The Role of Fibronectin in the Adherence and Inflammatory Response Induced by Enteroaggregative Escherichia coli on Epithelial Cells.

Enteroaggregative Escherichia coli (EAEC) infections are still one of the most important etiologic pathogens of diarrhea in children worldwide. EAEC pathogenesis comprises three stages: adherence and colonization, production of toxins, and diarrhea followed by inflammation. Previous studies have demonstrated that EAEC strains have the ability to bind to fibronectin (FN); however, the role this extracellular matrix protein plays in the inflammatory response induced by EAEC remains unknown. In this study, we postulated that FN-mediated adherence of EAEC strains to epithelial cells increases the expression of pro-inflammatory genes. To verify this hypothesis, we infected HEp-2 and HT-29 cells, in both the presence and absence of FN, with EAEC reference strain 042. We quantified IL-8 secretion and the relative expression of a set of genes regulated by the NF-κB pathway. Although FN increased EAEC adherence, no changes in IL-8 protein secretion or IL8 gene expression were observed. Similar observations were found in HEp-2 cells transfected with FN-siRNA and infected with EAEC. To evaluate the involvement of AAF/II fimbriae, we infected HEp-2 and HT-29 cells, in both the presence and absence of FN, with an EAEC 042aafA mutant strain transformed with a plasmid harboring the native aafA gene with a site-directed mutation in Lys72 residue (K72A and K72R strains). No changes in IL-8 secretion were observed. Finally, SEM immunogold assay of cells incubated with FN and infected with EAEC revealed that AAF fimbriae can bind to cells either directly or mediated by FN. Our data suggests that FN participates in AAF/II fimbriae-mediated adherence of EAEC to epithelial cells, but not in the inflammatory response of cells infected by this pathogen.

Identification of cell surface-exposed proteins involved in the fimbria-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC.

TNF-α is involved in the abnormal thymocyte migration during experimental Trypanosoma cruzi infection and favors the export of immature cells.

Previous studies revealed a significant production of inflammatory cytokines together with severe thymic atrophy and thymocyte migratory disturbances during experimental Chagas disease. Migratory activity of thymocytes and mature T cells seem to be finely tuned by cytokines, chemokines and extracellular matrix (ECM) components. Systemic TNF-α is enhanced during infection and appears to be crucial in the response against the parasite. However, it also seems to be involved in disease pathology, since it is implicated in the arrival of T cells to effector sites, including the myocardium. Herein, we analyzed the role of TNF-α in the migratory activity of thymocytes in Trypanosoma cruzi (T. cruzi) acutely-infected mice. We found increased expression and deposition of TNF-α in the thymus of infected animals compared to controls, accompanied by increased co-localization of fibronectin, a cell migration-related ECM molecule, whose contents in the thymus of infected mice is also augmented. In-vivo studies showed an enhanced export of thymocytes in T. cruzi-infected mice, as ascertained by intrathymic injection of FITC alone or in combination with TNF-α. The increase of immature CD4(+)CD8(+) T cells in secondary lymphoid organs was even more clear-cut when TNF-α was co-injected with FITC. Ex-vivo transmigration assays also revealed higher number of migrating cells when TNF-α was added onto fibronectin lattices, with higher input of all thymocyte subsets, including immature CD4(+)CD8(+). Infected animals also exhibit enhanced levels of expression of both mRNA TNF-α receptors in the CD4(+)CD8(+) subpopulation. Our findings suggest that in T. cruzi acute infection, when TNF-α is complexed with fibronectin, it favours the altered migration of thymocytes, promoting the release of mature and immature T cells to different compartments of the immune system. Conceptually, this work reinforces the notion that thymocyte migration is a multivectorial biological event in health and disease, and that TNF-α is a further player in the process.

Interação entre neutrófilos humanos e macrófagos contribui no desfecho da infecção por Leishmania amazonensis / Interaction between human neutrophils and macrophages contributes to the outcome of infection by Leishmania amazonensis

