Detection and neutralization of venom by ovine antiserum in experimental envenoming by Bothrops jararaca

In this study we optimized an enzyme-linked immunosorbent assay (ELISA) to evaluate bothropic venom levels in biological samples. These samples were obtained by two distinct protocols. In the first one, Swiss mice were injected with 1 LD 50 of Bothrops jararaca (B. jararaca) venom and 15 minutes later, animals were treated with ovine antibothropic serum. Blood and spleen homogenate samples were obtained 6 hours after antiserum therapy. Ovine antibothropic serum significantly neutralized venom levels in serum and spleen. In the second protocol, BALB/c mice were injected with 1 LD 50 of bothropic venom by either intraperitoneal (IP) or intradermal (ID) route and venom levels were evaluated 1, 3 and 6 hours after, in blood, spleen homogenates and urine. Serum and splenic venom levels were significantly higher in animals envenomed by IP route comparing with animals envenomed by ID route. Higher venom levels were also detected in urine samples from animals envenomed by IP route. However, these differences were not statistically significant. These results demonstrated that the optimized ELISA was adequate to quantify venom levels in different biological samples. This assay could, therefore, substitute the in vivo neutralizing assay and also be useful to evaluate the severity of human and experimental envenomations.

Porphyromonas gingivalis LPS stimulation downregulates DNMT1, DNMT3a, and JMJD3 gene expression levels in human HaCaT keratinocytes

Objective: The role of epigenetic regulation in inflammatory diseases such as periodontitis is poorly known. The aim of this study was to assess whether Porphyromonas gingivalis lipopolysaccharide (LPS) can modulate gene expression levels of the some enzymes that promote epigenetic events in cultures of the human keratinocytes and gingival fibroblasts. In addition, the same enzymes were evaluated in gingival samples from healthy and periodontitis-affected individuals. Materials and methods: Primary gingival fibroblast and keratinocyte (HaCaT) cultures were treated with medium containing P. gingivalis LPS or P. gingivalis LPS vehicle for 24 h. After this period, cell viability was assessed by MTT test and total RNA extracted to evaluate gene expression levels of the following enzymes by qRT-PCR: DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), histone demethylases Jumonji domain containing 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX). To evaluate gene expression in healthy and periodontitis-affected individuals, total RNA was extracted from biopsies of gingival tissue from healthy and periodontitis sites, and gene expression of DNMT1, DNAMT3a, JMJD3, and UTX was evaluated by qRT-PCR. Results: No significant differences were found in the gene expression analysis between healthy and periodontitis-affected gingival samples. The results showed that LPS downregulated DNMT1 (p < 0. 05), DNMT3a (p < 0. 05), and JMJD3 (p < 0. 01) gene expression in HaCaT cells, but no modulation was observed in gingival fibroblasts. Conclusion: P. gingivalis LPS exposure to human HaCaT keratinocytes downregulates gene expression of the enzymes that promote epigenetic events. Clinical relevance: The advance knowledge about epigenetic modifications caused by periodontopathogens may to possibly led to the development of new periodontal therapies. © 2012 Springer-Verlag.

Biostimulatory effect of low-level laser therapy on keratinocytes in vitro

Epithelial cells play an important role in reparative events. Therefore, therapies that can stimulate the proliferation and metabolism of these cells could accelerate the healing process. To evaluate the effects of low-level laser therapy (LLLT), human keratinocytes were irradiated with an InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm; 40 mW) using 0.5, 1.5, 3, 5, and 7 J/cm2 energy doses. Irradiations were done every 24 h totaling three applications. Evaluation of cell metabolism (MTT assay) showed that LLLT with all energy doses promoted an increase of cell metabolism, being more effective for 0.5, 1.5, and 3 J/cm2. The highest cell counts (Trypan blue assay) were observed with 0.5, 3, and 5 J/cm2. No statistically significant difference for total protein (TP) production was observed and cell morphology analysis by scanning electron microscopy revealed that LLLT did not promote morphological alterations on the keratinocytes. Real-time polymerase chain reaction (qPCR) revealed that LLLT also promoted an increase of type I collagen (Col-I) and vascular endothelial growth factor (VEGF) gene expression, especially for 1.5 J/cm2, but no change on fibroblast growth factor-2 (FGF-2) expression was observed. LLLT at energy doses ranging from 0.5 to 3 J/cm2 promoted the most significant biostimulatory effects on cultured keratinocytes. © 2012 Springer-Verlag London Ltd.

