Walnut ointment promotes full-thickness burning wound healing: role of linoleic acid

Purpose: To investigate the active ingredients of walnut ointment (WO) and its mechanism in repairing wounds. Methods: The ingredients of WO were detected by gas chromatography­mass spectrometry. The effect of linoleic acid (LA) was tested by in vitro Alamar Blue (AB) reagent. Image J software, histological and immunohistochemical analysis were used to confirm the healing effect of LA in the porcine skin model. The animals were euthanized after the experiment by injection of pentobarbital sodium. Results: LA, 24% in WO, promotes keratinocytes and fibroblasts proliferation, which were 50.09% and 15.07% respectively higher than control (p < 0.05). The healing rate of the LA group (96.02% ± 2%, 98.58% ± 0.78%) was higher than the saline group (82.11% ± 3.37%, 88.72% ± 1.73%) at week 3 and week 4 (p < 0.05). The epidermal thickness of the LA was 0.16 ± 0.04 mm greater and the expression of the P63 and CK10 proteins was stronger in the LA group than the control (p < 0.05). Conclusions: LA, which is the main components in WO can promote full-thickness burning wounds (FBWs) by stimulating cell proliferation and differentiation.

Isolation, cultivation and immunofluorescence characterization of lamellar keratinocytes from equine hoof by using explants / Isolamento, cultivo e caracterização por imunofluorescência de queratinócitos lamelares de casco equino utilizando explantes

The importance of the hoof to the horse health is clear, and the current knowledge regarding the cellular aspects of hoof keratinocytes is poor. Studies on equine keratinocyte culture are scarce. Developing keratinocyte cultures in vitro is a condition for studies on molecular biology, cell growth and differentiation. Some methods have already been established, such as those for skin keratinocyte culture. However, few methodologies are found for lamellar keratinocytes. The objective of this study was to standardize the equine hoof keratinocyte isolation and cultivation, and then characterize the cell immunophenotype. For this, the primary culture method used was through explants obtained from three regions of the equine hoof (medial dorsal, dorsal, and lateral dorsal). After the cell isolation and cultivation, the cell culture and its explants were stained with anti-pan cytokeratin (pan-CK) (AE1/AE3), vimentin (V9), p63 (4A4), and Ki-67 (MIB-1) antibodies. Cells were grown to third passage, were positive for pan-CK, p63 and Ki-67, and few cells had vimentin positive expression. As for the explants, the epidermal laminae were not stained for vimentin or Ki-67. However, some cells presented positive pan-CK and p63 expression. This study demonstrated the viability of lamellar explants of equine hooves as a form of isolating keratinocytes in primary cultures, as well as characterized the proliferation ability of such keratinocytes in monolayers.(AU)

É notória a importância do casco na saúde dos equinos, mas o conhecimento em nível celular é pouco entendido. Estudos envolvendo o cultivo de queratinócitos equinos são escassos. Sabe-se que o desenvolvimento de cultivos de queratinócitos in vitro é uma condição para estudos sobre a biologia molecular, crescimento e diferenciação celular. Alguns métodos já estão estabelecidos, como para cultivo de queratinócitos de pele, mas poucas metodologias são encontradas para queratinócitos lamelares. O objetivo desse estudo foi padronizar o cultivo de queratinócitos provenientes de casco equino visando futuramente associar ao estudo da medicina regenerativa para assim estabelecer um modelo experimental in vitro e indicar o uso criterioso de terapias regenerativas para a laminite equina. Desta forma, o cultivo em monocamada e a caracterização de queratinócitos lamelares foram realizados. Para isso, o método de cultura primária utilizado foi através de explantes obtidos de três regiões do casco (dorso-medial, dorsal e dorso-lateral). As células foram caracterizadas para os marcadores anti pan-cytokeratin (AE1/AE3), vimentin (V9), p63 (4A4) e Ki-67 (MIB-1) nos cultivos e nos explantes. As células foram cultivadas até terceira passagem, tendo marcação positiva para pan-CK, p63 e Ki-67 e fraca marcação para vimentina. Já as lâminas epidermais não tiveram marcação de vimentin e Ki-67, porém marcaram acentuadamente para pan-CK e p63. Este estudo demonstrou a exiquibilidade do uso de explantes lamelares do casco de equinos, como forma de isolamento de queratinócitos em cultivos primários, bem como caracterizou a habilidade de proliferação desses queratinócitos em monocamada.(AU)

