Novel technique of development of human derived acellular dermal matrix.

Reconstructive surgery techniques have evolved exponentially in last decades. From regional flaps to free tissue transfer, tissue movilization has become the gold standart treatment in many reconstructive procedures. Main disadvantage from these techniques lies in the possibility of sequels in donor zone. Furthermore, raising comorbidities in general population and growing indications for reconstructive surgery in elder people, have triggered the development of new biomaterials which can offer support in the reconstruction while elicit donor zone morbidity. Advances in tissue decellularization techniques have brought numerous matrices which have shown effectivity in many reconstructive procedures. Use of acellular dermal matrices may become an eligible solution for many reconstructive procedures. From breast reconstruction assisted by matrices to complex wound coverage passing throught tendon repair techniques, acellular dermal matrices have shown effectiveness in last studies. Local production of this biomaterial leads to cost minimization derived from harvesting and manufacturing matrices in our centre and avoid out-of-stock and storage issues. Current original protocol proposed by our group include all steps from harvesting samples from cadaveric donors till matrix storage after decellularization proccess. The result is a high valued biomaterial in terms of biocompatibility and security profile available.

Polymerizable Skin Hydrogel for Full Thickness Wound Healing.

The skin is the largest organ in the human body, comprising the main barrier against the environment. When the skin loses its integrity, it is critical to replace it to prevent water loss and the proliferation of opportunistic infections. For more than 40 years, tissue-engineered skin grafts have been based on the in vitro culture of keratinocytes over different scaffolds, requiring between 3 to 4 weeks of tissue culture before being used clinically. In this study, we describe the development of a polymerizable skin hydrogel consisting of keratinocytes and fibroblast entrapped within a fibrin scaffold. We histologically characterized the construct and evaluated its use on an in vivo wound healing model of skin damage. Our results indicate that the proposed methodology can be used to effectively regenerate skin wounds, avoiding the secondary in vitro culture steps and thus, shortening the time needed until transplantation in comparison with other bilayer skin models. This is achievable due to the instant polymerization of the keratinocytes and fibroblast combination that allows a direct application on the wound. We suggest that the polymerizable skin hydrogel is an inexpensive, easy and rapid treatment that could be transferred into clinical practice in order to improve the treatment of skin wounds.

Fibrin-Plasma Rich in Growth Factors Membrane for the Treatment of a Rabbit Alkali-Burn Lesion.

The purpose of this work is to describe the use of Fibrin-Plasma Rich in Growth Factors (PRGF) membranes for the treatment of a rabbit alkali-burn lesion. For this purpose, an alkali-burn lesion was induced in 15 rabbits. A week later, clinical events were evaluated and rabbits were divided into five treatment groups: rabbits treated with medical treatment, with a fibrin-PRGF membrane cultured with autologous or heterologous rabbit Limbal Epithelial Progenitor Cells (LEPCs), with a fibrin-PRGF membrane in a Simple Limbal Epithelial Transplantation and with a fibrin-PRGF membrane without cultured LEPCs. After 40 days of follow-up, corneas were subjected to histochemical examination and immunostaining against corneal or conjunctival markers. Seven days after alkali-burn lesion, it was observed that rabbits showed opaque cornea, new blood vessels across the limbus penetrating the cornea and epithelial defects. At the end of the follow-up period, an improvement of the clinical parameters analyzed was observed in transplanted rabbits. However, only rabbits transplanted with cultured LEPCs were positive for corneal markers. Otherwise, rabbits in the other three groups showed positive staining against conjunctival markers. In conclusion, fibrin-PRGF membrane improved the chemically induced lesions. Nonetheless, only fibrin-PRGF membranes cultured with rabbit LEPCs were able to restore the corneal surface.

Repair of segmental mandibular bone defects in sheep using bone marrow stromal cells and autologous serum scaffold: a pilot study.

AIM: The aim of this study was to evaluate effective bone regeneration using an autologous serum scaffold (alone or seeded with autologous bone marrow-mesenchymal stem cells, BM-MSCs), when implanted in a 30 mm length segmental mandibular defect in sheep. MATERIALS AND METHODS: The bone defect was filled either with serum scaffold alone (control group; n = 5) or combined with BM-MSCs (experimental group; n = 10). Bone regeneration was determined at 12 (T12; 2 control sheep and 4 experimental sheep) and 32 weeks (T32; 3 control and 6 experimental sheep), as measured by computed and microcomputed tomography and histological examination. RESULTS: Two sheep of the Experimental group died after surgery. While complete bone union in the control group was only observed at T32, it was observed both at T12 (1/4 sheep) and T32 (3/4 sheep) in the experimental group. When properties/characteristics of new bone where compared, a better bone quality, similar to native bone, was observed in the scaffold combined with BM-MSCs. CONCLUSIONS: Based on these results, we conclude that the serum scaffold can promote efficient repair of large bone defects, but the combination with BM-MSCs accelerates this process, increasing significantly the amount and quality of bone formed.

