Elevated concentrations of CCR7 ligands in patients with eosinophilic pneumonia.

BACKGROUND: Previous studies suggest that dendritic cells and macrophages play an important role in inflammation of eosinophilic pneumonia. The mechanism of dendritic cell and macrophage accumulation into the lung, however, is unknown. Here, we hypothesized that CCR7 ligands, CCL19 and CCL21, contribute to the accumulation of dendritic cells and alveolar macrophages in the inflamed lung of patients with eosinophilic pneumonia. METHODS: Concentrations of the CCR7 ligands as well as CCL16, CCL17 and CCL22 in the bronchoalveolar lavage fluid of 53 patients with eosinophilic pneumonia, 29 patients with sarcoidosis, 18 patients with idiopathic pulmonary fibrosis and 12 healthy volunteers were measured by enzyme-linked immunosorbent assay. Cell sources of CCR7 ligands and CCR7-expressing cells in the bronchoalveolar lavage fluid were evaluated by immunocytochemistry. RESULTS: CCL19 and CCL21 levels in the bronchoalveolar lavage fluid were significantly higher in patients with eosinophilic pneumonia than in controls. Levels of CCL19, but not CCL21, were statistically correlated with the levels of CCL16, CCL17 and CCL22 in patients with eosinophilic pneumonia. Immunocytochemistry revealed CCL19 expression in dendritic cells, macrophages and T-lymphocytes harvested from patients with eosinophilic pneumonia, and CCR7 expression in dendritic cells and macrophages. Levels of CCL19, but not CCL21, were significantly decreased after remission in patients with eosinophilic pneumonia. After provocation tests, CCL19 levels were elevated in all patients with eosinophilic pneumonia. CONCLUSIONS: These findings indicate that CCL19 rather than CCL21 may contribute to the accumulation of dendritic cells and macrophages in the inflamed lungs of patients with eosinophilic pneumonia.

Increased production of cysteinyl leukotrienes and prostaglandin D2 during human anaphylaxis.

BACKGROUND: Anaphylaxis is a life-threatening syndrome resulting from the sudden release of mast cell- and basophil-derived mediators into the circulation. However, pathological evidence of the association between inflammatory mediators and human anaphylaxis is insufficient. OBJECTIVE: The aim of this study was to better understand the relationship between in vivo production of inflammatory mediators and the pathogenesis of anaphylaxis. We also sought to evaluate mast cell activation in anaphylaxis. METHODS: We measured the concentrations of various inflammatory mediators in urine samples, which were collected from 32 anaphylactic patients during the onset of anaphylaxis and during clinical remission, 21 patients with asthma on acute exacerbation and 15 healthy control subjects. Blood and urine specimens were collected from the patients after provocation test. Urinary leukotriene E4 (LTE4), 9alpha, 11beta-prostaglandin F2 (9alpha, 11beta-PGF2), eosinophil-derived neurotoxin (EDN) and leukotriene B4 glucuronide (LTBG) concentrations were determined by enzyme immunoassay, and the activity of plasma platelet-activating factor acetylhydrolase and serum tryptase concentration were measured using commercially available kits. RESULTS: Significantly higher concentrations of urinary LTE4 and 9alpha, 11beta-PGF2, which immediately decreased during clinical remission, were observed in the anaphylactic patients than in asthmatic patients on acute exacerbation and healthy control subjects. Concentrations of EDN and LTBG were not significantly different among the anaphylactic patients, asthmatic patients on acute exacerbation and healthy subjects. There was a significant correlation between urinary LTE4 and 9alpha, 11beta-PGF2 concentrations in the anaphylactic patients (r=0.672, P=0.005, n=32). In addition, LTE4 concentration in patients with anaphylactic shock is significantly elevated compared with that in patients without anaphylactic shock. CONCLUSIONS: This is a report on the significant increase in urinary LTE4 and 9alpha, 11beta-PGF2 concentrations during anaphylaxis. Urinary LTE4 and 9alpha, 11beta-PGF2 concentrations may be a reliable marker of endogenous production of inflammatory mediators associated with anaphylaxis.

Concentration of 14,15-leukotriene C4 (eoxin C4) in bronchoalveolar lavage fluid.

