Comparative transcriptomic adaptations of Staphylococcus aureus to the wound environment in non-diabetic and diabetic mice.

Infection is a major source of complications in delayed diabetic wound healing. Increased understanding of differential bacterial responses to diabetic wounds will enable us to better understand chronic wound pathogenesis. Here we create delayed-healing wounds infected with Staphylococcus aureus in non-diabetic and diabetic mice and used RNA-seq to compare bacterial gene expression profiles 3 or 7 days after infection. Analysis at day 3 demonstrated substantial transcriptomic differences between bacteria colonising non-diabetic and diabetic wound beds. Most of these transcriptional differences resolved by day 7, suggesting normalisation of many bacterial phenotypes later in the diabetic wound healing process. Lingering differentially expressed genes at day 7 were enriched for genes related to carbohydrate metabolism, which includes genes of the lac operon, and capsular polysaccharide synthesis, which includes the cap8 locus. These data encourage further research into host-pathogen interactions in wound healing and how they influence differential outcomes in the diabetic wound environment.

Genome Sequences of Two Pseudomonas aeruginosa Isolates with Defects in Type III Secretion System Gene Expression from a Chronic Ankle Wound Infection.

Effector proteins translocated into host cells by the Pseudomonas aeruginosa type III secretion system (T3SS) are critical for phagocytic avoidance and systemic spread of the microorganism. The T3SS genes are present in virtually all P. aeruginosa strains. When examined in environmental isolates and clinical specimens, expression of the T3SS genes is the rule. Isolates from the airways of cystic fibrosis (CF) patients are one exception, and these isolates usually carry mutations that disable T3SS gene expression. In this study, we describe two P. aeruginosa isolates, one pigmented brown and one green, from a keratitis-ichthyosis-deafness (KID) syndrome patient with a chronic cutaneous ankle wound. Similar to most isolates from CF, both of the KID isolates were defective for T3SS gene expression. Providing the primary activator of T3SS transcription (exsA) in trans restored T3SS function. Since the exsA sequences were identical to that of a reference strain with active T3SS gene expression, we examined the cAMP-Vfr system, a critical regulator of T3SS gene expression. Vfr is a cAMP-dependent transcription factor that activates exsA expression. Whereas T3SS activity was corrected in the brown isolate by restoring cAMP synthesis, the same was not observed for the green isolate. These findings suggest that distinct mechanisms resulted in loss of T3SS gene expression in the KID isolates. The mutations responsible for the T3SS defects were not clearly evident by comparison of the whole-genome sequences to a reference strain. Our findings suggest that loss of T3SS gene expression may be a trait common to both CF and non-CF chronic infections. IMPORTANCE A common feature of microorganisms that cause chronic infections is a stealthy lifestyle that promotes immune avoidance and host tolerance. During chronic colonization of cystic fibrosis (CF) patients, Pseudomonas aeruginosa acquires numerous adaptations that include reduced expression of some factors, such as motility, O antigen, and the T3SS, and increased expression of other traits, such as biofilm formation. In this study, we report loss of T3SS gene expression in non-CF chronic isolates. This finding suggests that loss of the T3SS may be a common and important trait that contributes to persistence and may open avenues to explore the significance further using non-CF chronic infection models.

Factors related to the composition and diversity of wound microbiota investigated using culture-independent molecular methods: a scoping review.

All open wounds are often colonized by commensal microbes as a loss of skin can provide a ready portal of entry for microorganisms. Although the wound microbiota is known to be associated with wound infection and with delayed healing, the factors related to the formations of wound microbiota contributing to such poor clinical outcomes are not clear and have not led to effective infection prevention interventions. This review aimed to scope the factors related to the composition and diversity of wound microbiota that have been investigated using culture-independent molecular methods. Original articles on wound microbiota published from January 1986 to February 2020 were included in this review. Thirty-one articles met the inclusion criteria and were grouped according to wound types: chronic, acute, and animal model wounds. The factors identified were categorized according to patient characteristics, wound characteristics, treatment, and sampling. Although some studies reported the effect size of the factors, the values were small. No studies elucidated the mechanism of wound microbiota formation. The results of this scoping review highlight that the factors associated with the diversity of wound microbiota are poorly understood and that further studies are needed.

Myeloid-derived growth factor regulates neutrophil motility in interstitial tissue damage.

Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage.

Inhibition of miR-139-5p by topical JTXK gel promotes healing of Staphylococcus aureus-infected skin wounds.

BACKGROUND: The inflammatory skin wound response is regulated by argonaute 2-bound microRNAs (Ago2-miRNAs) such as miR-139-5p, which inhibit transcription of their target mRNAs. Jiang Tang Xiao Ke (JTXK) is a traditional Chinese medicine that reduces miR-139-5p expression, suggesting that topical application of JTXK may have effects on wound healing. METHODS: miR-139-/- mice and wild-type (WT) mice were employed to characterize the in vivo effects of miR-139-5p on sterile wound healing. Neutrophil migration and activation into the wound site were examined by live imaging analysis in lys-EGFP mice and myeloperoxidase/aminophenyl fluorescein assays, respectively. In silico and in vitro studies in differentiated HL60 cells were performed to identify miR-139-5p's downstream mediator(s). miR-139-/- neutrophil transplantation (with or without Eif4g2-knockdown rescue) or a topical JTXK gel preparation (with or without miR-139-5p mimic rescue) were employed to characterize the in vivo effects of miR-139-5p and JTXK, respectively, on Staphylococcus aureus (S. aureus)-infected wound healing. RESULTS: miR-139-/- mice display impaired sterile wound healing but improved S. aureus-infected wound healing. Eif4g2, a protein that supports neutrophil proliferation and differentiation, was identified as a key downstream mediator of miR-139-5p. miR-139-/- mice show elevated neutrophilic activation and Eif4g2 upregulation. miR-139-/- neutrophils enhanced S. aureus-infected wound healing in an Eif4g2-dependent manner. Moreover, topical JTXK gel therapy also enhanced S. aureus-infected wound healing in a miR-139-5p-dependent manner. CONCLUSIONS: miR-139-5p negatively regulates the neutrophilic response during S. aureus-infected wound healing, suggesting that JTXK or other miR-139-5p suppressants may be effective for treating infected skin wounds.

Patient genetics is linked to chronic wound microbiome composition and healing.

The clinical importance of microbiomes to the chronicity of wounds is widely appreciated, yet little is understood about patient-specific processes shaping wound microbiome composition. Here, a two-cohort microbiome-genome wide association study is presented through which patient genomic loci associated with chronic wound microbiome diversity were identified. Further investigation revealed that alternative TLN2 and ZNF521 genotypes explained significant inter-patient variation in relative abundance of two key pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis. Wound diversity was lowest in Pseudomonas aeruginosa infected wounds, and decreasing wound diversity had a significant negative linear relationship with healing rate. In addition to microbiome characteristics, age, diabetic status, and genetic ancestry all significantly influenced healing. Using structural equation modeling to identify common variance among SNPs, six loci were sufficient to explain 53% of variation in wound microbiome diversity, which was a 10% increase over traditional multiple regression. Focusing on TLN2, genotype at rs8031916 explained expression differences of alternative transcripts that differ in inclusion of important focal adhesion binding domains. Such differences are hypothesized to relate to wound microbiomes and healing through effects on bacterial exploitation of focal adhesions and/or cellular migration. Related, other associated loci were functionally enriched, often with roles in cytoskeletal dynamics. This study, being the first to identify patient genetic determinants for wound microbiomes and healing, implicates genetic variation determining cellular adhesion phenotypes as important drivers of infection type. The identification of predictive biomarkers for chronic wound microbiomes may serve as risk factors and guide treatment by informing patient-specific tendencies of infection.

Comparative RNA-Seq analyses of Drosophila plasmatocytes reveal gene specific signatures in response to clean injury and septic injury.

Drosophila melanogaster's blood cells (hemocytes) play essential roles in wound healing and are involved in clearing microbial infections. Here, we report the transcriptional changes of larval plasmatocytes after clean injury or infection with the Gram-negative bacterium Escherichia coli or the Gram-positive bacterium Staphylococcus aureus compared to hemocytes recovered from unchallenged larvae via RNA-Sequencing. This study reveals 676 differentially expressed genes (DEGs) in hemocytes from clean injury samples compared to unchallenged samples, and 235 and 184 DEGs in E. coli and S. aureus samples respectively compared to clean injury samples. The clean injury samples showed enriched DEGs for immunity, clotting, cytoskeleton, cell migration, hemocyte differentiation, and indicated a metabolic reprogramming to aerobic glycolysis, a well-defined metabolic adaptation observed in mammalian macrophages. Microbial infections trigger significant transcription of immune genes, with significant differences between the E. coli and S. aureus samples suggesting that hemocytes have the ability to engage various programs upon infection. Collectively, our data bring new insights on Drosophila hemocyte function and open the route to post-genomic functional analysis of the cellular immune response.

