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Although it has been well documented that aberrant major histocompatibility complex class II molecules may contribute to the development of autoimmune disorders, the precise mechanisms responsible for their tissue-specific expression remain unknown. Here we show that estrogen deficiency induces aberrant class II major histocompatibility complex expression in exocrine glands via interactions between epithelial cells and plasmacytoid dendritic cells. Relatively modest but functionally significant expression levels of major histocompatibility complex class II and class II transactivator molecules were observed in the exocrine glands of ovariectomized (Ovx) C57BL/6 (B6) mice, but were not seen in the exocrine glands of control B6 mice. We observed that the salivary dendritic cells adjacent to the apoptotic epithelial cells positive for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, were activated in Ovx mice, but were not activated in control mice. We obtained evidence that the salivary gland cells express both interferon regulatory factor-1 and class II transactivator type IV molecules in Ovx mice. Salivary gland cells from Ovx mice were also capable of inducing the activation of antigen-specific T cells from OT-II transgenic mice. These findings indicate that estrogen deficiency initiates class II transactivator type IV mRNA expression in exocrine glands via interactions between epithelial cells and plasmacytoid dendritic cells, suggesting that plasmacytoid dendritic cells play a pivotal role in gender-based autoimmune disorders in postmenopausal women.

Although several autoimmune diseases are known to develop in postmenopausal women, the mechanisms by which estrogen deficiency influences autoimmunity remain unclear. Recently, we found that retinoblastoma-associated protein 48 (RbAp48) induces tissue-specific apoptosis in the exocrine glands depending on the level of estrogen deficiency. In this study, we report that transgenic (Tg) expression of RbAp48 resulted in the development of autoimmune exocrinopathy resembling Sjögren's syndrome. CD4(+) T cell-mediated autoimmune lesions were aggravated with age, in association with autoantibody productions. Surprisingly, we obtained evidence that salivary and lacrimal epithelial cells can produce interferon-gamma (IFN-gamma) in addition to interleukin-18, which activates IFN regulatory factor-1 and class II transactivator. Indeed, autoimmune lesions in Rag2(-/-) mice were induced by the adoptive transfer of lymph node T cells from RbAp48-Tg mice. These results indicate a novel immunocompetent role of epithelial cells that can produce IFN-gamma, resulting in loss of local tolerance before developing gender-based autoimmunity.

A mutant strain with defective thymic selection of the Long-Evans Cinnamon (LEC) rat was found to spontaneously develop inflammatory bowel disease (IBD)-like colitis. The secretion of Th1-type cytokines including IFN-gamma and IL-2 from T cells of mesenteric lymph node cells (MLNs) and lamina propria mononuclear cells, but not spleen cells, in LEC rats was significantly increased more than that of the control Long-Evans Agouti rats through up-regulated expression of T-bet and phosphorylation of STAT-1 leading to NF-kappaB activation. In addition, the number of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells of the thymus, MLNs, and lamina propria mononuclear cells from LEC rats was significantly reduced, comparing with that of the control rats. Moreover, bone marrow cell transfer from LEC rats into irradiated control rats revealed significantly reduced CD25(+)Foxp3(+) Treg cells in thymus, spleen, and MLNs compared with those from control rats. Indeed, adoptive transfer with T cells of MLNs, not spleen cells, from LEC rats into SCID mice resulted in the development of inflammatory lesions resembling the IBD-like lesions observed in LEC rats. These results indicate that the dysfunction of the regulatory system controlled by Treg cells may play a crucial role in the development of IBD-like lesions through up-regulated T-bet, STAT-1, and NF-kappaB activation of peripheral T cells in LEC rats.

Objective: To determine whether oral administration of rebamipide, a mucosal protective agent, is effective in the treatment of Sjögren's syndrome (SS) in the NFS/sld mouse model of the disease., Methods: NFS/sld mice were given daily oral doses of rebamipide (0.3 mg/kg of body weight or 3 mg/kg) or vehicle alone starting from the age of 4 weeks to the age of 8 weeks. The volume of saliva and tears was monitored during and after treatment. After the final dose, histologic features of the tissues, TUNEL+ apoptotic duct cells in affected glands, T cell and cytokine function, and levels of immunoglobulin isotypes and serum autoantibodies were examined., Results: The 3-mg/kg dose of rebamipide prevented the development of autoimmune lesions. The average volume of saliva in rebamipide-treated mice was significantly higher than that in control mice. We found decreased TUNEL+ apoptotic duct cells in the salivary and lacrimal glands of rebamipide-treated mice as compared with control mice. Rebamipide treatment suppressed the activation of CD4+ T cells and Th1 cytokines (interleukin-2, interferon-gamma) associated with impaired NF-kappaB activity. Production of serum autoantibodies, IgM, and IgG1 was clearly inhibited., Conclusion: Our findings demonstrate the efficacy of oral administration of rebamipide in the treatment of SS. Rebamipide represents a new therapeutic strategy for the treatment of patients with sicca symptoms caused by SS, as well as for patients with other diseases.

