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Lactate dehydrogenase activity staining demonstrates time-dependent immune cell infiltration in human ex-vivo burn-injured skin

ABSTRACT: Burn injuries constitute one of the most serious accidental injuries. Increased metabolic rate is a hallmark feature of burn injury. Visualising lactate dehydrogenase (LDH) activity has been previously used to identify metabolic activity differences, hence cell viability and burn depth in burn skin. LDH activity was visualised in injured and uninjured skin from 38 sub-acute burn patients. LDH activity aided the identification of spatially correlating immunocompetent cells in a sub-group of six patients. Desorption Electrospray Ionisation Mass Spectrometry Imaging (DESI MSI) was used to describe relative lactate and pyruvate abundance in burned and uninjured tissue. LDH activity was significantly increased in the middle and deep regions of burnt skin compared with superficial areas in burnt skin and uninjured tissue and positively correlated with post-burn time. Regions of increased LDH activity showed high pyruvate and low lactate abundance when examined with DESI-MSI. Areas of increased LDH activity exhibited cellular infiltration, including CD3 + and CD4 + T-lymphocytes and CD68 + macrophages. Our data demonstrate a steady increase in functional LDH activity in sub-acute burn wounds linked to cellular infiltration. The cell types associated are related to tissue restructuring and inflammation. This region in burn wounds is likely the focus of dysregulated inflammation and hypermetabolism.

SUBMITTER: Cuddihy J

PROVIDER: S-EPMC8553775 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This study utilizes Raman spectroscopy to analyze the burn-induced collagen conformational changes in ex vivo porcine skin tissue. Raman spectra of wavenumbers 500-2000?cm-1 were measured for unburnt skin as well as four different burn conditions: (i) 200?°F for 10?s, (ii) 200?°F for the 30?s, (iii) 450?°F for 10?s and (iv) 450?°F for 30?s. The overall spectra reveal that protein and amino acids-related bands have manifested structural changes including the destruction of protein-related functional groups, and transformation from ?-helical to disordered structures which are correlated with increasing burn severity. The deconvolution of the amide I region (1580-1720?cm-1) and the analysis of the sub-bands reveal a change of the secondary structure of the collagen from the ?-like helix dominated to the ?-aggregate dominated one. Such conformational changes may explain the softening of mechanical response in burnt tissues reported in the literature.

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A novel human ex vivo skin model to study early local responses to burn injuries.

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| S-EPMC7801530 | biostudies-literature

Active neutrophil responses counteract Candida albicans burn wound infection of ex vivo human skin explants.

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| S-EPMC7732850 | biostudies-literature

Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to simulate histology-like appearance.

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Novel lactate dehydrogenase inhibitors with in vivo efficacy against Cryptosporidium parvum.

Project description:Cryptosporidium parvum is a highly prevalent zoonotic and anthroponotic protozoan parasite that causes a diarrheal syndrome in children and neonatal livestock, culminating in growth retardation and mortalities. Despite the high prevalence of C. parvum, there are no fully effective and safe drugs for treating infections, and there is no vaccine. We have previously reported that the bacterial-like C. parvum lactate dehydrogenase (CpLDH) enzyme is essential for survival, virulence and growth of C. parvum in vitro and in vivo. In the present study, we screened compound libraries and identified inhibitors against the enzymatic activity of recombinant CpLDH protein in vitro. We tested the inhibitors for anti-Cryptosporidium effect using in vitro infection assays of HCT-8 cells monolayers and identified compounds NSC158011 and NSC10447 that inhibited the proliferation of intracellular C. parvum in vitro, with IC50 values of 14.88 and 72.65 μM, respectively. At doses tolerable in mice, we found that both NSC158011 and NSC10447 consistently significantly reduced the shedding of C. parvum oocysts in infected immunocompromised mice's feces, and prevented intestinal villous atrophy as well as mucosal erosion due to C. parvum. Together, our findings have unveiled promising anti-Cryptosporidium drug candidates that can be explored further for the development of the much needed novel therapeutic agents against C. parvum infections.

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Novel lactate dehydrogenase inhibitors with in vivo efficacy against Cryptosporidium parvum.

| S-EPMC6687188 | biostudies-literature

Supplementation with antithrombin III ex vivo optimizes enoxaparin responses in critically injured patients.