Isolation, cultivation and immunofluorescence characterization of lamellar keratinocytes from equine hoof by using explants / Isolamento, cultivo e caracterização por imunofluorescência de queratinócitos lamelares de casco equino utilizando explantes

The importance of the hoof to the horse health is clear, and the current knowledge regarding the cellular aspects of hoof keratinocytes is poor. Studies on equine keratinocyte culture are scarce. Developing keratinocyte cultures in vitro is a condition for studies on molecular biology, cell growth and differentiation. Some methods have already been established, such as those for skin keratinocyte culture. However, few methodologies are found for lamellar keratinocytes. The objective of this study was to standardize the equine hoof keratinocyte isolation and cultivation, and then characterize the cell immunophenotype. For this, the primary culture method used was through explants obtained from three regions of the equine hoof (medial dorsal, dorsal, and lateral dorsal). After the cell isolation and cultivation, the cell culture and its explants were stained with anti-pan cytokeratin (pan-CK) (AE1/AE3), vimentin (V9), p63 (4A4), and Ki-67 (MIB-1) antibodies. Cells were grown to third passage, were positive for pan-CK, p63 and Ki-67, and few cells had vimentin positive expression. As for the explants, the epidermal laminae were not stained for vimentin or Ki-67. However, some cells presented positive pan-CK and p63 expression. This study demonstrated the viability of lamellar explants of equine hooves as a form of isolating keratinocytes in primary cultures, as well as characterized the proliferation ability of such keratinocytes in monolayers.(AU)

É notória a importância do casco na saúde dos equinos, mas o conhecimento em nível celular é pouco entendido. Estudos envolvendo o cultivo de queratinócitos equinos são escassos. Sabe-se que o desenvolvimento de cultivos de queratinócitos in vitro é uma condição para estudos sobre a biologia molecular, crescimento e diferenciação celular. Alguns métodos já estão estabelecidos, como para cultivo de queratinócitos de pele, mas poucas metodologias são encontradas para queratinócitos lamelares. O objetivo desse estudo foi padronizar o cultivo de queratinócitos provenientes de casco equino visando futuramente associar ao estudo da medicina regenerativa para assim estabelecer um modelo experimental in vitro e indicar o uso criterioso de terapias regenerativas para a laminite equina. Desta forma, o cultivo em monocamada e a caracterização de queratinócitos lamelares foram realizados. Para isso, o método de cultura primária utilizado foi através de explantes obtidos de três regiões do casco (dorso-medial, dorsal e dorso-lateral). As células foram caracterizadas para os marcadores anti pan-cytokeratin (AE1/AE3), vimentin (V9), p63 (4A4) e Ki-67 (MIB-1) nos cultivos e nos explantes. As células foram cultivadas até terceira passagem, tendo marcação positiva para pan-CK, p63 e Ki-67 e fraca marcação para vimentina. Já as lâminas epidermais não tiveram marcação de vimentin e Ki-67, porém marcaram acentuadamente para pan-CK e p63. Este estudo demonstrou a exiquibilidade do uso de explantes lamelares do casco de equinos, como forma de isolamento de queratinócitos em cultivos primários, bem como caracterizou a habilidade de proliferação desses queratinócitos em monocamada.(AU)

Squamous cell carcinoma in chinese hamsters (Cricetulus griseus)

Background: The companion animal market has changed over the years. The number of people living in small apartments has increased; as a result, the demand for small pets such as exotics, fish, and small rodents has also increased due to their smaller space requirements and ease of handling and care. Pets help relieve anxiety and stress in people suffering from social issues. Small rodents are usually bred in specific cages with cellulose or wood shaving bedding, and fed with commercially available diets. Small rodent clinics struggle due to the lack of scientific reports on some diseases and therapies. To date, the oncology literature is too limited to develop better diagnosis and treatment methods. Here, we report three cases of squamous cell carcinoma in the mandibular region of Chinese hamsters (Cricetulus griseus).Case: Three adult male hamsters averaging 1.5 years old, from different pet stores, bred under home conditions by different owners in Sergipe, Brazil, were brought to the Dr. Vicente Borelli Hospital at Pio X University for exotic veterinary care. Each animal had been bred alone in a specific breeding cage. Each had a history of apathy, loss of appetite, and rapid deformity of the facial region. Radiographs showed areas of bone involvement and extensive injury, with partial resorption of the left ramus and angle of the mandibular region. Due to the location of the tumor mass, the clinical status, and limitations in systemic treatment, euthanasia was recommended for each animal. After anamnesis, the animals were subjected to clinical assessment. A firm and well-circumscribed mass was identified on palpation. In case A, it compromised the left mandible from the angle to the body and extended to the maxillary soft tissues and left superior lips. In case B, it extended from the ramus to the symphysis on the left side and to the maxillary region, similar to case A.[...]

