Retinoic acid signalling is a candidate regulator of the expression of pituitary-specific transcription factor Prop1 in the developing rodent pituitary.

Development of the anterior pituitary proceeds via spatiotemporal patterning of transcription factors and signalling molecules. Among them, retinoic acid (RA) functions as an important signalling molecule for vertebrate organogenesis in many tissues. However, little is known regarding the target genes in the developing pituitary. The present study aimed to clarify the relationship between endogenous RA signalling and mRNA expression of the pituitary-specific transcription factor Prop1 in the pituitary primordium of Rathke's pouch. Gene expression analysis and in situ hybridisation demonstrated that retinaldehyde dehydrogenases (Raldhs) and all types of RA receptors (Rars) are expressed at the level of transcription in the rat Rathke's pouch. Ex vivo organ culture using Rathke's pouch and an in vitro reporter assay demonstrated that RA signalling increases the expression level of Prop1 via RARα. Moreover, a reporter assay using serial truncated constructs of the 5'-upstream region of mouse Prop1 revealed a predicted cis-regulatory element of RARα. This is the first report of a relationship between RA signalling and Prop1-expression during early pituitary development.

Management of young patients with lupus nephritis using tacrolimus administered as a single daily dose.

AIMS: Studies on immunosuppressive treatment with tacrolimus (Tac) in subjects with lupus nephritis (LN) are limited. Here, we report our experience with Tac administered daily as a single-dose for maintenance therapy in young patients with pediatric-onset, long-standing LN. METHODS: Eleven consecutive patients with long-standing biopsy-proven LN were recruited for at least 6 months or longer (6 - 24 months) as part of an open-label trial for the single-daily-dose administration of Tac (3 mg/day, 0.04 - 0.075 mg/kg) without dose increases of concomitantly administered prednisolone (PDN). Tac treatment was started at the time of the most recent flare. Data on clinical parameters and serologic lupus activity were collected prospectively. RESULTS: The baseline characteristics of the patients were as follows: mean age, 18 years; urinary protein/creatinine ratio (Up/cr), 0.74 +/- 1.49; serum C3 level, 69.5 +/- 26.5 mg/dl (normal 79 - 152 mg/dl); serum complement hemolytic activity (CH50), 23.0 +/- 8.9 U/ml (normal 23 - 46 U/ml); serum anti-dsDNA antibody titer, 58.9 +/- 54.2 IU/ml (normal < 12.0 IU/ml); serum creatinine, 0.54 +/- 0.13 mg/dl; and European Consensus Lupus Activity Measurement (ECLAM) index, 4.4 +/- 2.2. Despite the gradual tapering of the PDN dose, a marked improvement, compared with the baseline values was observed in the ECLAM index even at 1 month and in the serological parameters at 3 months after the start of treatment. These favorable results persisted until the end of the study. The Up/cre ratio gradually decreased and had dropped significantly at 24 months after the start of treatment. After a mean of 18 months of treatment, complete responses were achieved in 8 patients (73%) and a partial response was achieved in 2 patients. The remaining one patient showed no response. No serious adverse effects were observed. CONCLUSION: These data suggest that low-dose Tac treatment, administered once daily, is an effective and safe method for managing selected young patients with pediatric-onset, long-standing LN. However, further studies involving a larger number of patients are needed to confirm these results.

Increased granzyme B production from peripheral blood mononuclear cells in patients with acute coronary syndrome.

OBJECTIVES: To clarify the role of granzyme B in acute coronary syndrome. DESIGN AND SETTING: Granzyme B is a member of the serine esterase family released from cytotoxic lymphocytes and plays an important role in cellular apoptosis by activating intracellular caspases. Granzyme B expression was compared between patients with stable and unstable angina pectoris (UAP). PATIENTS: 173 patients with coronary artery disease (CAD) were enrolled. 84 patients were found to have stable angina pectoris (SAP) and 89 patients to have UAP. METHODS: Peripheral blood was drawn from the patients. Peripheral blood mononuclear cells (PBMCs) isolated by gradient centrifugation were cultured at a density of 2x106 cells/ml for 24 hours. The supernatants were collected 24 hours after incubation and the granzyme B level was measured by enzyme-linked immunosorbent assay. Polychromic flow cytometric analysis was performed to evaluate the expression of granzyme B in the cells. RESULTS: Granzyme B production from PBMCs of UAP patients was significantly higher than from those of patients with SAP (39.1 (SEM 6.6) versus 17.0 (SEM 1.8) pg/ml, p<0.05). Granzyme B production from PBMCs increased with the increasing TIMI risk score in UAP patients. The percentage of granzyme B-positive lymphocytes to CD3-positive lymphocytes in UAP patients was significantly higher than in SAP (32.1% (SEM 1.6%) versus 18.4% (SEM 0.9%), p<0.01). CONCLUSIONS: These results suggest that granzyme B might play an important role in triggering acute coronary events by inducing apoptosis and the degradation of atherosclerotic coronary plaques.

