Udenafil Induces the Hair Growth Effect of Adipose-Derived Stem Cells

Udenafil, which is a PDE5 inhibitor, is used to treat erectile dysfunction. However, it is unclear whether udenafil induces hair growth via the stimulation of adipose-derived stem cells (ASCs). In this study, we investigated whether udenafil stimulates ASCs and whether increased growth factor secretion from ASCs to facilitate hair growth. We found that subcutaneous injection of udenafil-treated ASCs accelerated telogen-to-anagen transition in vivo. We also observed that udenafil induced proliferation, migration and tube formation of ASCs. It also increased the secretion of growth factors from ASCs, such as interleukin-4 (IL-4) and IL12B, and the phosphorylation of ERK1/2 and NFκB. Furthermore, concomitant upregulation of IL-4 and IL12B mRNA levels was attenuated by ERK inhibitor or NFκB knockdown. Application of IL-4 or IL12B enhanced anagen induction in mice and increased hair follicle length in organ culture. The results indicated that udenafil stimulates ASC motility and increases paracrine growth factor, including cytokine signaling. Udenafil-stimulated secretion of cytokine from ASCs may promote hair growth via the ERK and NFκB pathways. Therefore, udenafil can be used as an ASC-preconditioning agent for hair growth.

Metformin, an Activator of AMPK, Promotes the Growth of Hair Follicles via the AMPK/β-catenin Signaling Pathway / 대한피부과학회지

BACKGROUND: AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that plays a pivotal role in the balance of cellular energy metabolism. Recent studies have reported that AMPK has numerous roles in physiological conditions, and dysregulation of AMPK induces pathological processes and diseases. However, the role of AMPK and its activators have not yet been studied in the context of hair growth regulation. OBJECTIVE: To investigate the effects of metformin on dermal papilla (DP) and outer root sheath (ORS) cells, as well as the role of the AMPK pathway in hair growth. METHODS: We evaluated whether metformin, a well-known AMPK activator, had any beneficial effects on hair growth. In addition, to evaluate the molecular and cellular mechanisms that were involved, protein levels of AMPK and β-catenin were analyzed. RESULTS: Metformin increased the cellular proliferation of human DP and ORS cells. Ki-67 expression was also significantly increased after metformin treatment in the ex vivo hair follicle organ culture. Furthermore, DP and ORS cells treated with metformin had a significant increase in AMPK phosphorylation, which in turn suppressed β-catenin degradation and enhanced its nuclear accumulation. CONCLUSION: We demonstrated that metformin promoted hair growth via the AMPK/β-catenin signaling pathway in vitro with DP and ORS cells. The hair-promoting effects of AMPK activators may potentially be used for the treatment of alopecia, and further investigation will be needed in the future.

Expression Pattern and Role of Klotho in Human Hair Follicles

BACKGROUND: Klotho protein plays a pivotal role in aging regulation. However, it is unclear whether klotho is expressed in human hair follicles and is correlated with hair growth. OBJECTIVE: The purpose of this study was to determine the expression pattern and role of klotho in human hair follicles. METHODS: We examined the klotho expression patterns in human hair follicles from young and aged donors. Furthermore, we examined the functional roles of klotho on human hair growth using klotho siRNA and klotho recombinant protein. RESULTS: Interestingly, klotho was expressed in human hair follicles at both gene and protein levels. In hair follicles, prominent klotho expression was mainly observed in the outermost regions of the outer root sheath and hair bulb matrix cells. Quantification of klotho protein expression in young and aged donors showed that klotho expression decreased with aging. In human hair follicle organ culture, klotho silencing promoted premature catagen induction and inhibited human hair growth. Otherwise, klotho protein prolonged human hair growth. CONCLUSION: These results indicate that klotho might be an important regulatory factor for human hair growth and hair cycle change.

Establishment and application of gastric and gastric cancer organoids / 中华胃肠外科杂志

Gastric organoid is the organotypic cultures of gastric stem cells or pluripotent stem cells. Gastric organoid is comprised of all major types of gastric epithelial cells and represent the architecture and function remarkably similar to those of the gastric epithelium, faithfully recapitulating the functional gastric epithelium ex vivo. As ideal basic experimental model, gastric organoid has advantages over animal models and conventional cell model in many aspects. Gastric organoid derived from human gastric tissue, in particular, allows the investigation of the function of human stomach in the ex vivo setting. It has now been applied in the field of formation and physiology of the stomach, Helicobacter pylori infection-associated diseases, research of the pathogenic gene, screening and development of drugs, and regenerative medicine. What is more, as an innovative pre-clinical cancer model, gastric cancer organoid has provided important insights in the development of gastric cancer and screening of antitumor drugs, such as simulating the occurrence and development of gastric cancer, screening and development of antitumor drugs, personalized medication and targeted therapy for gastric cancer, and combined application with patient-derived xenograft. In this review, we summarize the establishment and application of gastric and gastric cancer organoids, especially in modeling gastric cancer, basic research and drug development.