Os neutrófilos são células que atuam na primeira linha de defesa do organismo contra patógenos e estão constitutivamente programadas para a morte celular por apoptose, sendo então, fagocitados por macrófagos. No presente estudo, avaliamos inicialmente o efeito da fagocitose de neutrófilos humanos em repouso: viáveis, apoptóticos ou necróticos, por macrófagos humanos infectados por Leishmania amazonensis. A interação com neutrófilos em repouso apoptóticos, mas não com células viáveis, leva a um aumento na taxa de infecção e carga parasitária em macrófagos. O favorecimento do crescimento intracelular do parasita em presença de neutrófilos apoptóticos foi dependente da produção de TGF-f3 e PG~. Por outro lado, houve uma diminuição da infecção dos macrófagos por L. amazonensis, assim como da carga parasitária, na presença de neutrófilos necróticos. A atividade leishmanicida nessas co-culturas foi dependente da produção de TNF-a, elastase neutrofilica (NE) e superóxido. A presença de neutrófilos ativados viáveis e apoptóticos pelo contato com fibronectina, mas não com outras proteínas de matriz extracelular, também reduz a taxa de infecção e a carga parasitária dos macrófagos infectados com L. amazonensis. O efeito microbicida nas co-culturas com neutrófilos ativados foi independente de contato e envolve a participação de enzimas neutrofilicas NE e MPO, bem como outros mediadores pró-inflamatórios como TNF-a, LTB4 e ROI. Os resultados apresentados neste estudo demonstram que a interação com neutrófilos humanos em repouso ou ativados é capaz de modular a resposta de macrófagos contribuindo para o desfecho da infecção por L. amazonensis.

The outer wall of small airways is a major site of remodeling in fatal asthma.

BACKGROUND: Structural and inflammatory changes in asthma involve both the large and small airways, with involvement of the distal lung being related to disease severity. We have previously shown that changes in the extracellular matrix (ECM) composition of the distal lung are associated with loss of alveolar attachments in patients with fatal asthma. However, major ECM elements, such as collagen I and fibronectin and their regulators, have not been addressed at the distal level. OBJECTIVE: We sought to evaluate ECM remodeling in the distal lungs of asthmatic patients. METHODS: Using immunohistochemistry and image analysis, we determined the content of collagen I and III, fibronectin, and matrix metalloproteinases (MMPs) 1, 2, and 9 and tissue inhibitors of metalloproteinase (TIMPs) 1 and 2 in the large and small airways and lung parenchyma of 24 patients with fatal asthma and compared the results with those of 11 nonasthmatic control subjects. Protein content was defined as the area of positive staining divided by basement membrane or septum length. RESULTS: We observed increased collagen I and decreased collagen III content in the small airways of asthmatic patients compared with that seen in control subjects. Greater fibronectin and MMP-1, MMP-2, and MMP-9 content was observed at the outer area of the small airways in asthmatic patients. MMP content was also increased in the peribronchiolar parenchyma in asthmatic patients. In contrast, TIMP expression was only increased in the large airways of asthmatic patients compared with that seen in control subjects. CONCLUSIONS: The outer area of the small airways is a major site of ECM remodeling in fatal asthma, potentially contributing to functional changes and the loss of airway-parenchyma interdependence observed in patients with fatal asthma.

Freeze-drying as an alternative method of human sclera preservation.

PURPOSE: To compare the effect of preserving sclera samples in either 95% ethanol or freeze-dried. METHODS: Ninety-six samples of human sclera were studied. Half of them were freeze-dried and half preserved in 95% ethanol. Preservation periods of 18, 45, 90 or 174 days were studied. Automated immunostaining was carried out in the Ventana BenchMarkR LT platform using collagen 1 and fibronectin antibodies. Histological staining was also performed with hematoxilin-eosin and Masson trichrome. Samples were classified according to the degree of collagen fiber parallelism (0-2), intensity of Masson staining (0-2), and the expression of both antibodies (0-3). Friedman and Wilcoxon tests were applied to compare preservation methods and p-values below 0.05 were considered to ensure statistical significance. RESULTS: Relevant results were found in three situations: (i) Friedman's test showed better collagen fiber integrity in the freeze-dried group rehydrated after 174-days as compared to the 90-day group; (ii) Wilcoxon's test showed better collagen fiber integrity in the freeze-dried group after 18 and 174 days as compared to the ethanol group; (iii) the freeze-dried group disclosed higher immunohistochemical expression for COL-1 antibody in the sclera samples rehydrated after 45, 90 and 174 days as compared to the ones rehydrated after 18 days. CONCLUSION: Histological and immunohistochemical analysis showed freeze-drying to be a superior method for sclera preservation as compared to 95% ethanol. This technique provides an easy method to manipulate tissue, with longer shelf life, and storage at room temperature.

Involvement of extracellular matrix proteins in the course of experimental paracoccidioidomycosis.