Human amniotic epithelial cells are morphologically homogeneous: enzymehistochemical, tracer, and freeze-substitution fixation study.

We examined the fine subcellular morphology of human amniotic epithelial cells and attempted to answer the question as to whether amniotic epithelial cells consist of heterogeneous or homogeneous cells, which has long been controversial. Study subjects were fetal membranes from pregnant women (n=18) who abdominally gave birth to healthy infants at term (37.9+/-0.7 weeks of gestation, mean+/-sd). The methods employed were transmission electron microscopy, enzymehistochemistry, tracer permeability analysis, and freeze-substitution fixation. The labelings for acid phosphatase, cytochrome c oxidase, and CA++ATPase were seen in the lysosomes, mitochondria, and lateral plasma membranes, respectively. The staining distribution pattern of these three enzymes and the morphology of the organelle highlighted by these enzymehistochemistry did not differ among cells. Freeze-substitution fixation revealed that intercellular spaces in the amniotic epithelial cells were narrower than previously thought, but the tracers (horse radish peroxidase and lanthanum nitrate) fully entered these spaces. There were no variations in the tracer permeability among cells. All cells from freeze-substitution fixation exhibited the same morphological features. From these morphological viewpoints, we conclude that human term amniotic epithelial cells consist of a homogeneous cell population.

Immunohistochemical classification of the localization of laminin in the thickened bronchial epithelial basement membrane of deceased bronchial asthma patients.

To ascertain histological changes in the basal lamina of the bronchial epithelial basement membrane in patients with severe bronchial asthma, an immunohistochemical study was conducted in 43 patients who died of bronchial asthma. Antibodies against laminin, a component of the lamina lucida, were utilized. The results revealed various patterns for immunoreactivity to laminin in the thickened basement membrane layer. We were able to classify these reactivities into four patterns. In Pattern A, laminin reactions branched vertically in relation to the thickened basement membrane layer. In Pattern B, laminin reactions formed lines along the lower margin of the thickened basement membrane layer. In Pattern C, laminin reactions formed lines along the upper margin of the thickened basement membrane layer. Finally, in Pattern D, no laminin reactions were observed. In addition, relationships between immunohistological characteristics of laminin and findings such as epithelial cell shedding, basal cell proliferation and basement membrane layer thickening were investigated. In many Pattern A patients, epithelial cell shedding was observed, but goblet cell hyperplasia and basal cell proliferation were barely detectable. Conversely, in numerous Pattern D patients, epithelial cell shedding was barely seen, but goblet cell hyperplasia and basal cell proliferation were marked. Hence, Patterns A and D were on opposite ends of the spectrum of morphological characteristics associated with severe bronchial asthma. In Patterns B and C, laminin reactions formed lines along the lower and upper margin of the thickened basement membrane layer, respectively. However, no marked differences existed in epithelial cell shedding and basement membrane layer thickening. The present study is thus the first to clarify that laminin reactions in the thickened basement membrane layer vary, and this feature is unique to the bronchi of patients with severe bronchial asthma.

Experimental small bowel transplantation using a newborn intestine in rats: IV. Effect of cold preservation on graft neovascularization.

BACKGROUND/PURPOSE: University of Wisconsin (UW) solution is one of the most superior organ preservation solutions for liver, kidney, and pancreas; however, it still is controversial for intestinal preservation. Here, the authors studied the efficacy of preservation with 2 kinds of solutions, UW and modified TOM (m-TOM) solutions in our experimental newborn intestinal transplantation model. UW solution was used as a standard intracellular and m-TOM solution as an extracellular preservation solution. Lactated ringer (LR) solution was used as a control. METHODS: Newborn intestine, which were preserved in these solutions for 24 or 48 hours, were transplanted in the subcutaneous spaces of the syngeneic recipients without surgical vascular anastomosis and histologically examined 14 days after grafting. The preserved grafts were evaluated histologically by use of light and electron microscopy just after preservation. The biochemical parameters such as LDH and serotonin also were measured in the supernatants of preservation solutions. RESULTS: Fresh newborn grafts were revascularized successfully at a rate of 80% (16 of 20). After 24 hours of preservation, 65% (13 of 20), 75% (15 of 20), and 85% (17 of 20) of the grafts were observed to be revascularized in LR, m-TOM, and UW solutions, respectively. After 48 hours of preservation, 60% (12 of 20), 80% (16 of 20), and 80% (16 of 20) of the grafts also were revascularized in the respective solutions (no statistic difference among the groups). The cold-preservation did not affect the neovascularization of newborn intestine until 48 hours. Histologic findings of the preserved intestine and biochemical analyses showed that UW and m-TOM solutions kept villous architectures of the preserved grafts, however, might be harmful to enterochromaffin cells. CONCLUSION: Long-time preservation of newborn intestine did not interfere with neovascularization and maturation. J Pediatr Surg 36:1805-1810.