BACKGROUND: There has been no information about the concentration of 14,15-leukotriene C4, which is generated by 15- and 12-lipoxygenase and has been recently named eoxin C4, in biological fluids. OBJECTIVE: To determine the clinical concentrations of eoxin C4 in various respiratory inflammatory diseases, we quantified eoxin C4 in relation to the concentrations of cysteinyl-leukotrienes (CysLTs) and 15-hydroxyeicosatetraenoic acid (15-HETE) in bronchoalveolar lavage fluid (BALF). METHODS: BALF fluid was obtained from patients with a number of inflammatory lung diseases. Eoxin C4 and CysLTs were quantified by enzyme immunoassay in combination with high-performance liquid chromatography. Eoxin C4 immunoassay does not detect eoxin D4 or eoxin E4. 15-HETE was quantified by gas chromatography-mass spectrometry using (18)O-labeled compounds as an internal standard. RESULTS: The concentration of eoxin C4 (median 1.4, range <1.12-6.7 pg/mL) was significantly lower than that of eoxin C4 or CysLTs (P<0.0001). The concentration of 15-HETE significantly correlated with those of LTC4 and CysLTs or the number and the percentage of eosinophils in BALF. On the other hand, eoxin C4 concentration did not correlate with eosinophil number or CysLTs concentration in BALF. CONCLUSIONS: This is the first study demonstrating the presence of eoxin C4 in human biological fluids. Further studies are necessary to elucidate the pathophysiological role of eoxin C4 in some respiratory inflammatory diseases.

Increased urinary leukotriene E4 concentration in patients with eosinophilic pneumonia.

Although eosinophils produce cysteinyl leukotrienes (CysLTs) in large quantities, information on the relationship between CysLTs and eosinophilic pneumonia (EP) is lacking. Inflammatory mediator concentrations in urine were quantified to clarify the relationship between CysLT concentrations and EP severity. Leukotriene (LT)E(4), eosinophil-derived neurotoxin (EDN), 9alpha,11beta-prostaglandin F2 and LTB(4) glucuronide concentrations were quantified in the urine of: EP patients during acute exacerbation and clinical remission; asthmatic patients during acute exacerbation and under stable conditions; and healthy control subjects. The urinary LTE(4) and EDN concentrations of EP patients during acute exacerbation were significantly higher than those of asthmatic patients and healthy subjects, and decreased immediately during clinical remission. The urinary LTE(4) concentration was associated with the urinary EDN concentration of EP patients during acute exacerbation. The urinary LTE(4) concentration significantly correlated with the diffusing capacity of the lung for carbon monoxide in EP patients during acute exacerbation. The increased urinary concentrations of leukotriene and eosinophil-derived neurotoxin were associated with acute exacerbation in eosinophilic pneumonia patients. The increased leukotriene concentration significantly correlated with diffusing capacity of the lung for carbon monoxide, suggesting that the monitoring of leukotriene concentration may aid in the management of eosinophilic pneumonia patients.

Comparison of cysteinyl leukotriene concentrations between exhaled breath condensate and bronchoalveolar lavage fluid.

BACKGROUND: Collection of exhaled breath condensate (EBC) is a simple, non-invasive method of obtaining samples from the airways and it can be repeated in short intervals without side effects; therefore, it provides an opportunity to monitor the changes in concentration of inflammatory mediators in the airways. However, EBC analysis still has several unresolved issues. OBJECTIVE: To better understand the characteristics of EBC, we compared cysteinyl leukotriene (CysLT) concentrations between bronchoalveolar lavage fluid (BALF) and EBC. We also attempted to correct CysLT concentrations in BALF and EBC diluted with saline and water vapour using biological markers. METHODS: EBC was collected from 14 patients with idiopathic pulmonary fibrosis before bronchoscopy. We measured CysLT concentrations and also quantified tyrosine, urea and total protein as possible biomarkers for correcting dilution. RESULTS: (1) We have validated the quantification of CysLTs in EBC. (2) Although a significant correlation was observed among tyrosine and urea concentrations in BALF, urea and total protein concentrations were below the detection limit in EBC. (3) CysLT concentrations were higher in BALF than in EBC (median, 15.96 pg/mL vs. 5.5 pg/mL; P=0.001) and there was no correlation of CysLT concentrations in BALF with those in EBC. A significant correlation of the ratio of total CysLT concentration to tyrosine concentration (CysLT/Y) in EBC with that in BALF was observed (r=0.547, P=0.043). (4) CysLT/Y in EBC correlated with serum KL-6 concentration and total cell count in BALF, and CysLT/Y in BALF also correlated with exhaled NO concentration and %VC. CONCLUSIONS: CysLT/Y in EBC significantly correlated with that in BALF and some clinical parameters correlated with CysLT/Y. Tyrosine concentration may be used to correct the dilution error for CysLT concentrations, and CysLT/Y in EBC can be a surrogate marker for CysLT concentrations in BALF.

