Megacariopoyesis humana in vitro: determinación de la concentración óptima de trombopoyetina / In vitro human megacaryocytopoiesis: determination of thrombopoietin optimal concentration / Megacariópoiesis humana in vitro: determinação da concentração ótima da trombopoietina

El estudio de la megacariopoyesis humana se ha visto obstaculizado por la relativa escasez de megacariocitos en la médula ósea (0,05-0,2 % de las células medulares), lo que ha llevado a la optimización de protocolos de expansión in vitro a partir de precursores de diversos orígenes (cordón umbilical, médula ósea y sangre periférica con o sin movilización previa). Los cultivos celulares a partir de precursores han permitido la producción y el estudio tanto de megacariocitos así como de proplaquetas y plaquetas Sin embargo, la producción in vitro óptima de megacariocitos que culminen todos los estadios de diferenciación es un reto aún no resuelto. En este trabajo reportamos los hallazgos concernientes a la determinación de las condiciones y concentraciones de trombopoyetina para lograr una óptima relación entre la cantidad de trombopoyetina empleada y el porcentaje y grado de diferenciación megacariocítica en muestras obtenidas de cinco donantes alogénicos aceptados para trasplante de médula ósea.

The study of human megakaryocytopoiesis has been hampered by the relative scarcity of megakaryocytes in bone marrow (0.05-0.2 % of medullary cells), which has led to the optimization of protocols of in vitro expansion of precursors from diverse sources (umbilical cord, bone marrow and peripheral blood with or without previous mobilization). Cell cultures from different precursors have allowed the production and study of megakaryocytes as well as proplatelets and platelets. However, the in vitro production of megakaryocytes that culminate all stages of differentiation is a challenge that has not yet been resolved. In this work we report the findings related to the determination of thrombopoietin treatment conditions and concentrations to achieve an optimal relationship between the amount of thrombopoietin and the percentage and degree of megakaryocytic differentiation in five allogeneic donors that were accepted for bone marrow transplantation.

O estudo da megacariopoiese humana tem sido dificultado pela relativa escassez de megacariócitos na medula óssea (0,05-0,2 % das células medulares), o que levou à otimização dos protocolos de expansão in vitro a partir de precursores de diversas origens (cordão umbilical, medula óssea e sangue periférico com ou sem mobilização prévia). Culturas de células a partir de precursores permitiram a produção e o estudo tanto de megacariócitos e de proplaquetas e plaquetas. No entanto, a produção ótima in vitro de megacariócitos que culminam em todas as fases de diferenciação é um desafio ainda não resolvido. Neste trabalho, relatamos as descobertas relativas à determinação das condições e concentrações de trombopoietina para obter uma relação ótima entre a quantidade de trombopoietina usada e a taxa e o grau de diferenciação megacariocítica em amostras obtidas de cinco doadores alogênicos aceitos para transplante de medula óssea.

Estimation on Formation Time of Thrombus / 法医学杂志

OBJECTIVES@#To observe the changes of the formation time of venous thrombus in rats, and to provide new ideas and methods for the estimation on thrombus formation time of the forensic cases died from thrombosis.@*METHODS@#Totally 80 rats were randomly divided into 10 groups （0 h, 3 h, 6 h, 12 h, 1 d, 3 d, 1 week, 2 weeks, 3 weeks and 4 weeks after operation）. A vein thrombosis model was established by the "narrow" method. The processes of thrombosis, organization, recanalization and the features of change on hemosiderin and calcium salt were observed by HE stain, Perls stain and Von Kossa stain. The expression changes of CD61, α-SMA and CD34 were observed by immunohistochemical staining technique.@*RESULTS@#Platelets adhered to the exposed blood vessel intima 3 h after operation, and platelet trabeculae were formed by the repeated accumulation of platelets 1 d after operation. The thrombus organization formed through the fibroblasts from vessel wall that grew into the interior of the thrombus 3 d after operation. Endothelial cells covered the surface of thrombus and then the new blood vessels were reformed, and the vessels were reconstructed. The expression of CD61 upregulated at the stages of the thrombus formation （3 h） and thrombus reformation （4 weeks）, and reached the peak 1 d after thrombus formation. The release of hemosiderin and the initial expression of α-SMA were detected 3 d later. Calcium deposit and expression of CD34 were observed 1 week later.@*CONCLUSIONS@#The hemosiderin, calcium salt, CD61, α-SMA and CD34 show time-dependent changing characteristics, which is expected to provide a reference for the estimation on thrombus formation time of the forensic cases died from thrombosis.

