C-kit Staining Research Articles

Identification and protective role of CD34+ stromal cells/telocytes in experimental autoimmune encephalomyelitis (EAE) mouse spleen.

Experimental autoimmune encephalomyelitis (EAE) is a classical animal model of human multiple sclerosis (MS) that is most commonly used to study the neuropathology and therapeutic effects of the disease. Telocytes (TCs) are a specialized type of interstitial or mesenchymal cell first identified by Popescu in various tissues and organs. However, the existence, distribution and role of CD34+ stromal cells (SCs)/TCs in the EAE-induced mouse spleen remain to be elucidated. We conducted immunohistochemistry, immunofluorescence (double staining for CD34 and c-kit, vimentin, F4/80, CD163, Nanog, Sca-1, CD31 or tryptase) and transmission electron microscopy experiments to investigate the existence, distribution and role of CD34+ SCs/TCs in the EAE-induced mouse spleen. Interestingly, immunohistochemistry, double-immunofluorescence, and transmission electron microscopy results revealed that CD34+ SCs/TCs were significantly upregulated in the EAE mouse spleen. Immunohistochemical or double-immunofluorescence staining of CD34+ SCs/TCs showed positive expression for CD34, c-kit, vimentin, CD34/vimentin, c-kit/vimentin and CD34/c-kit, and negative expression for CD31 and tryptase. Transmission electron microscopy (TEM) results demonstrated that CD34+ SCs/TCs established close connections with lymphocytes, reticular cells, macrophages, endothelial cells and erythrocytes. Furthermore, we also found that M1 (F4/80) or M2 (CD163) macrophages, and haematopoietic, pluripotent stem cells were markedly increased in EAE mice. Our results suggest that CD34+ SCs/TCs are abundant and may play a contributing role in modulating the immune response, recruiting macrophages and proliferation of haematopoietic and pluripotent stem cells following injury to promote tissue repair and regeneration in EAE mouse spleens. This suggests that their transplantation combined with stem cells might represent a promising therapeutic target for the treatment and prevention of multiple autoimmune and chronic inflammatory disorders.

Repair mechanism of radiation-induced salivary gland injury by hypoxia-pretreated human urine-derived stem cell exosomes.

To explore the protective effect of human urine-derived stem cell exosomes (hUSC-Exos) on radiation-induced salivary gland (SG) injuries in Sprague Dawley rats. Fresh adult urine was collected, and primary hUSCs were isolated and identified. The hUSCs were hypoxia-pretreated with 1% oxygen for 24 h and then transferred to a normoxic culture environment for 24 h. The hUSC-Exos were collected and identified for exosomes. A radiation-induced injury model was established in the rats, and exosomes were introduced by local injection in the SG and tail vein. The submandibular gland was excised for morphological observation 1 week later. Immunohistochemical detection of the glandular tissue was conducted by α-smooth muscle actin (a-SMA), stem cell growth factor receptor (c-Kit) staining, and periodic acid-Schiff staining. Qualitative polymerase chain reaction and western blot analysis were adopted to detect the gene and protein expression of Wnt3a, GSK3β, and Axin. In both the normoxic and hypoxic hUSC-Exo groups, microvesicular structures with bilayer membranes of approximately 80 nm in diameter were detected, and the expressions of CD9 and CD63 were detected by nanoflow cytometry. Compared with the control group, in the radiation-induced injury model group, the expression of a-SMA was significantly higher, the expression of c-Kit was significantly lower, and the expressions of Wnt3a, GSK3β, and Axin were significantly upregulated; the differences were statistically significant (p < 0.05). Compared with the model group, in the normoxic and hypoxic hUSC-Exo groups, the expression of a-SMA was significantly decreased, the expression of c-Kit was significantly increased, and the expressions of Wnt3a, GSK3β, and Axin were significantly upregulated; the differences were statistically significant (p < 0.05). Hypoxia-pretreated hUSC-Exos could repair radiation-induced SG injuries by activating the Wnt3a/GSK3β pathway to suppress the expressions of a-SMA and c-Kit.

Gastric Spindle Cell Neoplasms With Mast Cells - A Pitfall In Diagnosis Of Gastric Biopsies.