Female urethral adenocarcinoma with a heterogeneous phenotype.

We here report a very rare case of female urethral adenocarcinoma. A 77-year-old woman presented with urinary retention. Cystoscopy showed a urethral tumor and the biopsy material showed adenocarcinoma. Macroscopically, the tumor measuring 3.0 x 3.0 x 2.4 cm was predominantly observed around the periurethral area on the proximal side. Histologically, patterns of columnar/mucinous adenocarcinoma, clear cell adenocarcinoma and papillary/micropapillary carcinoma were observed, but there was no evidence of a cribriform pattern. Immunohistochemically, neoplastic cells of at least one of three components were positive for CK7 and CK20 or CA125. We suggest that female urethral adenocarcinoma with a histologically and immunohistochemically heterogeneous phenotype may originate from cells within urethral or paraurethral tissue, such as urethritis glandularis or intestinal metaplastic epithelium and Mullerian tissue.

Gastric carcinosarcoma with neuroendocrine differentiation as the carcinoma component and leiomyosarcomatous and myofibroblastic differentiation as the sarcomatous component.

Gastric carcinosarcoma with neuroendocrine differentiation is a very rare neoplasm. In this article we present such a case. The gastroendoscopic examination of a 59-year-old Japanese man disclosed gastric cancer during follow-up after operation for rectal cancer. Subsequently, total gastrectomy was carried out because of gastric cancer. A large tumor measuring 9.2 x 8.4 cm was observed in the posterior wall of the upper portion of the stomach. The tumor was composed of carcinoma and sarcomatous cells, and the histological transition of both components was observed. Immunohistochemically, carcinoma and sarcomatous cells were positive for cytokeratin CAM5.2. The carcinoma contained adenocarcinoma and malignant cells with neuroendocrine differentiation. The sarcomatous component showed leiomyosarcomatous and myofibroblastic differentiation. The present tumor is the fifth case of gastric carcinosarcoma with neuroendocrine differentiation and the first case of gastric carcinosarcoma with myofibroblastic differentiation. Pathologists should bear in mind that gastric carcinosarcoma may show various types of differentiation.

Family Members, Transplantation Candidates, and Patients Who Underwent Liver Transplantation Had Insufficient Information About the Procedure.

INTRODUCTION: Adherence to treatment is essential for a successful liver transplantation (LT) because LT requires information, abilities, and competencies of patients and family members. OBJECTIVES: This study sought to identify whether the information received about the LT process was enough for either patients or family members who attended a liver transplant center in a school hospital. METHODS: This was a transversal study using questionnaires to verify received information on LT. It included 50 patients on the waiting list for LT, 50 transplanted patients, and 50 family members. RESULTS: There was a prevalence of men (82%) among patients, age range from 19 to 67 years (average: 46.87 ± 10.99), and of women (74%) among family members, age range from 18 to 80 years (average: 43.5 ± 11.77). The majority of subjects (88%) had a low education level. The most frequent etiology of hepatic cirrhosis was viral hepatitis associated with alcohol. A significant number of the listed and transplanted patients as well as all family members reported insufficient information about the process of the transplantation. The kind of insufficient information varied according to the period of treatment. The best way to obtain information, as reported by patients and family members, was a combination of oral and written information. CONCLUSIONS: Our data show the need for improvement in the means of delivering information to patients and family members, and an explanatory manual was created from this study.

Pulmonary adenocarcinoma with micropapillary component: an immunohistochemical study. Case report.

