Expression of autoimmune regulator during differentiation of mouse embryonic stem cells into thymic epithelial progenitor cells / 中国组织工程研究

BACKGROUND: Autoimmune diseases are a class of diseases that cause a strong immune response to the continuous lack of self-tissue-specific antigens in the thymus. Hypothyroidism and unstable expression of tissue-specific antigens in the thymus can limit the therapeutic effect. The thymus is mainly composed of thymic epithelial cells, but the limited number of mature thymic epithelial cells and thymic epithelial progenitor cells in the thymus has greatly limited related research. OBJECTIVE: To detect the expression of autoimmune regulator (AIRE) when mouse embryonic stem cells were transformed into thymic epithelial progenitor cells. METHODS: A two-step differentiation method was used to induce the differentiation of mouse embryonic stem cells into endoderm and then into thymic epithelial progenitor cells. The cells were collected at 0, 3, and 13 days of induced differentiation. Immunofluorescence, flow cytometry, western blot and real-time PCR were used to detect the expression of cell-associated genes and proteins. RESULTS AND CONCLUSION: Positive expression of OCT4 and SSEA1 was detected by immunofluorescence at 0 day of induction. The double positive expression of SOX17 and FoxA2 was measured by immunofluorescence at 3 days of induction. The positive expression of EpCAM, K5 and K8 were analyzed by flow cytometry at 13 days of induction. During the directional differentiation of mouse embryonic stem cells, real-time PCR indicated that the expression of PAX1, PAX9, FOXN1 and PLET1 showed an increasing trend. The expression of AIRE gene increased significantly at 0, 3, and 13 days of induction. At the same time, the expression of INS2 gene and GAD67 gene also increased. Western blot assay showed that the expression of AIRE protein gradually decreased at 0, 3, and 13 days of induction; however, insulin protein and GAD67 protein were not detected. Overall findings indicate that mouse embryonic stem cells can successfully differentiate into thymic epithelial progenitor cells with highly expressed AIRE gene, which promotes the expression of INS2 and GAD67 genes, and provides an evaluation basis for cell transplantation in the treatment of autoimmune diseases.

Melanoma spitzoide simulando lesão vascular - Relato de caso / Spitzoid melanoma simulating vascular lesion - Case report

O melanoma maligno é um tumor de melanócitos responsável por mais de 75% dos óbitos por câncer de pele. As variantes raras desta patologia são responsáveis por 5% dos casos e podem mimetizar outras patologias. Relatamos caso de paciente com melanoma spitzoide e discutimos os achados dermatoscópicos, histopatológicos e estudo imuno-histoquímico, assim como o seguimento desta rara variante de melanoma.

Malignant melanoma is a melanocyte tumor responsible for more than 75% of skin cancer deaths. The rare variants of this pathology are responsible for 5% of the cases and may mimic other pathologies. We report the case of a patient with spitzoid melanoma and we discuss the dermoscopic, histopathological, and immunohistochemical findings, as well as the follow-up of this rare variant of melanoma.

Desmoplastic melanoma: a rare variant with challenging diagnosis

Abstract: Desmoplastic melanoma, a distinct and uncommon variant, is characterized as an invasive lesion with proliferation of fusiform melanocytes in the dermis and subcutaneous tissue, associated with varying patterns of desmoplasia. Neurotropism and neural differentiation may occur. The clinical presentation is variable and nonspecific, easily confused with other fibrous neoplasms. The disease is locally aggressive and shows lower metastasis rates than other types of melanoma. Histopathology may be insufficient, requiring positive immunohistochemistry for S-100 protein and other antigens of melanocytic differentiation. Because desmoplastic melanoma represents a true clinical, dermoscopic, and histopathological diagnostic challenge, a case of invasive desmoplastic melanoma is reported, affecting a photoexposed area in an elderly woman after histological revisions and an initial diagnosis of fibroma.

Identification of IgE and IgG1 specific antigens in Echinococcus granulosus cyst fluid

Cystic echinococcosis (CE) is an anthropozoonotic disease with worldwide distribution and is caused by the cestode Echinococcus granulosus. Anaphylactic shock induced by CE rupture is a serious complication especially in patients with hydatid infections, as the resulting leakage of fluid contains highly toxic endogenous antigen. We aimed to isolate and identify the antigens of specific IgE and IgG1 (sIgE and sIgG1) in E. granulosus cyst fluid (EgCF). Crude antigen for EgCF was prepared from E. granulosus-infected sheep liver. Antigens were separated and identified by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE), two-dimensional gel electrophoresis (2-DE), and immunoblotting. Results of 1D SDS-PAGE and immunoblotting showed that 40.5 kDa protein was the major antigen of sIgE, and 35.5 kDa protein was the major antigen of sIgG1 in EgCF. Results of 2-DE and immunoblotting showed that main antigens of sIgE in EgCF were four proteins with pI values ranging from 6.5 to 9.0 and a molecular weight of 40.5 kDa. Main antigens of sIgG1 in EgCF were five proteins with pI values ranging from 6.5 to 9.0 and a molecular weight of 35.5 kDa. The antigens identified for sIgE and sIgG1 can provide critical insights into cellular and molecular mechanisms underlying anaphylactic shock induced by CE.

