the development of IC (95% in patients with IC vs. 64% in patients with HSIL; p = 0.015). Mean time the CD4/CD8 ratio was < 0.5 was significantly longer in patients with IC compared with patients with HSIL (7.7 years vs. 3.8 years; p = 0.002). Similarly, the mean percentage of time the CD4/CD8 ratio was < 0.5 was higher in those with IC versus those with HSIL (80% vs. 55%; p = 0.009). On multivariate analysis, duration CD4/CD8 ratio was < 0.5 was associated with increased odds of developing IC (odds ratio 1.25, 95% confidence interval 1.02-1.53; p = 0.034). CONCLUSIONS: In this retrospective, single-institution study of a cohort of people living with HIV and HSIL, increasing duration the CD4/CD8 ratio was < 0.5 was associated with increased odds of developing IC. Monitoring the number of years the CD4/CD8 ratio is < 0.5 could inform decision making in patients with HIV infection and HSIL.

Risk factors and outcomes for delayed kidney graft function in simultaneous heart and kidney transplant recipients: A UNOS/OPTN database analysis.

There are no prior studies assessing the risk factors and outcomes for kidney delayed graft function (K-DGF) in simultaneous heart and kidney (SHK) transplant recipients. Using the OPTN/UNOS database, we sought to identify risk factors associated with the development of K-DGF in this unique population, as well as outcomes associated with K-DGF. A total of 1161 SHK transplanted between 1998 and 2018 were included in the analysis, of which 311 (27%) were in the K-DGF (+) group and 850 in the K-DGF (-) group. In the multivariable analysis, history of pretransplant dialysis (OR: 3.95; 95% CI: 2.94 to 5.29; p < .001) was significantly associated with the development of K-DGF, as was donor death from cerebrovascular accident and longer cold ischemia time of either organ. SHK recipients with K-DGF had increased mortality (HR: 1.99; 95% CI: 1.52 to 2.60; p < .001) and death censored kidney graft failure (HR: 3.51; 95% CI: 2.29 to 5.36; p < .001) in the multivariable analysis. Similar outcomes were obtained when limiting our study to 2008-2018. Similar to kidney-only recipients, K-DGF in SHK recipients is associated with worse outcomes. Careful matching of recipients and donors, as well as peri-operative management, may help reduce the risk of K-DGF and the associated detrimental effects.

Accelerated complete human skin architecture restoration after wounding by nanogenerator-driven electrostimulation.

BACKGROUND: Electrostimulation (ES) therapy for wound healing is limited in clinical use due to barriers such as cumbersome equipment and intermittent delivery of therapy. METHODS: We adapted a human skin xenograft model that can be used to directly examine the nanogenerator-driven ES (NG-ES) effects on human skin in vivo-an essential translational step toward clinical application of the NG-ES technique for wound healing. RESULTS: We show that NG-ES leads to rapid wound closure with complete restoration of normal skin architecture within 7 days compared to more than 30 days in the literature. NG-ES accelerates the inflammatory phase of wound healing with more rapid resolution of neutrophils and macrophages and enhances wound bed perfusion with more robust neovascularization. CONCLUSION: Our results support the translational evaluation and optimization of the NG-ES technology to deliver convenient, efficient wound healing therapy for use in human wounds.

Indeterminate-Depth Burn Injury-Exploring the Uncertainty.

In the management of indeterminate-depth burns (IDB), common challenges include the ability to predict time to healing and regenerative potential, risk of burn wound progression, and timing of excision. Several technologies exist to aid in determination of the depth of a burn injury, yet surgeons continue to rely on the naked eye-visual assessment-as the standard of care. Newer and improved imaging technologies are closing in on the goal of inexpensive, accurate, noninvasive modalities for depth determination. Likewise, management of IDB is becoming more sophisticated as newer wound healing technologies continue to be developed. By describing what is meant by "indeterminate" depth burns, and their associated challenges, we hope to stimulate interest in research to develop new therapies and management strategies. The ultimate goal is to treat IDB without the need for autografts.

Evolution of ischemia and neovascularization in a murine model of full thickness human wound healing.