We aimed at determining involvement of extracellular matrix proteins (ECMp) and an ECM-binding adhesin (32-kDa protein) from Paracoccidioides brasiliensis, in the course of experimental paracoccidioidomycosis. BALB/c mice were infected with P. brasiliensis conidia previously incubated with soluble laminin, fibronectin and fibrinogen or a mAb against the fungal adhesin. Inflammatory response, chitin levels and cytokine production at different postinfection periods were determined. Chitin was significantly decreased in lungs of mice infected with ECMp-treated conidia when compared with controls at week 8, especially with laminin and fibrinogen. Contrariwise, when animals were infected with mAb-treated conidia no differences in chitin content were found. The observed inflammatory reaction in lungs was equivalent in all cases. IFN-gamma increased significantly in lungs from mice infected with soluble ECMp - (at day 4 and week 12) or mAb-treated conidia (at week 12) when compared with animals infected with untreated conidia. Significant increased levels of tumour necrosis factor-alpha were observed at 8 weeks in animals infected with ECMp-treated conidia while no differences were observed during the remaining periods. These findings point toward an inhibitory effect of ECMp on P. brasiliensis conidia infectivity and suggest that these proteins may interfere with conidia initial adhesion to host tissues probably modulating the immune response in paracoccidioidomycosis.

Freeze-drying as an alternative method of human sclera preservation / Liofilização como alternativa à preservação de esclera humana

PURPOSE: To compare the effect of preserving sclera samples in either 95 percent ethanol or freeze-dried. METHODS: Ninety-six samples of human sclera were studied. Half of them were freeze-dried and half preserved in 95 percent ethanol. Preservation periods of 18, 45, 90 or 174 days were studied. Automated immunostaining was carried out in the Ventana BenchMarkR LT platform using collagen 1 and fibronectin antibodies. Histological staining was also performed with hematoxilin-eosin and Masson trichrome. Samples were classified according to the degree of collagen fiber parallelism (0-2), intensity of Masson staining (0-2), and the expression of both antibodies (0-3). Friedman and Wilcoxon tests were applied to compare preservation methods and p-values below 0.05 were considered to ensure statistical significance. RESULTS: Relevant results were found in three situations: (i) Friedman's test showed better collagen fiber integrity in the freeze-dried group rehydrated after 174-days as compared to the 90-day group; (ii) Wilcoxon's test showed better collagen fiber integrity in the freeze-dried group after 18 and 174 days as compared to the ethanol group; (iii) the freeze-dried group disclosed higher immunohistochemical expression for COL-1 antibody in the sclera samples rehydrated after 45, 90 and 174 days as compared to the ones rehydrated after 18 days. CONCLUSION: Histological and immunohistochemical analysis showed freeze-drying to be a superior method for sclera preservation as compared to 95 percent ethanol. This technique provides an easy method to manipulate tissue, with longer shelf life, and storage at room temperature.

OBJETIVO: Comparar dois métodos de preservação de esclera humana, liofilização e álcool 95 por cento, em diferentes períodos de tempo. MÉTODOS: Foram avaliados 96 fragmentos de seis escleras humanas. Metade das amostras foi submetida ao processo de liofilização e metade conservada em álcool 95 por cento. Dois fragmentos de cada grupo foram avaliados pelas colorações de hematoxilina-eosina e tricrômio de Masson e submetidos a técnica de imuno-histoquímica para os anticorpos fibronectina e colágeno 1, após 18, 45, 90 e 174 dias de preservação. Os espécimens foram classificados de acordo com o paralelismo (PF:0-2) e integridade (IF:0-1) das fibras de colágeno e expressão imuno-histoquímica para os anticorpos fibronectina (FIB:0-3) e colágeno 1 (COL-1:0-3). A análise estatística foi realizada por meio dos testes de Friedman e Wilcoxon e o valor de p menor que 0,05 foi considerado estatisticamente significante. RESULTADOS: Verificaram-se diferenças significantes em três situações: (i) maior integridade das fibras de colágeno das escleras liofilizadas após 174 dias quando comparado aos 90 dias; (ii) maior expressão imuno-histoquímica para o anticorpo COL-1 nas amostras de escleras liofilizadas após os 18 dias iniciais de preservação; (iii) maior integridade das fibras de colágeno das escleras liofilizadas após 18 e 174 dias quando comparado às escleras preservadas em álcool. CONCLUSÕES: A preservação de tecido escleral por liofilização mostrou-se técnica tão eficaz quanto a preservação em álcool, apresentado vantagem quando considerada a integridade das fibras de colágeno. A liofilização mostra-se benéfica por permitir a estocagem do tecido em temperatura ambiente e com prazo de validade estendido.

Altered thymocyte migration during experimental acute Trypanosoma cruzi infection: combined role of fibronectin and the chemokines CXCL12 and CCL4.