Abstract Introduction/Objective Undifferentiated sarcoma, previously known as malignant fibrous histiocytoma (MFH), is a very rare primary neoplasm of stomach. It usually arises from soft tissue of extremities with an incidence of 1- 2/100,000, typically presenting in the 5th-7th decades of life with a slight male predominance. We report a case of gastric undifferentiated sarcoma which was interpreted as gastrointestinal stromal tumor (GIST) on initial biopsy. Methods/Case Report A 84 year old female presented to the Danbury Hospital emergency department with hematemesis and melena. Upper gastrointestinal endoscopy revealed a pedunculated mass in the cardia of stomach with ulceration, followed by a computed tomographic scan of chest and abdomen revealing a 2.9 cm mass along the proximal lesser curvature of the stomach. An endoscopic biopsy revealed a spindle cell neoplasm with immunohistochemical staining for c-kit consistent with GIST. She later underwent partial gastrectomy. Histopathology of the resected gastric tumor showed tumor cells arranged in sheets and storiform pattern, exhibiting spindled to polygonal morphology with marked nuclear pleomorphism, high mitotic activity (20 mitoses per 10 high power fields) and Touton type giant cells. Immunohistochemical stains demonstrated absence of staining for c-kit, DOG-1, CD34, actin, desmin, S100, cytokeratin AE1/AE3, CD3, CD5, CD20, CD21, CD15, CD30, ALK1, myeloperoxidase, CD1a and LCA. Numerous mast cells within the tumor stained for c-kit. Molecular testing for c-kit and PDGFRA mutations were negative. A final diagnosis of undifferentiated sarcoma with features of myxofibrosarcoma was rendered. Results (if a Case Study enter NA) NA. Conclusion Undifferentiated sarcomas are high grade malignant mesenchymal neoplasms without clear cellular differentiation or immunohistochemical marker. Only 4% of the total cases of undifferentiated sarcoma have been reported to be arising primarily from the gastrointestinal tract. They carry a poor prognosis with a 5 year survival of 47%. The main pitfall in our case was the positive uptake of c-kit stain by mast cells which led to the diagnosis of GIST in first gastric biopsy. We want to stress the importance of entertaining this diagnosis in the differential when encountered with a malignant gastric spindle cell neoplasm along with careful interpretation of c-kit stains in small biopsy specimens as the treatment and prognosis differs greatly from GIST.

Comparison of immunohistochemical characteristics of endometriomas with non-endometriotic benign ovarian cysts.

The aim of the study was to investigate the existence of neuroendocrine cells and to compare the density of those in normal ovarian tissue, endometriotic and non-endometriotic benign ovarian cysts. Twenty patients with the diagnosis of endometrioma and 30 control subjects consisting of ovarian serous cystadenoma (n=10), ovarian mucinous cystadenoma (n=10) and normal ovarian tissue (n=10) were included. The tissues were prepared and assessed according to staining density by using the H-score method. Tissues with mucinous cystadenoma were significantly more stained with PAS and VanGieson, when compared to women with endometrioma. Macrophage deposition was higher in cyst samples with endometrioma and in normal ovarian tissue when compared to serous cystadenoma and mucinous cystadenoma. Normal ovarian tissue was significantly more stained with PGP9.5, NSE and SYN when compared to endometrioma and non-endometriotic benign ovarian cyst. PGP9.5 staining was higher in normal ovarian tissue when compared with endometriotic lesions (p<.001). Endometrioma samples were significantly more stained with p53 when compared to non-endometriotic cysts and normal ovarian tissue. c-Kit staining was mild and not statistically significant among all groups. During endometrioma transformation, expression intensity of neuroendocrine markers decreases compared to normal ovarian tissue and other benign ovarian cysts.

Eosinophil and Mast Cell Counts in the Stomach and Duodenum of Patients with Functional Dyspepsia without a Helicobacter pylori infection.

Symptom-based subtyping of functional dyspepsia (FD) is used to segregate patients into groups with homogenous pathophysiological mechanisms. This study examined whether subtyping could reflect the duodenal and gastric microinflammation in FD patients. Twenty-one FD patients without Helicobacter pylori infection were recruited. An endoscopic biopsy was performed in the duodenum 2nd portion, stomach antrum, and body. The eosinophil and mast cell counts per high-power field (×40) were investigated by H&E and c-kit staining, respectively. The degree of inflammatory cell infiltration, atrophy, and intestinal metaplasia was also determined by H&E staining in the stomach. The baseline characteristics and eosinophil and mast cell infiltrations were compared among the three groups (epigastric pain syndrome, postprandial distress syndrome, and overlap). According to the symptom assessment, seven subjects were classified into the epigastric pain syndrome group, 10 into the postprandial syndrome group, and four into the overlap group. The baseline variables were similar in the three groups. Eosinophil infiltration was more prominent in the duodenum than in the stomach. In contrast, mast cell infiltration was similar in the duodenum and stomach. The eosinophil counts in the duodenum were similar in the three groups. The eosinophil counts in the stomach and mast cell counts in the duodenum and stomach were also similar in the three groups. Duodenal eosinophil infiltration was prominent in FD patients, but the eosinophil counts were similar regardless of the symptom-based subtypes of FD. Hence, the current symptom-based subtyping of FD does not reflect duodenal eosinophil and mast cell infiltration.