Klippel-Trenaunay-Weber syndrome (KTS) is an unusual congenital anomaly characterized by cutaneous hemangiomas, varicosities and bony hypertrophy of the extremities. Herein the case is reported of a 24-year-old man with urethral bleeding from hemangiomas associated with KTS that were successfully managed by endoscopic sclerotherapy. A 23-G puncture needle was inserted into the bleeding vein to inject a 5% solution of monoethanolamine oleate (Oldamine), which is typically used for sclerotherapy of esophageal vasix. At a 4-month follow-up, the patient only had slightly bloodstained urethral discharge, and is doing well. This is the first case reporting endoscopic sclerotherapy for a KTS-associated urethral hemangioma.

Although receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) signaling has been shown to prolong the survival of mature dendritic cells (DCs), the association of RANKL pathway with Fas-mediated apoptosis is obscure. Here, we found that bone marrow-derived DCs (BMDCs) from the Fas-deficient strain MRL/lpr mice, could survive much longer than normal DCs. The expressions of Bcl-x and Bcl-2 and the nuclear transport of NF-kappaB of RANKL-stimulated BMDCs from MRL/lpr mice were significantly up-regulated. By contrast, Fas expression of BMDCs from normal C57BL/6 and MRL(+/+) mice was increased by RANKL stimulation, and an enhanced DC apoptosis was found when stimulated with both RANKL and anti-Fas mAb, which was associated with activation of caspase-3 and caspase-9. Furthermore, the expression of FLIP(L), an inhibitory molecule against Fas-mediated apoptosis, in normal DCs was significantly decreased by RANKL and anti-Fas mAb. Indeed, the adoptive transfer of RANKL-stimulated DCs resulted in rapid acceleration of autoimmunity in MRL/lpr recipients. These findings indicate that the crosstalk between RANKL and Fas signaling in DCs might control immune tolerance.

We previously reported the results of a pilot study of intermittent androgen deprivation (IAD) therapy in which surveillance was performed when PSA level fell below 0.3 ng/ml and androgen deprivation was resumed when PSA level exceeded 2.0 ng/ml. In the present study, we compared the duration of androgen dependence in patients treated with IAD with that in patients with continuous androgen deprivation (CAD) therapy. Forty-six patients with clinically localized or metastatic prostate cancer, or biochemical recurrence after radical prostatectomy were treated with IAD from 1995 to 2003. Patients in or after the second cycle of IAD (30 patients) were evaluated for duration of androgen dependence. Patients whose serum PSA nadir became <0.3 ng/ml (33 patients) represented a control group of CAD. The overall 5-year biochemical progression-free rate was 58% and 89% in the IAD and CAD groups, respectively; there was no significant difference between the two groups (p=0.5). Subgroup analysis showed that, irrespective of metastasis, the 5-year biochemical progression-free survival rate in the IAD group was not significantly different from that in the CAD group. However, IAD offered significantly better results for well-differentiated prostate cancer, whereas CAD offered significantly better results for moderately or poorly differentiated prostate cancer. The results obtained from this retrospective and nonrandomized study suggested that IAD may be a feasible treatment for well-differentiated prostate cancer.

Although tissue-specific apoptosis in the exocrine glands in estrogen-deficient mice may contribute to the development of autoimmune exocrinopathy, the molecular mechanism responsible for tissue-specific apoptosis remains obscure. Here we show that RbAp48 overexpression induces p53-mediated apoptosis in the exocrine glands caused by estrogen deficiency. RbAp48-inducible transfectant results in rapid apoptosis with p53 phosphorylation (Ser9) and alpha-fodrin cleavage. Reducing the expression of RbAp48 through small interfering RNA inhibits the apoptosis. Prominent RbAp48 expression with apoptosis was observed in the exocrine glands of C57BL/6 ovariectomized (OVX) mice but not in OVX estrogen receptor alpha(-/-), p53(-/-), and E2F-1(-/-) mice. Indeed, transgenic expression of the RbAp48 gene induced apoptosis in the exocrine glands but not in other organs. These findings indicate that estrogen deficiency initiates p53-mediated apoptosis in the exocrine gland cells through RbAp48 overexpression and exerts a possible gender-based risk of autoimmune exocrinopathy in postmenopausal women.

Immature CD4+CD8+ thymocytes, which are generated in the thymic cortex, are induced upon positive selection to differentiate into mature T lymphocytes and relocate to the thymic medulla. It was recently shown that a chemokine signal via CCR7 is essential for the cortex-to-medulla migration of positively selected thymocytes in the thymus. However, the role of the cortex-to-medulla migration in T cell development and selection has remained unclear. The present study shows that the developmental kinetics and the thymic export of mature thymocytes were undisturbed in adult mice lacking CCR7 or its ligands (CCR7L). The inhibition of sphingosine-1-phosphate-mediated lymphocyte egress from the thymus led to the accumulation of mature thymocytes in the cortex of CCR7- or CCR7L-deficient mice, unlike the accumulation in the medulla of normal mice, thereby suggesting that mature thymocytes may be exported directly from the cortex in the absence of CCR7 signals. However, the thymocytes that were generated in the absence of CCR7 or CCR7L were potent in causing autoimmune dacryoadenitis and sialadenitis in mice and were thus incapable of establishing central tolerance to organ-specific antigens. These results indicate that CCR7-mediated cortex-to-medulla migration of thymocytes is essential for establishing central tolerance rather than for supporting the maturation or export of thymocytes.