We previously showed migration disturbances in the thymus during experimental infection with Trypanosoma cruzi, the causative agent of Chagas disease. These changes were related to the enhanced expression of extracellular matrix ligands and receptors, leading to the escape of immature cells to the periphery. Here, we analyzed the expression and role of selected chemokines (CXCL12 and CCL4) and their receptors (CXCR4 and CCR5) in regulating thymocyte migration in conjunction with extracellular matrix during acute T. cruzi infection. We found increased chemokine deposition in the thymus of infected mice when compared to controls, accompanied by enhanced co-localization with fibronectin as well as up-regulated surface expression of CXCR4 and CCR5 in thymocytes. We also noticed altered thymocyte migration towards the chemokines analyzed. Such an enhancement was even more prominent when fibronectin was added as a haptotatic stimulus in combination with a given chemokine. Our findings suggest that thymocyte migration results from a combined action of chemokines and extracellular matrix (ECM), which can be altered during pathological conditions such as T. cruzi infection, and may be at the origin of the changes in the T cell repertoire seen in this pathological process.

Autoantibodies directed to extracellular matrix components in patients with different clinical forms of periodontitis.

BACKGROUND: Periodontal disease occurs in different clinical forms, from mild and easily controllable to more aggressive inflammatory manifestations, with difficult clinical or surgical control. There is evidence that a local autoimmune reaction may participate in the onset and persistence of the aggressive forms of periodontal disease. As the underlying mechanism in this process is not fully understood, we decided to investigate whether patients bearing this form of disease presented higher levels of antibodies directed to extracellular matrix (ECM) components such as type I collagen, fibronectin, and laminin. METHODS: Three groups of patients were selected by clinical criteria: 22 subjects with aggressive periodontitis (AgP) = group A; 18 subjects with chronic periodontitis (CP) = group B; and 10 healthy (H) volunteers without periodontal disease = group C. Autoantibody levels were evaluated in the sera of these patients using the enzyme-linked immunosorbent assay (ELISA) method. RESULTS: The levels of autoantibodies directed to ECM components (type I collagen, fibronectin and laminin) in the sera of patients with AgP and CP were shown to be statistically different (P <0.05). CONCLUSIONS: Although the present findings suggest an involvement of autoantibodies directed to ECM components per se in the pathogeny of certain types of periodontal disease, the available data do not support the classification of the lesions as autoimmune. Nevertheless, the findings open a possibility for the development of an additional method for a differential diagnosis of the aggressive forms of periodontal disease.

Modulation of fibronectin expression in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis.

Fibronectin (FN), a large family of plasma and extracellular matrix (ECM) glycoproteins, plays an important role in leukocyte migration. In normal central nervous system (CNS), a fine and delicate mesh of FN is virtually restricted to the basal membrane of cerebral blood vessels and to the glial limitans externa. Experimental autoimmune encephalomyelitis (EAE), an inflammatory CNS demyelinating disease, was induced in Lewis rats with a spinal cord homogenate. During the preclinical phase and the onset of the disease, marked immunolabelling was observed on the endothelial luminal surface and basal lamina of spinal cord and brainstem microvasculature. In the paralytic phase, a discrete labelling was evident in blood vessels of spinal cord and brainstem associated or not with an inflammatory infiltrate. Conversely, intense immunolabelling was present in cerebral and cerebellar blood vessels, which were still free from inflammatory cuffs. Shortly after clinical recovery minimal labelling was observed in a few blood vessels. Brainstem and spinal cord returned to normal, but numerous inflammatory foci and demyelination were still evident near the ventricle walls, in the cerebral cortex and in the cerebellum. Intense expression of FN in brain vessels ascending from the spinal cord towards the encephalon preceded the appearance of inflammatory cells but faded away after the establishment of the inflammatory cuff. These results indicate an important role for FN in the pathogenesis of CNS inflammatory demyelinating events occurring during EAF.

Dermal extracellular matrix in cutaneous leprosy lesions.

Thirty-eight biopsies of cutaneous lesions from leprosy patients [borderline tuberculoid (BT) 14, borderline lepromatous (BL) 18, lepromatous (LL) 6] were processed for staining of some extracellular matrix (ECM) components (collagen, proteoglycans, elastic fibers and fibronectin). Specific histological staining and the indirect immunofluorescence method with antibodies to collagen and fibronectin were utilized. The ECM of the normal dermis was strikingly modified in the inflammatory infiltrate. By Gomori's reticulin and anti-fibronectin immunostaining, replacement of the dense interlaced collagen fibers with a reticular mesh was observed in the infiltrate. The immunoreactivity obtained with anti-type I and anti-type III collagens showed positive fibrils and a lumpy pattern in the lepromatous and tuberculoid lesions with a higher amount in the lepromatous lesions. The lack of clear-cut boundaries between the normal dermis and the inflammatory infiltrate in the lepromatous (BL, LL) lesions was correlated with the blurred limits of the clinical lesions of this pole of the leprosy spectrum. Absence of elastic fibers in the infiltrate was a constant finding, and fuchsin-positive microfibrils were found in some infiltrates. The clear zone of lepromatous lesions was devoid of oxytalan fibers. Elaunin fiber rings around sweat gland acini were present even when the leprosy infiltrate was seen enveloping them. The original ECM is replaced by a newly assembled one, which is suited for the dynamic nature of the inflammatory process. The trophic effects of the ECM upon the cutaneous epithelial structures are modified so that atrophy and late degeneration ensues. These ECM modifications contribute, therefore, to the biological alterations of the skin functions in leprosy.