Thrombocidin-1-derived antimicrobial peptide TC19 combats superficial multi-drug resistant bacterial wound infections.

Antimicrobial peptides are considered promising candidates for the development of novel antimicrobial agents to combat infections by multi-drug-resistant (MDR) bacteria. Here, we describe the identification and characterization of the synthetic peptide TC19, derived from the human thrombocidin-1-derived peptide L3. Biophysical experiments into the interaction between TC19 and mimics of human and bacterial plasma membranes demonstrated that the peptide is highly selective for bacterial membranes. In agreement, TC19 combined low cytotoxicity towards human fibroblasts with efficient and rapid killing in human plasma of MDR strains of several bacterial species of the ESKAPE panel. In addition, TC19 induced minor resistance in vitro, neutralized pro-inflammatory activity of bacterial cell envelope components while displaying slight chemotactic activity for human neutrophils. Importantly, topical application of TC19-containing hypromellose gel significantly reduced numbers of viable methicillin-resistant Staphylococcus aureus (MRSA) and MDR Acinetobacter baumannii in a superficial wound infection in mice. Together, TC19 is an attractive candidate for further development as a novel agent against (MDR) bacterial skin wound infections.

miR-23c regulates wound healing by targeting stromal cell-derived factor-1α (SDF-1α/CXCL12) among patients with diabetic foot ulcer.

Diabetic Foot Ulcer (DFU) is the most common in patients who have diabetic peripheral neuropathy and angiopathy as well as a foot deformity. The delayed process of wound healing in diabetic condition is mainly due to reduced expression of the growth factors, persistent inflammatory response and endothelial dysfunction. Emerging evidence indicate that miRNAs play a crucial role in regulating angiogenesis, collectively called as "angiomiRs". The present study aimed to screen the expressions of angiomiRs particularly miR23 family and its association with the various angiogenic factors including SDF-1α in the tissue biopsies isolated from DFU patients. Among the 40 enrolled subjects for this study, 10 were subjected in each group as healthy controls, type 2 diabetic subjects (T2DM), T2DM subjects with uninfected DFU, and T2DM subjects with infected DFU. The expression of both the miR23 family such as hsa-miR-23a, hsa-miR-23b, hsa-miR-23c and angiogenic factors such as SDF-1α, HIF-1α, VEGF, eNOS were investigated in peripheral blood mononuclear cells and tissue biopsy samples using qPCR. We found that the angiogenic factor SDF-1α was significantly decreased in both the circulation and tissue biopsies of patients with T2DM and infected DFU. The SDF-1α at the 3'-untranslated region pairs with target miRNAs namely hsa-miR-23a-3p, hsa-miR-23b-3p and hsa-miR-23c as established using miRNA target prediction algorithm. Further, the tissue-specific expressions of miR-23a and miR-23b were found to be low whereas miR-23c was increased in patients with infected DFU. Moreover, correlation analysis showed that SDF-1α was found to have a significant inverse association with miR-23c. In conclusion, miR-23c may function as a new regulator to inhibit angiogenesis by targeting SDF-1α.

MC1R gene polymorphisms are associated with dysfunctional immune responses and wound infection after burn injury.