Mitogen-activated Protein Kinases in the Development of Normal and Diseased Kidneys

Mitogen-activated protein kinases (MAPKs) play important roles in various cellular functions including proliferation, differentiation, and apoptosis. We showed that MAPKs are developmentally regulated in the rat kidney. p38 MAPK (p38) and extracellular signal-regulated kinase (ERK) were strongly expressed in the fetal kidney, whereas c-Jun N-terminal kinase (JNK) was detected predominantly in the adult kidney. The inhibition of p38 or ERK in organ culture resulted in reduced nephron formation with or without reduced kidney size. On the other hand, persistent fetal expression pattern of MAPKs, i.e., upregulation of p38 and ERK and downregulation of JNK, was observed in the cyst epithelium of human renal dysplasia, ovine fetal obstructive uropathy, and pcy mice, a model of polycystic kidney disease. Furthermore, activated p38 and ERK induced by cyclic stretch mediated proliferation and TGF-β1 expression in ureteric bud cells, probably leading to cyst formation and dysplastic changes. Inhibition of ERK slowed the disease progression in pcy mice. Finally, ERK and p38 were inactivated in the early embryonic kidney subjected to maternal nutrient restriction, characterized by reduced ureteric branching and nephron number. Thus, MAPKs mediate the development of normal and diseased kidney. Their modulation may result in novel therapeutic strategies against developmental abnormalities of the kidney.

Role of Arachidonic Acid in Promoting Hair Growth

BACKGROUND: Arachidonic acid (AA) is an omega-6 polyunsaturated fatty acid present in all mammalian cell membranes, and involved in the regulation of many cellular processes, including cell survival, angiogenesis, and mitogenesis. The dermal papilla, composed of specialized fibroblasts located in the bulb of the hair follicle, contributes to the control of hair growth and the hair cycle. OBJECTIVE: This study investigated the effect of AA on hair growth by using in vivo and in vitro models. METHODS: The effect of AA on human dermal papilla cells (hDPCs) and hair shaft elongation was evaluated by MTT assay and hair follicle organ culture, respectively. The expression of various growth and survival factors in hDPCs were investigated by western blot or immunohistochemistry. The ability of AA to induce and prolong anagen phase in C57BL/6 mice was analyzed. RESULTS: AA was found to enhance the viability of hDPCs and promote the expression of several factors responsible for hair growth, including fibroblast growth factor-7 (FGF-7) and FGF-10. Western blotting identified the role of AA in the phosphorylation of various transcription factors (ERK, CREB, and AKT) and increased expression of Bcl-2 in hDPCs. In addition, AA significantly promoted hair shaft elongation, with increased proliferation of matrix keratinocytes, during ex vivo hair follicle culture. It was also found to promote hair growth by induction and prolongation of anagen phase in telogen-stage C57BL/6 mice. CONCLUSION: This study concludes that AA plays a role in promoting hair growth by increasing the expression of growth factors in hDPCs and enhancing follicle proliferation and survival.

Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in Ex vivo Organ Culture / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The development of mechanically active culture systems helps increase the understanding of the role of mechanical stress in intervertebral disc (IVD) degeneration. Motion segment cultures allow for preservation of the native IVD structure, and adjacent vertebral bodies facilitate the application and control of mechanical loads. The purpose of this study was to establish loading and organ culture methods for rabbit IVD motion segments to study the effect of static load on the whole disc organ.</p><p><b>METHODS</b>IVD motion segments were harvested from rabbit lumbar spines and cultured in no-loading 6-well plates (control conditions) or custom-made apparatuses under a constant, compressive load (3 kg, 0.5 MPa) for up to 14 days. Tissue integrity, matrix synthesis, and the matrix gene expression profile were assessed after 3, 7, and 14 days of culturing and compared with those of fresh tissues.</p><p><b>RESULTS</b>The results showed that ex vivo culturing of motion segments preserved tissue integrity under no-loading conditions for 14 days whereas the static load gradually destroyed the morphology after 3 days. Proteoglycan contents were decreased under both conditions, with a more obvious decrease under static load, and proteoglycan gene expression was also downregulated. However, under static load, immunohistochemical staining intensity and collagen Type II alpha 1 (COL2A1) gene expression were significantly enhanced (61.54 ± 5.91, P = 0.035) and upregulated (1.195 ± 0.040, P = 0.000), respectively, compared with those in the controls (P < 0.05). In contrast, under constant compression, these trends were reversed. Our initial results indicated that short-term static load stimulated the synthesis of collagen Type II alpha 1; however, sustained constant compression led to progressive degeneration and specifically to a decreased proteoglycan content.</p><p><b>CONCLUSIONS</b>A loading and organ culture system for ex vivo rabbit IVD motion segments was developed. Using this system, we were able to study the effects of mechanical stimulation on the biology of IVDs, as well as the pathomechanics of IVD degeneration.</p>

Probabilistic graphic models applied to identification of diseases / Modelos probabilísticos gráficos aplicados à identificação de doenças

ABSTRACT Decision-making is fundamental when making diagnosis or choosing treatment. The broad dissemination of computed systems and databases allows systematization of part of decisions through artificial intelligence. In this text, we present basic use of probabilistic graphic models as tools to analyze causality in health conditions. This method has been used to make diagnosis of Alzheimer´s disease, sleep apnea and heart diseases.

RESUMO A tomada de decisões é um aspecto fundamental na conduta de um diagnóstico ou tratamento. A ampla difusão dos sistemas computacionais e dos bancos de dados permite sistematizar, por meio do uso da inteligência artificial, parte dessa tomada de decisão. Neste texto, é apresentada, de modo básico, a possibilidade de uso dos modelos gráficos probabilísticos como ferramenta de análise na causalidade das condições de saúde. Essa metodologia vem sendo utilizada para diagnósticos da doença de Alzheimer, apneia do sono e doenças cardiológicas.

Isolation and culture of renal glomeruli from rats / 生理学报

The aim of the study was to establish a method for isolation and culture of rat renal glomeruli. The renal glomeruli of Sprague Dawley (SD) rats were isolated by a sieving method, and Bowman's capsule was removed by digesting the glomeruli in 0.5% type IV collagenase. The inverted phase contrast microscope was used to observe the structure of isolated renal glomeruli, and trypan blue staining was used to identify the activity of cells in glomeruli. Expressions of nephrin and α-smooth muscle actin (α-SMA) were observed by double-labeling immunofluorencence. Effects of Ang II on reactive oxygen species (ROS) generation were detected by dihydroethidium (DHE) staining. The levels of transforming growth factor-beta 1 (TGF-β1) and collagen IV mRNA were measured by real-time PCR. The results showed that the renal glomeruli with high purity and intact capillary structure were isolated by the modified protocol. The cells in the isolated glomeruli remained alive after 48 h of culture in DMEM. Confocal microscopy observations showed that nephrin and α-SMA were highly expressed in the isolated glomeruli. Treatment of the isolated renal glomeruli with Ang II enhanced ROS production. Furthermore, Ang II increased the mRNA levels of TGF-β1 and collagen IV. In conclusion, we have established a modified method that can isolate glomeruli from rat kidney, and the isolated glomeruli can be used for further observation in cultured condition. The protocol will provide a useful method for preclinical research on kidney diseases.

Influence of 7-dehydrocholesterol reductase gene silencing on the fusion of mouse palatal shelves / 华西口腔医学杂志

