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.

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.

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.

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.

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.

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.

Sealing of the follicular lumen of the anterior pituitary gland of the male rat.

It is commonly accepted that follicular lumina of the adult rat anterior pituitary gland are tightly sealed by junctional complexes, especially tight junctions. In this report, we describe the presence of follicular lumina that are unsealed. Peroxidase (HRP) was used to study such structures and when injected through the femoral vein, was observed in association with a few follicular lumina, on their microvilli and around the cilia of folliculo-stellate cells. The existence of peroxidase-positive follicles clearly shows that follicles of the hypophysis are not always firmly sealed by tight junctions. The folliculo-stellate cells which faced the peroxidase-positive follicles displayed HRP deposits which were membrane bound within their cytoplasm. These findings suggest an absorptive function for the folliculo-stellate cells.

N-ras mutation: an independent prognostic factor for aggressiveness of papillary thyroid carcinoma.

BACKGROUND: The clinical importance of point mutations of ras oncogene in differentiated thyroid cancers has not been fully clarified. The purpose of this study is to determine the prognostic importance of ras mutation in papillary thyroid carcinoma. METHODS: Tumors of 91 patients with papillary carcinoma were studied; mean follow-up was 14.1 years (range, 1 to 40 years). Patients were grouped as follows: class I, intrathyroidal disease, n = 21; class II, cervical node metastases, n = 22; class III, extrathyroidal disease, n = 19; and class IV, distant metastases, n = 29. DNA was analyzed with polymerase chain reaction, oligonucleotide hybridization, and DNA sequence analysis techniques. RESULTS: Thirteen (14.3%) of 91 tumors showed an N-ras point mutation: 4.8% (1 of 21) patients in class I; 4.5% (1 of 22) patients in class II; 15.8% (3 of 19) patients in class III; and 27.8% (8 of 29) patients in class IV. Each mutation changed codon 61 from glutamine to arginine. Patients with distant metastases (8 of 29) had a significantly higher incidence of ras mutations than others (5 of 62, p = 0.01). Patients in classes III and IV also had a higher incidence of mutations (11 of 48) than patients in classes I and II (2 of 43, p = 0.01). The incidence of ras mutations was significantly higher in patients who died of papillary cancer (5 of 15, 33.3%) than in patients who are still alive (8 of 76, 10.5%) (p = 0.02). Kaplan-Meier survival curves also showed a greater mortality from tumor (p < 0.05) and a higher recurrence rate (p < 0.01) in ras-positive tumors than in the ras-negative group. Finally, in studies by multivariate analyses, positive ras mutation and age were shown to be two independent prognostic factors for prediction of death from papillary cancer and recurrence of cancer. CONCLUSIONS: Mutation of N-ras gene at codon 61 is an independent prognostic factor for aggressiveness of papillary thyroid carcinomas.

Point mutations of ras genes in human adrenal cortical tumors: absence in adrenocortical hyperplasia.

Point mutations of ras genes (K-, H-, and N-ras) at codons 12, 13, and 61 and of the Gi2 alpha gene at codons 179 and 205, were studied in 56 primary adrenal cortical tumors and 6 adrenal cortical hyperplasias. Of 56 tumors, 24 were carcinomas and 32 were benign. The 24 carcinomas and 20 of the benign tumors were from American patients; the 12 remaining adenomas were from Japanese patients. Of the benign tumors 12 were cortisol-producing adenomas, 15 were aldosterone-producing adenomas, 3 were nonfunctioning adenomas, and 2 were adenomas that produced a virilizing syndrome. Tumor DNA obtained from archival formalin-fixed, paraffin-embedded tissue or fresh frozen tissue was amplified by polymerase chain reaction; and point mutations were detected by sequence-specific oligonucleotide hybridization. Activating ras mutations were found in 7 of 56 (12.5%) of all tumors: 3 of 24 (12.5%) carcinomas and 4 of 32 (12.5%) adenomas. Of adenomas from an American population, 4 of 20 (20%) exhibited positive ras mutations, whereas none was present in the Japanese tumors. All mutations detected were adenine to guanine transitions at the second position of N-ras codon 61, resulting in a conversion from glutamine to arginine. No mutations were found in K-ras or H-ras genes. Furthermore, no mutations of the Gi2 alpha gene were identified. These findings demonstrate that N-ras mutations at codon 61 may contribute to the genesis of both benign and malignant human adrenal cortical tumors. Finally, no mutations of the ras or Gi2 alpha genes were identified in hyperplastic adrenocortical tissues.

[Clinical diagnosis of early breast cancer].

During the recent 5 years between 1988 and 1992, 254 cases of breast cancer were experienced in the Tsukuba University Hospital. Of them, 80 cases were palpable cancers measuring less than 2cm and 37 cases were nonpalpable cancers. The total of early cancers was 117 cases, 43.3% of all cases. Noninvasive carcinoma without minimally invasive carcinomas were 35 cases, 13.2% (ductal ca: 27, lobularca: 2, Paget ca: 6). According to the palpability of tumour mass, the sensitivity and accuracy rate of palpable early masses are 56.6% and 88.4% by physical examination, 57.9% and 96.4% by mammography, 77.6% and 82.5% by echography respectively. Those of nonpalpable diseases are 29.0% and 88.8% by physical examination, 42.9% and 91.2% by mammography, and 32.1% and 76.8% by echography. The sensitivity of echography for palpable breast cancer is extremely high. On the other hand, mammography is the most effective for nonpalpable cancer. For the detection of noninvasive carcinoma, characteristics of nipple discharge, measurement of CEA in nipple discharge and erosion of the nipple are other important factors.

Immunohistochemical study of the post-natal development of the folliculo-stellate cells in the rat anterior pituitary gland.

We investigated the post-natal development of cell-to-cell communication within the rat anterior pituitary gland cells using immunohistochemistry of the S-100 protein. Tissues of animals from 10 to 60 days of age were analyzed. At 10 days of age, S-100 protein-containing cells were rarely observed. With age, the population of S-100 immunostained cells increased until day 40 when they were found to be quite numerous. No further changes were noted from day 40 through day 60. From our previous studies, we conclude that the cells which reacted with the S-100 antiserum were folliculo-stellate cells and their developmental pattern parallels that of the hypophyseal-gonadal axis.