Highly restricted T-cell receptor repertoire in the CD8+ T-cell response against an HIV-1 epitope with a stereotypic amino acid substitution.

OBJECTIVE: In peripheral blood mononuclear cells (PBMCs) from HIV-1-positive patients, we sought to identify CD8+ T-cell populations and the corresponding T-cell receptor (TCR) repertoires that react to an immunogenic cytotoxic T lymphocyte (CTL) epitope with or without an escape mutation. METHODS: PBMCs from HLA-A*2402(A24)-positive patients were stimulated with peptides representing a wild-type CTL epitope in the HIV-1 Nef protein [Nef138-10(wt)] or an escape mutant with a Y to F (Y139F) substitution at the second position [Nef138-10(2F)]. Cultured PBMCs were stained with peptide-major histocompatibility complex tetramers containing Nef138-10(wt) or Nef138-10(2F) sequences. After in-vitro stimulation of PBMCs with cognate peptides, the CD8+ T-cell population was sorted into different fractions: positive only to the wild-type tetramer (wt-positive), positive only to the mutant tetramer (2F-positive), and positive to both wt-tetramers and mutant-tetramers (dual-positive). TCR repertoires of sorted epitope-specific CD8+ T-cell populations were determined by sequencing. RESULTS: A 2F-positive population was rarely observed under our culture and staining conditions. The wt-positive CD8+ T-cell populations had a diverse TCR repertoire, but the TCR repertoires in dual-positive CD8+ populations were highly restricted. In the dual-positive CD8+ T-cell populations, most clonotypes used the TRBV4-1 and TRBJ2-7 gene segments for the TCR beta-chain and the TRAV8-3 and TRAJ40-1 for the TCR alpha-chain. The CDR3 region of the TCR beta-chain showed little variation. CONCLUSION: These results provide an example of restricted TCR repertoire in a specific CTL response against the escaping epitope. We speculate that impairment of antigen presentation in escaping viruses may underlie the restricted repertoire.

Molecular mechanisms in two cell death-types, necrosis and apoptosis, induced by 5-fluoro-2'-deoxyuridine.

We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analysis. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FudR-treated original F28-7. This finding suggests a new role for lamin B1 as a regulator in the cell death.

Gene expression profiles of necrosis and apoptosis induced by 5-fluoro-2'-deoxyuridine.

5-Fluoro-2'-deoxyuridine (FUdR), a potent anticancer agent, exerts its effects by inhibiting thymidylate synthase, an essential machinery for DNA synthesis in cell proliferation. Also, cell death is caused by FUdR, primarily due to an imbalance in the nucleotide pool resulting from this enzyme inhibition. We have investigated the cancer cell death induced by FUdR, focusing on its molecular mechanisms. Using mouse mammary tumor FM3A cell lines, the original clone F28-7 and its variant F28-7-A cells, we previously reported an interesting observation that FUdR induces a necrotic morphology in F28-7, but induces, in contrast, an apoptotic morphology in F28-7-A cells. In the present study, to understand the molecular mechanisms underlying these differential cell deaths, i.e., necrosis and apoptosis, we investigated the gene expression changes occurring in these processes. Using the cDNA microarray technology, we found 215 genes being expressed differentially in the necrosis and apoptosis. Further analysis revealed differences between these cell lines in terms of the expressions of both a cluster of heat shock protein (HSP)-related genes and a cluster of apoptosis-related genes. Notably, inhibition of HSP90 in F28-7 cells caused a shift from the FUdR-induced necrosis into apoptosis. These findings are expected to lead to a better understanding of this anticancer drug FUdR for its molecular mechanisms and also of the general biological issue, necrosis and apoptosis.

Association of nuclear membrane protein lamin B1 with necrosis and apoptosis in cell death induced by 5-fluoro-2'-deoxyuridine.

We report that anticancer 5-fluoro-2 '-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells was performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Thus, the swelling feature for the necrosis was no longer observable, and instead cell shrinkage typical of apoptosis took place in almost all the cells examined. This finding suggests a new role for lamin B1 as a regulator in cell death.

Possible identification of third stromal component in extraadrenal paraganglioma: myofibroblast in fibrous band and capsule.