Role of telocytes in the pathogenesis of ectopic pregnancy.

The aim of this study is to investigate the effect of telocytes on tubal motility in ectopic pregnancies. This study included patients with ectopic pregnancy (EP) (n=10) and control patients (n=10) (partial salpingectomy for contraception). Immunohistochemical staining for c-Kit, vimentin, CD34 and S100A was performed to quantify telocytes in the mucosa, muscular layer and serosa of fallopian tubes of control and EP group. Spontaneous and KCl- (80 mM) induced contraction and cumulative progesterone dose-relaxation (10-11-10-5 M) and cumulative oxytocin dose-contraction (10-10-10-4 M) responses were recorded. The groups were comparable in terms of age, gravida, parity, delivery type and gestational week (p>0.05). The homogenous distribution of telocytes in the mucosa and muscular layers of the control group, changed to heterogeneous localization the EP group. Immunohistochemical staining with vimentin, S100A, c-Kit and CD34, revealed increased telocyte counts in the muscular layer and serosa of the tubal tissues of EP. The frequency of the spontaneous contractions was higher in the control group (p<0.001); contrarily, the amplitude of the contractions was higher in ectopic pregnancies (p<0.001). Although the cumulative oxytocin dose-contraction curves were similar at all concentrations (p>0.05), the cumulative progesterone dose-relaxation curves exhibited higher relaxation response in the EP group at all concentrations (p<0.001). Increased telocyte count in the fallopian tube may decrease tubal motility and may affect the transfer of the blastocyst to the uterus and possibly contribute to the pathogenesis of EP.

S3189 Not Just a GIST: A Rare Case of Gastric Leiomyosarcoma

Introduction: Leiomyosarcoma (LMS) is a malignant neoplasm showing smooth muscle differentiation. Before the recognition of gastrointestinal stromal tumors (GISTs), leiomyoarcomas were commonly reported in the gastrointestinal tract due to misclassification. Now, these tumors are considered rare with only 16 cases of gastric LMS reported in the post-GIST era. We present the 17th case of primary gastric LMS. Case Description/Methods: An 81F with a history of GERD,CHF and Afib presented for evaluation of syncope. She had also suffered with increased fatigue, poor appetite, 30lb unintentional weight loss, and a “gnawing” post-prandial epigastric pain. Her last EGD was 4 years prior which showed non bleeding erosive gastropathy and a non obstructing Schatzki’s ring. Biopsies showed chronic gastritis without evidence of dysplasia or H. Pylori. On admission, pertinent labs included Hg of 11.5 g/dL, MCV 99.7 fL, WBC 11.02 k/uL, and albumin 2.8 g/dL. She underwent an EGD which showed a large, infiltrative, transmural, ulcerated, non-circumferential mass in the greater curvature of the stomach. EUS showed a hypoechoic lesion which appeared to extend to the cardia by extrinsic involvement of the gastric wall. Biopsies showed an ulcerated spindle cell neoplasm which stained negative for C-kit and DOG1, but were strongly positive for desmin. This conformed the diagnosis of gastric leiomyosarcoma. Discussion: Before the development of immunohistochemical staining for C-kit, many GISTs were misdiagnosed as LMSs and leiomyoblastomas. Now these tumors are considered rare with only 16 prior case reports. The diagnosis mainly relies on pathologic examination with SMA, desmin, and h-caldesmon are positive in the majority ( >70%) of LMSs. CD117 (KIT), DOG 1 and CD34 are negative. It is important to rule out Carney tried, which is a type of multiple endocrine neoplasia which encompasses LMSs, pulmonary chondroma, and extra-adrenal paragangliomas. Complete for LMS surgical resection with negative margins remains the gold standard of treatment. It is important to note that not every spindled lesion is a GIST, as diagnosis will change management. As opposed to GISTs, LMSs are not likely to respond to Imatinib. Due to concern for splenic invasion, neoadjuvant chemotherapy was pursued in our patient with Ifosfamide and doxorubicin prior to surgical resection. Due to her other comorbidities, she was felt to be too high risk for surgery and she eventually chose to pursue hospice.Figure 1.: Figure 1 1a: Ulcer in the incisura of the stomach 1b: Ulcer status post hemostatic clip and argon plasma coagulation (APC).

Telocytes in the human ascending aorta: Characterization and exosome-related KLF-4/VEGF-A expression.

