ABSTRACT
The administration of soluble growth factors (GFs) to injured tendons and ligaments (T/L) is known to promote and enhance the healing process. However, the administration of GFs is a complex, expensive and heavily-regulated process and only achieved by employing supraphysiological GF concentrations. In addition, for proper healing, specific and spatial immobilization of the GFs (s) is critical. We hypothesized that biomaterials functionalized with GF-binding peptides can be employed to capture endogenous GFs in a spatially-controlled manner, thus overcoming the need for the exogenous administration of supraphysiological doses of GFs. Here we demonstrate that the modification of films of polycaprolactone (PCL) with transforming growth factor ß1 (TGF-ß1)-binding peptides allows GFs to be captured and presented to the target cells. Moreover, using a TGF-ß reporter cell line and immunocytochemistry, we show that the GFs retained their biological activity. In human primary tendon cells, the immobilized TGF-ß1 activated TGF-ß target genes ultimately lead to a 2.5-fold increase in total collagen matrix production. In vivo implantation in rats clearly shows an accumulation of TGF-ß1 on the polymer films functionalized with the TGF-ß1-binding peptide when compared with the native films. This accumulation leads to an increase in the recruitment of inflammatory cells at day 3 and an increase in the fibrogenic response and vascularization around the implant at day 7. The results herein presented will endow current and future medical devices with novel biological properties and by doing so will accelerate T/L healing. STATEMENT OF SIGNIFICANCE: Our study describes the possibility to deliver hTGF-ß1 to human derived hamstring cells using a non-covalent bioactive strategy. The significance of our results in vivo with our functionalized biomaterial with TGF-ß1-binding peptides lies in the fact that these materials can now be employed to capture endogenous TGF-ß1 in a spatially-controlled manner, overcoming the need for exogenous administration of supra-physiological TGF-ß1 doses. Our method is different from current solutions that rely on global TGF-ß1 administration, soaking the devices with TGF-ß1, etc. Therefore we believe that our method is a significant change from current state-of-the-art in the types of devices that are used for ligament/tendon repair and that following our method can endow current and future medical devices with TGF-ß1 binding properties.
Subject(s)
Tendons/drug effects , Tendons/metabolism , Transforming Growth Factor beta1/administration & dosage , Transforming Growth Factor beta1/metabolism , Animals , Biocompatible Materials , Cells, Cultured , Collagen/biosynthesis , Drug Delivery Systems , Drug Implants , Gene Expression , Humans , Immobilized Proteins/administration & dosage , Immobilized Proteins/metabolism , Male , Materials Testing , Mink , Polyesters , Rats , Rats, Sprague-Dawley , Recombinant Proteins/administration & dosage , Recombinant Proteins/metabolism , Smad Proteins/metabolism , Tendons/cytology , Wound Healing/drug effects , Wound Healing/physiologyABSTRACT
H. pylori is a potent pathogen due to its capacity to successfully evade host defence mechanisms. Despite inducing immune responses in infected individuals, sometimes these responses fail to clear the infection and the bacterium establishes a persistent infection leading to chronic inflammation. In this context, we hypothesized that human leucocyte antigen G (HLA-G), a non-classical major histocompatibility complex molecule that has the ability to regulate immune responses both in physiological and in pathological conditions, may play an important role in promoting tolerance and helping H. pylori to subvert host defence and consequently establish a chronic infection. Therefore, we evaluated the expression of HLA-G 14-bp Ins/Del polymorphism in patients harbouring H. pylori infection, as well as their relationship with histological and demographic variables, to gain a better understanding of the actual role of HLA-G and its genetic polymorphisms in bacterial infection. Sixty-eight patients with clinical symptoms suggestive of H. pylori infection were enrolled to assess HLA-G 14-bp Ins/Del polymorphism allele and genotype frequencies. After adjustment for covariates (age and gender), the odds of having the genotype Ins/Ins, compared to Del/Del, were 3.77 times greater among HP+ cases than among controls. These findings suggest that the 14-bp Ins/Ins genotype, already associated with inflammatory and autoimmune diseases as well as some viral and parasitic infections, could confer a greater risk of developing H. pylori infection.