Sustentacular and dendritic cells are known as the stromal components of extraadrenal paraganglioma. We identified a third stromal component in such a case. A 66-year-old Japanese woman complained of abdominal pain. The tumor was discovered near the right adrenal gland in the retroperitoneum. Histologically, the tumor consisting of round to oval neoplastic cells with eosinophilic cytoplasm proliferating with a "zellballen" pattern. Sustentacular cells were positive for S-100. Dendritic cells positive for HLA-DR were seen among the neoplastic nests. Additionally, many alpha-smooth muscle actin (ASMA)-positive and hcaldesmon-negative stromal cells, namely, myofibroblasts, were distributed in the capsule and fibrous band. Ultrastructurally, myofibroblasts contained many myofilaments and dense bodies in the cytoplasm. Finally, we identified the third stromal component, namely, myofibroblasts, in the extraadrenal paraganglioma. These myofibroblasts may play a role in the stromal response of host against neoplasm or the regulation of tumor growth.

Examination of the relationship between the working conditions of young workers and the content of the meals they buy at convenience stores.

The aim of this study was to investigate the relationship between the working conditions of young workers and the meals they buy at convenience stores, and to consider the prevention of obesity. The subjects of this study were 284 workers under 29 yr of age employed at a transportation company in Ishikawa prefecture. Questionnaires were sent to participants, and 193 valid responses were obtained. Working types, night duty and working time were correlated with convenience store patronage, although working situations were not obviously associated with the content of meal selection at convenience stores. The study results revealed a tendency for the proportion of the fat in meals to be greater than 25% for the majority of working situations. It was also shown that foods selected with high frequency contained a high proportion of fat. There results suggest that this situation increases the risk of obesity. In addition, the more days per week convenience stores were patronized, the greater the number of participants felt "The need to improve meals." Therefore, we believe it is essential that young workers consider the prevention of obesity by observing nutritional information when selecting foods. An environment in which such information is easy to obtain at convenience stores should be arranged.

Distribution and role of myofibroblasts in human normal seminal vesicle stroma.

We studied the distribution of myofibroblasts in the stroma of normal seminal vesicles. Twelve normal seminal vesicles obtained by surgery on the diagnosis of some diseases were selected, and we evaluated the distribution of myofibroblasts in the seminal vesicles using immunohistochemical and electron and immunoelectron microscopic techniques. Immunohistochemically, myofibroblasts, which were positive for alpha-smooth muscle actin (ASMA) and negative for high molecular weight caldesmon (h-CD), were observed in the stroma just beneath the epithelium of normal seminal vesicles. Moreover, an electron microscope examination revealed the presence of spindle or stellate cells in the stroma of the lamina propria beneath the seminal vesicle epithelium, and an immunoelectron microscopic examination showed that these cells were positive for ASMA. Finally, myofibroblasts are distributed in the lamina propria of human normal seminal vesicles and may play an important role in the ejection of sperm plasm.

Proteome and transcriptome analysis of cell death induced by 5-fluoro-2'-deoxyuridine.

5-fluoro-2'-deoxyuridine (FUdR) inhibits thymidylate synthase. We have been investigated the molecular mechanisms of cell death in mouse mammary tumor FM3A cells, F28-7 strain and its mutant F28-7-A strain, after treated with FUdR. Previously, we have been reported that F28-7 strain induced DNA cleavage into chromosomal sized fragments and subsequently develop necrosis, but F28-7-A strain induced DNA cleavage into oligonucleosomal sized fragments and subsequently develop apoptosis after treated with FUdR. To understand the molecular mechanisms of regulate of two differential cell death necrosis and apoptosis, we identify cell death regulator by using proteome and transcriptome analysis. When compared with the proteome of F28-7 and F28-7-A strain after treated with FUdR, it was found that 5 proteins were up-regulated and 11 proteins were down-regulated in F28-7-A strain. Furthermore, transcriptome analysis shows that 94 genes were up-regulated and 164 genes were downregulated in F28-7-A strain. Identified proteins and genes were involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. Our results suggested that numerous features indicated the coordinated regulation of molecular networks from various aspects of necrosis or apoptosis at the proteome and transcriptome levels.

Effect of selenium-deficient diet on tubular epithelium in normal rats.