Thiazolidinediones (TZD) are effective agents for the treatment of hyperglycemia, and appear ideal in diabetic patients with progressive or end-stage renal disease because of its predominant hepatic clearance. Troglitazone, the first available TZD for clinical use, was withdrawn due to safety concerns; however, studies completed with this agent can provide a better understanding of the class effect of TZDs. This study was an open-label, controlled clinical trial examining the safety and efficacy of troglitazone in type 2 diabetic patients with end-stage renal disease (ESRD). Twelve subjects were randomized to parallel study groups and treated for 6 mo with or without troglita-zone at a maximum dose of 600 mg/d in addition to continuing their previous diabetes medications (insulin or sulfonylurea). The results showed no significant differences in glycemic control with or without troglit-azone treatment for 6 mo. However, there was a significant reduction in insulin dosage with troglitazone treatment (22.9 +/- 7.3 units/d) than without troglita-zone treatment (54 +/- 12.9 units/d) (p < 0.05), as well as the change in the insulin dosage from baseline between the two groups (troglitazone, -8.4 units vs control, +4.3 units, p < 0.05). Weight changes and aspartate amino-transferase levels greater than 1.5 times the upper limit of normal were not observed in participants of either treatment group. This study demonstrates that troglit-azone was safe and effective for the treatment of hyper-glycemia in patients requiring dialysis, and strongly supports the clinical use of currently available TZDs in diabetic patients with renal failure.

The alpha-fodrin N-terminal portion (AFN) autoantigen mediates in vivo immunoregulation of autoimmune responses in primary Sjögren's syndrome (SS). We further examined this process and found that cleavage products of AFN were frequently detected in the salivary gland duct cells of SS patients. In in vitro studies using human salivary gland HSY cells, anti-Fas-induced apoptosis resulted in specific cleavage of alpha-fodrin into the 120-kd fragment, in association of alpha-fodrin with mu-calpain, and activation of caspase 3. Significant proliferative responses against AlphaFN autoantigen were observed in the peripheral blood mononuclear cells (PBMCs) from SS patients with higher pathological score (grade 4) and with short duration from onset (within 5 years). In vivo roles of AFN peptides were investigated using PBMCs from patients with SS, systemic lupus erythematosus, and rheumatoid arthritis. Significant proliferative T-cell responses of PBMCs to AFN peptide were detected in SS but not in systemic lupus erythematosus or rheumatoid arthritis. AFN peptide induced Th1-immune responses and accelerated down-regulation of Fas-mediated T-cell apoptosis in SS. Our data further elucidate the in vivo role of AFN autoantigen on the development of SS and suggest that the AFN autoantigen is a novel participant in peripheral tolerance.

Insulin resistance and beta-cell dysfunction, two factors central to the pathogenesis of type 2 diabetes, were studied in relation to the development of diabetes in a group of participants with impaired glucose tolerance in the Diabetes Prevention Program (DPP) at baseline and after specific interventions designed to prevent diabetes. Participants were randomly assigned to placebo (n = 1,082), metformin (850 mg twice a day) (n = 1,073), or intensive lifestyle intervention (n = 1,079). The diabetes hazard rate was negatively associated with baseline insulin sensitivity (hazard rate ratio = 0.62-0.94 per SD difference, depending on treatment group and measure of sensitivity) and with baseline insulin secretion (hazard rate ratio = 0.57-0.76 per SD). Improvements in insulin secretion and insulin sensitivity were associated with lower hazard rates in all treatment arms (hazard rate ratio = 0.46-0.95 per SD increase and 0.29-0.79 per SD increase, respectively). In multivariate models that included the three metabolic variables (changes in body weight, insulin sensitivity, and insulin secretion) each significantly and independently predicted progression to diabetes when adjusted for the other two variables. The intensive lifestyle intervention, which elicited the greatest reduction in diabetes incidence, produced the greatest improvement in insulin sensitivity and the best preservation of beta-cell function after 1 year, whereas the placebo group, which had the highest diabetes incidence, had no significant change in insulin sensitivity and beta-cell function after 1 year. In the metformin group, diabetes risk, insulin sensitivity, and beta-cell function at 1 year were intermediate between those in the intensive lifestyle and placebo groups. In conclusion, higher insulin secretion and sensitivity at baseline and improvements in response to treatment were associated with lower diabetes risk in the DPP. The better preventive effectiveness of intensive lifestyle may be due to improved insulin sensitivity concomitant with preservation of beta-cell function.

Nephrogenic adenoma is a relatively rare, benign tumor of the urinary tract. We experienced two cases of nephrogenic adenoma originating in the bladder. The first patient was a 61-year-old man. Two papillary tumors were found on the bladder. Transurethral resection of the bladder tumor (TUR-Bt) was performed. The second patient was a 72-year-old man who had a history of TUR-Bt for the bladder tumor and a history of left nephroureterectomy for left ureteral tumor. Cystoscopy showed a papillary tumor on the top of the bladder wall and TUR-Bt was performed. In both cases, the histopathological diagnosis was the nephrogenic adenoma. Our cases are the 40th and 41st cases of the nephrogenic adenoma of the bladder reported in the Japanese literature.