Squamous cell carcinoma in chinese hamsters (Cricetulus griseus)

Background: The companion animal market has changed over the years. The number of people living in small apartments has increased; as a result, the demand for small pets such as exotics, fish, and small rodents has also increased due to their smaller space requirements and ease of handling and care. Pets help relieve anxiety and stress in people suffering from social issues. Small rodents are usually bred in specific cages with cellulose or wood shaving bedding, and fed with commercially available diets. Small rodent clinics struggle due to the lack of scientific reports on some diseases and therapies. To date, the oncology literature is too limited to develop better diagnosis and treatment methods. Here, we report three cases of squamous cell carcinoma in the mandibular region of Chinese hamsters (Cricetulus griseus).Case: Three adult male hamsters averaging 1.5 years old, from different pet stores, bred under home conditions by different owners in Sergipe, Brazil, were brought to the Dr. Vicente Borelli Hospital at Pio X University for exotic veterinary care. Each animal had been bred alone in a specific breeding cage. Each had a history of apathy, loss of appetite, and rapid deformity of the facial region. Radiographs showed areas of bone involvement and extensive injury, with partial resorption of the left ramus and angle of the mandibular region. Due to the location of the tumor mass, the clinical status, and limitations in systemic treatment, euthanasia was recommended for each animal. After anamnesis, the animals were subjected to clinical assessment. A firm and well-circumscribed mass was identified on palpation. In case A, it compromised the left mandible from the angle to the body and extended to the maxillary soft tissues and left superior lips. In case B, it extended from the ramus to the symphysis on the left side and to the maxillary region, similar to case A.[...](AU)

Aldefluor protocol to sort keratinocytes stem cells from skin

Purpose: To investigate the use Aldefluor® and N, N - Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients. Methods: Activated Aldefluor® aliquots were prepared and maintained at temperature between 2 to 8°C, or stored at -20°C. Next, the cells were collected following the standard protocol of sample preparation. Results: Best results were obtained with Aldefluor® 1.5µl and DEAB 15 µl for 1 x 106 cells, incubated at 37°C for 15 minutes. Flow cytometer range for keratinocyte stem cells separation was evaluated. There were 14.8% of stem cells separated in one sample of keratinocyte culture used to pattern the protocol. After being defined the ideal concentration, the same test pattern was performed in other keratinocyte samples. We observed a final mean of 10.8%. Conclusion: Aldefluor® has been shown as a favorable marking of epidermal keratinocyte stem cells for subsequent separation on a flow cytometer, with detection of 10.8% of epidermal keratinocyte stem cells, in this protocol.(AU)

Use of Staphylococcus aureus phage Lysate Staphage Lysate (SPL)® for the control of recurrent pyoderma eczema in dogs with atopic dermatitis

Background: Recurrent staphylococcal infections are frequent in dogs with atopic dermatitis (AD). Many factors seem to contribute to making bacterial pyoderma refractory to treatment. Short-term systemic antibiotic therapy is effective for the treatment of acute symptoms, and may, along with pulsatile therapy, contribute to the long-term control of the disease. However, microbial resistance has become a growing and alarming problem. The aim of this study was to evaluate whether the use of Staphylococcus aureus Phage Lysate Staphage Lysate (SPL)®, can minimize the symptoms of recurrent pyoderma and increase the interval between acute atopic manifestations in dogs. Materials, Methods & Results: Thirteen dogs with a history of Canine Atopic Dematitis (CAD) and recurrent bacterial pyoderma received SPL at increasing intervals for 23 weeks. The contents of an intact pustule of each dog was collected and submitted to microbiological analysis. Systemic antibiotic therapy was established for the first 4-6 weeks of SPL protocol, based on the antibiotic sensitivity tests. The animals included in the study underwent a therapeutic protocol receiving shots of 0.5 mL of SPL subcutaneously (SC) twice a week for the first 12 weeks; 1.0 mL of SPL (SC) once a week for four weeks; 1.0 mL of SPL (SC) once every 15 days; 1.0 mL of SPL (SC) after a three-week interval from the last dose on week [...](AU)

