The natural killer T-cell ligand alpha-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice.

Diabetes in non-obese diabetic (NOD) mice is mediated by pathogenic T-helper type 1 (Th1) cells that arise because of a deficiency in regulatory or suppressor T cells. V alpha 14-J alpha 15 natural killer T (NKT) cells recognize lipid antigens presented by the major histocompatibility complex class I-like protein CD1d (refs. 3,4). We have previously shown that in vivo activation of V alpha 14 NKT cells by alpha-galactosylceramide (alpha-GalCer) and CD1d potentiates Th2-mediated adaptive immune responses. Here we show that alpha-GalCer prevents development of diabetes in wild-type but not CD1d-deficient NOD mice. Disease prevention correlated with the ability of alpha-GalCer to suppress interferon-gamma but not interleukin-4 production by NKT cells, to increase serum immunoglobulin E levels, and to promote the generation of islet autoantigen-specific Th2 cells. Because alpha-GalCer recognition by NKT cells is conserved among mice and humans, these findings indicate that alpha-GalCer might be useful for therapeutic intervention in human diseases characterized by Th1-mediated pathology such as Type 1 diabetes.

Effects of ML-236b (compactin) on sterol synthesis and low density lipoprotein receptor activities in fibroblasts of patients with homozygous familial hypercholesterolemia.

We studied biochemical genetics of low density lipoprotein (LDL) receptor mutations in fibroblasts from six homozygous and five heterozygous patients with familial hypercholesterolemia (FH). Three of six homozygotes are receptor-negative type and the other three homozygotes are receptor-defective type. In the cells from three receptor-negative homozygotes, the receptor binding, internalization, and degradation of (125)I-LDL were 0.5+/-0.3 ng/mg protein (mean+/-SEM), 14+/-8 and 8+/-6 ng/mg protein per 6 h (four normal cells; 44+/-3, 386+/-32, and 1,335+/-214 ng/mg protein per 6 h), respectively. In the cells from three receptor-defective homozygotes, the receptor binding, internalization, and degradation of (125)I-LDL were 6+/-2, 29+/-8, and 90+/-32 ng/mg protein per 6 h, respectively. In these six homozygotes, two pairs of siblings are included. Two siblings in the same family were classified as receptor-negative and two siblings in another family were classified as receptor-defective. The receptor-negative phenotypes and the receptor-defective phenotypes bred true in individual families. The cells from five heterozygotes showed approximately 46% of the normal activities of receptor.ML-236B, competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), completely inhibited the incorporation of [(14)C]acetate into digitonin-precipitable sterols in fibroblasts from normal subjects and heterozygous and homozygous patients with FH with the concentration of 0.5 mug/ml. However, at 0.05 mug/ml of ML-236B sterol synthesis in fibroblasts from homozygotes was not completely suppressed in contrast to normal and heterozygous cells. Moreover, after preincubation with 0.05 mug/ml of ML-236B for 24 h in medium containing lipoproteins, sterol synthesis in the cells from receptor-negative homozygote showed 75% of the initial activity compared with that of 25% without preincubation. In the cells from a normal subject and a heterozygote, sterol synthesis was inhibited even after preincubation. These results suggest that (a) the inhibitory effect of ML-236B is overcome in homozygote cells by their high intracellular levels of HMG-CoA reductase and (b) that a higher dose of ML-236B may be required to lower serum cholesterol levels in FH homozygotes than in heterozygotes.

Molecular evidence that most but not all carcinosarcomas of the uterus are combination tumors.

The pathogenesis of carcinosarcoma is still a subject of controversy. In the present study, molecular techniques were applied to determine the pathogenesis of uterine carcinosarcomas. The patterns of chromosome X inactivation were analyzed, targeting a portion of exon 1 of the human androgen receptor (HUMARA) in malignant epithelial and mesenchymal components. The presence of p53 and K-ras mutations were also analyzed. H&E-stained sections of paraffin-embedded, formalin-fixed tissues were microdissected to obtain both epithelial and nonepithelial lesions from 25 carcinosarcomas, and DNAs were extracted by proteinase K digestion. Following treatment with methylation-sensitive restriction endonuclease (HhaI or HpaII), PCR amplification was performed using nested primers targeted to the HUMARA locus. Mutations in the p53 gene and K-ras gene were found in eight (32%) and six (24%) tumors, respectively. The patterns of chromosome X inactivation were different between the carcinomatous and sarcomatous components of three carcinosarcomas, indicating that these three tumors represent collision tumors. By contrast, the patterns of chromosome X inactivation, K-ras sequence, and p53 sequence were identical in both carcinomatous and sarcomatous components in 21 carcinosarcomas, indicating that these 21 tumors represent combination tumors. One case produced equivocal results that precluded determination of whether it represented a collision or combination tumor. These observations show that although most carcinosarcomas are combination tumors, some develop as collision tumors. The determination of histogenesis in individual cases of carcinosarcoma using molecular markers may be worthwhile, because the result could help predict the prognosis of individual cases and help guide clinical management.