BACKGROUND: Systemic inflammatory response syndrome (SIRS) is associated with organ failure and infectious complications after major burn injury. Recent evidence has linked melanocortin signaling to anti-inflammatory and wound-repair functions, with mutations in the melanocortin 1 receptor (MC1R) gene leading to increased inflammatory responses. Our group has previously demonstrated that MC1R gene polymorphisms are associated with postburn hypertrophic scarring. Thus, we hypothesized that MC1R single nucleotide polymorphisms (SNPs) would be associated with increased burn-induced SIRS and increased infectious complications. METHODS: We performed a retrospective cohort study of adults (>18 y of age) admitted to our burn center with >20% total body surface area (TBSA) partial/full thickness burns between 2006 and 2013. We screened for five MC1R SNPs (V60L, V92M, R151C, R163Q, T314T) by polymerase chain reaction from genomic DNA isolated from blood samples. We performed a detailed review of each patient chart to identify age, sex, race, ethnicity, %TBSA burned, burn wound infections (BWIs), and 72-hr intravenous fluid volume, the latter a surrogate for a dysfunctional inflammatory response to injury. Association testing was based on multivariable regression. RESULTS: Of 106 subjects enrolled, 82 had complete data for analysis. Of these, 64 (78%) were male, with a median age of 39 and median burn size of 30% TBSA. A total of 36 (44%) subjects developed BWIs. The median total administered IV crystalloid in first 72h was 24.6 L. In multivariate analysis, the R151C variant allele was a significant independent risk factor for BWI (adjusted prevalence ratio 2.03; 95% CI: 1.21-3.39; P = 0.007), and the V60L variant allele was independently associated with increased resuscitation fluid volume (P = 0.021). CONCLUSIONS: This is the first study to demonstrate a significant association between genetic polymorphisms and a nonfatal burn-induced SIRS complication. Our findings suggest that MC1R polymorphisms contribute to dysfunctional responses to burn injury that may predict infectious and inflammatory complications.

Identification and functional analysis of inflammation-related miRNAs in skin wound repair.

Inflammation at a wound site is essential for preventing infection. However, misregulated inflammation leads to pathologies of the healing process, including chronic non-healing wounds and scarring. MicroRNAs (miRNAs) are key regulators of the inflammatory response and tissue repair, acting by translational processing of target mRNAs. In the final step of miRNA processing, Argonaute 2 (Ago2)-bound mature miRNA complexes bind to target mRNAs and inhibit their translation. A variety of wound healing-related miRNAs have been identified and their misregulation likely contributes to wound pathologies, including scarring and chronic healing. Recently, we have developed an Ago2-bound mature miRNA purification system that uses Ago2 antibody to analyze the expression of miRNAs from wound tissues by microarray and next generation sequencing. We have identified several wound inflammation-related miRNAs via Ago2-target immunoprecipitation assays and next generation sequencing of wound tissues from wild-type and PU.1 knockout mice, which exhibit no inflammatory response because of a lack of immune cell lineages. We demonstrated that miR-142, an identified inflammation-related miRNA, is essential role for neutrophilic chemotaxis via inhibition of small GTPase translation; its misregulation leads to susceptibility to infection against Staphylococcus aureus at skin wound sites. In this review, we summarize recent advances of miRNA studies in skin wound healing, introduce our miRNA purification system using an immunoprecipitation assay method, and discuss the function of miR-142 in skin wound healing.

Distribution of adeB and NDM-1 genes in multidrug resistant Acinetobacter baumannii isolated from infected wound of patients admitted in a tertiary care hospital in Bangladesh.

BACKGROUND: The adeB gene in Acinetobacter baumannii regulates the bacterial internal drug efflux pump that plays a significant role in drug resistance. The aim of our study was to determine the occurrence of adeB gene in multidrug resistant and New Delhi metallo-beta-lactamase-1 (NDM- 1) gene in imipenem resistant Acinetobacter baumannii isolated from wound swab samples in a tertiary care hospital of Bangladesh. METHODS: A total of 345 wound swab samples were tested for bacterial pathogens. Acinetobacter baumannii was identified by culture and biochemical tests. Antimicrobial susceptibility pattern was determined by the disc diffusion method according to CLSI standards. Extended spectrum beta-lactamases were screened using the double disc synergy technique. Gene encoding AdeB efflux pump and NDM-1 were detected by Polymerase Chain Reaction (PCR). RESULTS: A total 22 (6.37%) Acinetobacter baumannii were identified from 345 wound swab samples and 20 (91%) of them were multidrug resistant. High resistance rates to some antibiotics were seen namely, cefotaxime (95%), amoxyclavulanic acid (90%) and ceftriaxone (82%). All the identified Acinetobacter baumannii were sensitive to colistin and 82% to imipenem. Two (9%) ESBL producing Acinetobacter baumannii strains were detected. adeB gene was detected in 16 (80%) out of 20 multidrug resistant Acinetobacter baumannii. 4 (18%) of 22 Acinetobacter baumannii were imipenem resistant. NDM-1 gene was detected in 2 (50%) of the imipenem resistant strains of Acinetobacter baumannii. CONCLUSION: The results of this study provide insight into the role of adeB gene as a potential regulator of drug resistance in Acinetobacter baumanni in Bangladesh. NDM-1 gene also contributes in developing such resistance for Acinetobacter baumannii.