Subject(s)
Genetic Predisposition to Disease/genetics , HLA-G Antigens/genetics , Helicobacter Infections/genetics , Adult , Female , Genotype , Humans , Male , Middle Aged , Polymorphism, Single Nucleotide , Reverse Transcriptase Polymerase Chain Reaction , Risk FactorsABSTRACT
The subversion mechanisms employed by Helicobacter pylori (H. pylori) to escape from immune surveillance and to establish persistent infection are poorly understood. Growing evidence indicates that expression of HLA-G, a non-classical major histocompatibility complex molecule, negatively regulates immune responses in pathological conditions, including infectious diseases. In this context, we aimed to evaluate HLA-G expression in the gastric microenvironment of individuals harbouring H. pylori and to correlate it with histological variables. Fifty-four gastric specimens from patients harbouring H. pylori infection were evaluated by immunohistochemistry using anti-HLA-G monoclonal antibody. As a result, HLA-G expression was detected in 43 of 54 specimens harbouring H. pylori. The presence of HLA-G was significantly associated with milder colonization by H. pylori (P < 0.02), milder inflammatory activity (P < 0.02) and bacterium histological location in the gastric antrum. This study is the first to explore HLA-G expression in the context of bacterial infection. Whether the biological role of HLA-G during H. pylori infection is beneficial or hazardous for patients remains to be defined.
Subject(s)
Gastric Mucosa/metabolism , HLA-G Antigens/biosynthesis , Helicobacter Infections/metabolism , Gastric Mucosa/immunology , Helicobacter Infections/immunology , Helicobacter pylori , Humans , Immunohistochemistry , Protein Isoforms/biosynthesis , Retrospective Studies , Up-RegulationABSTRACT
SETTING: Ribeirão Preto, São Paulo, Brazil. OBJECTIVE: To develop and validate a preliminary instrument for assessing social support and tuberculosis (TB) stigma in families of TB patients. DESIGN: A literature review on social support and TB stigma was used to generate the theoretical domains for the instrument. A focus group was then conducted with TB patients and their families to revise the domains. Reviewers were invited to judge the appropriateness of the items in the instrument. A cross-sectional survey was carried out among 110 family members to assess the factorial structure using principal component analysis and confirmatory factor analysis to assess construct validity. Reliability was assessed in terms of internal consistency using Cronbach's alpha. RESULTS: After semantic validation and a pilot study, 23 items were selected for the scale. Examination of the factorial structure of the 16 items that were factorable using principal component analysis led to the extraction of two factors. The 16-item instrument was assessed for construct validity with confirmatory factor analysis, which confirmed a model with four items for each dimension. CONCLUSION: The study analysed the psychometric properties of an instrument that is still in its preliminary stages. Other studies on a similar scale in the Brazilian setting are required.
Contexte : Ribeirão Preto, Brésil.Objectif : Elaborer et valider un instrument préliminaire afin d'évaluer le soutien social et la stigmatisation liée à la tuberculose (TB) dans les familles des patients.Schéma : Une revue de littérature relative au soutien social et à la stigmatisation liée à la TB a permis de générer les domaines théoriques de l'instrument. Un groupe focal a ensuite été réalisé avec des patients tuberculeux et leurs familles afin de revoir ces domaines. Les réviseurs ont été invités à juger le caractère approprié des éléments de l'instrument. Une enquête traversable auprès de 110 membres des familles a été effectuée afin d'évaluer la structures factorielles grâce à l'analyse du composant principal, avec une analyse du facteur de confirmation qui a permis de vérifier la validité de la construction. La fiabilité a été évaluée par la cohérence interne grâce au coefficient α de Cronbach.Résultats : Vingt-trois éléments composaient l'échelle après la validation sémantique et l'étude pilote. L'exploration de la structure factorielle des 16 items qui étaient favorables grâce à l'analyse du composant principal a extrait deux facteurs. L'instrument à 16 items a été évalué en termes de validité de la construction avec une analyse du facteur de confirmation qui a confirmé un modèle à quatre items pour chaque dimension.Conclusion : L'étude a démontré les propriétés psychométriques d'un instrument encore au stade préliminaire. Cette échelle doit être testée à travers d'autres études dans le contexte brésilien.