Ectopic PTH secretion by tumor cells has been described as the cause of hypercalcemia associated with malignancy in the absence of osteolytic bone lesions. Although there have been case reports of elevated PTH and hypercalcemia in patients with rhabdomyosarcoma, to date ectopic PTH secretion by malignant cells has not been definitively shown. The possibility of PTH production by pleural-based metastatic nasopharyngeal rhabdomyosarcoma cells in a 62-yr-old Japanese male with hypercalcemia was investigated. The patient's serum PTH level was found to be elevated at 62.22 pmol/L, and pleural fluid PTH level was 47.28 pmol/L and PTHrP level was 3.7 pmol/L. RT-PCR of mRNA extracted from rhabdomyosarcoma cells in the pleural fluid was performed with the addition of PTH and PTHrP exonic primer sets yielded only a cDNA fragment of approx 150 bp consistent with the expected PTH fragment. Sequence analysis of a nested primer PCR fragment confirmed PTH mRNA sequence. We believe this patient to have had hypercalcemia secondary to ectopic PTH secretion, as we have identified the presence of PTH mRNA in tumor cells. We speculate that the overexpression of PTH in rhabdomyosarcoma cells results from molecular rearrangement of the PTH gene. The finding of a normal PTH DNA sequence of the PCR fragment suggests the likelihood of alterations in regulatory sequences.

A 52-year-old man had bilateral ureteral stents placed before treatment for ureteral and renal stones, but did not return for treatment and follow-up. Three years later, he complained of hematuria and vertigo. An abdominal X-ray revealed large renal and ureteral stones rising from and enveloping the stent. A bilateral percutaneous nephrostomy was placed. The right ureteral stent was easily removed with a cystoscope. The left ureteral stone was separated from the stent by ureteroscopic lithotripsy (TUL) and percutaneous nephroscopic lithotripsy (PNL). The left stent was torn off and difficult to remove because of encrustation. It was finally removed through an endoscopic procedure. Right PNL and extracorporeal shock wave lithotripsy (ESWL) were performed and all stones and stents were extracted. He was stone-free at 4 months.

Older adults often cite nocturia as one of the most bothersome lower urinary tract symptoms (LUTS). We investigated the efficacy and safety of intranasal desmopressin in the treatment of nocturia due to nocturnal polyuria on 12 patients (ten men, two women) ranging in age from 53 to 77 years (mean 67 years). All patients experienced more than two episodes of nocturia per night, and had a nocturnal urine volume greater than 35% of the daily voided volume, measured using a 3-day voiding diary with a frequency-volume chart. They began taking intranasal desmopressin (10 microg) at bedtime. When compared with the baseline data, the nocturnal urine volume, (928 +/- 307 versus 469 +/- 251 ml, p = 0.0007) and nocturnal frequency (4.8 +/- 2.0 versus 2.8 +/- 1.8, p = 0.0009) were significantly decreased. The daytime urine volume (1,008 +/- 458 versus 930 +/- 419 ml, p = 0.49) did not change significantly. The unine osmolarity (420 +/- 143 versus 598 +/- 158 mOsm/kg, p = 0.0065), and urine sodium levels (100 +/- 32 versus 140 +/- 60 mEq/l, p = 0.007) increased significantly, whereas the serum sodium levels (141 +/- 3 versus 135 +/- 7 mEq/l, p = 0.048) decreased significantly. Among the 12 patients, 5 (41.6%) patients reported side effects, including headache in 1, edema in 1 and hyponatremia in 3. The patient with edema discontinued medication, but the other 4 patients continued their medication and the side effects subsided. In conclusion, desmopressin is an effective treatment for adult patients complaining of nocturia due to nocturnal polyuria. One should be aware of the potential side effects including hyponatremia.

Autoimmune regulator (AIRE) gene mutation is responsible for the development of organ-specific autoimmune disease with monogenic autosomal recessive inheritance. Although Aire has been considered to regulate the elimination of autoreactive T cells through transcriptional control of tissue-specific Ags in thymic epithelial cells, other mechanisms of AIRE-dependent tolerance remain to be investigated. We have established Aire-deficient mice and examined the mechanisms underlying the breakdown of self-tolerance. The production and/or function of immunoregulatory T cells were retained in the Aire-deficient mice. The mice developed Sjogren's syndrome-like pathologic changes in the exocrine organs, and this was associated with autoimmunity against a ubiquitous protein, alpha-fodrin. Remarkably, transcriptional expression of alpha-fodrin was retained in the Aire-deficient thymus. These results suggest that Aire regulates the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus, at least against this ubiquitous protein. Rather, Aire may regulate the processing and/or presentation of self-proteins so that the maturing T cells can recognize the self-Ags in a form capable of efficiently triggering autoreactive T cells. With the use of inbred Aire-deficient mouse strains, we also demonstrate the presence of some additional factor(s) that determine the target-organ specificity of the autoimmune disease caused by Aire deficiency.