Micropapillary carcinoma has been described in various organs, including the breast, urinary bladder, ovary and lung. We here present a case of pulmonary micropapillary carcinoma in a 72-year-old Japanese man who died of respiratory failure and septic shock, following which autopsy was performed. A mass measuring 2.5 x 2.5 x 2.5 cm was observed in the left lower lobe of the lung. The tumor showed moderately differentiated papillary adenocarcinoma with a focal micropapillary component. Carcinomatous lymphangiosis was also observed in the left lung and metastatic lesions were observed in the bilateral lung, liver, vertebra, muscle layer of the urinary bladder, right adrenal gland, spleen and lymph nodes. The micropapillary component was predominant at some metastatic sites. Immunohistochemically, both the adenocarcinoma and micropapillary components were positive for cytokeratin (CK) 7, CK19, TTF (thyroid transcription factor)-1, carcinoembryonic antigen (CEA) and surfactant apoprotein A (SP-A), and negative for CK20, estrogen receptor, progesterone receptor, uroplakin III, and CA125. The invasive area of the conventional adenocarcinoma component contained a large number of myofibroblasts, whereas the stroma of the micropapillary component contained a small number of myofibroblasts. However, no myofibroblasts were observed in the stroma of the central core of the non-invasive micropapillary carcinoma. Several lymphatic invasions by neoplastic cells were identified in the peripheral area of the micropapillary component using D2-40 antibody. The immunohistochemical profile may be helpful in determining the primary location of the neoplasm containing micropapillary features. Myofibroblasts are present in the stroma of the invasive neoplastic nests in the micropapillary component as well as the conventional adenocarcinoma component, and D2-40 monoclonal antibody may be useful for evaluating the lymphatic invasion of pulmonary micropapillary carcinoma.

Consistent lack of CD34-positive stromal cells in the stroma of malignant breast lesions.

To examine the distribution of CD34-positive and ASMA-positive stromal cells in various breast lesions, we performed immunohistochemical assays (using a streptavidin-biotin immunoperoxidase technique) of tissue specimens, obtained by excisional biopsy and partial or total mastectomy, from 62 patients with breast lesions. Specimens were obtained from 64 lesions as follows: fibrocystic disease (n=12), intraductal papilloma (n=4), fibroadenoma (n=17), invasive lobular carcinoma (n=6), invasive ductal carcinoma (n=20) and invasive micropapillary carcinoma (n=5). In normal breast tissue (controls), CD34-positive spindle cells were abundant in the intralobular stroma, but no ASMA-positive stromal cells were identified except myoepithelial cells. Small to large numbers of CD34-positive cells were observed in the stroma of 29 of 33 benign diseases. In all invasive carcinomas (lobular, ductal and micropapillary), no CD34-positive stromal cells were observed in the stroma. In the stroma of benign lesions, the number of ASMA-positive stromal cells was various, but the stroma of all invasive breast cancers contained ASMA-positive stromal cells. The present results indicate that disappearance of CD34-positive stromal cells consistently occurs in the stroma of invasive carcinoma of the breast, irrespective of histological type and may be associated with the presence of ASMA-positive stromal cells.

The distribution of CD34-positive stromal cells and myofibroblasts in colorectal carcinoid tumors.

In order to understand the stromal reaction associated with colorectal neoplasms, we examined specimens from 26 patients including normal colorectal tissues (n=15), carcinoid tumors (n=12), well differentiated adenocarcinomas (n=10), and poorly differentiated adenocarcinomas (n=4), using an immunohistochemical method. Myofibroblasts and CD34-positive stromal cells were distributed in the mucosa and in the area between the submucosal and subserosal layers, respectively. However, the distribution of these cells markedly changed with the invasion of neoplasms. Namely, myofibroblasts were abundant in the invasive stroma of all colorectal neoplasms. CD34-positive stromal cells were completely absent from the invasive stroma of colorectal cancers. On the other hand, CD34-positive stromal cells were absent from four out of five carcinoid tumor cases with lesions measuring less than 2 mm in size, but were present in all seven cases of carcinoid tumors measuring more than 2 mm. Double-immunostaining identified stromal cells expressing both ASMA and CD34 in several carcinoid tumor cases. Finally, no CD34-positive stromal cells were observed in the invasive stroma of colorectal cancers. However, the distribution of these cells in carcinoid tumors may depend on the lesion size. Namely, CD34-positive stromal cells existed between neoplastic nests in large-sized carcinoid tumors. Myofibroblasts in the stroma of colorectal neoplasms may originate from CD34-positive stromal cells.

The appearance of myofibroblasts and the disappearance of CD34-positive stromal cells in the area adjacent to xanthogranulomatous foci of chronic cholecystitis.