<p><b>OBJECTIVE</b>RNA interference was applied to knockdown the Dhcr7 gene in mouse embryonic palatal shelves to facilitate understanding of the function of Dhcr7 gene variants in the fusion of palatal shelves.</p><p><b>METHODS</b>The pAdTrack-CMV-siDhcr7 was constructed using the specific siRNA sequence of Dhcr7 from C57BL/6J mouse. The pAdTrack-CMV- siDhcr7 of positive clones was reconstructed in vitro, and the recombinant adenovirus pAdEasy-1-siDhcr7 of kanamycin resistance was screened. The adenovirus vector DNA was then prepared for transfecting the embryonic palatal shelves. Thirty pairs of embryonic palatal shelves at 13.5 d gestational age were harvested and then randomly divided into the following three groups: normal control group (n = 10), which included palatal shelves inculture medium without cholesterol; blank adenovirus control group (n = 10), which included palatal shelves in culture medium without cholesterol and blank adenovirus; and experimental group (n = 10), which included palatal shelves in culture medium without cholesterol and adenovirus encoding Dhcr7 siRNA. At 48 h after in vitro cultivation, the mRNA and protein of the palatal shelves were obtained for scanning electron microscopy (SEM), reverse transcription polymerase chain reaction (RT-PCR), and Western blot analyses.</p><p><b>RESULTS</b>SEM showed that the palatal shelves of the normal control and blank adenovirus control groups fused and formed continuous palates, whereas those of the experimental group was almost undeveloped but exhibited large gaps between the two palatal shelves. RT-PCR and Western blot analyses showed that the mRNA and protein of Dhcr7 in the experimental group decreased compared with those in the normal control group with a significant difference (P < 0.05).</p><p><b>CONCLUSION</b>Results indicate that Dhcr7 gene silencing affects the fusion of palatal shelves. Thus, Dhcr7 gene may serve a function in the normal development of palates.</p>

Changes of nucleus pulposus after in vitro culture of rabbit whole intervertebral disc and spinal motion segment / 中国骨伤

<p><b>OBJECTIVE</b>To compare the changes of nucleus pulposus after in vitro culture of rabbit whole intervertebral disc and spinal motion segment.</p><p><b>METHODS</b>Twenty-one New Zealand white rabbits which were randomly divided into organ group with 8 rabbits and segment group with 13 rabbits. Fifty intervertebral discs and 50 spinal motion segments were harvested respectively under aseptic conditions from two groups. These specimens were maintained in organ culture with hyperosmotic media (410 mOsm/kg), then 10 discs of the two groups were observed respectively by HE staining, immunohistochemistry of collagen type III, proteoglycan content and cells viability of nucleus pulposus before culture and at 3, 7, 14, 21 days after culture.</p><p><b>RESULTS</b>HE staining showed the intervertebral disc tissue structure remained intact after culture of 21 days organ group and 14 days segment group,but there was severely degenerated of 21 days segment group. The intensity value of type II collagen immunohistochemical staining in the nucleus pulposus were not changed significantly between 21 days organ group and 14 days segment group (P > 0.05), but the staining of segment group at 21 days became shallower, there was significant difference compared with before each time points and organ group at 21 days (P < 0.05). PAS/AB staining of proteoglycan of nucleus pulposus showed that there were not decrease of tinting strength of two groups within 7 days, but the strength weakened slightly of two groups at 14 days, and the tinting strength became weaker at 21 days segment group, the change is more obvious than the organ group. The intensity value of fluorescence staining of nucleus pulposus cells was not changed significantly within 7 days of two groups (P > 0.05), the intensity value decreased slightly at 21 days organ group and 14 days segment group, but there were no significant difference compared with before time points (P > 0.05) however at 21 days segment group the intensity decreased as cells viability of nucleus pulposus decreased,and there was a significant difference compared with before each time points and organ group at 21 days (P < 0.05).</p><p><b>CONCLUSION</b>It is not obviously degenerated of the discs of organ group cultured within 21 days and segment group cultured within 14 days, but there was significant degeneration of the intervertebral disc of segment group after cultured 21 days, so the rabbit spinal motion segment can be used on research about the biomechanics of intervertebral disc as a vitro experimental model within 14 days.</p>

Establishment of a rotary aerobic culture system for in vitro culture of mouse testis / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish an in vitro model of cultured mouse testis using rotary aerobic culture.</p><p><b>METHODS</b>Rotary aerobic incubation with optimized culture conditions was used for in vitro culture of mouse testis, and the morphology of the cultured testicular tissues was compared with that cultured in Transwell chambers. The changes in the testicular tissue structure were examined using HE staining, and the cell proliferation was assessed with BrdU staining. Testosterone concentrations in the culture medium were tested with radioimmunoassay and the expression of the functionally related proteins in the testis was detected using immunohistochemistry.</p><p><b>RESULTS</b>The testicular tissue cultured by optimized rotary aerobic culture presented with more intact histological structure with the size of the testis ranged from 0.3 to 0.8 mm(3). In the two culture systems, the prolifeation index of the spermatogonia increased and that of Sertoli cells decreased with time, and such changes in spermatogonia and Sertoli cell proliferation indices became statistically significant at 3 days (P<0.05) and 5 days (P<0.05) of culture, respectively, as compared with those at 1 day. The concentration of testoerone in the culture media decreased significantly with incubation time (P<0.05). At 3 days of culture, the protein expression of 3β-hydroxysteroid dehydrogenase, cytochrome P450 17α-hydroxylase and cholesterol side-chain cleavage enzyme was detected in Leydig cell cytoplasm and vimentin expression in Sertoli cell cytoplasm.</p><p><b>CONCLUSION</b>An in vitro model of cultured mouse testis has been successfully established using rotary aerobic incubation.</p>