Marco de referencia: Ribeirão Preto, en Brasil.Objetivo: Validar una herramienta preliminar de evaluación del apoyo social y los estigmas relacionados con la tuberculosis (TB) en las familias de los pacientes.Método: Mediante una recensión bibliográfica sobre el apoyo social y los estigmas relacionados con la TB se definieron los dominios teóricos de la herramienta. Luego, en un grupo de opinión conformado por pacientes con diagnóstico de TB y sus familiares se verificaron los dominios. Se invitaron revisores con el objeto de juzgar la idoneidad de los elementos de la escala. Se llevó a cabo una encuesta transversal a 110 miembros de las familias de pacientes con el propósito de evaluar la estructura factorial, mediante un análisis de componentes principales y se aplicó un análisis factorial confirmatorio a fin de apreciar la validez conceptual de la escala. Mediante un análisis de la consistencia interna se examinó la fiabilidad de la escala con medición del coeficiente α de Cronbach.Resultados: Tras la validación semántica y el estudio preliminar, la escala estaba compuesta por 23 elementos. Se exploró la estructura factorial de los 16 elementos factorizables mediante el análisis de componentes principales y se extrajeron dos factores. La validez conceptual del instrumento de 16 elementos se verificó mediante un análisis factorial confirmatorio, el cual ratificó un modelo con cuatro elementos en cada dimensión.Conclusión: El estudio puso en evidencia las propiedades psicométricas de un instrumento que se encuentra en fases preliminares de desarrollo. Es necesario poner a prueba esta escala en otros estudios en el contexto del Brasil.
ABSTRACT
As the mechanisms leading to the persistence of hepatitis B virus (HBV) infection are poorly understood and as the histocompatibility leucocyte antigen (HLA)-G is well described as a tolerogenic molecule, we evaluated HLA-G expression in 74 specimens of HBV liver biopsies and in 10 specimens obtained from previously healthy cadaver liver donors. HBV specimens were reviewed and classified by the METAVIR score, and HLA-G expression was assessed by immunohistochemistry. No HLA-G expression was observed in control hepatocytes. In contrast, 57 (77%) of 74 HBV specimens showed soluble and membrane-bound HLA-G expression in hepatocytes, biliary epithelial cells or both. No associations between the intensity of HLA-G expression and patient age or gender, HBeAg status, severity of liver fibrosis, and grade of histological findings were observed. Although significance was not reached (P = 0.180), patients exhibiting HLA-G expression presented a higher median HBV DNA viral load (105 copies/mL) than those who did not express HLA-G (10(3.7) copies/mL). These results indicate that HLA-G is expressed in most cases of chronic HBV infection in all stages and may play a role in the persistency of HBV infection.
Subject(s)
HLA Antigens/biosynthesis , HLA Antigens/immunology , Hepatitis B virus/immunology , Hepatitis B, Chronic/immunology , Hepatitis B, Chronic/pathology , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/immunology , Liver/immunology , Liver/pathology , Adolescent , Adult , Biopsy , Epithelial Cells/chemistry , Female , Gene Expression , HLA-G Antigens , Hepatocytes/chemistry , Humans , Immunohistochemistry , Male , Microscopy , Middle Aged , Severity of Illness Index , Young AdultABSTRACT
BACKGROUND: Despite advances in immunosuppressive therapy in the past decade, allograft rejection remains an important cause of kidney graft failure. Cytokines play a major role in the inflammatory and immune responses that mediate allograft outcomes. Several studies have shown that the production of cytokines varies among individuals. These variations are determined by genetic polymorphisms, most commonly within the regulatory region of cytokine genes. The aim of the present study was to assess the effect of allelic variation on acute rejection episodes (ARE) or chronic allograft nephropathy (CAN) after kidney transplantation. METHODS: To determine a possible correlation between the interferon (INF)-γ +874 polymorphism and kidney allograft outcome, we isolated genomic DNA from 74 patients who underwent isolated kidney allografts and were classified into 2 groups--a rejection and a nonrejection group--for comparison with a control group of 163 healthy subjects. RESULTS: We genotyped INF-γ +874 polymorphisms in all groups. The transplant group showed a significantly increased homozygous genotype T/T (P = .0118) compared with healthy controls. Similarly, considering only patients with CAN, the homozygous genotype T/T (P = .0067) was significantly increased compared with the healthy controls. The rejection group indicated a significant increased homozygous genotype T/T compared with the control group (P = .0061). CONCLUSION: Homozygous genotype T/T was associated with increased levels of INF-γ and greater numbers among the rejection and CAN cohorts.
