Hidrogel de quitosana com nanofibras de Policaprolactona (PCL) associado a extratos fitoterápicos no metabolismo celular e ação antimicrobiana: estudo in vitro / Evaluation of the chitosan hydrogel with PCL nanofibers associated with phytotherapeutic extracts in cellular and microbiological activity: in vitro study

O objetivo neste estudo foi produzir hidrogel de quitosana (CH) com PCL e fitoterápicos para uso preventivo de úlcera de pressão. Os hidrogéis de CH foram produzidos com glicerofosfato (GP) e com xantana (X), associados ao PCL e foram caracterizados por estereomicroscopio, intumescimento, molhabilidade e MEV. Posteriormente foram submetidos ao teste de viabilidade (MTT) com fibroblastos HFF-1 e queratinócitos HaCat. O hidrogel que apresentou melhor resultado foi escolhido para continuar na pesquisa. Posteriormente, extratos de Pfaffia panculata K, Juglans regia L, Rosmarinus officinalis L, Zingiber officinale, Própolis e Hamamelis foram colocados em contato com cepas de Staphylococcus aureus (S.a) (ATCC 6538), Streptococcus pyogenes (S.p) (ATCC 19615), Staphylococcus epidermidis (S.e) (ATCC 12228), Pseudomonas aeruginosa (P.a) (ATCC 15442), Escherichia coli (E.c) (ATCC 25922) e Klebsiella Pneumoniae (K.p) (ATCC 4352) na forma planctônica nos testes de CIM e CMM. Os dois melhores extratos fitoterápicos foram avaliados quanto ao sinergismo no teste checkerboard e posteriormente associados ao hidrogel anteriormente eleito. A seguir, o comportamento da HaCat e HFF-1 com os hidrogéis foi analisado por MTT, proteína total, ELISA, genotoxicidade e formação de biofilme monotípico com suspensões padronizadas (107 cel/mL) de S.a, S.e, S.p, P.a, E.c e K.p. Na caracterização e viabilidade o hidrogel CHX PCL apresentou os melhores resultados. Os extratos selecionados após CIM, CMM e checkerboard foram gengibre (G) e própolis (P). O extrato G se destacou na CIM com inibição de K. p e P. a. Os extratos de G e P demonstraram ação microbicida para K. p e P. a e somente o extrato P obteve ação microbicida para S. a na CMM. Houve ação aditiva dos extratos associados no checkerboard para S.p e ação aditiva e sinérgica para S. e. Os grupos de hidrogéis foram compostos por: quitosana xantana (CHX), CHX própolis (CHXP), CHX gengibre (CHXG) e CHX própolis e gengibre associados (CHXPG), todos associados ao PCL. Todos os hidrogéis demonstraram viabilidade celular acima de 70% do grupo controle, permitindo metabolismo celular observado na proteína total. Houve quantificação de IL-6 maior no grupo CHX nas duas linhagens de células enquanto a quantificação de IL-10 não exibiu diferença estatística entre os grupos. Todos os hidrogéis promoveram redução acentuada de biofilme de K.p e E.c. Os grupos CHX, CHXP e CHXG reduziram biofilme de S.e. O grupo CHXG reduziu biofilme de S.p. Para S.a e P.a o grupo CHXPG foi mais eficaz reduzindo biofilme. Concluímos que os hidrogéis apresentaram resultados satisfatórios e promissores, trazendo inovação por associação de biopolímeros e associação de extratos fitoterápicos pouco estudados. Os resultados positivos justificam a continuidade dos estudos com esse biomaterial(AU)