Dexamethasone Induces Apoptosis of Nasal Polyp-Derived Tissue Cultures Through JNK and p38 MAPK Activation

OBJECTIVES: Glucocorticoids, such as dexamethasone (DEX), increase apoptosis in a variety of white cells in nasal polyps and apoptosis is an important factor in the resolution of inflammation. However, the mechanism of glucocorticoids induced apoptosis in nasal polyp remains unclear. In this study the authors evaluated which pathways were engaged in apoptosis induced by DEX in an ex vivo model of nasal polyps. METHODS: Nasal polyp tissues were cultured using an air-liquid interface method. Cultures were maintained in the absence or presence of DEX (10 or 100 microM) for 24 hours. To investigate the involvement of the apoptotic signaling pathways in nasal polyp, such as caspase cascades, Fas-FasL signaling pathway, mitochondrial pathway and p38 mitogen-activated protein kinase (MAPK)/JNK pathway, the authors performed reverse transcription-polymerase chain reaction and Western blotting. RESULTS: The expression ratios of FasL, activated form of caspase-8, caspase-9, and caspase-3 were significantly higher in DEX-treated polyps (P<0.01). In the Bcl-2 family expression, the anti-apoptotic molecules, Bcl-2 and Bcl-XL decreased, but pro-apoptotic molecules, Bax increased, and Bid and Bad were activated. In the conventional MAPKs, JNK, and the phospho-p38 MAPK were significantly higher, but phospho-extracellular signal-regulated kinase (ERK)1/2 was significantly lower in DEX-treated polyps (P<0.01). CONCLUSION: DEX induces apoptosis of nasal polyp via caspase cascades, Fas-FasL signaling pathway, mitochondrial pathway and p38 MAPK/JNK pathway.

Compatibilidad in vitro de un bioplaguicida a base de Lecanicillium lecanii (Hypocreales: Clavicipitaceae) con agroquímicos empleados en los cultivos de algodón y berenjena / In vitro compatibility of the biopesticide based on Lecanicillium lecanii (Hypocreales: Clavicipitaceae) with agrochemicals used in cotton and eggplant crops

El aislamiento colombiano de Lecanicillium lecanii Vl026 formulado como un granulado dispersable WG, ha demostrado una alta eficiencia para el control de Bemisia tabaci en los cultivos de algodón y berenjena. Teniendo en cuenta el potencial de este bioinsumo, el objetivo del siguiente fue determinar la compatibilidad in vitro del bioplaguicida a base de L. lecanii con agroquímicos (insecticidas y fungicidas) que se utilizan con mayor frecuencia en dichos cultivos. La compatibilidad in vitro se estableció determinando el porcentaje de germinación (%) y las Unidades Formadoras de Colonia (UFC) en presencia de los plaguicidas y estimando la concentración inhibitoria 10 (CI10). Cada plaguicida se evaluó a la dosis recomendada, a la mitad y a un cuarto de ésta. Los fungicidas químicos (Benomil®, Vitavax®, Ridomil® y Manzate®) no fueron compatibles con L. lecanii, ya que inhibieron la germinación y las Unidades Formadoras de Colonia del hongo en las tres dosis evaluadas. En cuanto a los insecticidas químicos, el producto Confidor® no inhibió la viabilidad en comparación con el tratamiento control, considerándose compatible con el bioplaguicida. Cuando se evaluó la dosis completa de los demás insecticidas (Oportune®, Actara®, Match® y Malathion®) se obtuvieron germinaciones inferiores al 80%, por lo que dichos productos se clasificaron como no compatibles con el bioplaguicida a base de L. lecanii. El único agroquímico que fue compatible en condiciones in vitro con L. lecanii fue el producto Confidor®. Sin embargo, se recomienda evaluar el efecto in vivo de los productos químicos habitualmente utilizados por los agricultores sobre L. lecanii, con el propósito de desarrollar y establecer estrategias de manejo integrado de la mosca blanca Bemisia tabaci.