El estadio amastigota precede la evolución del epimastigota durante la epimastigogénesis in vitro de Trypanosoma cruzi / The amastigote stadium precedes epimastigote evolution during in vitro Trypasnosoma cruzi epimastigogenesis

La transformación de tripomastigotas sanguíneos de Trypanosoma cruzi en epimastigotas, ocurre naturalmente en el intestino del insecto y se identifica como epimastigogénesis. Aquí reportamos que durante la epimastigogénesis in vitro, los tripomastigotas se transforman en formas redondeadas, biológica y antigénicamente equivalentes al estadio amastigota; con un tiempo de persistencia dependiente de la altura del medio de cultivo. Formas tripomastigotas procedentes de cultivo de tejidos infectados se incubaron en envases con diferentes alturas de medio LITB (3 y 83 mm), y se compararon las cinéticas de transformación hacia epimastigotas. Para la obtención de antígenos se colectaron masas de parásitos a diferentes tiempos de diferenciación. Los cambios morfológicos, incremento del inóculo y resistencia a complemento, se estudiaron por microscopía de contraste de fases y extendidos coloreados con Giemsa. Los cambios antigénicos se analizaron mediante Western Blot usando anticuerpo IgY anti-amastigotas. Los resultados mostraron que las formas redondeadas resisten la lisis por complemento, expresan antígenos amastigota-específicos y la velocidad de transformación hacia epimastigotas depende de la altura del medio sobrenadante en el cultivo. Las evidencias sugieren que además de caída de la temperatura, una baja tensión de oxígeno disuelto y una alta densidad de parásitos por área, aceleran el proceso de diferenciación.

Transformation of blood Trypanosoma cruzi tripomastigotes into epimastigotes occurs naturally at the insect gut and is identified as epimastigogenesis. Here we report that during in vitro epimastigogenesis, tripomastigotes transform into rounded forms, biologically and antigenically equivalent to the amastigote stadium, with a persistance time which depends on the height of the culture medium. Tripomastigote forms from cultures of infected tissues were incubated in flasks with different LITB medium heights (3 and 83 mm), and kinetics of transformation to epimastigotes were compared. Masses of parasites at different differentiation times were collected for antigen production. Morphological changes, inoculum increase and resistance to complement were studied by phase contrast microscopy and Giemsa stained smears. Antigenic changes were analyzed through Western Blot using an IgY anti-amastigote antibody. Results showed that rounded forms resist lysis by complement, express amastigote-specific antigens, and that the speed of transformation to epimastigotes depends on the height of the supernatant medium in the culture. Evidences suggest that apart from a temperature fall, a low dissolved oxygen tension and a high parasite per area density accelerate the differentiation process.

Melanoma desmoplástico / Desmoplastic melanoma

Descrito pela primeira vez em 1971 por Conley et al., o melanoma desmoplásico (MD) representa menos de 4% dos melanomas cutâneos. Trata-se de variante distinta e incomum,que se caracteriza por tumor fibroso de células fusiformes liberadoras de colágeno em matriz fibrosa. Seu diagnóstico é propenso a erro, principalmente por sua semelhança com outras neoplasias fibrosas. Em cerca de 600 casos descritos na literatura, observou-se apresentação clínica não pigmentada, profunda e de aspecto fibroso, associada à lesão precursora. Seu reconhecimento é de grande importância devido ao comportamento de caráter recidivante e à consequente necessidade de abordagem cirúrgica distinta.

First described in 1971 by Conley and colleagues, desmoplastic melanomas represent less than 4% of cutaneous melanomas. It is a distinctive and uncommon variant characterized by a spindle cell fibrous tumor of collagen forming cells, isolated by dense fibrous matrix.Desmoplastic melanomas are frequently misdiagnosed, mainly due to their similarity to other fibrous neoplasms. A review of about 600 reported cases unexpectedly revealed the pigmented clinical presentation of a deep and fibrous nodule that is classically associated with a precursor lesion. The ability to recognise this lesion?s characteristics is very important, due to its recurrent behaviour and subsequent need for a specialized surgical approach.