The aim of this study was to produce chitosan hydrogel (CH) with PCL and herbal medicines for preventive use of pressure ulcers. The CH hydrogels were produced with glycerophosphate (GP) and xanthan (X), associated with PCL and were characterized by stereomicroscope, swelling, wettability and SEM. Subsequently, they were submitted to a viability test (MTT) with HFF-1 fibroblasts and HaCat keratinocytes. The hydrogel that presented the best result was chosen to continue the research. Subsequently, extracts of Pfaffia panculata K, Juglans regia L, Rosmarinus officinalis L, Zingiber officinale, Propolis and Hamamelis were placed in contact with strains of Staphylococcus aureus (Sa) (ATCC 6538), Streptococcus pyogenes (Sp) (ATCC 19615), epidermidis (Se) (ATCC 12228), Pseudomonas aeruginosa (Pa) (ATCC 15442), Escherichia coli (Ec) (ATCC 25922) and Klebsiella Pneumoniae (Kp) (ATCC 4352) in planktonic form in CIM and CMM tests. The two best herbal extracts were evaluated for synergism in the checkerboard test and subsequently associated with the previously elected hydrogel. Next, the behavior of HaCat and HFF-1 with hydrogels was analyzed by MTT, total protein, ELISA, genotoxicity and monotypic biofilm formation with standardized suspensions (107 cel / mL) of Sa, Se, Sp, Pa, Ec and Kp In the characterization and viability the CHX PCL hydrogel presented the best results. The extracts selected after MIC, CMM and checkerboard were ginger (G) and propolis (P). The G extract stood out in the MIC with inhibition of K. p and P. a. The extracts of G and P showed microbicidal action for K. p and P. a and only the extract P obtained microbicidal action for S. a in CMM. There was an additive action of the associated extracts on the checkerboard for S.p and an additive and synergistic action for S. e. The hydrogel groups were composed of: xanthan chitosan (CHX), CHX propolis (CHXP), CHX ginger (CHXG) and CHX propolis and ginger associated (CHXPG), all associated with PCL. All hydrogels demonstrated cell viability above 70% of the control group, allowing cellular metabolism observed in the total protein. There was a greater quantification of IL-6 in the CHX group in the two cell lines while the quantification of IL-10 did not show statistical difference between the groups. All hydrogels promoted a marked reduction in the biofilm of K.p and E.c. The CHX, CHXP and CHXG groups reduced S.e biofilm. The CHXG group reduced S.p. For S.a and P.a, the CHXPG group was more effective in reducing biofilm. We conclude that the hydrogels presented satisfactory and promising results, bringing innovation through association of biopolymers and association of phytotherapic extracts little studied. The positive results justify the continuity of studies with this biomaterial(AU)

Las células guardianes residentes de la piel y su papel en la respuesta inmune. Parte 1 / Cells resident guardians of the skin and its role in the immune response

La piel constituye la primera barrera del sistema inmune contra potenciales agentes patógenos y nocivos externos. Evidencia importante sugiere que las células inmunológicas requieren de funciones conjuntas con los queratinocitos, para alertar y ensamblar una respuesta inmune adecuada, que incluye la formación del sistema de alerta denominado inflamosoma. Adicionalmente, nuevos fenotipos funcionales de células presentadoras de antígenos (CPA) en la piel como las células dendríticas han demostrado tener gran importancia en ensamblar la respuesta inmune incluso mayor que las células T circulantes. La primera entrega de este artículo describe la funcionalidad de los queratinocitos y las células dendriticas en la piel, para en la segunda parte discriminar sus roles juntos a los linfocitos T y las fallas de la regulación durante las interacciones en la formaión del inflamosoma.

Human skin is the first shield which provides essential protection of the human body from injury and infection. Important evidence reinforces the importance of keratinocytes as sensors of danger through alert systems such as the inflammasome and key components in the appropriate immune response. In addition, newly identified antigen-presenting cells (APCs), as dendritic cells, have demonstrated to have a key role of assembling the immune response even major than circulating T cells in skin. The first part of this review focuses on dissecting the functional role of keratinocytes and dendritic cells in skin in order to, in the second part, analyze their roles and interactions together with the T lymphocytes during the inflammasome formation.