A colombian isolate of Lecanicillium lecanii formulated as dispersible granules WG, has shown high efficiency to control Bemisia tabaci in cotton and eggplant crops. Considering that, the objetive of this work was to determine the in vitro compatibility of biopesticide based on L. lecanii with agrochemicals (insecticides and fungicides) that are most frequently used in tobacco and eggplant crops. In vitro compatibility of L. lecanii with agrochemicals was determinated by germination (%) and Colony Forming Units (CFU) in the presence of pesticides and also estimating the inhibitory concentration 10 (IC10). Each agrochemical was evaluated at the recommended dose, a half and a quarter of it. For the three doses tested (Benomyl®, Vitavax®, Ridomil® and Manzate®) were not compatible with L. lecanii, because these inhibited germination and Colony Forming Units of the fungus. Confidor® did not inhibit viability compared to control treatment, and it was considered compatible with the biopesticide. When the recommended dose (Oportune®, Actara®, Match® and Malathion®)was used, the germination of the L. lecanii was lower than 80%, then these products were classified as non-compatible with the biopesticide based on L. lecanii. The only agrochemical that was compatible in vitro with L. lecanii was Confidor®. However, is necesary to evaluate the in vivo effect of agrochemicals commonly used by farmers on L. lecanii, in order to develop and establish integrated management strategies for the control of the whitefly Bemisia tabaci.

Histopathological kidney alterations in rats naturally infected with Leptospira / Alteraciones histopatológicas en ratas naturalmente infectadas con Leptospira

Introduction: Histopathological changes by Leptospira in naturally infected rodent reservoirs have been poorly described. Objective: The aim of the current study is to describe renal histopathology associated with leptospirosis infection of naturally infected rodents captured in the urban area of the city of Medellin, Colombia. Materials and methods: We performed hematoxilin-eosin (H-E) on kidney samples collected from 254 captured rodents. The positive samples were processed by Warthin Starry (W-S) staining and PCR- LipL 32. Results: Fifty one rodent kidneys showed H-E histopathological changes that consisted of inflammatory infiltrate with lympho-plasmocitary cells and histiocytes. We performed W-S staining and PCR- LipL 32 to 67 kidney samples, including the 51 that had shown detectable changes by H-E and 16 (8%) of 203 rodents with negative results. Eight of the samples that tested positive for H-E (15.7%) were also positive for W-S staining. All negative for H-E were also negative for W-S staining. Of the W-S positive samples also tested for culture only three tested positive for both. Additionally, 47 (92.1%) samples positive for H-E were positive for PCR; while eleven of the 16 (68.8%) negative for H-E were positive for PCR. The samples positive for PCR were subsequently tested for culture and 11 (23.4%) were positive. Seven samples were positive for PCR and W-S and three were positive for PCR, W-S and culture. All of the PCR- LipL 32 fragments were sequenced and showed specific amplicons for L. interrogans . Conclusions: The Leptospira infection was confirmed in all of the animals tested. The only histological kidney lesion attributable to leptospiral infection in the reservoir was interstitial nephritis.

Introducción. Los hallazgos histopatológicos ocasionados por Leptospira spp. han sido poco estudiados en poblaciones de roedores naturalmente infectados. Objetivo. Describir la histopatología renal asociada con las infecciones naturalmente adquiridas en un grupo de roedores capturados en el área urbana de Medellín, Colombia. Materiales y métodos. Se llevaron a cabo coloraciones de hematoxilina y eosina de los riñones de 254 roedores recolectados en el área de estudio. Las muestras positivas se procesaron con la coloración de Warthin-Starry y mediante reacción en cadena de la polimerasa (PCR)-LipL32. Results. Se observaron cambios histopatológicos con hematoxilina y eosina en 51 riñones de roedores, que consistieron en infiltrado inflamatorio con linfoplasmocitos e histiocitos. Se utilizó coloración de Warthin-Starry y PCR-LipL32 en 67 muestras de riñón que incluyeron las 51 muestras que tuvieron cambios detectables por hematoxilina y eosina y 16 de 203 (8 %) muestras con resultados negativos. Ocho de las muestras positivas por hematoxilina y eosina (15,7 %) también fueron positivas por la coloración de Warthin-Starry. Las muestras negativas por hematoxilina y eosina (8 %) también fueron negativas con la coloración de Warthin-Starry. Tres de las ocho muestras positivas por esta última, también lo fueron por cultivo. Además, 47 (92,1 %) muestras positivas por hematoxilina y eosina fueron positivas por PCR. Del grupo de 16 negativos por hematoxilina y eosina, 11 (68,8 %) fueron positivos por PCR. De las muestras positivas por PCR, 11 también lo fueron por cultivo (23,4 %). Siete muestras fueron positivas por PCR y Warthin-Starry y tres lo fueron por PCR, Warthin-Starry y cultivo. Todos los fragmentos de la PCR-LipL32 fueron secuenciados y mostraron secuencias específicas de L. interrogans . Conclusiones. Se confirmó la infección por Leptospira y la única lesión presente en el reservorio atribuible fue la nefritis intersticial.