Translation of wound healing research is limited by the lack of an appropriate animal model, due to the anatomic and wound healing differences in animals and humans. Here, we characterize healing of grafted, full-thickness human skin in an in vivo model of wound healing. Full-thickness human skin, obtained from reconstructive operations, was grafted onto the dorsal flank of NOD.Cg-KitW41J Tyr + Prkdcscid Il2rgtm1Wjl /ThomJ mice. The xenografts were harvested 1 to 12 weeks after grafting, and histologic analyses were completed for viability, neovascularization, and hypoxia. Visual inspection of the xenograft shows drying and sloughing of the epidermis starting at week four. By week 12, the xenograft appears healed but has lost 63.05 ± 0.24% of the initial graft size. There is histologic evidence of epidermolysis as early as 2 weeks, which progresses until week 4, when new epidermis appears from the wound edges. Epidermal regeneration is complete by week 12, although the epidermis appears hypertrophied. An initial increase of infiltrating immune mouse cells into the xenograft normalizes to baseline 6 months after grafting. Neovascularization, as evidenced by positive staining for the proteins human CD31 and alpha smooth muscle actin, is present as early as 2 weeks after grafting at the interface between the xenograft and the mouse tissue. CD31 and alpha smooth muscle actin staining increased throughout the xenograft over the 12 weeks, leading to greater viability of the tissue. Likewise, there is increased Hypoxia Inducible Factor 1-alpha expression at the interface of viable and nonviable tissue, which suggest a hypoxia-driven process causing early graft loss. These findings illustrate human skin wound healing in an ischemic environment, providing a timeline for use of full thickness human skin after grafting in a murine model to study mechanisms underlying human skin wound healing.

Optical imaging of collagen fiber damage to assess thermally injured human skin.

Surgery is the definitive treatment for burn patients who sustain full-thickness burn injuries. Visual assessment of burn depth is made by the clinician early after injury but is accurate only up to 70% of the time among experienced surgeons. Collagen undergoes denaturation as a result of thermal injury; however, the association of collagen denaturation and cellular death in response to thermal injury is unknown. While gene expression assays and histologic staining allow for ex vivo identification of collagen changes, these methods do not provide spatial or integrity information in vivo. Thermal effects on collagen and the role of collagen in wound repair have been understudied in human burn models due to a lack of methods to visualize both intact and denatured collagen. Hence, there is a critical need for a clinically applicable method to discriminate between damaged and intact collagen fibers in tissues. We present two complementary candidate methods for visualization of collagen structure in three dimensions. Second harmonic generation imaging offers a label-free, high-resolution method to identify intact collagen. Simultaneously, a fluorophore-tagged collagen-mimetic peptide can detect damaged collagen. Together, these methods enable the characterization of collagen damage in human skin biopsies from burn patients, as well as ex vivo thermally injured human skin samples. These combined methods could enhance the understanding of the role of collagen in human wound healing after thermal injury and potentially assist in clinical decision-making.

Discordance between histologic and visual assessment of tissue viability in excised burn wound tissue.

The regenerative capacity of burn wounds, and the need for surgical intervention, depends on wound depth. Clinical visual assessment is considered the gold standard for burn depth assessment but it remains a subjective and inaccurate method for tissue evaluation. The purpose of this study was to compare visual assessment with microscopic and molecular techniques for human burn depth determination, and illustrate differences in the evaluation of tissue for potential regenerative capacity. Using intraoperative visual assessment, patients were identified as having deep partial thickness or full thickness burn wounds. Tangential excisions of burn tissue were processed with hematoxylin and eosin to visualize tissue morphology, lactate dehydrogenase assay to ascertain cellular viability, and Keratin-15 and Ki67 to identify epidermal progenitor cells and proliferative capacity, respectively. RNA from deep partial and full thickness burn tissue as well as normal tissue controls were submitted for RNA sequencing. Lactate dehydrogenase, Keratin-15, and Ki67 were found throughout the excised burn wound tissue in both deep partial thickness burn tissues and in the second tangential excision of full thickness burn tissues. RNA sequencing demonstrated regenerative capacity in both deep partial and full thickness burn tissue, however a greater capacity for regeneration was present in deep partial thickness compared with full thickness burn tissues. In this study, we highlight the discordance that exists between the intraoperative clinical identification of burn injury depth, and microscopic and molecular determination of viability and regenerative capacity. Current methods utilizing visual assessment for depth of injury are imprecise, and can lead to removal of viable tissue. Additionally, hematoxylin and eosin microscopic analysis should not be used as the sole method in research or clinical determination of depth, as there are no differences in staining between viable and nonviable tissue.