Telocytes (TCs), a novel interstitial cell entity promoting tissue regeneration, have been described in various tissues. Their role in inter‐cellular signalling and tissue remodelling has been reported in almost all human tissues. This study hypothesizes that TC also contributes to tissue remodelling and regeneration of the human thoracic aorta (HTA). The understanding of tissue homeostasis and regenerative potential of the HTA is of high clinical interest as it plays a crucial role in pathogenesis from aortic dilatation to lethal dissection. Therefore, we obtained twenty‐five aortic specimens of heart donors during transplantation. The presence of TCs was detected in different layers of aortic tissue and characterized by immunofluorescence and transmission electron microscopy. Further, we cultivated and isolated TCs in highly differentiated form identified by positive staining for CD34 and c‐kit. Aortic‐derived TC was characterized by the expression of PDGFR‐α, PDGFR‐β, CD29/integrin β‐1 and αSMA and the stem cell markers Nanog and KLF‐4. Moreover, TC exosomes were isolated and characterized for soluble angiogenic factors by Western blot. CD34+/c‐kit+ TCs shed exosomes containing the soluble factors VEGF‐A, KLF‐4 and PDGF‐A. In summary, TC occurs in the aortic wall. Correspondingly, exosomes, derived from aortic TCs, contain vasculogenesis‐relevant proteins. Understanding the regulation of TC‐mediated aortic remodelling may be a crucial step towards designing strategies to promote aortic repair and prevent adverse remodelling.

Immunolocalization of stem/progenitor cell biomarkers Oct-4, C-kit and Musashi-1 in endometriotic lesions.

Human endometrium harbors stem/progenitor cells (SPCs) that may contribute to the establishment of endometriosis when seeded outside the uterus. Oct-4, C-kit and Musashi-1 are some of the many proteins used to characterize SPCs, but their association with endometriosis is uncertain. In this study, specimens of normal endometrium (n = 12), eutopic endometrium from women with endometriosis (n = 9), superficial peritoneal endometriosis (SUP, n = 12) and deep endometriosis (DE, n = 13) lesions were evaluated for localization and intensity of immunostaining for Oct-4, C-kit and Musashi-1. The three markers were abundantly expressed in normal endometrium, eutopic endometrium from endometriosis patients, SUP and DE specimens. Oct-4 and C-kit expression did not vary across groups as regards intensity or frequency. C-kit staining signal was seldom detected in vascular endothelium of normal or eutopic endometrium from endometriosis patients; however, it was positive in 67% of the SUP lesions and in 25% of the DE lesions (p = 0.042). Musashi-1 was expressed in some endometriotic glands as cell clusters, but its signal was similar between the four types of tissue (p = 0.971) CONCLUSION: The wide distribution of Oct-4, C-kit and Musashi-1 in endometria of patients with and without endometriosis and in SUP and DE endometriotic lesions suggests that these markers are not suitable for the in situ characterization of endometrial SPCs and should not be taken as surrogates for the study of SPCs in the pathogenesis of endometriosis.

Outcome, prognostic factors and histological characterization of canine gastrointestinal sarcomas.

Canine gastrointestinal sarcomas, a group of tumours that includes leiomyosarcomas (LMSAs), gastrointestinal stromal tumours (GISTs) and other rarer sarcomas, comprise about 10-30% of all gastrointestinal tumours. This study aims to characterize the histologic characteristics and clinical behaviour in order to identify prognostic factors predictive of outcome. A single institution database search for surgically treated gastrointestinal sarcomas yielded 47 cases with adequate tissue remaining for histologic analysis and 42 cases available for analysis of clinical outcome. Tumours were then prospectively evaluated for mitotic count, necrosis, haemorrhage and inflammation, as well as categorized via immunohistochemical (IHC) staining for smooth muscle actin, c-KIT and DOG-1. IHC analysis defined 32 tumours as GISTs, 14 as LMSAs and one as a sarcoma not otherwise specified. For both GISTs and LMSAs, the overall median survival time (MST) is 1024 days (range 31-1456), which did not differ statistically between tumour types (p=.92). The overall metastatic rate of GISTs in this study was 32.1% (n=9) which was not significantly different to that of LMSAs at 15.3% (n=2, p=.45). In multivariate analysis, mitotic count under 9 in GIST patients and complete surgical excision in all tumour types correlated with improved MST. For patients with GISTs, the intensity of c-KIT staining also correlated positively with survival, with an MST of 250 days in cases with weak staining and an MST of 1418 days in cases with moderate or strong c-KIT staining (p=.005).

A Close Relationship Between Networks of Interstitial Cells of Cajal and Gastrointestinal Transit In Vivo.

The interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are located in the same area as the myenteric plexus. ICC-MP networks are linked to the generation of electrical pacemaker activity that causes spontaneous gastrointestinal (GI) contractions; however, its role in GI transit is not clear. The aim of this study was to comprehensively investigate the effect of ICC-MP disruption on GI transit in vivo using W/W v mice, partially ICC-deficient model mice. In this study, we measured GI transit using a 13C-octanoic acid breath test, an orally administered dye and a bead expulsion assay. ICC were detected by immunohistochemical staining for c-Kit, a specific marker for ICC. Interestingly, we found that gastric emptying in W/W v mice was normal. We also found that the ability of small intestinal and colonic transit was significantly reduced in W/W v mice. Immunohistochemical staining using whole-mount muscularis samples revealed that c-Kit-positive ICC-MP networks were formed in wild-type mice. In contrast, ICC-MP networks in W/W v mice were maintained only in the gastric antrum and were significantly reduced in the ileum and colon. No significant changes were observed in the nerve structures of the myenteric plexus in W/W v mice. These findings suggest that ICC-MP contribute to GI transit as a powerful driving function in vivo.

S2327 Single Incision Needle Knife (SINK) Biopsy for Diagnosis of a Large Gastric Subepithelial Lesion After Multiple Failed Fine Needle Aspirations

INTRODUCTION: Subepithelial lesions (SELs) are a group of heterogenous tumors arising from layers below the gastrointestinal (GI) mucosa and are a common incidental finding on upper endoscopy. Although fewer than 15% of SELs are malignant, accurate diagnosis is essential to identify tumors that will need definitive management. Endoscopic ultrasound with fine needle aspiration (EUS-FNA) is an established technique frequently used to sample SELs, but diagnosis can still remain elusive in many instances. We present a case of a large gastric SEL where single incision needle knife (SINK) biopsy successfully produced a histopathologic diagnosis after multiple failed attempts with EUS-FNA. CASE DESCRIPTION/METHODS: A 66-year-old woman with epigastric pain, weight loss and early satiety underwent CT imaging that showed a 4.5 cm exophytic gastric mass. Initial EUS revealed a subepithelial, hypoechoic lesion with smooth borders and intraluminal convexity along the greater curvature of the stomach. It originated from the muscularis propria and measured 27 mm in depth. Two attempts with EUS-FNA at an outside hospital were non-diagnostic. At our center, EUS-FNA involving multiple passes with 22 and 25-gauge needles was also inconclusive. EUS was then repeated using the SINK biopsy technique. After exclusion of overlying vasculature and circumferential epinephrine injection, a 10 × 5 mm (length x depth) needle knife incision was made at the area of highest convexity over the lesion. A biopsy forceps was inserted into the defect for direct tissue sampling. Minor bleeding post-biopsy was treated with additional epinephrine and two hemostatic clips. A histologic diagnosis of GIST was made and the patient was referred for surgical resection as definitive management. DISCUSSION: This case highlights the limitations of EUS-FNA, while also providing further evidence to support SINK biopsy as a safe and effective technique. The reported yield for histologic diagnosis in upper GI SELs using SINK biopsy is between 81% and 92%. In cases with failed conventional biopsy or EUS-FNA, subsequent SINK biopsy has success rates ranging from 75% to 92%. No instances of perforation have been reported with SINK biopsy. As current ASGE guidelines do not endorse a standard biopsy method, the increased diagnostic yield and strong safety profile of SINK biopsy performed by experienced endoscopists suggests it should be the preferred technique over repeated EUS-FNA in cases of SELs with intraluminal convexity and prior non-diagnostic sampling.Figure 1.: Endoscopic Ultrasound (EUS) image of large gastric (greater curvature) subepithelial lesion arising from the muscularis propria with intraluminal convexity.Figure 2.: 10 × 5 mm (length x depth) incision of overlying mucosa using needle knife (Endo Cut).Figure 3.: Histology of sample obtained by SINK biopsy showing (A) proliferative and atypical spindle cells (hematoxylin and eosin, 20x), (B) positive DOG-1 staining (20x) and (C) positive C-KIT staining (20x).

The effect of imatinib administered in the prenatal period on testis development in rats.

The purpose of this study was to examine the effects of exposure to imatinib in the prenatal period on testis development in rats. Although all the study groups received intraperitoneal imatinib on prenatal days 1-8, no pregnancy occurred in the Imatinib-80 group. Immunohistochemical analysis, TUNEL, c-kit and PDGF staining revealed no difference between the groups in terms of positivity scoring. A significant decrease was detected in total sperm counts in the Imatinib-20 group compared to the control group, but the sperm count was higher in the Imatinib-60 group than in the Imatinib-20 group. At biochemical measurements, the drug increased oxidative stress in the testis and serum in the Imatinib-20 group, but caused a decrease in tissue in the Imatinib-60 group. Thiol measurements revealed a decrease in the testis and serum in the Imatinib-60 group, while an increase in serum measurements was observed in the Imatinib-40 group. Analysis revealed no difference between the groups in terms of protamine and histone gene expression levels in testis tissue exposed to imatinib. Our findings show that prenatal exposure to imatinib can lead to histopathological and biochemical changes in testis tissue, but that no adverse effect occurs in nuclear maturation of germ cells during spermiogenesis.