[Immunohistochemical study of KL-6 in pulmonary tuberculosis].

KL-6 is a glycoprotein antigen derived from the cell line of human lung adenocarcinoma. Although KL-6 is known to be a serum marker of interstitial pneumonia, elevated KL-6 serum levels have also been reported in some cases of pulmonary tuberculosis. To elucidate the mechanism of KL-6 elevation in pulmonary tuberculosis, we stained pulmonary tissue samples from five clinical cases for immunohistochemical analyses. In the two cases showing productive changes, KL-6 immunoreactivity was localized in the area of type II pneumocytes showing strong interstitial changes surrounding caseous necrosis. In the two cases showing exudative changes, KL-6 immunoreaction was observed not only to surround caseous necrosis but also to appear within it, particularly in the remaining alveolar lumen septa. On the other hand, the one patient with old pulmonary tuberculosis that showed slight interstitial changes presented with weak KL-6 immunoreactivity on the surface of the alveolar lumen surrounding the tuberculotic region. These results suggest that serum elevation of KL-6 in pulmonary tuberculosis originates from the proliferation of type II pneumocytes along with interstitial changes that surround the tuberculous region.

Tissue distribution and subcellular localization of a variant form of the human ST2 gene product, ST2V.

The human ST2 gene has been known to encode three splice variants; namely, a soluble secreted form of ST2, a transmembrane form of ST2L, and ST2V of undetermined localization. Therefore, analysis of tissue distribution and subcellular localization of ST2V is important to elucidate functional relationships among the three splice variants of the human ST2 gene. RT-PCR procedure revealed that ST2V is predominantly expressed in the stomach, small intestine, and colon. Transfection of ST2V cDNA into COS7 cells in the presence of [(35)S] methionine and cysteine produced radiolabeled 40 kDa protein, which is recognized by specific monoclonal antibody against human ST2. Subcellular fractionation analysis showed that ST2V protein was distributed in the insoluble fraction of the cell lysate. Finally, ST2V protein was detected on the plasma membrane of COS7 cells, which had been transfected with ST2V cDNA, by confocal laser microscopic analysis. These findings taken together, indicate that ST2V protein localizes on the plasma membrane, suggesting its possible role in modification of the ST2L-signaling pathways.

Ultrasonographic features of parathyroid carcinoma.

Although several authors have reported single cases illustrative of some ultrasonographic characteristic of parathyroid carcinoma, the value of ultrasonography for diagnosing this entity remains to be determined. The purpose of our study was to investigate the ultrasonographic features of parathyroid carcinoma in a large number of cases. We assessed the shape, contour, echogenicity, and depth-width (DW) ratio of 16 parathyroid carcinomas and 61 parathyroid adenomas. Ultrasonography showed that parathyroid carcinomas tend to be large, inhomogeneous, hypoechoic masses with lobulated contours. In contrast, parathyroid adenomas were small, homogeneous, hypoechoic masses with smooth borders. The mean (range) DW ratios for parathyroid carcinomas were 1.21 (0.91-2.5) and 0.64 (0.33-1.47) for adenomas; the difference was statistically significant (p<0.0001). The DW ratio was > or =1 in 15 (94%) of the 16 cases of carcinoma, whereas only 3 (5%) of the 61 adenomas had a similar ratio. Ultrasonographic examination is useful not only for preoperative localization but also for differentiating parathyroid carcinoma from adenoma. Parathyroid tumors with irregular margins, inhomogeneous echogenicity, and a DW ratio > or =1 are likely to be malignant.

Non-invasive lobular carcinoma within a fibroadenoma, a preoperatively diagnosed case.