Selenium (Se) deficiency reduces glutathione peroxidase (GPx) activity, resulting in increased oxidative stress. We examined how Se deficiency induces renal injury via oxidative stress over time during the Se-deficient period. Seventy-two male Wistar rats were divided into two groups and fed either a control or Se-deficient diet. Rats were sacrificed on weeks 1, 2, 4, 6, 9, and 12. Blood and urine samples were collected, and the kidneys were removed. Urinalysis was performed, and creatinine clearance (Ccr) was calculated. Expressions of cellular GPx (cGPx) and phospholipid hydroperoxidase GPx (PHGPx) mRNA and GPx activity were measured. Histology was evaluated by light microscopy with immunohistochemistry for 4-hydroxy-2-nonenal (HNE) and vimentin. The Se-deficient diet caused significant decreases in GPx activity and cGPx mRNA expression but no change in PHGPx mRNA, together with significant proteinuria and glucosuria and slight decline in Ccr. The Se-deficient diet induced calcification in the kidney and increased the distribution of HNE and vimentin immunostaining in proximal tubuli, particularly around the outer medulla stripe. However, the histological damage did not progress after 6 weeks of deficiency. Se deficiency induces proteinuria and glucosuria with renal calcification, which may be primarily induced by injury of proximal tubuli via oxidative stress.

CAM5.2-positive subserosal myofibroblasts in appendicitis.

In this study, we examined the distribution and origin of myofibroblasts around the perforations of appendicitis. Stromal cells of 45 cases were studied by immunohistochemistry. In the normal appendix, myofibroblasts were restricted to the mucosa, and CD34-positive stromal cells were distributed in the submucosal and subserosal layers. Some mesothelial cells were positive for cytokeratin CAM5.2, cytokeratin 5, or mesothelial cells (HBME-1). In perforation of appendicitis with both abscess and granulation tissue, a small to moderate or a moderate to large number of myofibroblasts appeared in the subserosal area around the perforation, respectively, but CD34-positive stromal cells were completely absent there. In the subserosal area of the perforation of appendicitis with abscess, cytokeratin 5-positive stromal cells were absent. However, a small to moderate number of cytokeratin CAM5.2-positive stromal cells were observed there. Double immunostaining showed the coexpression of alpha-smooth muscle actin (ASMA) and cytokeratin CAM5.2 and the coexpression of cytokeratin 5 and cytokeratin CAM5.2 in many or some stellate-shaped or spindle-shaped stromal cells existing in the subserosal area with granulation tissue around the perforation of appendicitis, respectively. Finally, many myofibroblasts appearing in the subserosal area of the perforation of appendicitis may be derived from submesothelial cells or mesothelial cells.

Askin tumor with metastasis to the scalp: a histochemical, immunohistochemical and ultrastructural study.

A 29-year-old woman presented with facial edema, and imaging disclosed a tumor extending from the anterior chest wall to the anterosuperior aspect of the mediastinum. Transbronchial cytology of the primary tumor and biopsy of the metastatic scalp lesion were performed. Histologically, the tumor consisted of closely packed small round cells. The neoplastic cells generally had round nuclei, finely dispersed chromatin, and small to prominent nucleoli. Histochemically, the cytoplasm of the neoplastic cells contained abundant glycogen and stained with Grimelius silver. Immunohistochemically, the neoplastic cell membranes reacted with CD99 (MIC2) and the neoplastic nuclei reacted with Fli-1, but various other markers, including lymphocyte and skeletal muscle markers, were not detected. No neoplastic cells were also reactive for chromogranin A, synaptophysin, and neurofilament. Ultrastructurally, some neoplastic cells had delicate cytoplasmic processes and contained membrane-bound dense core granules in the cytoplasm. Even if results are immunohistochemically negative for neuroendocrine markers, the combination of immunohistochemistry of CD99 (MIC2) and Fil-1 may be useful in diagnosing Askin tumor or its metastatic lesion.

Proteome and transcriptome analysis of 5-fluoro-2'-deoxyuridine-induced cell death mechanisms.