Temporal dynamics of relative abundances and bacterial succession in chronic wound communities.

Polymicrobial bacterial infection is an important factor contributing to wound chronicity. Consequently, clinicians frequently adopt a biofilm-based wound care approach, in which wounds are treated utilizing DNA sequencing information about microbial communities. While more successful than treatment not using community information, there is little information about temporal dynamics of wound communities and optimal approaches over the course of treatment. To characterize these dynamics, temporal analysis over three sampling points was conducted for 167 chronic wounds. Across sampling intervals, wound communities from the same patients changed in composition, and most commonly shared less than 50% of observed species. There was a significant relationship between community similarity and time between sampling. Classifying wounds into state types, we found that communities frequently transitioned from Pseudomonas or Staphylococcus dominated, into a highly variable state type. Although low abundance microbial species are typically disregarded due to uncertainty of biological importance, we found that 80% of wound microbiomes included common or dominant species at subsequent time points that were in low abundance in earlier samples. Moreover, these species were often those known to frequently infect wounds. Results document compositional shifts through the course of treatment and suggest that routine consideration of low abundance species may improve biofilm-based wound care. Moreover, findings indicate that integrating ecological modeling to understand wound microbiome succession may lead to more informed therapy.

IL-22R Ligands IL-20, IL-22, and IL-24 Promote Wound Healing in Diabetic db/db Mice.

Diabetic foot ulcers (DFU) are one of the major complications in type II diabetes patients and can result in amputation and morbidity. Although multiple approaches are used clinically to help wound closure, many patients still lack adequate treatment. Here we show that IL-20 subfamily cytokines are upregulated during normal wound healing. While there is a redundant role for each individual cytokine in this subfamily in wound healing, mice deficient in IL-22R, the common receptor chain for IL-20, IL-22, and IL-24, display a significant delay in wound healing. Furthermore, IL-20, IL-22 and IL-24 are all able to promote wound healing in type II diabetic db/db mice. Mechanistically, when compared to other growth factors such as VEGF and PDGF that accelerate wound healing in this model, IL-22 uniquely induced genes involved in reepithelialization, tissue remodeling and innate host defense mechanisms from wounded skin. Interestingly, IL-22 treatment showed superior efficacy compared to PDGF or VEGF in an infectious diabetic wound model. Taken together, our data suggest that IL-20 subfamily cytokines, particularly IL-20, IL-22, and IL-24, might provide therapeutic benefit for patients with DFU.

Effects of Sequence Variations in Innate Immune Response Genes on Infectious Outcome in Trauma Patients: A Comprehensive Review.

Infectious complications, sepsis, and multiple organ dysfunction syndrome (MODS) remain important causes for morbidity and mortality in patients who survive the initial trauma. Increasing evidence suggests that genetic variants, particularly single nucleotide polymorphisms (SNPs), are critical determinants for interindividual differences in both inflammatory responses and clinical outcome in sepsis patients. Although the effect of SNPs on sepsis and MODS has been studied in many populations and diseases, this review aimed to summarize the current knowledge on the effect of SNPs on infectious complication specifically in trauma patients. A review of available literature was performed in PubMed database. The following genes have been studied in populations of trauma patients: CD14, HMGB1, IFNG, IL1A, IL1B, IL1RN, IL4, IL6, IL8, IL10, IL17F, IL18, MBL2, MASP2, FCN2, TLR1, TLR2, TLR4, TLR9, TNF, LTA, GR, MYLK, NLRP3, PRDX6, RAGE, HSPA1B, HSPA1L, HSP90, SERPINE1, IRAK1, IRAK3, VEGFA, LY96, ANGPT2, LBP, MicroRNA, and mtDNA. In this review, we discuss the genes of the Pattern Recognition Receptors, Signal Transducing Adaptor Proteins, and Inflammatory Cytokines of the innate immune system. A number of genetic variations have so far been studied in cohorts of trauma patients. Studies are often unique and numbers sometimes small. No definitive conclusions can be reached at this time about the influence of specific sequence variations on outcome in trauma patients.

Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing.

Clinical diagnosis of infection in chronic wounds is currently limited to subjective clinical signs and culture-based methods that underestimate the complexity of wound microbial bioburden as revealed by DNA-based microbial identification methods. Here, we use 16S rRNA next generation sequencing and quantitative polymerase chain reaction to characterize weekly changes in bacterial load, community structure, and diversity associated with a chronic venous leg ulcer over the 15-week course of treatment and healing. Our DNA-based methods and detailed sampling scheme reveal that the bacterial bioburden of the wound is unexpectedly dynamic, including changes in the bacterial load and community structure that correlate with wound expansion, antibiotic therapy, and healing. We demonstrate that these multidimensional changes in bacterial bioburden can be summarized using swabs taken prior to debridement, and therefore, can be more easily collected serially than debridement or biopsy samples. Overall, this case illustrates the importance of detailed clinical indicators and longitudinal sampling to determine the pathogenic significance of chronic wound microbial dynamics and guide best use of antimicrobials for improvement of healing outcomes.

Decreased expression of heat shock proteins may lead to compromised wound healing in type 2 diabetes mellitus patients.

BACKGROUND: Heat shock proteins (HSPs) are inducible stress proteins expressed in cells exposed to stress. HSPs promote wound healing by recruitment of dermal fibroblasts to the site of injury and bring about protein homeostasis. Diabetic wounds are hard to heal and inadequate HSPs may be important contributors in the etiology of diabetic foot ulcers (DFU). OBJECTIVE: To analyze the differential expression of HSPs and their downstream molecules in human diabetic wounds compared to control wounds. METHODS: Expressional levels of HSP27, HSP47 and HSP70 and their downstream molecules like TLR4, p38-MAPK were seen in biopsies from 101 human diabetic wounds compared to 8 control subjects without diabetes using RT-PCR, western blot and immunohistochemistry. RESULTS: Our study suggested a significant down regulation of HSP70, HSP47 and HSP27 (p value=<0.001 for HSP70; p value=0.007 for HSP47; p value=0.007 for HSP27) in DFU along with their downstream molecules TLR4 and p38-MAPK (p value=0.006 for p38-MAPK; p value=0.02 for TLR4). HSP70 levels were significantly lower in male subjects and their levels increased significantly with the grades of wound on Wagner's scale. Infection status of the wounds was found to be significantly associated with the increased levels of HSP70 and HSP27 in infected diabetic wounds. CONCLUSIONS: Our study demonstrates that the down regulation of HSPs in diabetic wounds is associated with wound healing impairment in T2DM subjects.

Fingerprinting of galectins in normal, P. aeruginosa-infected, and chemically burned mouse corneas.

PURPOSE: In this study, we aimed to assess whether the expression pattern of galectins is altered in Pseudomonas aeruginosa-infected and chemically burned mouse corneas. METHODS: Galectin (Gal) fingerprinting of normal, P. aeruginosa-infected, and silver nitrate-cauterized corneas was performed by Western blotting, immunofluorescence staining, and qRT-PCR. RESULTS: In normal corneas, Gal-1 was distributed mainly in the stroma, Gal-3 was localized mainly in epithelium, and Gal-7, -8, and -9 were detected in both corneal epithelium and stroma. Expression levels of the five galectins were drastically altered under pathological conditions. In both infected and cauterized corneas, overall Gal-3 expression was downregulated, whereas overall Gal-8 and -9 were upregulated. Changes in the expression level of Gal-7, -8, and -9 were distinct in the epithelium of infected and cauterized corneas. Expression of these three galectins was upregulated in corneal epithelium of infected corneas but not in cauterized corneas. Consistent with the changes in protein expression: (1) Gal-7, -8, and -9 mRNA expression was upregulated in cauterized corneas, and (2) Gal-3 mRNA was downregulated and Gal-9 mRNA expression was upregulated in infected corneas. CONCLUSIONS: Our data demonstrate differential regulation of various members of the galectin family in the course of corneal infection and neovascularization. The emerging functionality of the sugar code of cell surface receptors via endogenous galectins reflect to the pertinent roles of the five tested galectins in the diseases of cornea.

Negative-pressure wound therapy enhances local inflammatory responses in acute infected soft-tissue wound.