IL-2, IL-5, TNF-α and IFN-γ mRNA expression in epidermal keratinocytes of systemic lupus erythematosus skin lesions

OBJECTIVE: To analyze cytokine gene expression in keratinocytes from patients with systemic lupus erythematosus (SLE). INTRODUCTION: Keratinocytes represent 95% of epidermal cells and can secrete several cytokines. METHODS: Keratinocytes were obtained by laser microdissection from 21 patients with SLE (10 discoid and 11 acute lesions) at involved and uninvolved sites. All patients were receiving a low/moderate prednisone dose and 18 were receiving chloroquine diphosphate. IL-2, IL-5, TNF-α and IFN-γ gene expression was evaluated by real-time PCR and expressed as the ratio (R) to a pool of skin samples from 12 healthy volunteers. RESULTS: Heterogeneity in cytokine gene expression was found among patients with SLE. Eighteen of 38 valid SLE samples (47%) presented overexpression (R>1) of at least one cytokine. Lesional skin samples tended to show higher cytokine expression than samples from uninvolved skin (p = 0.06). IL-5 and IFN-γ were the most commonly overexpressed cytokines. Samples with cytokine overexpression corresponded to more extensive and severe lesions. Prednisone dose did not differ between samples without cytokine overexpression (15.71±3.45 mg/day) and those with overexpressed cytokines (12.68±5.41 mg/day) (p = 0.216). Samples from all patients not receiving diphosphate chloroquine had at least one overexpressed cytokine. CONCLUSIONS: The heterogeneous keratinocyte cytokine gene expression reflects the complex immunological and inflammatory background in SLE. Patients with severe/extensive skin lesions showed a higher frequency of cytokine gene overexpression. Increased IFN-γ and IL-5 expression suggests that Th1 and Th2 cells are involved in SLE skin inflammation. The possibility that prednisone and antimalarial drugs may have contributed to low cytokine gene expression in some samples cannot be ruled out.

Effects of Soft Denture Liners on L929 Fibroblasts, HaCaT Keratinocytes, and RAW 264.7 Macrophages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of hydrogen peroxide on thawed ovine sperm motility

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fate of non O157 Shigatoxigenic Escherichia coli in ovine manurecomposting

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Changes in nuclear morphology and chromatin texture of basal keratinocytes in melasma

BackgroundThe pathogenesis of melasma and the role of keratinocytes in disease development and maintenance are not completely understood. Dermal abnormalities, the expression of inflammatory mediators, growth factors, epithelial expression of melanocortin and sexual hormones receptors suggest that not only melanocytes, but entire epidermal melanin unit is involved in melasma physiopathology.ObjectivesTo compare nuclear morphological features and chromatin texture between basal keratinocytes in facial melasma and adjacent normal skin.MethodsWe took facial skin biopsies (2mm melasma and adjacent normal skin) from women processed for haematoxylin and eosin. Thirty non-overlapping basal keratinocyte nuclei were segmented and descriptors of area, highest diameter, perimeter, circularity, pixel intensity, profilometric index (Ra) and fractal dimension were extracted using ImageJ software.ResultsBasal keratinocyte nuclei from facial melasma epidermis displayed larger size, irregular shape, hyperpigmentation and chromatin heterogeneity by fractal dimension than perilesional skin.ConclusionBasal keratinocytes from facial melasma display changes in nuclear form and chromatin texture, suggesting that the phenotype differences between melasma and adjacent facial skin can result from complete epidermal melanin unit alterations, not just hypertrophic melanocytes.

Activation of the low molecular weight protein tyrosine phosphatase in keratinocytes exposed to hyperosmotic stress

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The oxidation of p-phenylenediamine, an ingredient used for permanent hair dyeing purposes, leads to the formation of hydroxyl radicals: oxidative stress and DNA damage in human immortalized keratinocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular confirmation of ovine herpesvirus 2-induced malignant catarrhal fever lesions in cattle from Rio Grande do Norte, Brazil

Molecular findings that confirmed the participation of ovine herpesvirus 2 (OVH-2) in the lesions that were consistent with those observed in malignant catarrhal fever of cattle are described. Three mixed-breed cattle from Rio Grande do Norte state demonstrated clinical manifestations that included mucopurulent nasal discharge, corneal opacity and motor incoordination. Routine necropsy examination demonstrated ulcerations and hemorrhage of the oral cavity, corneal opacity, and lymph node enlargement. Significant histopathological findings included widespread necrotizing vasculitis, non-suppurative meningoencephalitis, lymphocytic interstitial nephritis and hepatitis, and thrombosis. PCR assay performed on DNA extracted from kidney and mesenteric lymph node of one animal amplified a product of 423 base pairs corresponding to a target sequence within the ovine herpesvirus 2 (OVH-2) tegument protein gene. Direct sequencing of the PCR products, from extracted DNA of the kidney and mesenteric lymph node of one cow, amplified the partial nucleotide sequences (423 base pairs) of OVH-2 tegument protein gene. Blast analysis confirmed that these sequences have 98-100% identity with similar OVH-2 sequences deposited in GenBank. Phylogenetic analyses, based on the deduced amino acid sequences, demonstrated that the strain of OVH-2 circulating in ruminants from the Brazilian states of Rio Grande do Norte and Minas Gerais are similar to that identified in other geographical locations. These findings confirmed the active participation of OVH-2 in the classical manifestations of sheep associated malignant catarrhal fever.