Breast cancer within a fibroadenoma is rare and usually diagnosed postoperatively from pathological specimens. This paper reports a 54-year-old female with non-invasive carcinoma within a fibroadenoma, diagnosed preoperatively. She underwent a medical examination and mastopathy was suspected. On physical examination a mass 2 cm in diameter was palpated in the left breast. Ultrasonography showed a mass with smooth margins and uniform internal echoes, but cytology showed malignancy. Mammography showed a round mass with distinct margins and no calcification. As fibroadenoma, diagnosed by ultrasonography and mammography, and breast cancer, diagnosed by cytology, were not consistent results several core biopsies were performed. Needle biopsy showed proliferation of atypical epithelial cells; breast cancer within a fibroadenoma was diagnosed. MRI showed a circular mass with distinct, smooth margins and in a dynamic study, the mass showed irregular staining and the presence of early staining. Left lumpectomy and dissection of the left axillary lymph nodes was performed. Histological examination showed non-invasive lobular carcinoma occurring within a fibroadenoma.

Interventricular methotrexate therapy for carcinomatous meningitis due to breast cancer: a case with leukoencephalopathy.

A 46-year-old woman presented with paraplegia and severe lumbago. She had had a radical mastectomy for left breast cancer 10 years earlier, and 6 months prior to presentation she completed CMF chemotherapy for treatment of retroperitoneal metastasis. CT and MRI to identify potential causes of the paraplegia and lumbago showed leptomeningeal carcinomatosis due to dissemination from invasive recurrence of the retroperitoneal tumor. An Ommaya reservoir was inserted, and infusion of intrathecal methotrexate (MTX; 5 mg twice weekly) began. Her clinical symptoms improved after receiving 53 mg MTX. However, after receiving 83 mg MTX, the patient became dizzy from leukoencephalopathy. Although administration of prednisolone mostly resolved her symptom, the patient died 9 months after the diagnosis of carcinomatous meningitis.

Enhanced expression of mRNA coding for the adrenaline-synthesizing enzyme phenylethanolamine-N-methyl transferase in adrenaline-secreting pheochromocytomas.

PURPOSE: In some pheochromocytomas, the tumors contain and secrete greater amounts of adrenaline than do normal adrenal medullas. It is not yet known how adrenaline synthesis is enhanced in the adrenaline-secreting pheochromocytomas. MATERIALS AND METHODS: As a first step toward understanding the molecular mechanisms by which adrenaline synthesis is controlled in these tumors, we measured the level of mRNA coding for the adrenaline-synthesizing enzyme phenylethanolamine N-methyl transferase (PNMT) and the content of adrenaline in the pheochromocytomas (n = 9), including 3 cases of the adrenaline-secreting type (one of the patients had bilateral pheochromocytomas), and in normal adrenal medullas (n = 7). We then measured the concentration of cortisol, which is thought to regulate the PNMT activity. Finally, we examined the expression of the mRNA for Egr-1, which was recently reported to be a transcriptional factor regulating PNMT gene expression. RESULTS: In the 4 tissue specimens from 3 adrenaline-secreting pheochromocytomas, the contents of adrenaline and the PNMT mRNA expression were considerably greater than those of the normal adrenal medullas. PNMT immunoreactivity was only detected in the adrenaline-secreting tumors. Three of the 4 specimens showed high concentrations of cortisol. To show the capacity for cortisol production locally in the pheochromocytoma tissues, we showed the expression of a glucocorticoid biosynthetic enzyme, 17alpha-hydroxylase, in the tumors by Western blotting. PNMT expression was found to be associated with 17alpha-hydroxylase expression in the tumors. The glucocorticoid receptor expression was also correlated with PNMT expression in the tumors and the expression of Egr-1 was also high in 3 of the 4 specimens. CONCLUSIONS: These findings indicate that adrenaline production in adrenaline-secreting pheochromocytomas is primarily controlled by the level of PNMT gene expression, and that the gene expression may be enhanced by both cortisol and Egr-1.

Breast cancer shortly after the diagnosis of interstitial pneumonia associated with polymyositis.

Polymyositis is sometimes associated with pulmonary fibrosis or malignant diseases, however, rarely with both simultaneously. For such patients, no criterion for initial treatment and tapering of steroid has been reported. We describe a patient who had breast cancer and interstitial pneumonia associated with polymyositis. In this case, slow tapering of the steroid was an important aspect of the treatment, and close clinical follow-up was necessary to monitor for disease exacerbation.

Ultracytochemical localization of glucose-6-phosphatase in the rat anterior pituitary cells.