Tissue-engineered oral mucosa for mucosal reconstruction in a pediatric patient with hemifacial microsomia and ankyloglossia.

Many types of soft tissue grafts have been used for the reconstruction of oral mucosal defects. The best results are achieved with mucosal grafts; however, when large areas must be grafted, sufficient donor tissue is not available. Tissue engineering represents an alternative method to obtain sufficient autologous tissue for reconstructing oral wounds. Herein we present a pediatric patient with hemifacial microsomia and congenital ankyloglossia requiring multiple surgical interventions, and in which an autologous full-thickness tissue-engineered oral mucosa was used for successful oral reconstruction. Our study demonstrates that even under challenging conditions, robust tissue-engineered products, such as the fibrin-based oral mucosa described here, can achieve successful tissue regeneration.

tPA Alu (I/D) polymorphism associates with bacterial osteomyelitis.

BACKGROUND: Coagulation and fibrinolysis are important in infections and systemic inflammatory response syndrome. Polymorphisms in plasminogen activator inhibitor-1 (PAI-1, SERPINE1) and tissue plasminogen activator (tPA, PLAT), such as PAI-1 (-675 4G/5G deletion/insertion) and tPA (Alu insertion/deletion [I/D]), are associated with strokes, myocardial infarctions, bacterial infections and septic shock severity, and trauma. Osteomyelitis is a mostly posttraumatic, Staphylococcal bone infection. PATIENTS AND METHODS: tPA Alu (I/D) (rs4646972) and PAI-1 (4G/5G) (rs1799889) polymorphisms were studied by DNA amplification with polymerase chain reaction in 261 patients with osteomyelitis and in 299 matched blood donors. Plasma PAI-1/tPA complex was assessed by enzyme-linked immuosorbent assay. RESULTS: II homozygotes (37.9% vs 19.1%) and I allele carriers (56.3% vs 46.3%) for the tPA Alu (I/D) polymorphism were significantly more frequent in osteomyelitis patients compared to controls (P < .001). II genotype carrier osteomyelitis patients had lower PAI-1/tPA complex levels compared to those with the D allele (P ≤ .04). There was no association between these genotypes and chronicity of osteomyelitis, post-traumatic etiology, or with a specific bacterial etiology. PAI-1 (4G/4G) homozygotes were not significantly different between osteomyelitis patients and controls (P = .1). CONCLUSIONS: We report for the first time to our knowledge an association between the tPA Alu (I/D) polymorphism and susceptibility to bacterial osteomyelitis, perhaps by fibrinolysis dysfunction.

The regenerative potential of fibroblasts in a new diabetes-induced delayed humanised wound healing model.

Cutaneous diabetic wounds greatly affect the quality of life of patients, causing a substantial economic impact on the healthcare system. The limited clinical success of conventional treatments is mainly attributed to the lack of knowledge of the pathogenic mechanisms related to chronic ulceration. Therefore, management of diabetic ulcers remains a challenging clinical issue. Within this context, reliable animal models that recapitulate situations of impaired wound healing have become essential. In this study, we established a new in vivo humanised model of delayed wound healing in a diabetic context that reproduces the main features of the human disease. Diabetes was induced by multiple low doses of streptozotocin in bioengineered human-skin-engrafted immunodeficient mice. The significant delay in wound closure exhibited in diabetic wounds was mainly attributed to alterations in the granulation tissue formation and resolution, involving defects in wound bed maturation, vascularisation, inflammatory response and collagen deposition. In the new model, a cell-based wound therapy consisting of the application of plasma-derived fibrin dermal scaffolds containing fibroblasts consistently improved the healing response by triggering granulation tissue maturation and further providing a suitable matrix for migrating keratinocytes during wound re-epithelialisation. The present preclinical wound healing model was able to shed light on the biological processes responsible for the improvement achieved, and these findings can be extended for designing new therapeutic approaches with clinical relevance.