Analysis of various potential prognostic markers and survival data in clear cell renal cell carcinoma

ABSTRACT Purpose Clear cell renal cell cancers frequently harbor Von Hippel-Lindau gene mutations, leading to stabilization of the hypoxia-inducible factors (HIFs) and their target genes. In this study, we investigated the relationship between vascular endothelial growth factor (VEGF), HIF-1α, HIF-2α, p53 positivity, microvessel density, and Ki-67 rates with prognostic histopathologic factors (Fuhrman nuclear grade, stage, and sarcomatoid differentiation) and survival in clear cell renal cell carcinomas. Material and Methods Seventy-two nephrectomy specimens diagnosed as clear cell renal cell carcinoma between 2000 and 2012 were reevaluated. Immunohistochemically VEGF, HIF-1α, HIF-2α, p53, CD34 (for microvessel density evaluation), and Ki-67 antibodies were applied to the tumor areas. The relationships of these antibodies with prognostic factors and survival rates were evaluated with statistical analyses. Results Mean survival time was 105.6 months in patients with ccRCC. Patients with high expression of VEGF, HIF-1α and HIF-2α positivity, a high Ki-67 proliferation index, and a high microvessel density evaluation score had a shorter survival time (p<0.05). Conclusions Our findings supported that with the use of these immunohistochemical markers, prognosis of renal cell carcinoma may be predicted at the first step of patient management. New treatment modalities targeted to HIF-1α and HIF-2α might be planned as well as VEGF-targeted therapies in the management of clear cell renal cell carcinomas.

Pleomorphic Hyalinizing Angiectatic Tumor: Immunohistochemical Study with Review of Literature.

Pleomorphic hyalinizing angiectatic tumor (PHAT) is relatively a new entity that was described first in 1996. Although it is often confused with neurilemmoma and low grade malignant fibrous histiocytoma, it is considered to be a distinctive neoplasm because of certain unique microscopic and immunohistochemical findings. It is a low grade, slowly growing tumor that recurs after surgical removal in about a third of all cases. It is not known to metastasize. In this review, we have immunohistochemically analyzed a case of gluteal soft tissue tumor in a 22-year-old woman using CD 34, CD 68, CD 99, S100 and desmin. Classical histological features and positivity for only CD 34 helped in establishing the diagnosis as PHAT. We have reviewed the available literature.

Fetal hematopoietic stem cells express MFG-E8 during mouse embryogenesis

The milk fat globule-EGF-factor 8 protein (MFG-E8) has been identified in various tissues, where it has an important role in intercellular interactions, cellular migration, and neovascularization. Previous studies showed that MFG-E8 is expressed in different cell types under normal and pathophysiological conditions, but its expression in hematopoietic stem cells (HSCs) during hematopoiesis has not been reported. In the present study, we investigated MFG-E8 expression in multiple hematopoietic tissues at different stages of mouse embryogenesis. Using immunohistochemistry, we showed that MFG-E8 was specifically expressed in CD34+ HSCs at all hematopoietic sites, including the yolk sac, aorta-gonad-mesonephros region, placenta and fetal liver, during embryogenesis. Fluorescence-activated cell sorting and polymerase chain reaction analyses demonstrated that CD34+ cells, purified from the fetal liver, expressed additional HSC markers, c-Kit and Sca-1, and that these CD34+ cells, but not CD34- cells, highly expressed MFG-E8. We also found that MFG-E8 was not expressed in HSCs in adult mouse bone marrow, and that its expression was confined to F4/80+ macrophages. Together, this study demonstrates, for the first time, that MFG-8 is expressed in fetal HSC populations, and that MFG-E8 may have a role in embryonic hematopoiesis.

Hepatic solitary fibrous tumor: Report of a rare case.

Solitary fibrous tumor (SFT) of the liver is an extremely rare neoplasm of mesenchymal origin. In the English literature, less than 40 cases of SFTs of the liver have been reported. The present case concerns a 34-year-old female who presented to us with complaint of dyspepsia. On examination, there was hepatomegaly. On ultrasound examination, an SOL in the liver was detected. Large tumor measuring 14.5 × 10 × 8 cm was resected. Microscopic evaluation of the tumor showed a well-circumscribed, low to moderately cellular tumor demonstrating spindle- and fibroblast-like cells within the collagenous stroma. Immunohistochemistry revealed diffuse strong cytoplasmic immunopositivity of CD34, Bcl2, and vimentin. A diagnosis of a benign SFT was given. The patient remained well 4 years after surgery. SFT is a rare mesenchymal neoplasm that occasionally involves the liver in adult patients. Most SFTs are benign, but some may have malignant histological features. With less than 40 reported cases in the literature, little can be said regarding its natural history or the benefits of adjuvant radio chemotherapy. Complete surgical resection remains the cornerstone of its treatment.