Clinical studies found that negative-pressure wound therapy (NPWT) displayed significant clinical benefits in the healing of infected wounds. However, the effect of NPWT on local inflammatory responses in acute infected soft-tissue wound has not been investigated thoroughly. The purpose of this study was to test the impact of NPWT on local expression of proinflammatory cytokines, amount of neutrophils, and bacterial bioburden in wound from acute infected soft-tissue wounds. Full-thickness wounds were created on the back of rabbits, and were inoculated with Staphylococcus aureus strain ATCC29213. The wounds were treated with sterile saline-moistened gauze dressings and NPWT with continuous negative pressure (-125 mmHg). Wound samples were harvested on days 0 (6 h after bacterial inoculation), 2, 4, 6, and 8 at the center of wound beds before irrigation for real-time PCR analysis of gene expression of IL-1ß, IL-8, and TNF-α. Wound biopsies were examined histologically for neutrophil quantification in different layers of tissue. Quantitative bacterial cultures at the same time point were analyzed for bacterial clearance. Application of NPWT to acute infected wounds in rabbits was compared with treatment with sterile saline-moistened gauze, over an 8-day period. NPWT-treated wounds exhibited earlier and greater peaking of IL-1ß and IL-8 expression and decrease in TNF-α expression over the early 4 days (P < 0.05). Furthermore, histologic examination revealed that significantly increased neutrophil count was observed in the shallow layer in wound biopsies of NPWT treatment at day 2 (P < 0.001). In addition, there was a statistically significant decrease of bacteria load from baseline (day 0) at days 2 and 8 in NPWT group (P < 0.05). In conclusion, this study demonstrates that NPWT of acute infected soft-tissue wounds leads to increased local IL-1ß and IL-8 expression in early phase of inflammation, which may trigger accumulation of neutrophils and thus accelerate bacterial clearance. Meanwhile, the success of NPWT in the treatment of acute wounds can attenuate the expression of TNF-α, and the result may partly explain how NPWT can avoid significantly impairing wound healing.

Single-nucleotide polymorphisms in the Toll-like receptor pathway increase susceptibility to infections in severely injured trauma patients.

BACKGROUND: Sepsis and subsequent multiple-organ failure are the predominant causes of late mortality in trauma patients. Susceptibility and response to infection is, in part, heritable. Single-nucleotide polymorphisms (SNPs) in Toll-like receptor (TLR) and cluster of differentiation 14 (CD14) genes of innate immunity may play a key role. The aim of this study was to assess if SNPs in TLR/CD14 predisposed trauma patients to infection. METHODS: A prospective cohort of trauma patients (age 18-80 years; injury severity score [ISS] ≥ 16) admitted to a Level I trauma center between January 2008 and April 2011 was genotyped for SNPs in TLR2 (T-16934A and R753Q), TLR4 (D299G and T399I), TLR9 (T-1486C and T-1237C), and CD14 (C-159T) using high-resolution melting analysis. Association of genotype with prevalence of positive culture findings (gram positive, gram negative, fungi), systemic inflammatory response syndrome (SIRS), sepsis, septic shock, and mortality was tested with χ(2) and logistic regression analysis. RESULTS: Genotyping was performed for 219 patients, of whom 51% developed positive culture findings in sputum, wounds, blood, or urine. SIRS developed in 64%, sepsis in 36%, and septic shock in 17%. The TLR2 T-16934A TA genotype increased the risk of a gram-positive infection (odds ratio, 2.816; 95% confidence interval, 1.249-6.348; p = 0.013) and SIRS (odds ratio, 2.386; 95% confidence interval, 1.011-5.632; p = 0.047). Trends were noted for TLR9 and CD14 SNPs but did not reach statistical significance. Sepsis and septic shock were unrelated to any of the SNPs studied. CONCLUSION: Aberrant functioning of the TLR/CD14 pathway of innate immunity changes the risk of infectious complications in severely injured trauma patients. Of the seven SNPs studied, the TLR2 T-16934A increased the risk, the TLR9 T-1486C SNPs may decrease the risk, and TLR4 variation seemed unrelated to outcome. Early genotyping may prove to be helpful in the future in identifying polytraumatized patients at risk for infectious outcome. LEVEL OF EVIDENCE: Prognostic and epidemiologic study, level II.