Glucose-6-phosphatase is generally accepted as a functional component of rough endoplasmic reticulum and has been histochemically examined in many organs. The aim of this study is to know the ultracytochemical localization of glucose-6-phosphatase in each type of hormone-producing cell constituting the anterior pituitary gland in the rat. Pituitaries of male Sprague-Dawley rats were perfused with 1.5% glutaraldehyde from the left ventricles. After buffer washing 40 microns sections were incubated in the medium of Hugon et al. for 60 min at 37 degrees C. The sections were then postfixed with 1% osmium tetroxide, embedded in epoxy resin and observed under an electron microscope. The reaction product for glucose-6-phosphatase was observed in the lumen of rough endoplasmic reticulum and nuclear envelope of all anterior pituitary cells. The enzyme activities in thyroid-stimulating hormone-producing cells and luteinizing hormone/follicle-stimulating hormone-producing cells (LH/FSH cells) were stronger than those in growth hormone-producing cells and prolactin-producing cells; adrenocorticotropic hormone-producing cells and folliculo-stellate cells presented intermediate activity. In LH/FSH cells, the activity in dilated cisternae of endoplasmic reticulum had weaker density than that in flattened cisternae. In addition, substantial reaction product was also frequently observed in the cis saccules of the Golgi apparatus. These findings suggest that glucose-6-phosphatase may play different functional roles in hormone synthesis within different types of anterior pituitary cells.

Expression of alpha-fetoprotein and prostate-specific antigen genes in several tissues and detection of mRNAs in normal circulating blood by reverse transcriptase-polymerase chain reaction.

BACKGROUND: alpha-Fetoprotein (AFP) and prostate-specific antigen (PSA) in serum are widely used as tumor markers in the evaluation of prognosis and management of patients with hepatocellular carcinoma and prostate cancer, respectively. To establish the molecular diagnosis of cancer, reverse transcriptase polymerase chain reaction (RT-PCR) for AFP and PSA was used to identify circulating cancer cells in the blood of cancer patients. Here, we examined the tissue-specificity of AFP and PSA and tested whether AFP and PSA are suitable targets in the detection of certain cancer cells by RT-PCR using peripheral blood samples. METHODS: Tissue specificity of AFP and PSA was analyzed by Northern blotting and RT-PCR. Probes for AFP and PSA were hybridized with poly A+ RNAs from 50 human tissues. RT-PCR for AFP and PSA mRNA was performed using several cancerous tissues and normal tissues and peripheral blood cells from seven healthy volunteers. RESULTS: Broad expression of AFP was observed in several tissues and a large amount of AFP mRNA was found in fetal liver. PSA was expressed in prostate, salivary gland, pancreas and uterus. By RT-PCR, AFP and PSA mRNA were detected in several tumors, including salivary pleomorphic adenoma, hilar bile duct carcinoma, pancreatic carcinoma, transitional cell carcinoma of urinary bladder and thyroid papillary carcinoma. Furthermore, AFP and PSA mRNAs were frequently detected by RT-PCR, even in peripheral blood cells from healthy volunteers. CONCLUSIONS: Neither AFP nor PSA showed tissue-specific expression. AFP and PSA mRNA were detected in several diseased and non-diseased tissues and normal circulating blood by RT-PCR.

[The storage technology of blood, the latest advances--cryopreservation of red blood cells].

In the field of red blood cell cryopreservation, the latest research informations were mainly divided into two parts as follows: 1) The improvement of the frozen storage bag. 2) The long-term storage of the frozen and thawed red blood cells. That was based on the research for changing the film of frozen storage bags from polyvinyl chloride to polyolefin. In consequent, the broken ratio of the bags in the period of frozen storage was expected to reduced, immediately. And this was studied for establishment of the closed bag system and introduction of the mannitol-adenine-phosphate(MAP) solution. From these results, it was made clear that the frozen and thawed red blood cells could be stored in MAP solution at 4 degrees C for 3 weeks.

Differential expression of PPAR gamma1 and gamma2 isoforms in human adipose tissue.

One of the essential factors for adipogenesis, peroxisome proliferator activated receptor gamma (PPAR gamma), is classified into two isoforms, gamma1 and gamma2 encoded by a single PPAR gamma gene. To examine the mode of expressions of both the PPAR gamma1 and gamma2 isoforms in human adipose tissue, we cloned a partial cDNA of the human PPAR gamma2 (hPPAR gamma2) from a human adipose tissue cDNA library. The sequence encoded an additional 28 amino acids amino-terminal to the first ATG codon of hPPAR gamma1. The simultaneous quantitation of hPPAR gamma1 and gamma2 mRNA in subcutaneous or visceral (mesenteric) fat tissue from 10 different individuals was performed by an RNase protection assay and revealed a relatively higher expression of gamma1 than gamma2 in all specimens examined.