We investigated the distribution of myofibroblasts and CD34-positive stromal cells in normal gallbladder and its pathological conditions (cholecystitis, n=25) using immunohistochemistry and in situ hybridization. In the wall of normal gallbladder, myofibroblasts were generally absent from all layers, but many CD34-positive stromal cells were observed in the connective tissue layer. In chronic cholecystitis with mild perimuscular fibrosis, a small to moderate number of myofibroblasts appeared in the mucosal layer. In chronic cholecystitis with marked perimuscular fibrosis, a small to large number of myofibroblasts appeared predominantly in the connective tissue layer, whereas the number of CD34-positive stromal cells decreased at the same location, although the number of myofibroblasts increased. In chronic cholecystitis with xanthogranulomatous foci, a small to large number of myofibroblasts were observed in the periphery of the xanthogranulomatous reaction and adjacent area. In contrast, CD34-positive stromal cells were completely absent or were limited to the area just around the xanthogranulomatous reaction. Induction of collagen type I and III mRNA was predominantly observed in the cytoplasm of myofibroblasts associated with the marked fibrosis, which consisted primarily of mature collagen fibers, and in the cytoplasm of myofibroblasts around the xanthogranulomatous reaction, respectively. Finally, myofibroblasts were observed in all subtypes. The increased number of myofibroblasts was most prominent in the connective tissue layer of chronic cholecystitis with marked perimuscular fibrosis or in the area adjacent to xanthogranulomatous foci of chronic cholecystitis. Under these conditions, CD34-positive stromal cells tended to disappear from the connective tissue layer, which exhibited an increase in myofibroblasts.

The participation of myofibroblasts in the capsular formation of human conventional and chromophobe renal cell carcinomas.

The presence of myofibroblasts has been elucidated in neoplastic capsules of various organs. In the present article, we examine the presence of myofibroblasts in the capsule of renal cell carcinoma (RCC) and discuss the origin of the myofibroblasts. Nineteen renal tumors (conventional RCC, n=17; chromophobe RCC, n=2) with evident and totally surrounded fibrous capsule were selected. Abundant myofibroblasts were immunohistochemically observed in the capsule of the RCCs. These findings were confirmed by electron and immunoelectron microscopic studies of three conventional RCCs. Type III and I collagens were predominant in the outer and inner layers of the RCC capsule, respectively. The cytoplasm of the tubular epithelial cells in the tissue surrounding the neoplastic capsule stained positively for transforming growth factor (TGF)-beta 1. In situ hybridization detected type I collagen mRNA in myofibroblasts of the capsule. Myofibroblasts may participate in the capsular formation of conventional and chromophobe RCCs through the collagen production.

Poor response of Wv/Wx mice to a grafted neutrophilia-inducing, colony-stimulating-factor-producing tumor.

Although mice possessing two mutant genes at the W locus have a defect in multipotential hematopoietic stem cells that form macroscopic colonies in the spleen of irradiated mice, the number of neutrophils in the blood of these mutant mice is normal or nearly normal. We investigated neutrophil production using the NFSA fibrosarcoma of C3H mouse origin, which induces neutrophilia accompanied by production of a neutrophil-macrophage colony-stimulating factor by the tumor. When the NFSA tumor was transplanted to (C57BL/6 X C3H/He)F1-Wv/Wx or to congenic +/+ mice, neutrophilia developed in mice of both genotypes. However, there was a significant difference between the degree of neutrophilia that developed in them; there was a 107-fold increase in the +/+ mice, but only a 28-fold increase in the Wv/Wx mice four weeks after tumor transplantation. This result is consistent with the concept that doubly heterozygous W mice have multipotential stem cells with diminished ability to respond to stimulation. The unperturbed condition may not provide a sufficient stimulus to demonstrate the defect in neutrophil production in doubly heterozygous W mutant mice.

Nav1.1 mutations cause febrile seizures associated with afebrile partial seizures.

Recent evidence has suggested that the neuronal voltage-gated sodium channel alpha(1)-subunit gene (Na(v)1.1: SCN1A) is responsible for generalized epilepsy with febrile seizures plus (GEFS+2). Here the authors report two novel disease mutations of Na(v)1.1 in patients with febrile seizures associated with afebrile partial seizures. One is a Val1428Ala substitution in the pore-forming region, and the other is Ala1685Val in the transmembrane helix. These results support the previous findings and contribute to the reliable diagnosis of epilepsy.

Frequent mutations of SCN1A in severe myoclonic epilepsy in infancy.

Mutations in the neuronal voltage-gated sodium channel alpha-subunit type I gene (SCN1A) were found responsible for severe myoclonic epilepsy in infancy (SMEI). The authors describe novel mutations of SCN1A in Japanese patients with SMEI. They screened 12 unrelated patients and a pair of monozygotic twins and detected 10 mutations that lead to truncation of the protein.