HPV16 Oncoproteins Induce MMPs/RECK-TIMP-2 Imbalance in Primary Keratinocytes: Possible Implications in Cervical Carcinogenesis

Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/ RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome.

Meccanismi evolutivi dei parapoxvirus: caratterizzazione genomica di pseudocowpoxvirus e messa a punto di sistemi per lo studio delle ricombinazioni

The Poxviruses are a family of double stranded DNA (dsDNA) viruses that cause disease in many species, both vertebrate and invertebrate. Their genomes range in size from 135 to 365 kbp and show conservation in both organization and content. In particular, the central genomic regions of the chordopoxvirus subfamily (those capable of infecting vertebrates) contain 88 genes which are present in all the virus species characterised to date and which mostly occur in the same order and orientation. In contrast, however, the terminal regions of the genomes frequently contain genes that are species or genera-specific and that are not essential for the growth of the virus in vitro but instead often encode factors with important roles in vivo including modulation of the host immune response to infection and determination of the host range of the virus. The Parapoxviruses (PPV), of which Orf virus is the prototypic species, represent a genus within the chordopoxvirus subfamily of Poxviridae and are characterised by their ability to infect ruminants and humans. The genus currently contains four recognised species of virus, bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) both of which infect cattle, orf virus (OV) that infects sheep and goats, and parapoxvirus of red deer in New Zealand (PVNZ). The ORFV genome has been fully sequenced, as has that of BPSV, and is ~138 kb in length encoding ~132 genes. The vast majority of these genes allow the virus to replicate in the cytoplasm of the infected host cell and therefore encode proteins involved in replication, transcription and metabolism of nucleic acids. These genes are well conserved between all known genera of poxviruses. There is however another class of genes, located at either end of the linear dsDNA genome, that encode proteins which are non-essential for replication and generally dictate host range and virulence of the virus. The non-essential genes are often the most variable within and between species of virus and therefore are potentially useful for diagnostic purposes. Given their role in subverting the host-immune response to infection they are also targets for novel therapeutics. The function of only a relatively small number of these proteins has been elucidated and there are several genes whose function still remains obscure principally because there is little similarity between them and proteins of known function in current sequence databases. It is thought that by selectively removing some of the virulence genes, or at least neutralising the proteins in some way, current vaccines could be improved. The evolution of poxviruses has been proposed to be an adaptive process involving frequent events of gene gain and loss, such that the virus co-evolves with its specific host. Gene capture or horizontal gene transfer from the host to the virus is considered an important source of new viral genes including those likely to be involved in host range and those enabling the virus to interfere with the host immune response to infection. Given the low rate of nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in poxviruses is intimately linked to DNA replication with both viral and cellular proteins participate in this recombination-dependent replication. It has been shown, in other poxvirus genera, that recombination between isolates and perhaps even between species does occur, thereby providing another mechanism for the acquisition of new genes and for the rapid evolution of viruses. Such events may result in viruses that have a selective advantage over others, for example in re-infections (a characteristic of the PPV), or in viruses that are able to jump the species barrier and infect new hosts. Sequence data related to viral strains isolated from goats suggest that possible recombination events may have occurred between OV and PCPV (Ueda et al. 2003). The recombination events are frequent during poxvirus replication and comparative genomic analysis of several poxvirus species has revealed that recombinations occur frequently on the right terminal region. Intraspecific recombination can occur between strains of the same PPV species, but also interspecific recombination can happen depending on enough sequence similarity to enable recombination between distinct PPV species. The most important pre-requisite for a successful recombination is the coinfection of the individual host by different virus strains or species. Consequently, the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology. At present there are no available data on the replication dynamics of PPV in permissive and non permissive hosts and reguarding co-infetions there are no information on the interference mechanisms occurring during the simultaneous replication of viruses of different species. This work has been carried out to set up permissive substrates allowing the replication of different PPV species, in particular keratinocytes monolayers and organotypic skin cultures. Furthermore a method to isolate and expand ovine skin stem cells was has been set up to indeep further aspects of viral cellular tropism during natural infection. The study produced important data to elucidate the replication dynamics of OV and PCPV virus in vitro as well as the mechanisms of interference that can arise during co-infection with different viral species. Moreover, the analysis carried on the genomic right terminal region of PCPV 1303/05 contributed to a better knowledge of the viral genes involved in host interaction and pathogenesis as well as to locate recombination breakpoints and genetic homologies between PPV species. Taken together these data filled several crucial gaps for the study of interspecific recombinations of PPVs which are thought to be important for a better understanding of the viral evolution and to improve the biosafety of antiviral therapy and PPV-based vectors.