Use of Staphylococcus aureus phage Lysate Staphage Lysate (SPL)® for the control of recurrent pyoderma eczema in dogs with atopic dermatitis

Background: Recurrent staphylococcal infections are frequent in dogs with atopic dermatitis (AD). Many factors seem to contribute to making bacterial pyoderma refractory to treatment. Short-term systemic antibiotic therapy is effective for the treatment of acute symptoms, and may, along with pulsatile therapy, contribute to the long-term control of the disease. However, microbial resistance has become a growing and alarming problem. The aim of this study was to evaluate whether the use of Staphylococcus aureus Phage Lysate Staphage Lysate (SPL)®, can minimize the symptoms of recurrent pyoderma and increase the interval between acute atopic manifestations in dogs. Materials, Methods & Results: Thirteen dogs with a history of Canine Atopic Dematitis (CAD) and recurrent bacterial pyoderma received SPL at increasing intervals for 23 weeks. The contents of an intact pustule of each dog was collected and submitted to microbiological analysis. Systemic antibiotic therapy was established for the first 4-6 weeks of SPL protocol, based on the antibiotic sensitivity tests. The animals included in the study underwent a therapeutic protocol receiving shots of 0.5 mL of SPL subcutaneously (SC) twice a week for the first 12 weeks; 1.0 mL of SPL (SC) once a week for four weeks; 1.0 mL of SPL (SC) once every 15 days; 1.0 mL of SPL (SC) after a three-week interval from the last dose on week [...]

Keratinocyte growth factor and the expression of wound-healing-related genes in primary human keratinocytes from burn patients

PURPOSE:To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients.METHODS:Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC.RESULTS:Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes.CONCLUSION:There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.(AU)

Toll like receptors gene expression of human keratinocytes cultured of severe burn injury

To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human Primary Epidermal keratinocytes of patients with severe burns. 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 TLR pathways PCR Arrays plates (SA Biosciences). After the analysis of gene expression we found that 21% of these genes were differentially expressed, of which 100% were repressed or hyporegulated. Among these, the following genes (fold decrease): HSPA1A (-58), HRAS (-36), MAP2K3 (-23), TOLLIP (-23), RELA (-18), FOS (-16), and TLR1 (-6.0). This study contributes to the understanding of the molecular mechanisms related to TLR pathways and 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.(AU)

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

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. 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). 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). 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.(AU)

Human beta defensin-4 and keratinocyte growth factor gene expression in cultured keratinocyte and fibroblasts of burned patients

To evaluate KGF and human beta defensin-4 (HBD-4) levels produced by dermic fibroblasts and keratinocytes cultivated from burned patients' skin samples. Keratinocytes and fibroblasts of 10 patients (four major burns, four minor burns and two controls) were primarily cultivated according to standard methods. HBD-4 and KGF genes were analyzed by quantitative PCR. In fibroblasts, KGF gene expression was 220±80 and 33.33±6.67 (M±SD; N=4), respectively for major and minor burn groups. In keratinocytes, KGF gene expression was 11.2±1.9 and 3.45±0.37 (M±SD; N=4), respectively for major and minor burn groups. In fibroblasts, HBD-4 gene expression was 15.0±4.0 and 11.5±0.5 (M±SD; N=4), respectively for major and minor burn. In keratinocyte, HBD-4 gene expression was 0.0±0.0 and 13.4±4.8 (M±SD; N=4), respectively for major and minor burn. KGF expression was increased in burn patient fibroblasts compared to control group. In keratinocytes culture, KGF suppression is inversely proportional to burn extension; it is active and increased in major burn but decreased in minor burn. HBD-4 expression was increased in fibroblasts and decreased in keratinocytes from all burned patients.(AU)

Keratinocyte growth factor, tumor necrosis factor-alpha and interleukin-1 beta gene expression in cultured fibroblasts and keratinocytes from burned patients