The effect of urinary protein excretion in post-renal transplant recurrent nephrotic syndrome on basiliximab pharmacokinetics and pharmacodynamics in a pediatric patient.

A pharmacokinetic and pharmacodynamic study of Basiliximab therapy has not been reported in recurrent post-renal transplant nephrotic syndrome. We assessed the effect of urinary protein in a focal segmental glomerulosclerosis patient. We measured the serum concentrations of basiliximab as well as the rate of activated CD25-positive T lymphocytes at fixed intervals in nephrotic versus eight nonnephrotic pediatric post-renal transplant patients. A significant reduction in the antibody concentrations was observed in focal segmental glomerulosclerosis. The CD25 expression rate showed a similar trend to the pharmacokinetic data. We conclude that cases of massive urinary protein excretion need special care to maintain immunosuppression in renal transplant using Basiliximab.

A classification of in vivo bone labels after double labeling in canine bones.

Labeling patterns were classified after double bone labeling of four male beagles, 10 months of age. Calcein and oxytetracycline were given on the 18th and the 7th day prior to simultaneous iliac and 11th rib biopsies. Undecalcified sections stained with the Villanueva bone stain were studied by epifluorescence microscopy. Five structures were identified and classified: the first or green label, the interlabel layer of mineralized bone, the second or yellow label, the post double-labeled mineralized bone layer, and osteoid seams. Doubly plus singly labeled surface equalled 40.8 +/- 8.4% of the total trabecular surface of the ilium. Doubly labeled surface as a percent of the total labeled surfaces equaled 55.5% in trabeculae and 68.1% in osteons, whereas green first-singly labeled surface equaled 24.2% and 11.9%, respectively, and yellow second-singly labeled surface equaled 20.3% and 20.0%, respectively. Unequivocal examples appeared in both biopsy sites of all four dogs of bone-forming systems that lacked one or the other label, or both, and also of systems in which cessation of mineralization or of new matrix formation occurred between the two labels and between the second label and the day of biopsy. The findings prove that the On-Off states in active bone-forming sites that have been postulated by other investigators do exist. Since widely different labeling patterns appeared in different bone-forming centers in the same bone and the same animal, a local factor rather than a systemic one should control those differences at the level of the BMU.

The effects of prostaglandin E2 in rapidly growing rats: depressed longitudinal and radial growth and increased metaphyseal hard tissue mass.

The effects of 0, 0.3, 1.0, 3.0, or 6.0 mg of prostaglandin E2 (PGE2)/kg/day administered subcutaneously for 3 weeks to triple fluorochrome-labeled weanling rats are reported. Microradiographs and undecalcified sections of proximal tibiae, tibial shafts, and seventh caudal vertebrae were evaluated by static and dynamic bone histomorphometry techniques. Significant changes were observed only at higher dose levels. Proximal tibial longitudinal growth rates were depressed in doses of 1, 3, or 6 mg PGE2/kg/day. Growth plate thickness and the size of hypertrophic cartilage cells were decreased in animals given 3 and 6 mg of PGE2/kg/day, but the calculated rate of cartilage cell production was unaffected. At doses of 6 mg PGE2/kg/day, periosteal bone apposition rates between Day -1 and Day +19 in both the tibial shafts and caudal vertebral cortices were depressed by less than 25%. Cortical bone mass and endosteal bone apposition rates in the tibial shaft and caudal vertebrae were unaffected. Hard tissue mass in the secondary spongiosa of the proximal tibial metaphysis increased dramatically (28%, 44%, and 60%, respectively) in rats treated with 1, 3, or 6 mg PGE2/kg/day. In addition, the secondary spongiosa contained numerous islands of woven trabecular bone along with an increased number of trabeculae. The study demonstrates that high doses of PGE2 stimulate new woven trabecular bone production and depress longitudinal and radial growth in rapidly growing rats.

Serum lipids and coronary heart disease in heterozygous familial hypercholesterolemia in the Hokuriku District of Japan.