MAGE Gene Expression in Bronchial Washing Fluid in Suspected Parenchymal Lung Cancer / 결핵및호흡기질환

BACKGROUND: The main goal of this study was to evaluate the diagnostic efficacy of reverse transcription-nested polymerase chain reaction (RT-nested PCR) in bronchial washing fluid with MAGE A1-6 common primers for the detection of lung cancers invisible by bronchoscopy. METHODS: To determine the expression of MAGE A1-6 gene in 189 lung cancers diagnosed by conventional fluoroscopy-guided lung biopsy and 89 cancer-free controls, RT-nested PCR was performed in bronchial washing specimens. We analyzed MAGE A1-6 RT-nested PCR data according to tumor histology, stage, size, and compared them with cytological data. RESULTS: 189 patients (111 cases in adenocarcinoma, 47 cases in squamous cell carcinoma, 22 cases in small cell lung carcinoma, and 9 cases in other cancers) and 89 benign patients were investigated. The expression of MAGE was performed by nested RT-PCR using common MAGE primer. Among 189 cancer patients, the expression rate of MAGE was 49.2%, and the positive predictive value was 89.4%. However, the expression rate of MAGE in patients with benign lesions was 12.4%. In peripheral lung cancer, the positive rate of MAGE expression was 57.4% in squamous cell carcinoma, 44.1% in adenocarcinoma and 59.1% in small cell lung cancer. Whereas the expression rate of bronchial washing cytology in peripheral lung cancer was 9.0% (p=0.011). CONCLUSION: MAGE RT-PCR in bronchial washing fluid gave us promising data for the detection of peripheral lung cancer. It could be a useful method for selecting diagnostic tools for peripheral lesions.

The Levels of Zn2+ and Prostate Specific Antigen in the Semen with Abnormal Liquefaction and Their Relationship with Spermatozoa Motility / 华中科技大学学报(医学版)

Objective To investigate the levels of seminal zinc and prostate specific antigen in abnormal liquefaction sperm and their relationship with spermatozoa motility. Methods Thirty cases of abnormal liquefaction sperm (abnormal group)and 30 cases of normal semen ]iquefaction(normal group)were selected. The semens were analyzed by the CASA,and the seminal plasma was separated and preserved at - 20℃. The levels of seminal zinc were measured by atomic absorption spectrometry. The levels of PSA in seminal plasma were detected by ELISA. Results Zn~(2+) and PSA levels in abnormal group were significantly lower than in normal group(P<0. 05). The levels of seminal Zn~(2+) in abnormal group and normal group were (82. 50±0. 72)and (120. 43±0. 52) fig/ml respectively,with the difference being significant between two group(P<0. 05). The levels of seminal PSA in abnormal group and normal group were (0. 68±0. 14) and (1. 21±0. 21) mg/ml respectively, with the difference being significant between two groups(P<0. 05). Sperm motility in abnormal group was lower than in normal group (P<0. 05). Conclusion The levels of seminal Zn~(2+) and PSA in the semens with abnormal liquefaction is low, which resulting in abnormal liquefaction sperm and inhibiting the sperm motility.

Lymphangiomyomatosis Arising in the Pelvic Cavity: A Case Report

Lymphangioleiomyomatosis (LAM) is a rare disease usually occurring in young women of child-bearing age. It is characterized by a distinctive proliferation of lymphatic smooth muscle cells, especially occurring in the pulmonary parenchyme. The majority of primary LAM occurs in the lung, but there are a few reports of extrapulmonary cases. We report a case of a 21-yr-old female who first complained of low abdominal pain and was referred from a local clinic with the impression of an ovarian cyst. Gynecologic ultrasonography revealed a large posterior pelvic mass with an irregular echogenicity measuring 9.7x4.2 cm in size. Pelviscopy showed a large, thin walled, partly cystic, pelvic mass. The mass was partly removed. Microscopically, the mass was characterized by a haphazard proliferation of smooth muscle cells arranged in fascicular, trabecular, and papillary patterns around a ramifying network of endothelium-lined spaces. The cells were plump or epithelioid with abundant eosinophilic cytoplasm and showed a positive reaction for both alpha-smooth muscle actin and HMB-45 antigen. Surgical and pathological findings were consistent with pelvic retroperitoneal LAM. Despite the numerous treatment attempts, the patient suffered from intractable chylous ascites and developed pulmonary LAM and died due to severe respiratory distress.

Effect of cupric-isonicotinic acid hydrazide complex on normal and avian myeloblastosis virus-infected cells cultivated in vitro.

The cupric complex of isonicotinic acid hydrazide was found to be nontoxic to normal yolk sac macrophages upto a concentration of 100 μΜ. At this concentration the complex did not significantly inhibit DNA, RNA or protein synthesis in these cells. The complex inhibited the avian myeloblastosis virus multiplication in these cells when added 0–4 h post-infection as demonstrated by the inhibition of both focus formation and expression of viral specific antigens. This inhibition was not observed when the complex was added 8 and 16 h after avian myeloblastosis virus infection. The studies carried out on avian myeloblastosis virus-transformed myeloblasts indicated that the complex had no effect on the colony (focus) formation. The results suggest that the complex inhibits the virus multiplication by interfering in an early event of viral growth cycle, possibly the process of reverse transcription.