PURPOSE: To evaluate the gene expression of KGF, TNF-alpha and IL-1 beta in skin fibroblasts and keratinocytes cultured from burned patients. METHODS: Three patients with large burns and three patients with small burns, as well as two controls, were included. The cell culture was initiated by the enzymatic method. After extraction and purification of mRNA, qPCR was used to assess the gene expression of KGF, TNF-alpha and IL-1 beta. RESULTS: The expression of KGF was increased on average 220-fold in large burns and 33.33-fold in small burns in fibroblasts, and 11.2-fold in large burns and 3.45-fold in small burns in keratinocytes compared to healthy patients (p<0.05). Expression of TNF-alpha was not observed. IL-1 beta is down-regulated in fibroblasts of burned patients, and much more repressed in small burns (687-fold, p<0.05). In keratinocytes, the repression of IL-1 beta expression occurs in patients with small burns (28-fold), while patients with large burns express this gene intensively (15-fold). CONCLUSIONS: The study showed a quantitative pattern in the expression of KGF gene, which is more expressed according to the size of the burn. TNF-alpha was not expressed. A qualitative pattern in the expression of IL-1 beta gene was demonstrated.(AU)

PADRONIZAÇÃO DA CULTURA DE QUERATINÓCITOS LAMELARES DE CASCO EQUINO

Os queratinócitos são células presentes na epiderme, constituída pelo epitélio estratificado pavimentoso queratinizado. Estas células organizam-se em camadas de diferentes estágios de maturação celular (camada basal ou germinativa, espinhosa, granulosa, lúcida e córnea) e tem a função de revestimento desses tecidos. Diversos estudos buscam elucidar e caracterizar tecidos hígidos de origem epitelial, como pele, olhos e casco, para que possam identificar os mecanismos de desenvolvimento de enfermidades nesses tecidos. O casco equino é formado por epiderme em diferentes estágios de queratinização, subdivididos em estrato externo ou parede do casco, estrato médio e estrato interno, nesse último estão localizadas as lâminas epidermais primárias (LEP) e secundárias (LES) onde os queratinócitos estão presentes junto a membrana basal (transição de epiderme e derme, junção que faz a adesão do casco com a falange distal). É notória a importância do casco na saúde dos equinos, mas o conhecimento em nível celular é pouco entendido. Estudos envolvendo o cultivo de queratinócitos equinos são escassos. Sabe-se que o desenvolvimento de cultivos de queratinócitos in vitro é uma condição para estudos sobre a biologia molecular, crescimento e diferenciação celular. Alguns métodos já estão estabelecidos, como para cultivo de queratinócitos de pele, mas poucas metodologias são encontradas para queratinócitos lamelares. O objetivo desse estudo, foi padronizar o cultivo de queratinócitos provenientes de casco equino, visando futuramente associar ao estudo da medicina regenerativa para assim estabelecer um modelo experimental in vitro e indicar o uso criterioso de terapias regenerativas para a laminite equina. Desta forma, foi realizado o cultivo em monocamada e caracterização de queratinócitos lamelares. Para isso, o método de cultivo primário utilizado foi através de explantes obtidos de três regiões do casco (dorso-medial, dorsal e dorso-lateral). As células foram caracterizadas para os marcadores anti pan-cytokeratin (AE1/AE3), vimentin (V9), p63 (4A4) e Ki-67 (MIB-1) nos cultivos e nos explantes. As células foram cultivadas até terceira passagem, tendo marcação positiva para pan-CK, p63 e Ki-67 e fraca marcação para vimentina. Já as lâminas epidermais não tiveram marcação de vimentin e Ki-67, porém marcaram acentuadamente para pan-CK e p63. Este estudo demonstrou a exiquibilidade do uso de explantes lamelares do casco de equinos, como forma de isolamento de queratinócitos em cultivos primários, bem como caracterizou a habilidade de proliferação desses queratinócitos em monocamada.