Immunohistochemical study of osteopontin, Ki-67, and CD34 of psoriasis in Mansoura, Egypt.

Background and Objective: Psoriasis is a chronic inflammatory skin disease characterized by hyper-proliferation, abnormal differentiation, and inflammatory infiltration in epidermis and dermis. We planned this study to analyze probable associations between Osteopontin (OPN), Ki-67, CD34, and histopathological features in psoriasis. Materials and Methods: We studied OPN expression and its correlation with Ki-67 and CD34 expression in lesional, non-lesional skin, and normal skin. Immunoreactivity for OPN and Ki-67 was based on the level of epidermal staining. CD34 expression was scored as mild, moderate, and strong, according to the number of stained dermal capillaries. Results: Our results showed statistically significant differences in the expression of OPN, Ki-67, and CD34 between lesional and non-lesional skin as well as between non-lesional skin and control group (P≤0.001). In addition, there was a significant difference in the expression of OPN, Ki-67, and CD34 between control and lesional group (P=0.02, P=0.02, and P=0.04, respectively). Conclusions: OPN expression seems to be related to Ki-67 (proliferation index) and CD34 expression (angiogenesis marker) confirming its role in the pathogenesis of psoriasis. Then "anti- OPN and anti-angiogenesis" may eventually become a useful therapeutic approach in psoriasis.

Primary glomus tumor of the liver: A rare case report and review of the literature.

Glomus tumor is a rare neoplasm of the distal extremities. It occurs very rarely in the deep visceral organs such as stomach, lung, pancreas, ovary, and liver. Herein, we report a very rare case of glomus tumor of the liver in a 50-year-old woman presenting with abdominal mass which was diagnosed after surgery. The tumor was large and cystic; however, the morphology was similar to the ordinary soft-tissue glomus tumor. Tumor cells were reactive with CD34 and SMA. The patient's follow up failed to show any evidence of malignant behavior or tumor recurrence. This report is the third primary glomus tumor of the liver in the English literature.

Angiogenesis in liver cirrhosis and hepatocellular carcinoma.

BACKGROUND: Angiogenesis has been well documented in hepatocellular carcinoma (HCC). As liver cirrhosis is considered preneoplastic condition, the aim of this study was to evaluate the process of angiogenesis using CD 34 as an endothelial cell marker in normal liver, cirrhosis and HCC. MATERIALS AND METHODS: A total of 111 cases were included in this study, which consisted of 30 cases each of normal liver and cirrhosis that were all autopsy cases. Twenty-one cases of HCC included 10 autopsy specimens, nine surgically resected specimens and two liver biopsies. Remaining were 30 cases of metastasis to the liver, which included 20 autopsy specimens, one surgically resected specimen and nine liver biopsies. The patients were between the age range from 17 to 80 years with 70 males and 11 females. Paraffin-embedded liver sections of all these cases were stained routinely by hematoxylin-eosin stain, while immunohistochemistry for CD 34 was performed for expression of endothelial cells. The positivity of CD 34 staining was evaluated by counting in 10 high-power field, grading was done from 0 to 4 and compared between normal liver, cirrhosis and HCC and metastasis. RESULTS: CD 34 was positive in 16/30 (53.3%) cases of cirrhosis, 18/21 (85%) cases of HCC and 26 (86.6%) of metastasis to the liver. None of the normal liver showed any positivity. Grade 3 to 4 positivity was seen in 4/16 (25%) and 13/18 (72%) cases of cirrhosis and HCC, respectively. Amongst these, 10 were moderately differentiated, one well differentiated and rest two were fibrolamellar and sarcomatoid variants of HCC. CONCLUSION: Over expression of endothelial cell marker CD 34 with gradual progression was found from normal liver to cirrhosis to HCC and metastasis. Understanding of this process of angiogenesis might help in the design of efficient and safe antiangiogenic therapy for these liver disorders.

Myofibroblastoma of breast.

Soft tissue neoplasms of breast thought to be derived from Myofibroblasts have been classified as myofibroblastomas. Myofibroblasts are spindle shaped mesenchymal cells derived from fibroblasts. These are rare neoplasms (more often seen in the male breast) and have same structural and immunohistochemical characteristics as those of solitary fibrous tumors. We present a case of an adult female presenting as firm breast lump diagnosed as myofibroblastoma and confirmed by diffuse positivity of CD34 immunohistochemical stain.

A study of proliferative activity, angiogenesis and nuclear grading in renal cell carcinoma.