5-Fluoro-2'-deoxyuridine (FUdR) inhibits thymidylate synthase. The inhibition of thymidylate synthase causes an imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools which subsequently induced cell death. We have been investigated the molecular mechanisms of cell death in mouse mammary tumor FM3A cells, F28-7 strain and its mutant F28-7-A strain, after treated with FUdR. We have previously been reported that F28-7 strain induced DNA cleavage into chromosomal sized fragments and subsequently develop necrosis, but F28-7-A strain induced DNA cleavage into oligonucleosomal sized fragments and subsequently develop apoptosis after treated with FUdR. In this report, in order to understand the molecular mechanisms of regulate of two differential cell death necrosis and apoptosis, we identify cell death regulator by using proteome and transcriptome analysis. When compared with the proteome from F28-7 strain and F28-7-A strain, it was found that ten proteins were increased and six proteins were decreased in F28-7-A strain. Furthermore, transcriptome analysis shows that 127 genes were increased and 181 genes were decreased in F28-7-A strain. These differentially expressed proteins and genes were involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. These two techniques clarified numerous features in F28-7 strain and F28-7-A strain. Our results revealed that numerous features indicated the coordinated regulation of molecular networks from various aspects of necrosis or apoptosis at the proteome and transcriptome levels.

Myofibroblasts in the stroma of metastatic pulmonary calcification in a patient with chronic renal failure.

We present a case of metastatic pulmonary calcification. Histologically, deposition of hematoxyphilic materials was seen along the alveolar and vessel walls. Fibrous tissues were also seen within the alveolar lumens, resulting in intra-alveolar fibrous pneumonia. Immunohistochemically, CD34-positive perivascular adventitial fibroblasts were seen in normal alveolar septa, whereas no myofibroblasts were observed. In contrast, perivascular adventitial fibroblasts were absent in the alveolar septa of the lesion of metastatic calcification, whereas many myofibroblasts were present in the fibrous tissue within alveolar lumens. No positive cells for TGF-(beta1) were observed in the lesion of metastatic calcification, but positive cells for PDGF-BB were focally seen in adveolar epithelial cells. Finally, many myofibroblasts appear in the alveolar lumens of metastatic pulmonary calcification, and we suggest that these myofibroblasts may be derived from CD34-positive perivascular adventitial fibroblasts and PDGF-BB may be involved in the pathogenesis of surrounding fibrosis.

Increased iron deposition in rat liver fibrosis induced by a high-dose injection of dimethylnitrosamine.

Using a developed rat model of hepatic necrosis and subsequent fibrosis induced by a high-dose intraperitoneal injection of dimethylnitrosamine (DMN), we studied iron deposition and expression of transforming growth factor-beta(1) (TGF-beta(1)) during the development of persistent liver fibrosis. Rats were sacrificed at several timepoints from 6 h to 10 months post-injection and the livers were examined for iron content and distribution, and for expression of alpha-smooth muscle actin, ED-1, TGF-beta(1), and collagen (alpha(2))I. Morphologic evidence of acute submassive hemorrhagic necrosis peaked at 36 h; on day 3 the residual parenchyma contained activated hepatic stellate cells (HSCs) and necrotic areas contained numerous macrophages; and on day 5, necrotic tissues and erythrocytes had been phagocytosed and macrophages contained abundant iron deposits. From days 7 to 10, iron-laden macrophages and activated HSCs (myofibroblasts) populated the fibrous septa in parallel. From week 2 to month 10, closely arranged macrophages and myofibroblasts were found in central-to-central bridging fibrotic tissue. TGF-beta(1) was strongly detected in both macrophages and HSCs during development of liver fibrosis. Our data suggest that increased iron deposition may be involved in the initiation and perpetuation of rat liver fibrosis. Iron-laden macrophages may influence HSCs through the action of TGF-beta(1) in DMN-induced liver fibrosis.

Invasive micropapillary carcinoma of the urinary bladder: an immunohistochemical study of neoplastic and stromal cells.

A 66-year-old man complained of hematuria. A cystoscopy revealed a non-papillary tumor and radical cystectomy was performed. Macroscopically, an ulcerative lesion was observed. Microscopically, the neoplasm showed a mixture of urothelial carcinoma, squamous cell carcinoma and micropapillary carcinoma. Immunohistochemically, micropapillary carcinoma cells were positive for cytokeratins 7 and 20, carcinoembryonic antigen and CA125. Additionally, myofibroblasts were distributed in a chicken-wire pattern in the stroma of micropapillary carcinoma. Subsequently, the patient died of carcinoma 1 year after the onset of symptoms. Our results support the previous hypothesis that bladder micropapillary carcinoma runs an aggressive clinical course and suggest that micropapillary carcinoma may show the glandular differentiation of urothelial carcinoma and show the stromal reaction by myofibroblasts resembling that of carcinoma in other anatomic sites.