Innate and adaptive immunity gene expression of human keratinocytes cultured of severe burn injury

PURPOSE: Evaluate the expression profile of genes related to Innate and Adaptive Immune System (IAIS) of human Primary Epidermal keratinocytes (hPEKP) of patients with severe burns. METHODS: After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific IAIS PCR Arrays plates (SA Biosciences). RESULTS: After the analysis of gene expression we found that 63% of these genes were differentially expressed, of which 77% were repressed and 23% were hyper-regulated. Among these, the following genes (fold increase or decrease): IL8 (41), IL6 (32), TNF (-92), HLA-E (-86), LYS (-74), CCR6 (- 73), CD86 (-41) and HLA-A (-35). CONCLUSIONS: This study contributes to the understanding of the molecular mechanisms underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome. .

Tumor necrosis factor-alpha and the cytokine network in psoriasis / Fator de necrose tumoral-alfa e a rede de citocinas na psoriase

New molecular methods of research have greatly expanded the knowledge about the role of cytokines in several diseases, including psoriasis. The work orchestrated by these peptides is essential for the communication between resident inflammatory cells (keratinocytes and endothelial cells) and infiltrating cells (neutrophils, lymphocytes, Langerhans cells). This is a complex network due to redundancy, synergism and, sometimes, the antagonism of cytokines, which prevents full understanding of the pathogenesis of the disease. Currently, it seems premature to try to establish a main actor, but TNFalpha participates in all stages of psoriatic plaque development, as we shall see.

A introdução de novos métodos moleculares de investigação ampliou muito o conhecimento sobre o papel das citocinas em diversas doenças, entre elas a psoríase. O trabalho orquestrado desses polipeptídeos é fundamental na comunicação entre as células inflamatórias residentes (queratinócitos e células endoteliais) e infiltrantes (neutrófilos, linfócitos, células de Langerhans). Trata-se de uma rede complexa devido à redundância, ao sinergismo e, por vezes, ao antagonismo das citocinas, o que dificulta a compreensão da fisiopatogenia da doença a partir de um mecanismo linear. No momento atual, parece precoce tentar estabelecer um regente, mas o TNF-alfa se destaca em todos os passos do desenvolvimento da placa psoriásica, como veremos a seguir.

Imunopatogênese da psoríase: revisando conceitos / Reviewing concepts in the immunopathogenesis of psoriasis

O conhecimento sobre a fisiopatogenia da psoríase possibilitou o desenvolvimento de ferramentas terapêuticas que visam ao bloqueio do seu gatilho imunológico. Paralelamente, citocinas como o TNF têm sido reconhecidas como integrantes da etiopatogenia da psoríase e comorbidades a ela relacionadas. Estudos genéticos e epidemiológicos contribuíram efetivamente para as conclusões a que se tem chegado atualmente sobre esta complexa patologia.

Insights into the pathogenesis of psoriasis led to the development of therapeutic tools aimed at blocking its immunological trigger. In parallel, cytokines such as the tumor necrosis factor (TNF) have been recognized as playing a crucial role in the pathogenesis of psoriasis and its associated comorbidities. Genetic and immunological studies have contributed effectively towards establishing the currently held concepts regarding this complex disease.

Expression of iNOS message in human buccal mucosal [keratinocytes] cell line TR146 using RT-PCR and immunocytochemistry

This study was carried out at the department of Oral Pathology, Queen Mary College of Medicine and Dentistry, Barts and The London to determine whether human oral buccal mucosal [keratinocytes] cell line, TR 146, expressed iNOS message and whether the expression of iNOS is varied when TR146 cells were exposed to different cytokines such as IL-15, IL-8, IL-1 beta, or TNF-alpha using RT-PCR and Immuno-cytochemistry and to determine the effect of the above mentioned cytokines on NO function by measuring nitrite production in TR146 cells. Immuno-cytochemistry analysis revealed that TR146 cells expressed iNOS proteins when incubated with IL-15 and IL-8 and a modest increase was seen with IL-1 beta /TNF-alpha. RT-PCR for iNOS indicated a marked increase in expression when the cells were exposed to IL-8, IL-15 or IL-1beta/ TNF-alpha. NOS activity was assessed by measuring nitrite activity. It was observed that treating the cells with cytokines caused significant increase in nitrite levels, except in the case of IL-8. These results suggest that TR146 cells expressed iNOS, the levels of which varied with various cytokines. It is clear that IL-15, IL-1beta /TNF-a and IL-8 are regulators of iNOS expression in oral keratinocytes and affect NOS activity

Imunopatogênese da psoríase / Immunopathogenesis of psoriasis

A psoríase é doença inflamatória comum, afetando cerca de 1 por cento da população brasileira. Os linfócito T auxiliares (Th17 e Th1) estão envolvidos na imunopatogênese da psoríase. Neste artigo é discutida a interação entre a imunidade inata (especialmente células dendríticas e queratinócitos) e adquirida (linfócitos T) na patogênese da psoríase.