Autosomal dominant aplasia of lacrimal and salivary glands (ALSG; OMIM 180920 and OMIM 103420) is a rare condition characterized by irritable eyes and dryness of the mouth. We mapped ALSG to 5p13.2-5q13.1, which coincides with the gene fibroblast growth factor 10 (FGF10). In two extended pedigrees, we identified heterozygous mutations in FGF10 in all individuals with ALSG. Fgf10(+/-) mice have a phenotype similar to ALSG, providing a model for this disorder. We suggest that haploinsufficiency for FGF10 during a crucial stage of development results in ALSG.

Objective: A wide spectrum of extraglandular manifestations may occur in patients with Sjogren's syndrome (SS), but the mechanisms responsible for in vivo progression are still obscure. We undertook this study to evaluate the age-related changes during the development of extraglandular autoimmune lesions, including arthritis, in the murine model of primary SS, and to evaluate the possible relationship between age-related disturbance of activation-induced cell death and the in vivo kinetics against autoantigens., Methods: A total of 126 NFS/sld mice were investigated at ages 2, 4, 6, 10, 12, 18, 20, and 24 months. Cytokine production was tested using culture supernatants from anti-CD3 monoclonal antibody-stimulated T cells. Anti-single-stranded DNA (anti-ssDNA) antibodies, Ig isotypes (IgG1, IgG2a), rheumatoid factor (RF), and anti-type II collagen (anti-CII) antibodies were detected by enzyme-linked immunosorbent assay. Proliferative T cell responses against each of 3 recombinant alpha-fodrin proteins and against CII were analyzed., Results: Autoimmune arthritis developed in SS model mice until age 24 months. Significant elevations in serum levels of RF, anti-ssDNA antibodies, and anti-CII antibodies were found in aging SS model mice. A high titer of serum autoantibodies against alpha-fodrin fragments (containing different epitopes that were originally identified in primary SS model mice) was frequently detected in young and aged SS model mice. Moreover, we found that alpha-fodrin autoantigen induced Th1 immune responses and accelerated disturbance of Fas-mediated T cell apoptosis in aged SS model mice., Conclusion: These results indicate that age-related disturbance of activation-induced cell death via bystander T cell activation may play a crucial role in the development of autoimmune arthritis in a murine model of SS.

A 53-year old woman was admitted with of sensory disturbance and weakness of lower limbs which had progressed slowly in the previous ten years. A diagnosis of sensory ataxic neuropathy associated with Sjögren's syndrome was made. A sural nerve biopsy showed marked loss of myelinated fibers. A muscle biopsy revealed atrophy of muscle fibers along with perivascular cellular infiltration. The dorsal root ganglia have been considered to be the main site affected in the ataxic neuropathy in Sjögren's syndrome. However, the evidence for that was meager. The perivascular inflammatory change observed in the muscle may also have existed in the peripheral nervous system including the dorsal root ganglia.

Objective: To determine whether eyedrop administration of an anti-CD4 monoclonal antibody (mAb) is effective in the treatment of Sjögren's syndrome (SS) using a mouse model of the disease., Methods: The anti-CD4 mAb was administered daily into the eyes of mice with SS from ages 4 to 8 weeks or ages 10 to 12 weeks. During treatment, tear volume was monitored and after final treatment, histologic features of the lacrimal and salivary glands, the phenotypes and function of T cells, and serum titers of anti-alpha-fodrin antibody were examined., Results: Eyedrop administration of anti-CD4 mAb before the onset of SS prevented the autoimmune pathology seen in the lacrimal glands but not that in the salivary glands. Furthermore, eyedrop administration of anti-CD4 mAb after the development of SS inhibited mononuclear cell infiltration and the destruction of parenchyma only in the lacrimal glands. Eyedrop administration of anti-CD4 mAb suppressed the local activation of CD4+ T cells rather than deleting CD4+ T cells, which reduced the expansion of pathologic CD4+ T cells against alpha-fodrin., Conclusion: These results demonstrate the remarkable efficacy of anti-CD4 mAb eyedrops in the treatment of SS eye symptoms, which illustrates a new antibody-based therapeutic strategy for patients with eye problems caused by SS as well as other diseases.

Purpose of Review: Primary Sjögren syndrome is an autoimmune disorder characterized by lymphocytic infiltrates and destruction of the salivary and lacrimal glands, and systemic production of autoantibodies to the ribonucleoprotein particles SS-A/Ro and SS-B/La. The purpose of this review is to discuss recent advances in the pathogenesis of primary Sjögren syndrome., Recent Findings: Although several candidate autoantigens including alpha-fodrin have been reported in Sjögren syndrome, the pathogenic roles of the autoantigens in initiation and progression of SS are still unclear. It is possible that individual T cells activated by an appropriate self antigen can proliferate and form a restricted clone. Recent evidence suggests that the apoptotic pathway plays a central role in tolerizing T cells to tissue-specific self antigen, and may drive the autoimmune phenomenon. Cleavage of certain autoantigens during apoptosis may reveal immunocryptic epitopes that could potentially induce autoimmune response. The studies reviewed imply that Fas-mediated cytotoxicity and caspase-mediated alpha-fodrin proteolysis are involved in the progression of tissue destruction in Sjögren syndrome. Fas ligand (FasL), and its receptor Fas are essential in the homeostasis of the peripheral immune system. It can be considered that a defect in activation-induced cell death of effector T cells may result in the development of autoimmune exocrinopathy in Sjögren syndrome., Summary: Although the mechanisms by which estrogen deficiency influences autoimmune lesions remain unclear, it is possible that antiestrogenic actions might be a potent factor in the formation of pathogenic autoantigens., (Copyright 2004 Lippincott Williams & Wilkins)