The serum cholesterol and triglyceride levels and the incidence of ischemic heart disease were studied in 122 (55 men and 67 women) consecutive heterozygous familial hypercholesterolemic patients in the Hokuriku district of Japan. (1) The mean +/-SD of serum cholesterol level was 354.0 +/- 71.0 mg/100 ml, which was lower than those of the Western countries by about 60--70 mg/100 ml. (2) The mean +/-SD of serum triglyceride level was 116.5 +/- 54.0 mg/100 ml. (3) The average serum cholesterol values in the 20--50-year-old group showed no differences from those of the Western countries. However, in the above 50 years of age group the serum cholesterol levels were much lower than those in the United States. (4) The occurrence of ischemic heart disease in 83 heterozygous familial hypercholesterolemic patients was 43.3%. The incidence of myocardial infarction was 20.5%. Thus, familial hypercholesterolemia is as highly atherogenic as that of the Western countries even in Japan where the low incidence of coronary heart disease in the general population has been attributed to the low level of serum cholesterol.

Serum lipids, lipoprotein lipids and coronary heart disease in patients with xanthelasma palpebrarum.

Serum lipids and lipoprotein lipids were studied in 53 patients (21 males and 32 females) with xanthelasma palpebrarum and 40 age-matched normal controls (20 males and 20 females). Patients were subdivided into patients with normolipidemia, hyperlipidemia or familial hypercholesterolemia (FH). In both male and female patients with hyperlipidemia or FH, the serum cholesterol (Chol) levels were significantly higher than in normal controls. In both male and female patients with normolipidemia or hyperlipidemia, the VLDL-Chol levels were significantly higher than in normal controls. Male patients with FH showed significantly higher levels of VLDL-Chol than normal controls. Both male and female patients with normolipidemia, hyperlipidemia or FH showed significantly higher levels of LDL-Chol, lower HDL-Chol levels and lower HDL-Chol/LDL-Chol ratios than normal controls. In both male and female patients with hyperlipidemia and in male patients with FH, the serum triglyceride (TG) levels were significantly higher than in normal controls. Both male and female hyperlipidemic patients showed significantly higher levels of VLDL-TG than normal controls. In male patients with FH, the VLDL-TG levels were significantly above the control levels. In male patients with normolipidermia, the LDL-TG levels were significantly higher than in normal controls. In both male and female patients with hyperlipidemia or FH, the LDL-TG levels were significantly higher than in normal controls. The HDL-TG levels in patients with normolipidemia (males) or FH (females) were significantly lower than in normal controls. The prevalence of coronary heart disease in patients with normolipidemia, hyperlipidemia or FH was 29.4%, 24.0% and 45.4%, respectively.

Clonal analysis of hepatocellular carcinoma and dysplastic nodule by methylation pattern of X-chromosome-linked human androgen receptor gene.

To investigate the monoclonality of hepatocellular carcinoma (HCC) and dysplastic nodule (DN) and the origin of multiple lesions, patterns of inactivation of X-linked human androgen receptor gene were studied. Fourteen of 15 patients (93%) were heterozygous in the size of the target, and were informative for clonal analysis. Monoclonal composition was demonstrated in all 17 HCCs and two DNs, whereas all non-cancerous hepatic tissues were polyclonal. Of four patients with more than two lesions of HCC or DN, two patients had two lesions with different patterns of X-chromosome inactivation, indicating that the two lesions were multicentric in origin.

More than 1,000 cases of total parathyroidectomy with forearm autograft for renal hyperparathyroidism.

Between March 1981 and December 2000, we performed 1,053 total parathyroidectomies with forearm autograft for advanced renal hyperparathyroidism (HPT). Based on histopathologic and pathophysiologic investigations, surgical treatment should be considered when parathyroid glands show nodular hyperplasia. Measuring parathyroid volume by ultrasonography was useful to detect nodular glands and to determine surgical indications. The clinical effect of parathyroidectomy on the symptoms and biochemical variables was striking. Skeletal deformity, progressive bone loss, and vessel calcification leading to high mortality risk could not be alleviated by even successful surgery, however. To prevent cardiovascular complications, parathyroidectomy should be performed in the relatively early stage of renal HPT. Total parathyroidectomy with forearm autograft is a suitable procedure for renal HPT, especially in patients who require long-term hemodialysis. For surgeons, it is important to remove all parathyroid glands, including supernumerary glands, at the initial operation and to choose adequate parathyroid tissue for the autograft to prevent persistent and recurrent HPT. Although the risk of graft-dependent recurrent HPT is not negligible, enlarged transplanted parathyroid tissue can be removed easily and noninvasively from the forearm under local anesthesia. There is no risk of hypofunction of the autograft.

Osteoprotegerin levels before and after renal transplantation.