Psoriasis is a common inflammatory disease affecting 1 percent of the Brazilian population. Th17 and Th1cells are involved with the immunopathogenisis of psoriasis. In this article it is discussed the interaction between the innate immunity (especially dendritic cells and keratinocytes) and adaptive immunity (T lymphocytes) in the pathogenesis of psoriasis.

Basal cell carcinoma: expression of apoptosis suppressing protein BCL-2

The Bcl-2 protein increases cell longevity by inhibiting apoptosis [programed cell death]. The aim of the study is to evaluate the expression and possible role of Bcl-2 in basal cell carcinoma [BCC]. Twenty two patients with 24 lesions [BCCs] were included in the study. After excision with safety margins, routine paraffin section of formalin-fixed BCCs were labeled with anti-Bcl-2 monoclonal antibodies using a biotin-avidin immunoperoxidase procedure. Apoptotic cells were counted in ten high power fields [HPFs] of each section. The results were compared with those in ten age and sex matched controls. Excision with reconstructive procedures was done with a safety margin [5mm]. Simple undermining and advancement was done in 8 patients, post-auricular full thickness skin graft in 7 patients, local flaps [V-Y advancement flap, rotation flap] in 9 patients. In normal skin samples, Bcl-2 was only expressed by the basal keratinocytes in one of ten [10%] samples. The mean apoptotic index [AI] was 0.5 cells/10 HPFs. In BCC, Bcl-2 was expressed by tumor cells in 21/24 cases [87.5%] with apoptotic index AI ranging between 2-11 cells/10 HPFs with a mean 4.94 +/- 1.2 cells [statistically highly significant]. Bcl-2 over-expression in the majority of BCCs may be the initial factor that predisposes to malignant transformation of keratinocytes by inhibiting apoptosis

Immunohistochemical detection of transforming growth factor beta 2 and insulin like growth factor 1 in labial tissues of treated and non treated diabetic rats

The purpose of this study was to detect transforming growth factor beta - 2 [TGF-beta2] and insulin like growth factor-1 [IGF-1] in the treated and non treated diabetic labial tissue in rat. Twenty four male's albino adult rats weighting between 150-200g each, were used throughout the experiment. They were housed under similar conditions. After determination of fasting blood glucose level for all animals, they were randomly divided into two main groups: Group I [control group] consisted of 8 rats, each animal received 0.2 ml citrate-buffered saline solution PH 4.5. Group II [diabetic group] this group consisted of 16 rats for induction of diabetes. The animals were given a single inter-peritoneal of streptozotocin dissolved in citrate-buffered saline solution PH 4.5 [0.2ml/rat] with equivalent dose of 50ml/kg body weight. This group was divided into two subgroups: Subgroup A, which consisted of 8 rats used as diabetic non treated group. Subgroup B, It consisted of 8 rats, each rat received gliclazide [diamicron] dissolved in distilled water [20 mg/kg p.o.] with gastric tube daily before feeding the animals. All rats in group 1, subgroup A and subgroup B were sacrificed at the same time after eight weeks by using a large dose of anesthesia. The specimens were taken from the lip of all rats, fixed, processed for paraffin section, cut and mounted on slides treated to enhance tissues adherence. Then the immuono-histochemical staining for TGF beta-2 and IGF-1 were processed to all specimens. The results of this study, In control group, TGF-beta 2 were detected in the basal cell.layer of epithelium and the lamina propri of the mucous membrane and skin and also were detected in the hair follicles, it were detected in the connective tissues but not the glandular epithelium of the labial minor salivary gland. In group II [sub group A], detection of TGF-beta 2 in the same sites as control group but with more reaction than it. In group II [sub group B], TGF-beta 2 detections were more than group 1 and group II [sub group A]. IGF-1 were detected in control group in the basal cell layer of epithelium and the lamina propri of the mucous membrane and skin but more than TGF-beta 2, it were also detected in the sebaceous gland itself but not in hair follicles, in the labial minor salivary gland neither detection of IGF-1 in glandular epithelium nor in the connective tissues. In group II [sub group A], the same results as control group in the mucous membrane and skin but with more reaction, in the labial minor salivary gland there were some detection in the glandular epithelium and connective tissues. In group II [sub group B], the same results as control group in the mucous membrane skin but with more reactions and as the labial minor salivary gland. Based on the previous results we can conclude that TGF-beta 2 and IGF-1 play an important role in regulation of proliferation and differentiation of keratinocytes, fibroblasts, and endothelial cells in the skin and mucous membrane of the lip in normal, diabetic none treated and treated rats. TGF-beta 2 controls hair cycling and regulates myogenesis of muscles and the activity of labial minor salivary gland in the lip of normal, diabetic none treated and treated rats. IGF-1 control the sebaceous gland secretion in the lip of normal, diabetic none treated and treated rats. It regulates the activity of labial minor salivary gland in the lip of diabetic none treated rats