Low doses of celecoxib stimulate human endometrium growth in a three-dimensional culture model

The endometrium plays a pivotal role in implantation and pregnancy. Cyclooxygenase II [COX-2] has an important function in biological processes such as cell proliferation and inflammation. Celecoxib is a selective inhibitor of COX-2 with numerous pharmacologic functions. The aim of present study is to investigate the effects of celecoxib on the human endometrium in a three-dimensional [3D] culture model. In this experimental study, normal human endometria [n=10] obtained from reproductive age women were cut into 1x1 mm sections. Endometrial explants were placed between two layers of fibrin gel. To create the fibrin gel, we poured a thin layer of fibrinogen solution [3 mg/ml in medium 199 [M199]] into each well of a 24-well culture dish and added thrombin enzyme. Endometrial fragments were placed in the center of each well and covered with a second layer of fibrinogen solution. M199 supplemented with L-glutamine, fetal bovine serum [FBS, 5%] and antibiotics were added to each well. The media in each experimental well contained either1, 10 or 50 micro M of celecoxib. At the end of the study, we calculated endometrial tissue growth changes by scoring methods and determined the percentage of angiogenesis. Data were analyzed by the Kruskal-Wallis method. P<0.05 was considered significant. The growth scores were as follows: control [1.37 +/- 0.16], 1 micro M [1.96 +/- 0.28], 10 micro M [2.01 +/- 0.25], and 50 micro M [1.17 +/- 0.14] celecoxib, all of which were significantly different. The angiogenesis percentages were: 25.56 +/- 6.72% [control], 31.98 +/- 6.18% [1 micro M], 42.67 +/- 7.27% [10 micro M] and 23.44 +/- 4.03% [50 micro M], which were not significantly different from each other. Lower celecoxib concentrations had stimulatory effects on the growth of normal endometrium

Endogenous Gastric-Resident Mesenchymal Stem Cells Contribute to Formation of Cancer Stroma and Progression of Gastric Cancer

BACKGROUND: Carcinoma-associated fibroblasts (CAFs) contribute to carcinogenesis and cancer progression, although their origin and role remain unclear. We recently identified and investigated the in situ identity and implications of gastric submucosa-resident mesenchymal stem cells (GS-MSCs) in the progression of gastric carcinogenesis. METHODS: We isolated GS-MSCs from gastric submucosa using hydrogel-supported organ culture and defined their identity. Isolated cells were assessed in vitro by immunophenotype and mesengenic multipotency. Reciprocal interactions between GS-MSCs and gastric cancer cells were evaluated. To determine the role of GS-MSCs, xenografts were constructed of gastric cancer cells admixed with or without GS-MSCs. RESULTS: Isolated cells fulfilled MSCs requirements in regard to plastic adherence, stromal cell immunophenotype, and multipotency. We demonstrated a paracrine loop that gastric cancer cells enhanced the migration, proliferation, and differentiation of GS-MSCs; additionally, GS-MSCs promoted the proliferation of gastric cancer cell in vitro. Xenograft experiments showed that GS-MSCs significantly promoted cancer growth and angiogenesis. GS-MSCs that integrated into gastric cancer became not only CAFs but also rarely endothelial cells which contributed to the formation of cellular and vascular cancer stroma. CONCLUSIONS: Endogenous GS-MSCs play an important role in gastric cancer progression.

A human corneal endothelium equivalent constructed with acellular porcine corneal matrix.