The potential role of mast cells and fibroblast growth factor-2 in the development of hypertension-induced renal damage

Hypertension-induced renal injury is a multifactorial process which plays a crucial role in the development of chronic kidney disease. Multiple studies have demonstrated that interstitial rather than glomerular changes correlate better with renal functional capacity. Recent evidence indicates that mast cells and cell signaling proteins such as fibroblast growth factor-2 may contribute to the progression of interstitial changes under hypertensive conditions. The aim of our study was to determine the localization of mast cells in the renal cortex and report on the changes in their number, to analyze the distribution of fibroblast growth factor-2, to assess the extent of renal fibrosis and to evaluate renal damage and correlate it with the changes in the number of mast cells in a model of hypertension-induced renal injury by comparing two age groups of spontaneously hypertensive rats. We used 6- and 12-month-old animals. A light microscopic study was conducted on sections stained with hematoxylin and eosin, periodic acid-Schiff stain, Mallory’s trichrome method and toluidine blue. For the immunohistochemical study we used monoclonal antibodies against mast cell tryptase and fibroblast growth factor-2 and a polyclonal antibody against c-kit. The expression of fibroblast growth factor-2 was assessed semi-quantitatively. The number of mast cells was evaluated on toluidine blue-, tryptase- and c-kit-stained sections, as well as double-stained sections and a comparative statistical analysis with the Mann-Whitney test was conducted between the two age groups. Our results showed that mast cells were located mainly in the peritubular and perivascular areas and were absent in the region of the renal corpuscles. Their number increased significantly in 12-month-old animals. Immunostaining for tryptase, c-kit and double staining for both molecules yielded identical results. The immunohistochemical expression of fibroblast growth factor-2 increased in the kidneys of older animals, as did the percentage of collagen fibers. In addition, we described more severe renal damage in 12-month-old spontaneously hypertensive rats and noted a positive correlation in both age groups between the number of mast cells on the one hand and glomerular sclerosis index and tubulointerstitial damage index, on the other. The results obtained in the present study support the pivotal role of mast cells in the development of hypertension-induced kidney damage.

SCF/c-kit signaling pathway participates in ICC damage in neurogenic bladder

ABSTRACT Neurogenic bladder (NB) is a type of double renal dysfunction caused by nerve lesions. The interstitial cells of Cajal (ICC) damage are involved in bladder dysfunction. The aim of this study is to investigate the effect of stem cell factor (SCF)/c-kit signaling pathway on ICC damage in NB model rats. Maximum cystometric capacity (MCC), bladder leak point pressures (BLPP), and bladder compliance (BC) were measured in sham-operated and NB model rats. Immunofluorescent staining for c-kit was performed to determine ICC count in rat bladder trigone. The morphology and ultrastructure changes of ICCs were observed under an electron microscope. The mRNA levels of c-kit and SCF in bladder tissues were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein levels of c-kit, SCF, p-JAK, p-STAT1, and p-STAT3 in bladder tissues were determined by western blot. ICC proliferation was detected by CCK-8 assay. NB resulted in changes in ultrastructure changes of ICCs and a decrease in the number of ICCs and in expression of c-kit, SCF, p-JAK, p-STAT1, and p-STAT3 in NB tissues. Inhibition of SCF/c-kit signaling pathway suppressed ICC proliferation by inhibiting JAK/STAT3 pathway. Moreover, inhibition of SCF/c-kit signaling pathway impaired the SCF-induced attenuation of ICC damage in NB model rats. Collectively, our data indicate that SCF/c-kit signaling pathway participates in ICC damage in NB.

Changes in the number of mast cells, expression of fibroblast growth factor-2 and extent of interstitial fibrosis in established and advanced hypertensive heart disease