Role of Staphylococcal Superantigen in Atopic Dermatitis: Influence on Keratinocytes

Staphylococcus aureus may perform an crucial function in atopic dermatitis (AD), via the secretion of superantigens, including staphylococcal enterotoxins (SE) A or B, and toxic shock syndrome toxin-1 (TSST-1). Dysregulated cytokine production by keratinocytes (KCs) upon exposure to staphylococcal superantigens (SsAgs) may be principally involved in the pathophysiology of AD. We hypothesized that lesional KCs from AD may react differently to SsAgs compared to nonlesional skin or normal skin from nonatopics. We conducted a comparison of HLA-DR or CD1a expression in lesional skin as opposed to that in nonlesional or normal skin by immunohistochemistry (IHC). We also compared, using ELISA, the levels of IL-1alpha, IL-1beta, and TNF-alpha secreted by cultured KCs from lesional, nonlesional, and normal skin, after the addition of SEA, SEB and TSST-1. IHC revealed that both HLA-DR and CD1a expression increased significantly in the epidermis of lesional skin versus nonlesional or normal skin in quite a similar manner. IL-1alpha, IL-1beta, and TNF-alpha secretion was also significantly elevated in the cultured KCs from lesional skin after the addition of SsAgs. Our results indicated that KCs from lesional skin appear to react differently to SsAgs and increased proinflammatory cytokine production in response to SsAgs may contribute to the pathogenesis of AD.

Harlequin baby--a rare case of keratinization disorder.

Harlequin icthyosis is a very rare inborn error of epidermal keratinization with autosomal recessive inheritance. Abnormal lipid metabolism in mitochondria with defective lamellar body formation is the main defect leading to hyperkeratosis. Prenatal diagnosis can be done by invasive procedures such as fetal skin biopsy and also by ultrasonography.

Calprotectina epidérmica en la reacción injerto-versus-huésped con depleción linfocitaria cutánea / Epidermal calprotectin in lymphocyte-depleted cutaneous graft-vs-host reaction