Keratinocyte cell lines derived from severe generalized recessive epidermolysis bullosa patients carrying a highly recurrent COL7A1 homozygous mutation: models to assess cell and gene therapies in vitro and in vivo.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by deficiency of type VII collagen due to COL7A1 mutations such as c.6527insC, recurrently found in the Spanish RDEB population. Assessment of clonal correction-based therapeutic approaches for RDEB requires large expansions of cells, exceeding the replication capacity of human primary keratinocytes. Thus, immortalized RDEB cells with enhanced proliferative abilities would be valuable. Using either the SV40 large T antigen or papillomavirus HPV16-derived E6-E7 proteins, we immortalized and cloned RDEB keratinocytes carrying the c.6527insC mutation. Clones exhibited high proliferative and colony-forming features. Cytogenetic analysis revealed important differences between T antigen-driven and E6-E7-driven immortalization. Immortalized cells responded to differentiation stimuli and were competent for epidermal regeneration and recapitulation of the blistering RDEB phenotype in vivo. These features make these cell lines useful to test novel therapeutic approaches including those aimed at editing mutant COL7A1.

Improved Tool for Predicting Skin Irritation on Reconstructed Human Epidermis Models Based on Electrochemical Impedance Spectroscopy.

The rabbit skin irritation test has been the standard for evaluating the irritation potential of chemicals; however, alternative methods that do not use animal testing are actively encouraged. Reconstructed human epidermis (RhE) models mimic the biochemical and physiological properties of the human epidermis and can be used as an alternative method. On RhE methods, the metabolic activity of RhE models is used to predict skin irritation, with a reduction in metabolic activity indicating a reduced number of viable cells and linking cell death to skin irritation processes. However, new challenges have emerged as the use of RhE models increases, including the need for non-invasive and marker-free methodologies to assess cellular states. Electrochemical impedance spectroscopy (EIS) is one such methodology that can meet these requirements. In this study, our results showed that EIS can differentiate between irritant and non-irritant chemicals, with a significant increase in the capacitance values observed in the irritant samples. A ROC curve analysis showed that the prediction method based on EIS met OECD TG 439 requirements at all time points and had 95% within-laboratory reproducibility. Comparison with the MTT viability assay showed that prediction using EIS achieved higher sensitivity, specificity, and accuracy. These results suggest that EIS could potentially replace animal testing in the evaluation of irritation potential and could be a valuable addition to in vitro testing strategies.

In-stent restenosis is associated with proliferative skin healing and specific immune and endothelial cell profiles: results from the RACHEL trial.

Introduction: In-stent restenosis (ISR) is a major challenge in interventional cardiology. Both ISR and excessive skin healing are aberrant hyperplasic responses, which may be functionally related. However, the cellular component underlying ISR remains unclear, especially regarding vascular homeostasis. Recent evidence suggest that novel immune cell populations may be involved in vascular repair and damage, but their role in ISR has not been explored. The aims of this study is to analyze (i) the association between ISR and skin healing outcomes, and (ii) the alterations in vascular homeostasis mediators in ISR in univariate and integrative analyses. Methods: 30 patients with ≥1 previous stent implantation with restenosis and 30 patients with ≥1 stent without restenosis both confirmed in a second angiogram were recruited. Cellular mediators were quantified in peripheral blood by flow cytometry. Skin healing outcomes were analyzed after two consecutive biopsies. Results: Hypertrophic skin healing was more frequent in ISR patients (36.7%) compared to those ISR-free (16.7%). Patients with ISR were more likely to develop hypertrophic skin healing patterns (OR 4.334 [95% CI 1.044-18.073], p=0.033), even after correcting for confounders. ISR was associated with decreased circulating angiogenic T-cells (p=0.005) and endothelial progenitor cells (p<0.001), whereas CD4+CD28null and detached endothelial cells counts were higher (p<0.0001 and p=0.006, respectively) compared to their ISR-free counterparts. No differences in the frequency of monocyte subsets were found, although Angiotensin-Converting Enzyme expression was increased (non-classical: p<0.001; and intermediate: p<0.0001) in ISR. Despite no differences were noted in Low-Density Granulocytes, a relative increase in the CD16- compartment was observed in ISR (p=0.004). An unsupervised cluster analysis revealed the presence of three profiles with different clinical severity, unrelated to stent types or traditional risk factors. Conclusion: ISR is linked to excessive skin healing and profound alterations in cellular populations related to vascular repair and endothelial damage. Distinct cellular profiles can be distinguished within ISR, suggesting that different alterations may uncover different ISR clinical phenotypes.