Expression of CD9/motility-related protein 1 (MRP-1) in renal parenchymal neoplasms: consistent expression in papillary and chromophobe renal cell carcinomas.

CD9 is a glycoprotein that is abundant in hematopoietic cells. Recently, it has been reported that CD9 is also present in the human kidney. In this article, we investigated the expression of CD9 using an immunohistochemical technique. We also studied the expression of CD9 protein and messenger RNA (mRNA) in tissue samples of some renal tumors using immunoblotting and reverse-transcription polymerase chain reaction (RT-PCR) analysis. Immunohistochemically, all tumors of papillary and chromophobe renal cell carcinomas (RCCs) and oncocytomas expressed CD9. In addition, CD9 was expressed in 31 of 66 conventional RCCs and 1 of 4 collecting duct carcinomas. On immunoelectron microscopy, CD9 was identified on the plasma membrane of a conventional RCC. The presence of CD9 protein in normal kidneys and various renal tumors, except for a collecting duct carcinoma and an oncocytoma, was confirmed by immunoblotting. On RT-PCR analysis, the expression of CD9 mRNA was observed in 1 normal kidney, 2 conventional RCCs, and 1 oncocytoma. The frequency of immunohistochemical CD9 positivity was significantly higher in papillary and chromophobe RCCs than in collecting duct carcinomas and conventional RCCs, respectively. These results suggest that CD9 may be a beneficial marker in the differential diagnosis between papillary RCCs and collecting duct carcinomas and also between chromophobe and conventional RCCs.

The disappearance of CD34-positive and alpha-smooth muscle actin-positive stromal cells associated with human intra-uterine and tubal pregnancies.

In order to elucidate the change in alpha-smooth muscle actin (ASMA)-positive and CD34-positive stromal cells associated with pregnancy, we examined endometrial and Fallopian tube tissues from 40 patients including normal endometrium (n=10), intra-uterine pregnancy (n=10), normal Fallopian tube (n=10), and tubal pregnancy (n=10), using immunohistochemistry. In normal endometrium, only a few ASMA-positive cells were focally observed. Additionally, a wide range of CD34-positive stromal cell abundance was observed. In normal Fallopian tube mucosa, a small to moderate number of both ASMA-positive and CD34-positive stromal cells was observed. Neither ASMA-positive nor CD34-positive stromal cells were observed anywhere in the decidual stroma during both intra-uterine and tubal pregnancies. Likewise, a varying abundance of ASMA-positive cells but no CD34-positive stromal cells were observed at the fetal side during both intra-uterine and tubal pregnancies. In conclusion, the disappearance of CD34-positive and ASMA-positive stromal cells may be an indicator of decidualisation induced change in the stroma during both intra-uterine and tubal pregnancies. ASMA-positive stromal cells at the fetal side associated with pregnancy may play a role in the production of villous extracellular matrix or regulation of blood flow.

Distribution and role of CD34-positive stromal cells and myofibroblasts in human normal testicular stroma.

CD34-positive stromal cells are distributed in various organs including breast, Fallopian tubes, thyroid gland, colon, pancreas, and uterine cervix. To elucidate the distribution of CD34-positive stromal cells, smooth muscle cells, and myofibroblasts in normal human testis, we examined 48 testes obtained by autopsy and operation, including five fetal, one neonatal, and 42 adult cases without evident testicular lesions, using a streptavidin-biotin immunoperoxidase technique. The expression of alpha-smooth muscle actin (ASMA), h-caldesmon, CD34, and CD31 were immunohistochemically examined in all cases. The tunica albuginea and the inner layer of seminiferous tubules in adult testis were predominantly composed of myofibroblasts. Smooth muscle cells were also scattered throughout these sites in some cases. CD34-positive stromal cells were abundant, and they formed a reticular network around the seminiferous tubules and Leydig cells as well as the outer layer of seminiferous tubules. Moreover, myofibroblasts and the CD34 reticular network were already present in the testicular stroma during fetal or neonatal development. Double immunostaining of fetal, neonatal and adult testes using ASMA and CD34 confirmed that myofibroblasts and CD34-positive stromal cells were present in the inner and outer layers of peritubular tissue, respectively. This distribution and cytological identification was also confirmed by an ultrastructural study of four cases. Finally, CD34-positive stromal cells and myofibroblasts are major components of human testicular stroma.