The aims of this study were to evaluate the in vivo effects of estrogen deficiency in MRL/lpr mice as a model for rheumatoid arthritis and to analyze the possible relationship between immune dysregulation and receptor activator of nuclear factor-kappaB ligand (RANKL)-mediated osteoclastogenesis. Experimental studies were performed in ovariectomized (Ovx)-MRL/lpr, Ovx-MRL+/+, sham-operated-MRL/lpr, and sham-operated-MRL+/+ mice. Severe autoimmune arthritis developed in younger Ovx-MRL/lpr mice until 24 wk of age, whereas these lesions were entirely recovered by pharmacological levels of estrogen administration. A significant elevation in serum rheumatoid factor, anti-double-stranded DNA, and anti-type II collagen was found in Ovx-MRL/lpr mice and recovered in mice that underwent estrogen administration. A high proportion of CD4(+) T cells bearing RANKL was found, and an enhanced expression of RANKL mRNA and an impaired osteoprotegerin mRNA was detected in the synovium. An increase in both osteoclast formation and bone resorption pits was found. These results indicate that estrogen deficiency may play a crucial role in acceleration of autoimmune arthritis associated with RANKL-mediated osteoclastogenesis in a murine model for rheumatoid arthritis.

Objective: The pathologic mechanisms responsible for organ-specific tissue damage in primary Sjögren's syndrome (SS) remain unclear, but it has been suggested that the pathology is mediated by autoreactive CD4+ T cells infiltrating the salivary and lacrimal glands. This study was undertaken to investigate whether alpha-fodrin autoantigen-specific autoreactive CD4+ T cells are capable of inducing autoimmune lesions., Methods: A total of 45 synthetic alpha-fodrin peptides designed to be 20 amino acid residues in length were generated. To establish an autoreactive T cell line, limiting dilution analysis (LDA) was performed on lymph node cells (LNCs) in the presence of alpha-fodrin peptides. The effects of adoptive transfer of autoreactive CD4+ T cells into normal syngeneic recipients were investigated., Results: Autoreactive CD4+ T cell lines that recognize synthetic alpha-fodrin peptide, which produced Th1 cytokines and showed cytotoxic activities, were established in a murine model for SS. T cell receptor V(beta) usage and third complementarity-determining region (CDR3) sequences indicated that in some cases V(beta)6-CDR3 genes matched between the tissue-infiltrating T cells and the autoreactive T cell lines. Adoptive transfer of the autoreactive CD4+ T cells into normal syngeneic recipients induced autoimmune lesions quite similar to those of SS., Conclusion: Our data help to elucidate the pathogenic mechanisms responsible for tissue destruction in autoimmune exocrinopathy and indicate that autoreactive CD4+ T cells play a pivotal role in the development of murine SS.

Background and Purpose: Moyamoya disease (MMD) is a rare entity that results in progressive occlusion of the arteries of the circle of Willis, but the pathogenesis of MMD is unknown., Methods: MMD sera (n=32) were tested for anti-endothelial cell antibodies by enzyme-linked immunoassays and flow cytometric analysis. Apoptosis was induced in human umbilical vein endothelial cells by tumor necrosis factor-alpha., Results: We found that a high proportion of MMD sera had anti-endothelial cell antibodies with apoptotic stimuli. Prominent reactivities of MMD sera (72%) with recombinant human alpha-fodrin were observed., Conclusions: Our study demonstrates that MMD sera contain a high incidence of anti-alpha-fodrin autoantibodies, providing new insight into the mechanisms of occlusion of MMD arteries.

Although a number of autoimmune diseases are known to develop in postmenopausal women, the mechanisms by which estrogen deficiency influences autoimmune lesions remain unclear. We speculate that antiestrogenic actions might be a potent factor in the formation of pathogenic autoantigens. Previously, we have identified 120-kd alpha-fodrin as an important autoantigen in Sjögren's syndrome (SS). When healthy C57BL/6 (B6) mice were treated with an ovariectomy (Ovx), we found a significant increase in TUNEL(+)-apoptotic epithelial cells in the salivary gland cells associated with alpha-fodrin cleavage during 2 and 3 weeks after Ovx. By contrast, no apoptotic cells were found in estrogen receptor-alpha knockout mice. In in vitro studies using primary cultured mouse salivary gland cells and human salivary gland cells, we found a cleavage product of 120-kd alpha-fodrin in cells that had undergone tamoxifen (Tam)-induced apoptosis through caspase activation, especially caspase-1. Adoptive transfer of alpha-fodrin-reactive T cells into Ovx-B6 and -SCID mice resulted in the development of autoimmune exocrinopathy quite similar to SS. These results suggest that estrogen deficiency exerts a crucial influence on autoantigen cleavage, and may cause, in part, autoimmune exocrinopathy in postmenopausal women.