Tissue bioengineering and artificial organs.

The scarcity of organs and tissues for transplant and the need of immunosuppressive drugs to avoid rejection constitute two reasons that justify organ and tissue production in the laboratory. Tissue engineering based tissues (TE) could allow to regenerate the whole organ from a fragment or even to produce several organs from an organ donor for grafting purposes. TE is based in: (1) the ex vivo expansion of cells, (2) the seeding of these expanded cells in tridimensional structures that mimic physiological conditions and, (3) grafting the prototype. In order to graft big structures it is necessary that the organ or tissue produced "ex vivo" bears a vascular tree to ensure the nutrition of its deep layers. At present, no technology has been developed to provide this vascular tree to TE derived products. Thus, these tissues must be thin enough to acquire nutrients during the first days by diffusion from surrounding tissues. This fact constitutes nowadays the greatest limitation of technologies for organ development in the laboratory.In this chapter, all these problems and their possible solutions are commented. Also, the present status of TE techniques in the regeneration of different organ systems is reviewed.

In-house performance assessment of 3D QobuR-Reconstructed Human Cornea-Like Epithelium (RhCE) for the evaluation of eye hazard.

To replace the Draize eye irritation test (OECD Test Guideline 404), several test methods based on reconstructed cornea-like epithelium (RhCE) have been developed and adopted in the OECD TG 492. The objective of this study was to stablish the experimental procedures and evaluate the performance assessment of QobuR-RhCE, an in-house RhCE model to be used for the evaluation of eye hazard. We define the essential structural, functional and procedural elements of the test method components to help assuring that the proposed test method is based on the same concepts as the validated reference methods. Performance assessment was evaluated in accordance with the revised performance standards for the assessment of proposed similar or modified in vitro reconstructed human cornea-like epithelium and the minimum list of reference chemicals was evaluated. As result, the proposed method scored 93.3% sensibility, 60% specificity, 76.7% accuracy and 96.7% within-laboratory reproducibility (WLR), providing a similar performance in comparison to the validated reference methods. Additionally, we describe a secondary endpoint based on Transepithelial Electrical Resistance (TEER) that could be of use to better discriminate between irritants and non-irritants. Taken together the results indicate that the QobuR-RhCE test method is an accurate screening tool that can be used as a standalone alternative to evaluate ocular irritation.

Impedance-based non-invasive assay for ocular damage prediction on in vitro 3D reconstructed human corneal epithelium.

Reconstructed human cornea-like epithelium (RhCE) holds unprecedented promise for toxicological analyses and the replacement of animal use. However, current standards to evaluate potential ocular irritancy present a major downfall, the need to invasively alter tissue samples to evaluate cell viability. In this study, the applicability of impedance analysis was validated by monitoring the change in cell capacitance during tissue maturation and before and after chemical application using coupled electrodes. Our results indicate that cell maturation on RhCE models can be evaluated during model production using capacitance sensing offering a faster and simpler quality control criteria for RhCE model usability. Additionally, cell capacitance resulted to be more sensitive in detecting slight cell damages than methods based on cell metabolism, and when integrated into OECD-approved testing strategies, capacitance sensing performed as good as currently accepted methodologies displaying 66% sensitivity, 100% specificity and 83% accuracy when evaluated at 300 Hz. In summary, a quantitative analysis to predict in vivo ocular irritation based on changes in RhCE capacitance by impedance spectroscopy is suggested. This methodology represents a non-invasive and non-destructive alternative that would enable the monitoring of reversible effects or repeated dose toxicity.

Eye bank versus surgeon prepared DMEK tissues: influence on adhesion and re-bubbling rate.