The keratinocytes are cells presented in the epidermis, constituted by the stratified keratinized squamous epithelium. These cells organized in layers of different stages of cell maturation (basal or germinative, prickly, granular, lucid and corneal layers) and have a tissue coating function. Several studies seek to elucidate and characterize healthy tissues of epithelial origin, such as skin, eyes and hoof, so that they can identify the mechanisms of development of diseases in these tissues. The equine hoof is composed of epidermis at different stages of keratinization, subdivided into external stratum, middle stratum and internal stratum, in which the primary epidermal lamellae (PEL) and secondary epidermal (SEL) are located and where keratinocytes are present along the basement membrane (Transition of the epidermis and dermis, which joins the hoof to the distal phalanx). It is evident the importance of the hoof health of horses, but the knowledge at the cellular level is poorly understood. Studies involving the culture of equine keratinocytes are scarce. The development of keratinocyte cultures in vitro is a condition for studies on molecular biology, cell growth and differentiation. Some methods are already established, such as for culture of skin keratinocytes, but few methodologies are found for lamellar keratinocytes. The aim of this study was to standardize the cultivation of keratinocytes from equine hoof, aiming future associate with the study of regenerative medicine so as to establish an experimental in vitro model and indicate the wise use of regenerative therapies for equine laminitis. In this way, monolayer culture and characterization of lamellar keratinocytes were performed. For this, the method of primary culture used was through explants obtained from three regions of the hoof (dorso-medial, dorsal and dorso-lateral). Cells were characterized for anti pan-cytokeratin (AE1 / AE3), vimentin (V9), p63 (4A4) and Ki-67 (MIB-1) markers in cultures and explants. Cells were grown to third passage, having positive marking for pan-CK, p63 and Ki-67 and poor labeling for vimentin. Already the epidermal laminae were not labeling for vimentin and Ki-67, but pan-CK and p63 had sharp labeling. This study demonstrated the feasibility of using lamellar explants in equine hoofs as a form of isolating keratinocytes in primary cultures, as well as characterizing the proliferation ability of these keratinocytes in monolayers. ¨¨

Human T-Lymphotropic virus (HTLV) type I in vivo integration in oral keratinocytes

Although the infection of HTLV-1 to cell components of the mouth have been previously reported, there was not until this report, a detailed study to show the characteristics of such infection. From 14 Tropical Spastic Paraparesis/ HTLV-1-Associated Myelopathy (HAM/TSP) patients and 11 asymptomatic carrier individuals (AC) coming from HTLV-1 endemic areas of southwest Pacific of Colombia, infected oral mucosa cells were primary cultured during five days. These cell cultures were immunophenotyped by dual color fluorescence cell assortment using different lymphocyte CD markers and also were immunohistochemically processed using a polyclonal anti-keratin antibody. Five days old primary cultures were characterized as oral keratinocytes, whose phenotype was CD3- /CD4-/CD8-/CD19-/CD14-/CD45-/A575-keratin+. From DNA extracted of primary cultures LTR, pol, env and tax HTLV-1 proviral DNA regions were differentially amplified by PCR showing proviral integration. Using poly A+ RNA obtained of these primary cultures, we amplify by RT-PCR cDNA of tax and pol in 57.14% (8/14) HAM/TSP patients and 27.28% (3/11) AC. Tax and pol poly A+ RNA were expressed only in those sIgA positive subjects. Our results showed that proviral integration and viral gene expression in oral keratinocytes are associated with a HTLV-1 specific local mucosal immune response only in those HTLV-1 infected individuals with detectable levels of sIgA in their oral fluids. Altogether the results gave strong evidence that oral mucosa infection would be parte of the systemic spreading of HTLV-1 infection.

Keratinocyte growth factor protected cultured human keratinocytes exposed to oxidative stress / Fator de crescimento de queratinócitos protegeu queratinócitos humanos cultivados expostos ao estresse oxidativo

PURPOSE: To evaluate effects of oxidative stress and supplementation of keratinocyte growth factor (KGF) on cultivated human keratinocytes. METHODS: Oxidative stress was produced through addition of hydrogen peroxide (H2O2) to the culture medium. Cultivated human keratinocytes were divided in 4 groups: Group control (G C), Group KGF (G KGF), Group H2O2 (G H2O2), Group H2O2 and KGF (G H2O2-KGF). Each experiment was accomplished with the same lineage cultivated keratinocytes, in triplicate. Cell viability was evaluated by trypan blue exclusion assay. RESULTS: The results showed that the culture medium supplemented with KGF presented a small rate of cell viability when compared to cells only in culture medium (p<0,001). It demonstrated that only the growth factor does not have protector effects for cells in vitro. However, in front of the oxidative stress produced by addition of hydrogen peroxide to the medium, KGF showed a beneficial effect, protecting cells when compared to the group that suffered hydrogen peroxide action but had not been exposed to KGF (p<0,001). CONCLUSION: KGF determined protection to the primary human keratinocytes exposed to oxidative stress.(AU)