Adult Clinical Trials Group Study 349 examined the immunology, virology, and safety of 40 mg/d prednisone as an adjunct to antiretroviral therapy in 24 HIV-infected subjects with >200 CD4+ T cells/mm in a randomized placebo-controlled trial. After 8 weeks, median lymphocyte and CD4+ cell numbers increased >40% above baseline values (p =.08). No effect was observed on markers of cell activation or apoptosis, although the proportion of CD28+ CD8+ T cells increased (p =.006). Prednisone inhibited monocyte TNFalpha production without affecting T-cell responses to antigens or mitogens. Two subjects assigned to prednisone were subsequently found to have asymptomatic osteonecrosis of the hip. Many questions remain regarding the role of activation-induced sequestration and apoptosis as causes of progressive CD4+ T-cell loss in AIDS. The potential role of corticosteroids as tools to examine this question will be limited by concerns regarding their toxicity; however, further studies of other agents to limit cellular activation in AIDS are warranted.

Primary Sjögren syndrome (SS) is an autoimmune disease characterized by diffuse lymphoid cell infiltrates in the salivary and lacrimal glands, resulting in symptoms of dry eye and dry mouth due to insufficient secretion. Previously, we have identified the 120 kDa alpha-fodrin as an important autoantigen on the development of SS in both animal model and SS patients, but the mechanism of a -fodrin cleavage leading to tissue destruction in SS remains unclear. In murine primary SS model, tissue-infiltrating CD4+ T cells purified from the salivary glands bear a large proportion of Fas ligand (FasL), and the salivary gland duct cells constitutively possess Fas. Infiltrating CD4+ T cells identified significant 51Cr release against mouse salivary gland (MSG) cells. In vitro studies demonstrated that apoptotic MSG cells result in a specific alpha-fodrin cleavage into 120 kDa, and preincubation with caspase-inhibitor peptides blocked alpha-fodrin cleavage. The treatment with caspase-inhibitors in vivo prevented the development of autoimmune lesions in the salivary and lacrimal glands. Thus, an increased activity in caspase cascade may be involved in the progression of alpha-fodrin proteolysis and tissue destruction on the development of SS.

The cysteine endoprotease cathepsin S mediates degradation of the MHC class II invariant chain Ii in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on autoantigen presentation and organ-specific autoimmune diseases in a murine model for Sjögren syndrome. Specific inhibitor of cathepsin S (Clik60) in vitro markedly impaired presentation of an organ-specific autoantigen, 120-kDa alpha-fodrin, by interfering with MHC class II-peptide binding. Autoantigen-specific T cell responses were significantly and dose-dependently inhibited by incubation with Clik60, but not with inhibitor s of cathepsin B or L. Clik60 treatment of mouse salivary gland cells selectively inhibited autopeptide-bound class II molecules. Moreover, the treatment with Clik60 in vivo profoundly blocked lymphocytic infiltration into the salivary and lacrimal glands, abrogated a rise in serum autoantibody production, and led to recovery from autoimmune manifestations. Thus, inhibition of cathepsin S in vivo alters autoantigen presentation and development of organ-specific autoimmunity. These data identify selective inhibition of cysteine protease cathepsin S as a potential therapeutic strategy for autoimmune disease processes.

The in vivo role of autoantigen cleavage during apoptosis in autoimmune diseases remains unclear. Previously, we found a cleavage product of 120-kDa alpha-fodrin as an important autoantigen in the pathogenesis of primary Sjögren's syndrome (SS). In the murine primary SS model, tissue-infiltrating CD4(+) T cells purified from the salivary glands bear a large proportion of Fas ligand, and the salivary gland duct cells constitutively possess Fas. Infiltrating CD4(+) T cells, but not CD8(+) T cells, identified significant (51)Cr release against mouse salivary gland cells. In vitro studies demonstrated that apoptotic mouse salivary gland cells result in a specific alpha-fodrin cleavage into 120 kDa and that preincubation with caspase inhibitor peptides blocked alpha-fodrin cleavage. In vivo treatment with caspase inhibitors N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone and N-acetyl-Asp-Glu-Val-Asp-al-CHO into the murine model results in dramatic inhibitory effects on the development of autoimmune lesions and in restoration of sicca syndrome. Furthermore, we found that immunization with recombinant alpha-fodrin protein identical with an autoantigen into normal recipients induced autoimmune lesions similar to SS. These data indicate that prevention and induction of autoimmune exocrinopathy is dependent on autoantigen cleavage via caspase cascade and that caspase inhibitors might provide a new therapeutic option directed at reducing tissue damage in the murine model for SS.