Disposal of Unused Postoperative Opioids: A Real-World Demonstration of Surgeon-initiated Strategies Using an Activated Charcoal Bag System.

Excessive opioid prescribing following surgery creates a reservoir of unused medications available for diversion and abuse. We conducted a cohort study examining the impact of clinic-based, surgeon-initiated strategies using an activated charcoal bag (ACB) system on disposal of unused opioids. Among patients undergoing a variety of general surgery procedures, 67% of those with unused opioids disposed of them using the ACB. Our findings demonstrate practical ways to incorporate opioid disposal into surgical practice as a complement to judicious opioid prescribing.

Modeling early thermal injury using an ex vivo human skin model of contact burns.

BACKGROUND: Early mechanisms underlying the progressive tissue death and the regenerative capability of burn wounds are understudied in human skin. A clinically relevant, reproducible model for human burn wound healing is needed to elucidate the early changes in the human burn wound environment. This study reports a reproducible contact burn model on human skin that explores the extent of tissue injury and healing over time, and defines the inter-individual variability in human skin to enable use in mechanistic studies on burn wound progression and healing. METHODS: Using a customized burn device, contact burns of various depths were created on human skin by two operators and were evaluated for histologic depth by three raters to determine reproducibility. Early burn wound progression and wound healing were also evaluated histologically after the thermally injured human skin was cultured ex vivo for up to 14 days. RESULTS: Burn depths were reproducibly generated on human skin in a temperature- or time-dependent manner. No significant difference in operator-created or rater-determined depth was observed within each patient sample. However, significant inter-individual variation was identified in burn depth in ten patient samples. Burn-injured ex vivo human skin placed into culture demonstrated differential progression of cell death and collagen denaturation for high and low temperature contact burns, while re-epithelialization was observed in superficial burn wounds over a period of 14 days. CONCLUSION: This model represents an invaluable tool to evaluate the inter-individual variability in early burn wound progression and wound healing to complement current animal models and enhance the translation of preclinical research to improvements in patient care.

Distinct Tissue Damage and Microbial Cues Drive Neutrophil and Macrophage Recruitment to Thermal Injury.

Tissue damage triggers a rapid innate immune response that mediates host defense. Previously we reported that thermal damage of the larval zebrafish fin disrupts collagen organization and induces a robust and potentially damaging innate immune response. The mechanisms that drive damaging versus protective neutrophil inflammation in interstitial tissues remain unclear. Here we identify distinct cues in the tissue microenvironment that differentially drive neutrophil and macrophage responses to sterile injury. Using live imaging, we found a motile zone for neutrophils, but not macrophages, in collagen-free regions and identified a specific role for interleukin-6 (IL-6) receptor signaling in neutrophil responses to thermal damage. IL-6 receptor mutants show impaired neutrophil recruitment to sterile thermal injury that was not present in tissues infected with Pseudomonas aeruginosa. These findings identify distinct signaling networks during neutrophil recruitment to sterile and microbial damage cues and provide a framework to limit potentially damaging neutrophil inflammation.

Significant Improvement in Rat Kidney Cold Storage Using UW Organ Preservation Solution Supplemented With the Immediate-Acting PrC-210 Free Radical Scavenger.