Project description:BackgroundThe high incidence of venous thromboembolism (VTE) following trauma persists in spite of aggressive thromboprophylaxis strategies. Approximately half of VTE patients do not achieve the recommended anti-FXa response to enoxaparin anticoagulation (0.1-0.4 IU/mL), however, research to explain or correct this phenomenon is lacking. We hypothesized that antithrombin III (AT) deficiency is associated with poor enoxaparin responsiveness in trauma patients that develop VTE which can be reversed through supplementation with AT.Methods and findingsA retrospective cohort study was performed on plasma collected from trauma patients who did and did not develop pulmonary embolism (PE) as well as healthy volunteers. AT levels, thrombin generation, and anti-FXa levels were measured in the collected plasma at baseline and in response to supplementation with AT concentrate at 120-200% or plasma (30% volume). A total of 54 PE patients and 46 non-PE patients were enrolled in this study for analysis. Compared to healthy volunteers, trauma patients had lower levels of AT, elevated thrombin generation, and lower anti-FXa levels in response to enoxaparin. Moreover, thrombin generation was higher and responses to enoxaparin were lower in patients who developed PE compared to those who did not develop PE. We found that supplementation with AT, but not plasma, increased AT levels and improved enoxaparin-mediated inhibition of thrombin generation.ConclusionsSupplementation with AT may provide a novel adjunct therapy to increase the effectiveness of enoxaparin thromboprophylaxis and reduce the incidence of VTE in the trauma population.

| S-EPMC7531030 | biostudies-literature

Ki-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo.

Project description:The capacity for robust proliferation upon re-infection is a hallmark of adaptive immunity and the basis of vaccination. A widely used animal model for the study of human disease is the rhesus macaque (RM), where capacity for proliferation can be assessed ex vivo using carboxyfluorescein succinimidyl ester (CFSE)-based dilution assays. However, we show over the course of the standard ex vivo proliferation assay that CFSE-labeling at commonly used dye concentrations induces significant cell death, but that this phenomenon is dose-dependent. Here, we describe an alternative semiquantitative method for estimating T cell proliferative responses that avoids the putative biases associated with chemical modification. RM peripheral blood mononuclear cells were stimulated ex vivo with cognate peptides for 5 days, immunostained for intracellular Ki-67, and then analyzed by flow cytometry. We describe a gating strategy using Ki-67 and side light scatter, also a marker of blastogenesis, which correlates strongly with data from CFSE dilution. We show that this method is a valid tool for measuring RM antigen-specific cellular proliferation ex vivo and can be used as an alternative to CFSE dilution assays.

| S-EPMC2939446 | biostudies-literature

Ex-Vivo Method to Quantifiably Evaluate the Staining Effectiveness of Anterior Lens Capsule Dyes.

Project description:PurposeHigh frequency of cataracts and the requirements of new European Union regulations for medical devices require the availability of preclinical models to adequately evaluate anterior lens capsule dyes before their use in patients. Herein, we describe an ex vivo method to quantifiably evaluate the macroscopic and microscopic staining effectiveness of anterior lens capsule dyes using porcine eyes.MethodsCommercially available trypan blue-based products or physiological saline solution (negative control) was injected into porcine eyes. Anterior pole and lens (after extraction) were macroscopically photographed, and the images were quantitatively analyzed. Lenses were histologically processed, and the staining intensity microscopically was semiquantified.ResultsMacroscopic evaluation of the anterior pole revealed bluish staining of the anterior capsule; however, this coloring cannot be macroscopically discerned after lens extraction. Quantitative image analyses showed significant (P < 0.01) staining of the lens capsule compared to the negative control, but not significant (P > 0.05) between the products tested. Quantitative analysis of dying on lens images could not be performed. Microscopic semiquantification of the capsule staining intensity allows us to appreciate differences between products.ConclusionsThe described method is a quick and useful tool for macroscopic evaluation by surgeons to choose an anterior capsule staining for use during everyday surgeries, and a more specific microscopic evaluation also allows us to determine the effectiveness and usefulness of these products.Translational relevanceThis method satisfies preclinical effectiveness evaluations required by European Union regulations and complements the safety and toxicity evaluations that new products must guarantee before they enter the market and are used in clinical practice.

| S-EPMC8685404 | biostudies-literature

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