Subject(s)
Interferon-gamma/genetics , Introns , Kidney Transplantation , Polymorphism, Genetic , Biopsy , Case-Control Studies , Humans , Transplantation, HomologousABSTRACT
UNLABELLED: Acute rejection episodes (ARE) are important complications that involve the interplay between mechanisms that maintain graft tolerance and promote rejection. The proinflammatory cytokine interleukin-17 (IL-17) has been implicated in many conditions in humans and mice. In kidney transplant patients, the evaluation IL-17 levels has been performed in only a few patients. We performed a cross-sectional study correlating quantitative IL-17 levels and clinical outcomes. PATIENTS AND METHODS: We studied 19 specimens from biopsies performed in patients (n = 19) who received isolated kidney grafts. ARE signs were present in 9 (47%) patients who provide specimens; whereas, 10 (53%) others showed no signs of rejection. Eighteen healthy control sample IL-17 underwent measurement, all of which were performed by an enzyme-linked immunosorbent assay method. We assessed other factors, such as the recipients demographic data, cold ischemia time, HLA mismatches, time elapsed from transplantation to the biopsy, posttransplantation status, antibody panel, donor type, and immunosuppressive treatment. RESULTS: IL-17 levels were clearly increased among samples derived from patients with ongoing rejection (125.7 +/- 27.06 pg/mL) in contrast, to the nonrejection group, (30 +/- 13.32 pg/mL) (P < .05). Healthy controls showed no detectable IL-17 levels. CONCLUSIONS: These findings suggested that IL-17 was important in the pathophysiology of acute kidney rejection.
Subject(s)
Biomarkers/blood , Graft Rejection/blood , Interleukin-17/blood , Adult , Animals , Biopsy , Female , Graft Rejection/immunology , Humans , Immunosuppressive Agents/therapeutic use , Male , Mice , Middle Aged , Reference ValuesABSTRACT
Chronic renal failure (CRF) leads in the majority of instances to end-stage renal disease (ESRD) requiring renal replacement therapy. Age, gender, genetics, race, hypertension, and smoking among others are factors associated with ESRD. Our interest was to evaluate the possible associations of class I and II HLA antigens with ESRD renal disease independent of other factors, among patients with CRF, having various diagnoses in the Brazilian population of the São Paulo state. So 21 HLA-A, 31 HLA-B, and 13 HLA-DR were detected in 105 patients who were compared with 160 healthy controls of both sexes who were not related to the patients evaluated until 2005. We calculated allelic frequencies, haplotypes frequencies, etiological fractions (EF), preventive fractions, and relative risks (RR). We compared demographic data of patients and controls. The antigens positively associated with ESRD were: HLA-A78 (RR = 30.31 and EF = 0.96) and HLA-DR11 (RR = 18.87 and EF = 0.65). The antigens HLAB14 (RR = 29.90 and EF = 0.75) was present at a significantly lower frequency among patients compared with controls. In contrast, no haplotype frequency showed statically significant associations. Further molecular studies may clarify types and subtypes of alleles involved with ESRD progression.
Subject(s)
HLA Antigens/genetics , Kidney Failure, Chronic/genetics , Kidney Failure, Chronic/immunology , Kidney Transplantation/immunology , Polymorphism, Genetic , Waiting Lists , Adult , Aged , Aged, 80 and over , Brazil , Disease Progression , Humans , Incidence , Kidney Failure, Chronic/epidemiology , Middle Aged , Reference ValuesABSTRACT
UNLABELLED: HLA-G is a non-classic Human Leukocyte Antigen (HLA-G) Class I of low polymorphism and restricted tissue distribution that displays tolerogenic functions. In heart transplantation and in combined liver/renal allograft transplantation, the expression of HLA-G has been associated with a lower incidence of acute graft rejection episodes and absence of chronic dysfunction. Since the expression of HLA-G in renal biopsies has been investigated only in few patients who received a combined kidney and liver transplant, in this study we performed a cross-sectional study, systematically comparing the expression of HLA-G in post-transplanted renal grafts, stratifying patients according to the presence or absence of rejection. PATIENTS AND METHODS: Seventy-three renal specimens (10 with acute rejection and 13 with chronic allograft nephropathy, and 50 with no signs of rejection) were immunohistochemically evaluated for HLA-G expression. RESULTS: In the group as a whole, HLA-G molecules were detected in 40 cases (54.8%). Among specimens that presented HLA-G expression, 2 out of 40 (5%) exhibited acute rejection, 2 (5%) exhibited chronic allograft nephropathy, and the remaining 36 (90%) exhibited no signs of rejection. The comparison between patients with rejection and those without rejection showed that the expression of HLA-G was significantly increased in specimens exhibiting no signs of rejection (p<0.0001). Considering only patients with acute rejection, 8 out of 10 patients showed no HLA-G expression in their kidney biopsies when compared to patients exhibiting no signs of rejection and absence of HLA-G was observed in 14 out of 50 (p=0.0032). Similarly, considering only patients with chronic allograft nephropathy, absence of HLA-G expression was observed in 11 out of 13 specimens, whereas in patients without rejection absence of HLA-G was observed in 14 out of 50 (p=0.003). Therapy with tacrolimus was significantly associated with the expression of HLA-G and a better graft prognosis. CONCLUSIONS: Our results suggest that HLA-G expression in the kidney allograft and the use of tacrolimus are associated with a lower frequency of acute renal rejection and chronic allograft nephropathy.