Modulation of fibronectin expression in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis

Fibronectin (FN), a large family of plasma and extracellular matrix (ECM) glycoproteins, plays an important role in leukocyte migration. In normal central nervous system (CNS), a fine and delicate mesh of FN is virtually restricted to the basal membrane of cerebral blood vessels and to the glial limitans externa. Experimental autoimmune encephalomyelitis (EAE), an inflammatory CNS demyelinating disease, was induced in Lewis rats with a spinal cord homogenate. During the preclinical phase and the onset of the disease, marked immunolabelling was observed on the endothelial luminal surface and basal lamina of spinal cord and brainstem microvasculature. In the paralytic phase, a discrete labelling was evident in blood vessels of spinal cord and brainstem associated or not with an inflammatory infiltrate. Conversely, intense immunolabelling was present in cerebral and cerebellar blood vessels, which were still free from inflammatory cuffs. Shortly after clinical recovery minimal labelling was observed in a few blood vessels. Brainstem and spinal cord returned to normal, but numerous inflammatory foci and demyelination were still evident near the ventricle walls, in the cerebral cortex and in the cerebellum. Intense expression of FN in brain vessels ascending from the spinal cord towards the encephalon preceded the appearance of inflammatory cells but faded away after the establishment of the inflammatory cuff. These results indicate an important role for FN in the pathogenesis of CNS inflammatory demyelinating events occurring during EAE

Antiaorta antibodies and Takayasu arteritis.

A search for antibodies reactive against a total human aorta extract and its main protein components such as elastin, fibronectin and collagen was attempted by electroimmunetransference and ELISA. Thirty five sera from clinically and angiographically proven diagnosis of Takayasu Arteritis patients were compared with 32 sera from people without abnormalities. Non specific binding was found on electroimmune transference and no difference was shown in optical density readings in ELISA, therefore, we did not demonstrate the presence of antiaorta specific antibodies in this vasculitic condition. Our findings are in agreement with several authors, the contribution of humoral immunity to pathogenesis of Takayasu Arteritis has not been proved yet.

Histological differentiation and basement membrane distribution in gastric adenocarcinomas.

To analyse the histological distribution of basement membranes (BM) in gastric adenocarcinomas, we produced a monoclonal antibody, BM909 (IgG 2b, kappa), which has been found useful for identifying the BM in routinely processed specimens. The BM 909 antibody was shown by ELISA to be negative against the three major BM components (type IV collagen, laminin and fibronectin). Gastric carcinomas from 78 patients including both differentiated and undifferentiated adenocarcinomas were classified into three types (tubular, trabecular, and isolated) based on the histological arrangement of the cancer cells. Histological distributions of the BM were also classified into four patterns (peritubular, peritrabecular, intratrabecular, and pericellular) based on the immunostaining results with the BM 909 antibody. Our results demonstrated a close relationship between these two parameters as follows: 1) the tubular type of gastric carcinomas showed a peritubular pattern of BM distribution in the differentiated adenocarcinomas; 2) The isolated type of gastric carcinomas showed a pericellular pattern of BM distribution in the undifferentiated adenocarcinomas; and 3) The trabecular type of gastric carcinomas showed either peritrabecular or intratrabecular patterns of BM distribution, in both differentiated and undifferentiated adenocarcinomas. In conclusion, we suggest that all gastric adenocarcinomas are accompanied by BM deposition regardless of the degree of histological differentiation.

Imunidade do feto e do recem-nascido / Immunity of the fetus and the newborn infant

O conhecimento da imunidade do feto e do recem-nascido e de grande importancia, por estar diretamente relacionado com a susceptibilidade deste, frente a processos infecciosos. Apresentamos uma revisao da literatura focalizando a imunidade humoral, celular, o sistema complemento e a atividade das celulas fagocitarias, chamando atencao para os aspectos mais importantes que poderao ser uteis na escolha de uma terapeutica imunologica adequada