AIM: To investigate the difference in adhesion and rebubbling rate between eye bank and surgeon prepared Descemet membrane endothelial keratoplasty (DMEK) tissues. METHODS: Laboratory and clinical retrospective comparative interventional case series. Research corneal tissues were obtained for laboratory investigation. The clinical study involved patients with endothelial dysfunction who underwent DMEK surgery and tamponade with air. Tissues were stripped using a standard DMEK stripping technique (SCUBA) and shipped as prestripped or loaded in a 2.2 intra-ocular lens cartridge with endothelium facing inwards (preloaded) before transporting from the eye bank to the surgeon. For surgeon prepared tissues, all the grafts were stripped in the theatre and transplanted or stripped in the laboratory and tested immediately. Adhesion force and elastic modulus were measured in the centre and mid-periphery in a laboratory ex vivo investigation using atomic force microscopy, while rebubbling rates were recorded in the clinical study. RESULTS: There was no difference in endothelial cell viability between surgeon or eye bank prepared tissue. Surgeon-stripped DMEK grafts in the laboratory investigation showed significantly higher elastic modulus and adhesion force compared to prestripped and preloaded tissues (p<0.0001). In the clinical data, rebubbling rates of 48%, 40% and 15% were observed in preloaded, prestripped and surgeon-stripped DMEK grafts, respectively. Rebubbling rates were significantly associated with combined cataract surgery (p=0.009) and with time from harvesting the graft to the surgery (p=0.02). CONCLUSIONS: Decreased adhesion forces and elastic modulus in eye bank prepared tissues may contribute to increased rebubbling rates.

A Simple, Safe, and Effective Method for Preparing Autologous Bio-Based Fibrin Glue for Ophthalmic Use.

This study proposes a method to prepare autologous bio-based fibrin glue (FG) for use in ophthalmic surgery. FGs containing three fibrinogen concentrations and a thrombin concentrate were prepared using human blood from five donors (FG1: physiological fibrinogen concentration; FG2 and FG3: concentrated fibrinogen). The adhesion strength was tested, and the clinical safety and efficacy were studied in rabbit eyes in conjunctival surgery. A commercial FG was used as a control. From each donor, 2 mL of FG was prepared, containing 1 mL of 3.49 ± 0.78 (FG1), 17.74 ± 4.66 (FG2), or 47.46 ± 9.36 mg/mL (FG3) of fibrinogen and 1 mL of 2248.12 ± 604.20 UI/mL of thrombin. The average adhesion strength increased with the fibrinogen concentration, from 1.49 ± 0.39 kPa (FG1) to 3.14 ± 1.09 kPa (FG3). FG1 showed poor results when used for autograft adhesion. In contrast, the conjunctival autografts were successfully grafted using FG2 and FG3, revealing equivalent adhesion properties compared with commercial FG, but with less inflammation. In conclusion, FGs could be prepared on demand within minutes from small volumes of human blood, using a method that results in FGs which exhibit good adhesion capacity and are also safe and effective in a preclinical study.

In vivo assessment of acute UVB responses in normal and Xeroderma Pigmentosum (XP-C) skin-humanized mouse models.

In vivo studies of UVB effects on human skin are precluded by ethical and technical arguments on volunteers and inconceivable in cancer-prone patients such as those affected with Xeroderma Pigmentosum (XP). Establishing reliable models to address mechanistic and therapeutic matters thus remains a challenge. Here we have used the skin-humanized mouse system that circumvents most current model constraints. We assessed the UVB radiation effects including the sequential changes after acute exposure with respect to timing, dosage, and the relationship between dose and degree-sort of epidermal alteration. On Caucasian-derived regenerated skins, UVB irradiation (800 J/m(2)) induced DNA damage (cyclobutane pyrimidine dimers) and p53 expression in exposed keratinocytes. Epidermal disorganization was observed at higher doses. In contrast, in African descent-derived regenerated skins, physiological hyperpigmentation prevented tissue alterations and DNA photolesions. The acute UVB effects seen in Caucasian-derived engrafted skins were also blocked by a physical sunscreen, demonstrating the suitability of the system for photoprotection studies. We also report the establishment of a photosensitive model through the transplantation of XP-C patient cells as part of a bioengineered skin. The inability of XP-C engrafted skin to remove DNA damaged cells was confirmed in vivo. Both the normal and XP-C versions of the skin-humanized mice proved proficient models to assess UVB-mediated DNA repair responses and provide a strong platform to test novel therapeutic strategies.

Development of a bioengineered skin-humanized mouse model for psoriasis: dissecting epidermal-lymphocyte interacting pathways.

Over the past few years, whole skin xenotransplantation models that mimic different aspects of psoriasis have become available. However, these models are strongly constrained by the lack of skin donor availability and homogeneity. We present in this study a bioengineering-based skin-humanized mouse model for psoriasis, either in an autologous version using samples derived from psoriatic patients or, more importantly, in an allogeneic context, starting from skin biopsies and blood samples from unrelated healthy donors. After engraftment, the regenerated human skin presents the typical architecture of normal human skin but, in both cases, immunological reconstitution through intradermal injection in the regenerated skin using in vitro-differentiated T1 subpopulations as well as recombinant IL-17 and IL-22 Th17 cytokines, together with removal of the stratum corneum barrier by a mild abrasive treatment, leads to the rapid conversion of the skin into a bona fide psoriatic phenotype. Major hallmarks of psoriasis were confirmed by the evaluation of specific epidermal differentiation and proliferation markers as well as the mesenchymal milieu, including angiogenesis and infiltrate. Our bioengineered skin-based system represents a robust platform to reliably assess the molecular and cellular mechanisms underlying the complex interdependence between epidermal cells and the immune system. The system may also prove suitable to assess preclinical studies that test the efficacy of novel therapeutic treatments and to predict individual patient response to therapy.