Ischemia-reperfusion injury, including injury from warm- and cold-ischemia (CI) organ storage, remains a significant problem for all solid organ transplants. Suppressing CI damage would reduce delayed graft function and increase the donor organ pool size. PrC-210 has demonstrated superior prevention of damage in several preclinical studies as an immediate-acting free-radical scavenger. Here, we describe its profound efficacy in suppressing CI injury in a rat kidney model. METHODS: Kidneys in 300 gm Sprague-Dawley rats were perfused in situ with UW solution with or without added PrC-210 and then stored at 4°C in the same solution for 0 to 48 hours. When procured, kidney-activated caspase-3 level (a marker of cell death) was measured, and direct histological analysis of kidneys was performed to assess PrC-210 protective efficacy. In vitro analyses of PrC-210-conferred protection to isolated rat kidneys or naked DNA were also performed. RESULTS: A single 15 seconds in situ perfusion of kidneys with 20 mmol/L PrC-210 in UW solution resulted in significant reductions in (1) 30-hour CI-induced kidney-activated caspase level (P < 0.0001); activated caspase was reduced to levels not significantly different than control activated caspase levels seen in unperturbed kidneys, (2) 30-hour CI-induced renal Tubular Injury Scores (P = 0.0004) where brush border and tubular necrosis were markedly reduced, (3) PrC-210 conferred 100% protection against ·OH damage to naked DNA and isolated kidney mitochondria while current UW solution antioxidants were without protective effect. CONCLUSIONS: A single PrC-210-UW solution perfusion of rat kidneys upon removal from the rat profoundly reduced caspase and renal tubular injury in kidneys exposed to 30 hours of CI organ storage. These findings support further development of the PrC-210 molecule to suppress or to prevent ischemia-reperfusion injury in organ transplant and other ischemia-reperfusion injury settings.

Interpreting Outcomes in DCDD Liver Transplantation: First Report of the Multicenter IDOL Consortium.

BACKGROUND: In the United States, 5% of adult liver transplant recipients receive a graft donation after circulatory determination of death (DCDD). Concerns for ischemic cholangiopathy (IC), a disease of diffuse intrahepatic stricturing limits broader DCDD use. Single-center reports demonstrate large variation in outcomes. METHODS: Retrospective deidentified data collected between 2005 and 2013 were entered electronically by 10 centers via a Research Electronic Data Capture database. Our primary outcome was development of intrahepatic biliary strictures consistent with IC. RESULTS: Within 6 months post-DCDD transplant, 162 (21.8%) patients developed a biliary stricture, of which 88 (11.8%) exhibited intrahepatic structuring consistent with IC. Unadjusted 6-month IC rate among the 10 centers varied significantly (P = 0.006) from 6.3% to 25.9%. The only factor associated with increased risk of IC within 6 months was Roux-en-Y hepaticojejunostomy (vs duct-to-duct) (odds ratio, 3.06; 95% confidence interval, 1.52-6.16; P = 0.002). Graft failure by 6 months was more than 3 times higher for DCDD recipients with IC (odds ratio for IC, 3.36; 95% confidence interval, 1.95-5.79). CONCLUSIONS: This first report of the large combined experience with DCDD from the Improving DCDD Outcomes in Liver Transplant consortium demonstrates significant differences in IC among centers, the importance of biliary strictures as a risk factor for graft failure, and does not validate other risk factors for IC found in smaller studies.

Strategy for microbiome analysis using 16S rRNA gene sequence analysis on the Illumina sequencing platform.

Understanding the identity and changes of organisms in the urogenital and other microbiomes of the human body may be key to discovering causes and new treatments of many ailments, such as vaginosis. High-throughput sequencing technologies have recently enabled discovery of the great diversity of the human microbiome. The cost per base of many of these sequencing platforms remains high (thousands of dollars per sample); however, the Illumina Genome Analyzer (IGA) is estimated to have a cost per base less than one-fifth of its nearest competitor. The main disadvantage of the IGA for sequencing PCR-amplified 16S rRNA genes is that the maximum read-length of the IGA is only 100 bases; whereas, at least 300 bases are needed to obtain phylogenetically informative data down to the genus and species level. In this paper we describe and conduct a pilot test of a multiplex sequencing strategy suitable for achieving total reads of > 300 bases per extracted DNA molecule on the IGA. Results show that all proposed primers produce products of the expected size and that correct sequences can be obtained, with all proposed forward primers. Various bioinformatic optimization of the Illumina Bustard analysis pipeline proved necessary to extract the correct sequence from IGA image data, and these modifications of the data files indicate that further optimization of the analysis pipeline may improve the quality rankings of the data and enable more sequence to be correctly analyzed. The successful application of this method could result in an unprecedentedly deep description (800,000 taxonomic identifications per sample) of the urogenital and other microbiomes in a large number of samples at a reasonable cost per sample.