OBJETIVO: Avaliar os efeitos do estresse oxidativo e da suplementação do fator de crescimento de queratinócitos (KGF) em queratinócitos humanos cultivados. MÉTODOS: O estresse oxidativo foi produzido através da adição de peróxido de hidrogênio (H2O2) ao meio de cultura. Os queratinócitos humanos cultivados foram divididos em quatro grupos: grupo controle (G C), grupo KGF (G KGF), grupo H2O2 (G H2O2), grupo H2O2 e KGF (G H2O2-KGF). Cada experimento foi realizado com a mesma linhagem celular, em triplicata. A viabilidade celular foi avaliada pelo ensaio da exclusão do azul de tripan. RESULTADOS: Os resultados mostraram que o meio de cultura suplementado com o KGF apresentou menor taxa de viabilidade celular quando comparado às células do grupo controle (p<0,001). Isso mostra que somente o fator de crescimento de queratinócitos não apresentou efeito protetor às células em cultura. Entretanto, frente ao estresse oxidativo produzido pela adição do peróxido de hidrogênio ao meio de cultura, o KGF mostrou efeito benéfico, protegendo as células quando comparado ao grupo que sofreu a ação do estresse oxidativo, mas que não foi exposta ao KGF (p<0,001). CONCLUSÃO: O KGF determinou a proteção aos queratinócitos humanos primários cultivados expostos ao estresse oxidativo.(AU)

Ultrastructural evaluation of human keratinocyte growth and differentiation on a fibrin substrate / Avaliação ultraestrutural do crescimento e da diferenciação de queratinócitos sobre um substrato de fibrina

PURPOSE: In order to circumvent several difficulties that have been met in the routine use of the in vitro keratinocyte cultures using the standard procedure described by Rheinwald and Green, and obtain a more resilient and the least possible immunogeneic skin substitute for a future clinical application, this work studied a new keratinocyte culture system, which envisages the utilization of a fibrin substrate in association with high densities of human keratinocytes. METHODS: Through light and transmission electron microscopy and immunohistochemical assays, long-term proliferative and differentiative characteristics of keratinocytes cultured onto a fibrin gel under immerse and air-liquid interface culture conditions were evaluated. RESULTS: Despite the absence of a dermal substitute, the results demonstrated that the proposed composite was constituted of a transparent and elastic fibrin film covered by a well-attached, multistratified epithelium with morphological characteristics that resemble human epidermis, including the neoformation, albeit incomplete, of the basement membrane. CONCLUSIONS: Increased mechanical resistance due to the presence of an easy handling substrate, the delivery of nonclonfluent keratinocytes as well as the removal of animal-derived cells from the culture system suggest its potential use for future transplantation purposes.(AU)

OBJETIVO: Com o intuito de contornar diversas dificuldades encontradas no uso rotineiro de queratinócitos cultivados in vitro pela técnica descrita por Rheinwald e Green, e obter um substituto cutâneo mais resistente e o menos imunogênico possível para futuras aplicações clínicas, este trabalho avaliou um novo sistema de cultura de queratinócitos que prevê a utilização de um substrato de fibrina em associação com queratinócitos humanos em alta densidade. MÉTODOS: Através de microscopia óptica e eletrônica e análise imunohistoquímica, foram avaliadas as características proliferativas e de diferenciação em longo prazo de queratinócitos cultivados em condição imersa e na interface ar-líquido. RESULTADOS: Apesar da ausência de um substituto dérmico, foi demonstrado que o composto proposto constituiu-se de um substrato de fibrina transparente e elástico coberto por epitélio multi-estratificado, bem aderido, com características morfológicas semelhantes à epiderme humana, incluindo a neo-formação, embora incompleta, da membrana basal. CONCLUSÕES: A maior resistência mecânica com a presença de um substrato de fácil manuseio, a possível liberação de queratinócitos não-confluentes, e a remoção de células com origem animal dos sistemas de cultura sugerem que o composto proposto neste estudo apresenta grande potencial para uso clínico futuro.(AU)

Piodermite bacteriana em cães com dermatite atópica - revisão de literatura / Bacterial pyoderma in dogs with atopic dermatitis- literature review