Osteoprotegerin (OPG) is a newly identified glycoprotein that belongs to the tumor necrosis factor receptor superfamily and regulates bone mass by inhibiting osteoclastic bone resorption. The regulatory mechanism of OPG is still unclear after successful renal transplantation (RTX), however, resulting in resolution of uremia. The present study was designed to clarify the potential role of OPG in uremia and after RTX under immunosuppressive therapy. We evaluated circulating OPG levels by measuring them before and after RTX (postoperative days 2, 14, and 28). Our protocol of immunosuppressive drugs was dual therapy using cyclosporine and steroids. Serum OPG was quantitated using enzyme-linked immunosorbent assay. After successful RTX, serum OPG levels decreased significantly on day 14 and day 28 compared with the baseline level (P < 0.05). Creatinine clearance dramatically increased until day 14 and decreased thereafter. Serum OPG declines for the first 2 weeks after RTX owing to functioning allograft and decreases again for the next 2 weeks because of steroids and possible immunosuppressive agents.

Normalization of low-density lipoprotein levels and disappearance of xanthomas during pregnancy in a woman with heterozygous familial hypercholesterolemia.

Serum lipids and lipoproteins were studied prior to conception, during pregnancy, and after delivery in a woman heterozygous for familial hypercholesterolemia. Prior to conception, serum and low-density lipoprotein (LDL) cholesterol levels were 613 and 528 mg/dL, respectively. At 37-week gestation, serum and LDL cholesterols decreased to the normal levels, 226 and 90 mg/dL, respectively. At two-week postpartum serum and LDL cholesterols returned to the preconception levels, 547 and 427 mg/dL, respectively. At delivery her cutaneous xanthomas almost disappeared. The patient was challenged by ethinyl estradiol of 120 micrograms/d for two months, as a result serum cholesterol decreased from 565 to 385 mg/dL, and LDL cholesterol fell from 460 to 208 mg/dL. During her second pregnancy, serum and LDL cholesterol decreased again significantly. Thus, this case, which showed dramatic reductions of serum and LDL cholesterol levels, may be considered a new variant of heterozygous familial hypercholesterolemia, and the reductions were probably brought about by the action of estrogens, which are known to increase LDL degradation through LDL receptors.

A case of heterozygous familial hypercholesterolemia associated with hyperthyroidism: effects of triiodothyronine on low-density lipoprotein receptor and cholesterol synthesis.

A 58-year-old patient with heterozygous familial hypercholesterolemia (FH) showed normal levels of serum cholesterol (193 mg/dL) in coexistence with hyperthyroidism. After hyperthyroidism therapy with radioiodine and methimazole, the patient's lipid profile showed high concentrations of cholesterol (whole serum 318 mg/dL, VLDL 35 mg/dL, LDL 217 mg/dL, HDL 44 mg/dL). There was a significant inverse correlation between serum cholesterol levels and serum thyroxine levels (r = -0.815, p less than 0.01). Effects of triiodothyronine on LDL degradation and cholesterol synthesis from 14C-labeled acetate were studied in cultured skin fibroblasts. Triiodothyronine (T3) stimulated both LDL degradation and cholesterol synthesis in the cells from normal subjects and patients with heterozygous FH. The T3 increased cellular cholesterol synthesis markedly in the cells from patients with homozygous FH but did not increase LDL receptor activity. These results suggest that normal serum cholesterol levels in our case result in part from an enhancement of LDL receptors by thyroid hormone.

Achilles tendon thickness and ischemic heart disease in familial hypercholesterolemia.

Achilles tendon thickness (ATT) of 112 patients with familial hypercholesterolemia (FH) with and without ischemic heart disease (IHD) was measured radiographically and was compared with that of normal subjects. The mean and SD of serum cholesterol in the heterozygotes (107 cases), the homozygotes (5 cases) and the normal subjects (36 cases) were 347 +/- 63, 589 +/- 69 and 187 +/- 30 mg/dl, respectively. The mean and SEM of ATT in the heterozygotes, the homozygotes and the normal subjects were 12.5 +/- 0.4 mm, 18.6 +/- 6.6 mm, and 6.3 +/- 0.2 mm, respectively. Cutaneous xanthomas were observed in 34 out of 112 patients (30.4%). Increased ATT was observed in 95 (84.8%). IHD was diagnosed in 39 (34.8%). The ATT of FH with IHD was significantly thicker than that of FH without IHD (P less than 0.05) and that of normal subjects (p less than 0.001). Thus, the increased ATT evaluated by x-ray was the earliest clinical sign of FH and the measurement of ATT seems to be a useful adjunctive procedure for detecting familial hypercholesterolemic patients and predicting IHD in them.