Background & objectives: Artificial corneal endothelium equivalents can not only be used as in vitro model for biomedical research including toxicological screening of drugs and investigation of pathological corneal endothelium conditions, but also as potential sources of grafts for corneal keratoplasty. This study was aimed to demonstrate the feasibility of constructing human corneal endothelium equivalents using human corneal endothelial cells and acellular porcine corneal matrix. Methods: Porcine corneas were decellularized with sodium dodecyl sulphate (SDS) solution. Human corneal endothelial cells B4G12 were cultured with leaching liquid extracted from the acellular porcine corneal matrix, and then cell proliferative ability was evaluated by MTT assay. B4G12 cells were transplanted to a rat corneal endothelial deficiency model to analyze their in vivo bio-safety and pump function, and then seeded and cultured on acellular porcine corneal matrix for two wk. Corneal endothelium equivalents were analyzed using HE staining, trypan blue and alizarin red S co-staining, immunofluorescence and corneal swelling assay. Results: The leaching liquid from acellular porcine corneal matrix had little influence on the proliferation ability of B4G12 cells. Animal transplantation of B4G12 cells showed that these cells had similar function to the native cells without causing a detectable immunological reaction and neoplasm in vivo. These formed a monolayer covering the surface of the acellular porcine corneal matrix. Trypan blue and alizarin red S co-staining showed that B4G12 cells were alive after two wk in organ culture and cell boundaries were clearly delineated. Proper localizations of ZO-1 and Na+/K+ ATPase were detected by immunofluorescence assay. Functional experiments were conducted to show that the Na+/K+ ATPase inhibitor ouabain could block the ionic-pumping function of this protein, leading to persistent swelling of 51.7 per cent as compared to the control. Interpretation & conclusions: Our findings showed that B4G12 cells served as a good model for native corneal endothelial cells in vivo. Corneal endothelium equivalents had properties similar to those of native corneal endothelium and could serve as a good model for in vitro study of human corneal endothelium.

In vitro meniscal culture of juvenile porcine / 中国骨伤

<p><b>OBJECTIVE</b>To observe the histological changes of juvenile porcine menisci in an in vitro organ culture system. It was hoped that the experiment can provide evidence for the study of menisci in an in vitro organ culture model.</p><p><b>METHODS</b>Eight juvenile swines of one month old were involves in the study. Thirty-two meniscal specimens with 8 mm in width were obtained from each meniscus in the two posterior extremities. The specimens were incubated in an in vitro organ culture model. At 0, 2, 4 and 6 weeks after culture, 8 specimens were observed. Hematoxylin eosin staining: the cell density of the inner 1/3 zone was measured. Safranin-O staining:the degree of staining in the inner zone was measured and semi-quantitative scores were obtained.</p><p><b>RESULTS</b>At 0, 2, 4 and 6 weeks, the cell density were (285.3 +/- 12.0)/HPF, (182.8 +/- 11.2)/HPF, (129.7+/- 9.9)/HPF, (92.3 +/- 9.3)/HPF respectively. Statistically significant differences were found between different time points (P < 0.05), Paired comparison showed statistical differences (P < 0.05). At 0, 2, 4 and 6 weeks, the semi-quantitative scores of Safranin-O staining were 2.0 +/- 0.0,1.5 +/- 0.5, 1.0 +/- 0.0, 0.5 +/- 0.5, statistically differences were found between different time points (P < 0.05). Paired comparison showed statistical differences (P < 0.05).</p><p><b>CONCLUSION</b>In the juvenile meniscus in vitro organ culture model, the cell density and viability diminished along with the cultural time. In vitro organ culture model is suitable for the study of menisci in short-term.</p>

Ginkgolide B promotes axonal growth of retina ganglion cells by anti-apoptosis in vitro / 生理学报

One common feature of glaucoma, optic neuritis and some other optic nerve diseases is sustained and irreversible apoptosis of retinal ganglion cells (RGCs). Ginkgolide B is believed to protect neurons in brain and contribute to neurite outgrowth and synapse formation. The aim of the present study was to explore the effects of Ginkgo biloba extract (EGB761) and ginkgolide B on axonal growth of RCGs. Retina explants were cultured in three-dimensional tissue culture system, and the number and length of neurites were analyzed. Immunohistochemistry staining was performed to confirm that the neurite observed was axon of RGCs. TUNEL and activated caspase-3 staining were also applied to observe RGCs apoptosis. The result shows that neurites of RGCs treated with EGB761 or ginkgolide B were more and longer than those in control. The neurite is proved to be the axon of RGCs by immunostaining. Furthermore, compared with control group, RGCs treated with ginkgolide B showed decreased cellular apoptosis and inhibited caspase-3 activation. These results suggest ginkgolide B can promote RGCs axon growth by protecting RGCs against apoptosis.