As infecções estafilocócicas de repetição são frequentes em cães com dermatite atópica. Vários fatores comoa diminuição da função da barreira epidérmica, diminuição da produção dos peptídeos antimicrobianos emaior colonização e aderência das bactérias aos queratinócitos parecem colaborar para tornar as piodermitesbacterianas refratárias aos tratamentos. A antibioticoterapia sistêmica em curto prazo é eficaz para o tratamentodas crises e esta em regime pulsátil pode colaborar para o controle em longo prazo. Bacterinas estafilocócicastambém têm sido estudadas. Objetiva-se neste trabalho reunir as principais informações sobre a piodermite bacteriana secundária a dermatite atópica, através de revisão de literatura. (AU)

Recurrent staphylococcal infections are frequent in dogs with atopic dermatitis. Several factors such decreaseof epidermal barrier function, decrease production of antimicrobial peptides and increased colonization and adherence of bacteria to keratinocytes seem to collaborate to make bacterial pyoderma refractory to treatment. Systemic antibiotic therapy in short-term is effective for the treatment of crisis and that, under pulsatile therapy, might contribute to the long-term control. Staphylococcal bacterins have also been studied. The aim of this work is to gather key information about the bacterial pyoderma secondary to atopicdermatitis, through literature review. (AU)

Piodermite bacteriana em cães com dermatite atópica - revisão de literatura / Bacterial pyoderma in dogs with atopic dermatitis- literature review

As infecções estafilocócicas de repetição são frequentes em cães com dermatite atópica. Vários fatores comoa diminuição da função da barreira epidérmica, diminuição da produção dos peptídeos antimicrobianos emaior colonização e aderência das bactérias aos queratinócitos parecem colaborar para tornar as piodermitesbacterianas refratárias aos tratamentos. A antibioticoterapia sistêmica em curto prazo é eficaz para o tratamentodas crises e esta em regime pulsátil pode colaborar para o controle em longo prazo. Bacterinas estafilocócicastambém têm sido estudadas. Objetiva-se neste trabalho reunir as principais informações sobre a piodermite bacteriana secundária a dermatite atópica, através de revisão de literatura.

Recurrent staphylococcal infections are frequent in dogs with atopic dermatitis. Several factors such decreaseof epidermal barrier function, decrease production of antimicrobial peptides and increased colonization and adherence of bacteria to keratinocytes seem to collaborate to make bacterial pyoderma refractory to treatment. Systemic antibiotic therapy in short-term is effective for the treatment of crisis and that, under pulsatile therapy, might contribute to the long-term control. Staphylococcal bacterins have also been studied. The aim of this work is to gather key information about the bacterial pyoderma secondary to atopicdermatitis, through literature review.

Experimental model of cultured skin graft

One of the most used animal models of cultured keratinocytes autografting is based on xenografting of human keratinocytes to the rat or athymic mice, immunological neutral recipient that acts as biological carrier. It could be studied in this model many facts that occur after transplant without the ethical aspect in the clinical study. The proposition of the experimental model is related to the sequence of the total or partial skin transplant, as autografting or xenografting, cultured or not, to the back of athymic mice. The model presents the possibility of study in vivo athymic animal, when the in vivo study in anima nobili is not ethical. It permits the xenografting evaluation of cultured cells graft or of the genetically modified cells and of the association of the cultured cells and the dermal substitutes, the composite grafts, and of the autografting.

Um dos modelos animais mais utilizados de auto-enxertia de queratinócitos cultivados é baseado em xeno-enxerto de queratinócitos humanos em rato atímico, um receptor imunologicamente neutro que atua como carreador biológico. Muitos fatos podem ser estudados nesse modelo que acontecem após o transplante sem os aspectos éticos do estudo clínico. A proposição do modelo experimental esta relacionada a sequência do transplante de pele parcial ou total como auto-enxerto ou xeno-enxerto, cultivado ou não, no dorso do rato atímico. O modelo apresenta a possibilidade do estudo in vivo do animal atímico, quando o estudo in vivo em anima nobili não é considerado ético. Isso permite a avaliação do xeno-enxerto de células humanas normais ou modificadas geneticamente modificadas cultivadas e a associação de células cultivadas e substitutes dérmicos, de enxertos compostos e de auto-enxerto.