Una de las complicaciones más importantes asociada a los trasplantes de médula ósea es la Reacción Injerto versus Huésped (RIVH). Los regímenes inmunosupresores reducen la severidad de las lesiones cutáneas determinando una forma peculiar de RIVH con deplesión linfocitaria (RIVH-DL), caracterizada por pocas células inflamatorias reconocibles y casi una total ausencia de daño epidérmico. Los recientemente actualizados criterios histopatológicos para la RIVH ayudan poco para el diagnóstico de la RIVH-DL. Como la calprotectina (L1-proteína) ha sido encontrada en diversos tipos de estress epitelial, analizamos la calprotectina epitelial en la RIVH-DL. La expresión de calprotectina se estudió por inmunohistoquímica usando Mac 387 moAb en 50 casos de RIVH-DL y 40 casos de reacciones tóxicas secundarias a los regímenes condicionantes pre-trasplante o por drogas post-trasplante. Se detectó calprotectina en queratinocitos de apariencia normal en todos los casos de RIVH-DL y en la mayoría de las dermatitis con citotoxicidad inducidas por drogas. Concluímos que la inmunoreactividad a la calprotectina aparenta ser una clave diagnóstica para la RVIH-DL. Se expresión aparece precozmente en el curso de la RIVH-DL independientemente del grado histológico de la reacción. Sin embargo, no puede ser usada en forma aislada para distinguir entre una RIVH-DL inicial y una dermatitis inducida por drogas

El queratinocito: una celula inmunologica / The keratinocyte: an inmunological cell

El queratinocito es una celula inmunologica que participa activamente en la respuesta inflamatoria e inmunologica. Se efectua una revision de los conocimientos sobre las multiples funciones de esta celula epitelial

Immunopathology of american cutaneous leishmaniasis. Modulation of MHC class II gene products by Keratinocytes before and after glucantime therapy

Epidermal changes from 32 cutaneous and 3 mucosal American leishmaniasis (ACL) active lesions were studied for HLA-DR, -DP expression, Lanerhans cells and lymphocyte infiltration. In addition to a DR and DQ positivity at the surface of the cells of the inflammatory infiltrate, a strong reaction for DR antigens was detected on keratinocytes. Hyperplasia of Langerhans cells was present in al cutaneous lesions and epidermis was infiltrated by T lymphocytes. When healed lesions of 14 of these subjects were re-biopsied 1 to 12 months after the end of pentavalent antimonial therapy, MHC class antigens could no longer be seen on keratinocytes. Our data represrn evidence for hhe reversibility of the abnormal HLA-DR expression by keratinocytes in ACL after Glucantime therapy or spontaneous scar formation, demonstrating that this expresion is restricted to the period of active lesions. The present findings can be regarded as an indirect evidence that keratinocytes may be involved in the immunopathology of ACL

Cellular response to the intradermal injection of recombinant human gamma-interferon in lepromatous leprosy patients

The local response to single intradermal injection of 10 ug recombinat gamma-interferon (rIFNgamma) has been studied in 17 patients with lepromatous leprosy. Of these, 2 patients additionally received two intradermal injections of 10 ug rIFNgamma at received another site. The results were compared with those of 3 patients who received three injections of the same dose at a single site in an earlier study. One to 7 days after lymphokine administration 4-mm pinch biopsies were obtained and axamined for cellular alterations in the dermis and epidermis. This allowed a kinetic analysis of mononuclear cell infiltration, keratinocyte proliferation and differentiation and Langerhans cell redistribution. At 24 hours, the migration of large numbers of helper T cells and monocytes was already prominent and associated with induration. Mononuclear cell eccumulation peaked at 72 hours but then persisted for 5-7 days. Only smal numbers (one-third or less of toal T cells) of suppressor/cytotoxic T cells were present at any time, and granulocytes were absent. Two daily injections of rIFNgamma led to a more intense accumulation of cells. Ten ug of rIFNgamma resulted in enhanced keratinocyte proliferation, Ia expression, and thickening of the epidermis. At 24-48 hours major histocompatibility Class II (Ia) antigen was first noted on the dividing cells of the basal layer. By 72-96 hours the entire epidemir exhibited strong expression of Ia antigen on cell surfaces. Repeated doses of lymphokine accentuated these changes and resulted in a more prompt keratinization and sloughing of this layer. Whereas a single dose of rIFNgamma resulted in the upward movement of T6+ Langerhans cells (LCs) in the epidermis, two injections led to a 50% reduction in their numbers and three doses were associated with an almost total loss of detectable T6+ LCs from the epidermis. These are probably aloughed along with keratinocytes. In contrast to the situation with a delayed immune response in the skin (purified protein derivative), no LCs accumulated in the dermis in association with helper T cells