Effect of selenium on Cisplatin-induced nephrotoxicity in rats.

Cisplatin (CP), a commonly used antineoplastic drug, is nephrotoxic. CP-induced nephrotoxicity involves oxidative pathways. A deficiency of selenium (Se) reduces glutathione peroxidase (GPx) activity resulting in oxidative stress. We investigated how Se deficiency or oral Se administration influences CP-induced nephrotoxicity. Thirty male Wistar rats were fed a Se-deficient or control diet for 4 weeks. Then they were given intraperitoneal (i.p.) CP alone, i.p. saline alone, or Se by gavage 24 and 1 h prior to i.p. CP. Blood and urine samples were collected and the kidneys were removed 5 days after CP treatment. Urinalysis, renal function, GPx activity, and expression of cellular GPx mRNA were measured. Histology was evaluated by light microscopy with immunohistochemistry for 4-hydroxy-2-nonenal (HNE), vimentin, and heme oxygenase (HO)-1. CP induced renal tubular damage with increased expression of vimentin, HO-1 and HNE staining, which represents lipid peroxidation. Se deficiency exacerbated CP-induced nephrotoxicity as shown by deterioration of the above parameters and depressed GPx activity and expression of GPx mRNA. Se treatment ameliorated CP-induced nephrotoxicity, but did not significantly improve renal function. These findings suggest that Se deficiency increases oxidative stress and enhances CP-induced nephrotoxicity, whereas oral Se treatment partially protects against the nephrotoxicity in rats.

[Prognosis of bronchial asthma in adults].

BACKGROUND: The prognosis of adult asthma, whether a long-term remission is available, is still unknown. In this paper we investigated the prognosis of adult asthma patients. METHODS We sent a questionnaire by mail to 1168 patients who had been taken care in our clinic until 1990-1992, but disappeared afterward. Those patients were asked their present status of clinical condition of asthma including symptoms, medication, etc. RESULTS: Delivery of mail was failed in 370 patients because of changed address. 430 of 798 patients replied the mail and 86 patients out of 430 patients were in remission state with no symptom without any medications. The characterictis of these patients in remission are early hospital visit after developing asthma, mild in severity, mild in obstructive lung function and mild in bronchial hypersensitivity to acetylcholine at the first visit hospital. CONCLUSION: We concluded that some of adult asthmatic patients might become in clinical remission.

Vascularization in tissue remodeling after rat hepatic necrosis induced by dimethylnitrosamine.

We observed postnecrotic tissue remodeling to examine vascularization in adult rat livers. Livers, bone marrow, and peripheral blood from rats at 24 h to 14 days after an injection of dimethylnitrosamine (DMN) were examined by light microscopic, immunohistochemical, and ultrastructural methods. Numerous ED-1 (a marker for rat monocytes/macrophages)-positive round mononuclear cells infiltrated in the necrotic areas at 36 h after DMN treatment. On day 5, when necrotic tissues were removed, some of the cells were transformed from round to spindle in shape. On day 7, these cells were contacted with residual reticulin fibers and became positive for SE-1, a marker of hepatic sinusoidal endothelial cells and Tie-1, an endothelial cell-specific surface receptor, associated with frequent occurrence of ED-1/SE-1 and ED-1/Tie-1 double-positive spindle cells. Ultrastructurally, the spindle cells simultaneously showed phagocytosis and endothelial cell-like morphology. With time necrotic areas diminished, and on day 14, the necrotic tissues were almost replaced by regenerated liver tissues and thin bundles of central-to-central bridging fibrosis. Bone marrow from 12 h to day 2 showed an increase of BrdU-positive mononuclear cells. Some of them were positive for ED-1. The BrdU-labeled and ED-1-positive cells appeared as early as 12 h after DMN injection and reached a peak in number at 36 h. They were similar in structure to ED-1-positive cells in necrotic liver tissues. These findings suggest that round mononuclear ED-1-positive cells proliferate first in bone marrow after DMN treatment, reach necrotic areas of the liver through the circulation, and differentiate to sinusoidal endothelial cells. Namely, hepatic sinusoids in DMN-induced necrotic areas may partly be reorganized possibly by vasculogenesis.