Cyclooxygenase-2 expression in an adenoid cystic carcinoma of the uterine cervix.

Adenoid cystic carcinoma (ACC) of the uterine cervix is extremely rare, frequently metastasizes to distant organs, and its prognosis is poorer than squamous cell carcinoma of the uterine cervix. The reasons for its poor prognosis are unclear. This case had both an ACC and a carcinoma in situ (CIS) of the uterine cervix, so the expressions of CD34, vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) were investigated in both tumors. Hysterectomy was performed on a 76-year old woman and the uterine cervical tissues immunohistochemically analyzed. Expressions of CD34 were positive in the ACC lesions but negative in the CIS ones and angiogenesis was confirmed in ACCs. Furthermore, expressions of VEGF and COX-2 were shown in ACC, but were absent in CIS. In conclusion, the expression of COX-2 in ACC may induce the expression of VEGF, increase angiogenesis and enhance tumor growth and invasion.

Platelet-derived endothelial cell growth factor in keratinizing-type squamous cell uterine cervical cancer.

The purpose of this study was to determine whether intratumor expression of platelet-derived endothelial cell growth factor (PD-ECGF) is a prognostic factor. We examined specimens from 61 cases of carcinoma of uterine cervix (Ca-Cx). Specimens were stained with periodic acid-Schiff preparation with diastase (d-PAS) and alcian blue (pH 2.5) to identify the presence of intracellular mucin. PD-ECGF expression and microvessel count (MVC) were assessed by immunohistochemical staining. Twenty specimens stained positive for mucin. MVC correlated with clinical stage (p<0.01). There was a correlation between PD-ECGF expression and survival time (p<0.05), but no correlation was seen between mucin staining positivity, MVC, and PD-ECGF expression and survival time by the Cox proportional hazard model. Intratumor PD-ECGF expression was not a prognostic factor in keratinizing-type squamous cell carcinoma of the uterine cervix.

Angiogenesis in metastatic verrucous carcinoma of the uterine cervix.

A 65-year old woman operated on for verrucous carcinoma of the uterine cervix 13 years previously was found to have multiple small recurrent tumors in the retroperitoneal space. Tumor cell expression of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) was investigated at the second operation. Expression of VEGF was strongly positive in the tumor cells and expression of PD-ECGF was strongly positive in both the tumor cells and the interstitial cells.

Clinical significance of mucin staining in keratinizing-type squamous cell carcinoma of the uterine cervix.

Tumor tissue from 57 cases of keratinizing-type squamous cell carcinoma of the uterine cervix that had been stored in the archives of Osaka City University Hospital between May 1986 and October 1994 were stained with periodic acid-Schiff (PAS) reagent following amylase and alcian blue to identify the presence of intracellular mucin and to assess the value and significance of demonstrating the presence of mucin. Nineteen specimens (33.3%) stained positive for mucin with alcian blue or PAS following diastase. The results of mucin staining were not significant in terms of survival by Kaplan-Meier's analysis.

Cyclooxygenase-2 expression in malignant mesenchymal tumors and related uterine lesions.

The aim of this study was to determine whether the expression of cyclooxygenase-2 (COX-2) in uterine sarcoma cells and carcinosarcoma cells is associated with cell type. Nineteen sections of tissues from uterine sarcomas, carcinosarcomas, and an adenosarcoma, and endometrial stromal sarcomas, were immunohistochemically analyzed for the cellular expression of COX-2. Positive immunostaining for COX-2 was observed in 88.9% (8/9) uterine carcinosarcomas, uterine adenosarcomas but was not observed in uterine sarcomas and the endometrial stromal sarcoma (0/10). But positive immunostaining for COX-2 was observed in some sarcomatoid cells in carcinosarcoma tissue. These findings suggest that some of the sarcoma cells in uterine carcinosarcomas resemble epithelial malignant cells in regard to the increase in COX-2 expression, and support the hypothesis that some uterine carcinosarcomas are combination tumors. This may serve as a basis for new chemoprevention and treatment strategies for uterine carcinosarcomas through the inhibition of COX-2 activity.

An unusual middle fossa interdural epidermoid tumor. Case report.

The authors report an unusual case of an intracranial, interdural epidermoid tumor and cyst in a 72-year-old woman who presented with longstanding, mild numbness over her right cheek. She was initially treated conservatively, but on follow-up review the mass was found to have grown and evidence of hemorrhage was present, and therefore a subtotal resection was performed. This case should probably be classified as a paratrigeminal, interdural epidermoid cyst; this is the first known report in which magnetic resonance and computerized tomography images of such an entity are presented and discussed.