Subject(s)
Graft Rejection/epidemiology , Graft Rejection/immunology , HLA Antigens/biosynthesis , HLA Antigens/genetics , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/genetics , Kidney Transplantation/immunology , Adolescent , Adult , Aged , Cross-Sectional Studies , Female , Graft Rejection/blood , Graft Rejection/prevention & control , HLA Antigens/blood , HLA-G Antigens , Histocompatibility Antigens Class I/blood , Humans , Incidence , Male , Middle Aged , Prospective StudiesABSTRACT
Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex (MHC) class Ib molecule predominantly expressed in cytotrophoblasts, where it acts as a specific immunosuppressor. Literature data have shown that grafts in some settings, such as cardiac and liver/kidney-associated transplantations, express HLA-G and this expression is associated with less severe rejection and also reduces the incidence of rejection. Fourteen-base pair deletion/insertion polymorphism has been reported in exon 8 of the 3'-untranslated region of HLA-G. This polymorphism within exon 8 of the HLA-G gene might influence transcription activity, which in turn may influence the stability of HLA-G transcripts. This influences the stability of the HLA-G protein and therefore is of potential functional relevance. In order to determine a possible correlation between the 14-bp insertion/deletion polymorphism and kidney allograft outcome, we isolated genomic DNA from 83 patients who had received isolated kidney allografts, and we classified the 83 specimens into two groups, grafts presenting Banff features of rejection group and a non-rejection group, and compared them with a control group of 97 healthy subjects. The 14-bp polymorphism at exon 8 was genotyped in all groups. There was no significant difference in allelic frequencies of 14-bp insertion/deletion polymorphism between normal controls and kidney transplant patients. In the RG, the homozygous genotype +14/+14 bp (P = 0.0238) was significantly increased in the group with acute rejection compared with the healthy control group. Analysis of other HLA-G polymorphisms and functional studies on immune regulation are essential to elucidate the role of HLA-G in kidney allografts.
Subject(s)
Exons/genetics , Gene Frequency , HLA Antigens/genetics , Histocompatibility Antigens Class I/genetics , Kidney Transplantation/immunology , Mutagenesis, Insertional/genetics , Polymorphism, Genetic , Sequence Deletion/genetics , Adolescent , Adult , Aged , Base Sequence , Female , Graft Rejection/genetics , Graft Rejection/immunology , HLA-G Antigens , Humans , Male , Middle Aged , Transplantation, HomologousABSTRACT
The presence of microchimerism in the peripheral blood of solid organ graft recipients has been associated with long-term solid organ acceptance, immunologic tolerance, and less aggressive immunosuppressive therapy. Molecular biology assays are among the most sensitive methods to detect microchimerism, primarily to evaluate Y chromosome sequences in females as indirect evidence of circulating male nucleated donor cells. We screened for the presence of the SRY sequence region in peripheral blood of 13 female recipients of male kidney grafts: 5 living-related and 8 deceased grafts. Only patients who received grafts from related living donors exhibited microchimerism. Five of 13 patients studied exhibited better graft outcomes, including the 4 who were positive for the SRY sequences.