Comparative analysis of plasma-derived albumin scaffold, alveolar osteoblasts and synthetic membrane in critical mandibular bone defects: An experimental study on rats.

Repair of bone deficiencies in the craniofacial skeleton remains a challenging clinical problem. The aim of this study was to evaluate and compare the effects of a plasma-derived albumin scaffold, alveolar osteoblasts and synthetic membrane implanted into experimental mandibular defects. Bilateral mandibular defects were created in twelve immunodeficient rats. The bone defect was filled with serum scaffold alone in left sides and scaffold combined with human alveolar osteoblast in right side defects. Implanted areas were closed directly in Group 1 (n = 6) and covered by a resorbable polyglycolic-polylactic acid membrane in Group 2 (n = 6). Bone regeneration was determined at 12 weeks as measured by and exhaustive multiplanar computed tomography analysis and histological examination. No significant differences in bone density were observed between defects transplanted with scaffold alone or scaffold seeded with osteoblasts. The use of membrane did not result in a determining factor in the grade of bone regeneration between Groups 1 and 2. Based on these results, it could be concluded that the albumin scaffold alone has osteoinductive capacity but presence of seeded ostogenic cells accelerates defect repair without being significantly influenced by covering the defect with a resorbable membrane.

Mini Cleanroom for the Manufacture of Advanced Therapy Medicinal Products (ATMP): Bioengineered Corneal Epithelium.

Among several requirements for the manufacture of Advanced Therapy Medicinal Products (ATMP) are: following the guidelines of a pharmaceutical quality system, complying with Good Manufacturing Practice (GMP) and access to a cleanroom fulfilling strict environmental conditions (Class A work area and Class B environment). This makes ATMP expensive. Moreover, the production of many of these therapeutic products may also be unprofitable, as in most cases their use is limited to a few patients and to a single batch per manufacturing unit. To reduce costs, ATMP may be produced in a scaled-down system isolated from the external environment (isolator), allowing for placement of this facility in a Class D environment, which is much more permissive and less costly. In this work, we confirm that it is possible to manufacture bioengineered corneal epithelium inside an isolator while fulfilling all the safety assurance standards at an affordable cost for patients. This small-scale ultra-clean working environment complies with GMP guidelines and could be a solution for the high costs associated with conventional cleanroom ATMP production.

The TNF-α (-238 G/A) polymorphism could protect against development of severe sepsis.

Primary responses in sepsis-mediated inflammation are regulated by pro-inflammatory cytokines. Variations in the cytokine genes might modify their transcription or expression, plasma cytokines levels and response to sepsis. Activation protein-1 (AP-1) and NF-κB regulate cytokines gene expression in sepsis. A total of 90 severely septic and 91 non-infected patients were prospectively studied. IL-1α (-889 C/T), IL-1ß (+3954 C/T), IL-6 (-174 G/C), TNF-α (-238 G/A), TNF-α (-308G/A), IL-8 (-251A/T) and IL-10 (-1082 G/A) SNPs, plasma IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-13, IFN-γ, TNF-α and monocyte chemoattractant protein 1 (MCP-1) levels, and AP-1 and NF-κB gene expression by neutrophils were assessed. A allele carriers of TNF-α (-238 G/A) SNP were less frequent among septic patients. IL-6, IL-8, IL-10, TNF-α and MCP-1 levels were higher, and AP-1 and NF-κB gene expressions lower in septic patients. Sepsis was independently associated with higher fibrinogen, neutrophils counts and IL-8 levels, lower prothrombin, absence of the variant A allele of the TNF-α (-238 G/A) SNP, and haemodynamic failure. Death was independently associated with a higher APACHE II score, higher IL-8 levels, and the diagnosis of sepsis. TNF-a (-238 G/A) SNP could protect against sepsis development. Higher IL-8 levels are predictive of sepsis and mortality.