IntroductionAn increasing number of studies have shed light on the role of cardiac mast cells in the pathogenesis of hypertension-induced myocardial remodeling. Mast cells promote fibroblast activation, myofibroblast differentiation and subsequent collagen accumulation through the action of tryptase, chymase, histamine and fibroblast growth factor-2. The aim of the present study was to report on the changes in the number of mast cells as evaluated through toluidine blue, tryptase and c-kit staining, to assess the extent of interstitial fibrosis and correlate it with the changes in the number of mast cells and to analyze the immunohistochemical expression of fibroblast growth factor-2 in two groups of spontaneously hypertensive rats indicative of established and advanced hypertensive heart disease. A novel aspect of our work was the analysis of all parameters in the right ventricle. Material and methodsFor the present study, we used 6- and 12-month-old spontaneously hypertensive rats. A light microscopic study was conducted on sections stained with hematoxylin and eosin and toluidine blue. For the immunohistochemical study we used monoclonal antibodies against mast cell tryptase and fibroblast growth factor-2 and a polyclonal antibody against c-kit. The expression of fibroblast growth factor-2 was assessed semi-quantitatively through ImageJ. The number of mast cells was evaluated on toluidine blue-, tryptase- and c-kit-stained sections and a comparative statistical analysis with the Mann-Whitney test was conducted between the two age groups. A separate statistical analysis between results obtained through immunostaining for tryptase and for c-kit was conducted in each age group with the Wilcoxon signed-rank test. The extent of fibrosis was assessed quantitatively on slides stained with Mallory’s trichrome stain as a percentage of the whole tissue and compared between the two age groups. Spearman’s correlation was used to test whether a correlation exists between the number of mast cells and the percentage of interstitial fibrosis. ResultsMast cells with typical cytoplasmic granules were visualized in the interstitial tissue and in the perivascular zone in both age groups. In both ventricles, their number increased significantly in 12-month-old animals as evaluated through all three staining methods. Moreover, immunostaining for tryptase and for c-kit yielded comparable results. The immunoreactivity of fibroblast growth factor-2 increased in both ventricles in older animals. Expression of this protein was particularly intensive in the cytoplasm of connective tissue cells with the characteristic features of mast cells mainly found in the areas of fibrotic alterations in 12-month-old spontaneously hypertensive rats. In both ventricles, interstitial fibrosis was more extensive throughout the myocardium of older animals and was positively correlated with the changes in the number of mast cells in both age groups. ConclusionThe present study reported for the first time that the increase in the number of mast cells, observed as hypertension-induced myocardial changes progress, is statistically significant and confirmed that this process takes place in both ventricles. This increase is accompanied by a higher expression of fibroblast growth factor-2 and is more strongly correlated with the more pronounced interstitial fibrosis in older animals, further supporting the role of mast cells in the structural changes taking place in the myocardium in response to systemic hypertension.

Pentoxifylline alleviated cardiac injury via modulating the cardiac expression of lncRNA-00654-miR-133a-SOX5 mRNA in the rat model of ischemia-reperfusion

Pentoxifylline (PTX) protects from many cardiovascular complications. It plays a critical role in stem cell proliferation and differentiation. Here, the effect of PTX administration on cardiac ischemia and dysfunction was explored. PTX in 3 doses (20, 30, and 40 mg/kg), was administered in vivo 5 min before a 45 min occlusion of the left anterior descending artery, followed by a 120 min reperfusion in male Wistar rats. The left ventricular end-diastolic pressure and dP/dtmax were assessed. Blood and cardiac tissue samples were collected for measuring the levels of cardiac enzymes and the expression of lncRNA-00654-miR-133a-SOX5. Samples of left ventricles were collected and processed for light microscopic, immunohistochemical staining for c-kit (a marker for cardiac progenitor cells) and transmission electron microscopic examination. PTX administration showed improvements in cardiac function tests, enzymes, and myocytes. Microscopic features showed minimal cardiac edema, hemorrhage, cellular inflammatory infiltration and fibrosis in addition to increased c-kit + cells in cardiac tissue samples. Notably, this treatment also produced a dose-dependent decrease in lncRNA-00654 with an increase in SOX5 mRNA and miRNA-133a-3p expressions. In conclusion, PTX has the potential to alleviate cardiac injury and increase the number of c-kit + cells following ischemia-reperfusion in the rat model via modulation of lncRNA-00654 and miR-133a-SOX5 mRNA expressions.

C-KIT Analysis and Targeted Molecular Sequencing of Mesonephric Carcinomas of the Female Genital Tract.

Mesonephric carcinoma is a rare malignancy, thought to derive from Wolffian remnants. To date, no targeted molecular therapeutic options have been identified. On the basis of limited case reports, c-KIT immunohistochemical expression has been reported in female adnexal tumors of Wolffian origin, and targeted therapy with Imatinib has been attempted with mixed success. Currently, it is unclear whether c-KIT immunohistochemical expression is seen in mesonephric carcinoma, a tumor that is thought to be related to female adnexal tumors of Wolffian origin, and how this correlates with KIT mutational status. In this study, we assessed the immunohistochemical expression of c-KIT and KIT mutational status, in a series of 13 mesonephric neoplasms (5 cervical [including 2 cervical carcinosarcomas], 3 uterine corpora, 4 ovarian, and 1 vaginal/pelvic). The intensity of staining and proportion of cells showing cytoplasmic/membranous staining for c-KIT were recorded. KIT was sequenced using a next-generation sequencing panel that targeted 120 hotspots and 17 exons in 33 known actionable cancer genes. This panel included KIT exons 9, 11, and 13, and 6 hotspots (T670, D816, D820, N822, Y823, A829). Although c-KIT immunohistochemical expression was observed in the majority of mesonephric carcinomas (10/12 cases; 83%), no KIT mutations were detected. This cautions pathologists against the use of c-KIT immunohistochemistry as a surrogate marker for KIT-activating mutations in this setting. Consistent with previous studies, the majority of mesonephric neoplasms (10/13; 77%) harbored KRAS mutations. Additional mutations were found in CTNNB1 (2/13, 15%), TP53 (2/13, 15%), and PIK3CA (1/13, 8%).