To evaluate the role of proliferative marker, proliferating cell nuclear antigen (PCNA) and microvessel density (MVD) as prognostic markers in renal cell carcinoma (RCC) and to see their relationship with the clinical stage and nuclear grades, we studied 30 cases of RCC for nuclear grading (Fuhrman's nuclear grade), MVD (using anti CD-34 antibody), and PCNA labeling index (using anti-PCNA antibody) over a period of 2.5 years. Staging was assessed by peroperative and radiologic findings. The area of highest MVD within the tumor was selected for microvessel count (MVC) per high-power field (0.1885 mm 2 area). PCNA labeling index was determined by counting percentage of positively stained tumor cell nuclei. PCNA labeling index above 60% was taken as high PCNA index and up to 60% was considered low. There was significant positive correlation between PCNA labeling index with both nuclear grade and clinical stage using Spearman's correlation coefficient. No association was noted between MVC with PCNA, nuclear grade, and clinical stages. Evaluation of proliferative status of RCC is a useful adjunct as a prognostic parameter as it is seen to correlate well with both clinical stage and nuclear grade. In our study, MVD was not seen to correlate with either of these.

Benign Schwannoma of the Liver: A Case Report

A primary benign schwannoma of the liver is extremely rare. Only nine cases have been reported in the medical literature worldwide and no case has been reported in Korea previously. A 36-yr-old woman was admitted to our hospital with vague epigastric pain. The ultrasound and computed tomography scan revealed a multiseptated cystic mass in the right lobe of the liver. The mass was resected; it was found to be a 5x4x2 cm mass filled with reddish yellow fluid. The histological examination confirmed the diagnosis of a benign schwannoma, proven by positive immunoreaction with the neurogenic marker S-100 protein and a negative response to CD34, CD117 and smooth muscle actin. This is the first report of a benign schwannoma of the liver parenchyma in a Korean patient.

Prostatic stromal proliferation of uncertain malignant potential (PSPUMP): a case report.

Proliferative lesions of specialized prostatic stroma are rare with only few isolated case reports documented in literature. These lesions are histologically characterized by exuberant proliferation of prostatic stroma with variable number of normal and hyperplastic glands. Recently this group of tumors have been labeled as prostatic stromal proliferations of uncertain malignant potential (PSPUMP). We report a case of PSPUMP in a 72 years old male who underwent radical prostatectomy for a mass in pelvis. Based on histological appearance and immunohistochemistry, a diagnosis ofPSPUMP was made.

Gastrointestinal stromal tumors: a demographic, morphologic and immunohistochemical study.

BACKGROUND AND AIM: There is limited published information on gastrointestinal stromal tumors (GIST) in the South Asian region. This study was conducted to describe the demographic characteristics, organ distribution and frequency of risk categories in cases of GIST referred to a tertiary hospital. METHODS: Data pertaining to 37 cases of gastrointestinal stromal tumors received at the histopathology section of the Aga Khan University Hospital between December 2004 and July 2005 were analyzed. Immunohistochemical stains including vimentin, CD34, CD117 (c-kit), ASMA, desmin and S-100 were performed. RESULTS: Of 37 tumors, 24 (64.9%) were from males. The mean age of the patients was 50.0 years (95% CI 45.3-54.6). Tumors were categorized as high risk (27 cases; 69.2%), intermediate risk (4 cases; 10.3%) and low risk (3 cases; 7.7%). The most common site of involvement was the stomach (29.7%), followed by small bowel (24.3%), mesentery (10.8%), pancreas (8.1%), rectum (2.7%) and retroperitoneum (2.7%). In 21.6% of cases, the site of origin was not specified. The mean age at diagnosis was 50.9 years (95% CI 45.5 - 56.3) in the high risk and 44.8 years (95% CI 28.6 - 60.9) in the intermediate risk category. CONCLUSIONS: Cases of GIST referred to us were more frequently from men, most commonly from stomach or small bowel, mostly in the high risk category, and presented a decade earlier than in other reported series.

A Case of Primary Hepatic Epithelioid Hemangioendothelioma Mimicking Metastatic Carcinoma / 대한소화기학회지

Epithelioid hemangioendothelioma is a rare vascular origin tumor which usually occurs in soft tissues, liver, and lung. It usually affects adult women and presents as multiple hepatic nodules with mainly peripheral distribution. It is difficult to diagnose and treat because of non-specific clinical manifestations and findings on the imaging study. Moreover, pathological misdiagnosis is common. We report a case of this rare tumor that was detected incidentally. Final diagnosis was based on histological evidence. A 52-years old man suffered from right upper quadrant abdominal pain for 3 months, and was initially misdiagnosed as a metastatic carcinoma. Physical examination revealed superior cervical lymphadenopathy with mild hepatomegaly. Finally, hepatic epithelioid hemangioendothelioma was diagnosed on the basis of positive immunohistochemical staining for factor VIII, CD34, and VEGF. Our case highlights the importance of a histological diagnosis to avoid misdiagnosis.