1999 Jejunal GIST: An Unusual Cause of Acute Massive Gastrointestinal Bleeding

INTRODUCTION: Gastrointestinal stromal tumors (GISTs) are rare, making up 0.1–3% of all gastrointestinal (GI) tumors. While most commonly found in the stomach, GISTs in jejunum are among the rarest subtypes. The majority of GISTs present with occult GI hemorrhage. Here we present a case of massive GI bleeding due to a jejunal GIST in a young woman with BRCA2 gene mutation. CASE DESCRIPTION/METHODS: A 30-year-old woman presented with 1 day of melena and symptomatic anemia. Medical history was notable for BRCA2 gene mutation. Family history was significant for colon cancer in her maternal grandfather and breast cancer in her maternal aunt. On admission, there was melena in the rectal vault and her abdomen was soft, non-tender with no palpable masses. She was anemic with hemoglobin 6.8 g/dL. Emergent EGD revealed no blood or source of bleeding identified. After a rapid bowel preparation, an urgent colonoscopy showed melena in the entire colon and examined terminal ileum, however no culprit lesion was identified. Patient was hemodynamically unstable subsequently so an urgent computed tomography angiogram was performed and revealed active GI bleeding from the jejunal artery. A 3.1 × 2.2 cm mass with peripheral hemorrhage in the left upper quadrant was seen. The patient then underwent emergent embolization of two jejunal branches. An exploratory laparotomy was subsequently performed with resection of a proximal jejunal mass. Histology confirmed a spindle-cell GIST with positive staining for c-kit (CD117). Given the size, low-grade, and mitotic rate of 1/50 high-power field, no adjuvant therapy was needed. Magnetic resonance imaging surveillance was recommended. DISCUSSION: Bleeding jejunal GIST is a rare cause of acute massive GI bleeding. GISTs can be particularly difficult to diagnose because of their endoscopic inaccessibility. The majority of GISTs are thought to be a result of activating mutations of proto-oncogenes c-KIT (CD117) or PDGFR-alpha. These then increase tyrosine kinase receptor activity and result in the proliferation of stem cells. No reports yet suggest a direct association between a BRCA2 germline mutation and small bowel neoplasms. This case highlights a dramatic presentation of massive hemorrhage from a GIST, especially when confined to the small bowel. Further studies are needed to establish whether an association exists between BRCA mutations and GIST as well as other small bowel neoplasms.

A study of residual lesions in horses that recovered from clinical signs of chronic equine dysautonomia.

BackgroundEquine dysautonomia (ED) causes degeneration and loss of autonomic neurons. Approximately 50% of chronic cases recover, but it is unclear how they survive neuronal loss.ObjectivesTo assess lesions, autonomic neuron numbers, interstitial cells of Cajal (ICC), and neurodegeneration in recovered cases.AnimalsThirteen cases (group ED), euthanized 10.3 ± 5.2 (1–16) years from diagnosis and 6 age‐matched controls (group C).MethodsProspective, case control; routine post mortem examination, neuron counts in peripheral and enteric ganglia and immunohistochemical assessment of neural networks (Protein gene product [PGP] 9.5), ICC (c‐kit), and neurodegeneration (beta‐amyloid precursor protein and ubiquitin) in intestine.ResultsPostmortem findings in group ED were small intestinal dilation (4/12, 33%) and muscular hypertrophy (4/12, 33%), and gastric mucosal hypertrophy (3/11, 27%) and ulceration (4/11, 36%). Neuron density was lower in group ED (mean 39% lower for cranial cervical ganglion [P < .001], median 44% lower in celiacomesenteric ganglion [P = .01]). In intestine, neuronal depletion was worst in ileum (median 100% lower in submucosal plexus [P < .001], 91% lower in myenteric plexus [P = .004]). Group ED had less PGP 9.5 staining in ileal myenteric plexus (mean 66% lower [P = .04]) and circular muscle (median 75% lower [P = .006]). In ileum, there was less c‐kit staining in myenteric plexus (median 57% lower [P = .02]) but not muscularis externa. Beta‐amyloid precursor protein and ubiquitin results were not indicitive of neurodegeneration.Conclusions and Clinical ImportanceIntact ICC in muscularis externa might help maintain motility after neuronal loss. Treatment supporting ICC function warrants investigation.