Cell-free translation/translocation systems are broadly applied to examine gene expression and characterize the structure-function relationship of gene products. We present the characterization of Xenopus egg extract (XEE) translocation and processing of proteins synthesized in rabbit reticulocyte lysate. The XEE was prepared from eggs laid by adult female frogs that received serial injections of gonadotropins. The eggs were then dejellied in 2% L-cysteine-HCl and the cytoplasm extracted by centrifugation at 10,000 rpm for 15 min. The in vitro translocation and processing of XEE was examined with a cell-free translation system containing reticulocyte lysate, and appropriate messenger ribonucleic acid (RNA) or complementary deoxyribonucleic acid plasmids with RNA polymerase. Cell-free production of the following proteins were used to assess posttranslational modifications: Escherichia coli beta-lactamase for signal sequence cleavage, Saccharomyces cerevisiae alpha-mating factor for translocation and N-linked glycosylation, the soluble protein luciferase for functional activity, and the membrane-bound human insulin receptor for translation efficiency. All translation products were identified by [35S]-methionine labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The results demonstrate that (1) XEE produces near-complete signal sequence and N-glycosylation processing of proteins synthesized in reticulocyte lysate, (2) XEE contains endoplasmic reticulum-equivalent microsomes, which allows for protein translocation and protease protection, (3) the addition of XEE in the translation reaction does not affect synthesis and chemiluminescence activity of luciferase, (4) XEE is efficient in processing the nascent 160-kDa human insulin receptor precursor, a transmembrane protein, and (5) as compared to canine pancreatic microsomes, XEE translocation efficiency is minimally decreased with the addition of dimethylsulfoxide. These results are the first description of the combined use of XEE with reticulocyte lysate and clearly demonstrate a higher efficiency of translocation and processing compared to canine pancreatic microsomes. This method of cell-free translation and processing allows for more extensive in vitro examination of posttranslational modifications of secretory and membrane-bound proteins.

The cell-free examination of the human insulin receptor during biogenesis may provide a greater understanding of the elements that contribute to the acquisition of receptor function. The insulin receptor precursor components were produced in a cell-free system and the insulin binding ability of the [35S]methionine-labeled translation products was determined. The processed proreceptor represented by a 190 kDa band was retained on insulin-linked biotin-streptavidin agarose or an insulin column. The insulin binding 190 kDa band migrated slower than the non-binding 190 kDa band on SDS-PAGE which suggests that covalent modifications account for these differences. The trypsin-digested product of the 190 kDa proreceptor was also retained on insulin-linked biotin-streptavidin agarose, however the alpha-subunit precursor was retained on insulin agarose to a much lesser degree. We conclude that a significant fraction of the processed, in vitro translated insulin proreceptor acquires insulin binding ability.

Objective: To identify metabolic and body composition changes associated with HIV-1 infection in a cross-sectional study of individuals stratified by immunologic status and body mass., Design: Metabolic abnormalities including glucose intolerance and changes in body morphology have recently been described in HIV-1-infected individuals following therapy with protease inhibitor-containing highly active anti-retroviral therapy. Although this is suggestive of a direct drug effect, the possibility that HIV infection may induce a tendency towards such underlying derangements should be considered. HIV-infected patients are heterogeneous with respect to immunologic status and body mass. In examining the underlying effect of HIV-1 on metabolic and body composition parameters, stratification by various immunologic and body mass categories may give divergent results that would not be detected otherwise., Methods: Thirty male participants were categorized into four cohorts: non-wasting HIV-seronegative controls, non-wasting HIV-infected patients with relatively intact immune function (CD4 cell count > 500 x 10(6)/l); non-wasting individuals with AIDS (CD4 cell count < 200 x 10(6)/l); and individuals with AIDS wasting., Results: Increased fasting plasma insulin and waist-to-hip ratios were found specifically in non-wasting individuals with AIDS compared with HIV-negative controls., Conclusions: Our study emphasises the importance of both body mass and immune function in studying metabolic and body composition abnormalities associated with HIV-1 infection. The association of increased waist-to-hip ratios and hyperinsulinemia suggestive of insulin resistance in non-wasting individuals with AIDS suggest that the tendency towards these metabolic abnormalities may be related to the HIV infectious process or to factors associated with immunologic dysfunction.

Recent studies have demonstrated that human herpesvirus 6 (HHV-6) and HHV-7 interact with HIV-1 and alter the expression of various surface molecules and functions of T lymphocytes. The present study was undertaken to clarify whether coreceptors for HIV-1, CXCR4 and CCR5, are necessary for HHV-6 and HHV-7 infection. Although CXCR4 and CCR5 appeared not to be the coreceptors for these viruses, marked down-regulation of CXCR4, but not CCR5, was detected in HHV-6 variant A (HHV-6A)-, HHV-6 variant B (HHV-6B)-, and HHV-7-infected cells. Down-regulation of CXCR4 resulted in impairment of chemotaxis and a decreased level of elevation of the intracellular Ca2+ concentration in response to stromal cell-derived factor-1. Northern blot analysis of mRNAs extracted from HHV-6A-, HHV-6B-, and HHV-7-infected CD4+ T lymphocytes demonstrated a markedly decreased level of CXCR4 gene transcription, but the posttranscriptional stability of CXCR4 mRNA was not significantly altered. These data demonstrate that unlike HIV-1, HHV-6 and HHV-7 infections do not require expression of CXCR4 or CCR5, whereas marked down-regulation of CXCR4 is induced by these viruses, suggesting that HHV-6 and HHV-7 infections may render CD4+ T lymphocytes resistant to T lymphocyte-tropic HIV-1 infection.

T22 ([Tyr5,12, Lys7]-polyphemusin II) is a strong anti-HIV compound. Six analogs of T22 and two natural forms were synthesized. Of them, all downsized peptides (14 residues; TW70, T131, T134, and T140) showed a higher selectivity index than did other, 17- or 18-residue peptides. In particular, T134 and T140 showed both lower cytotoxicity and higher antiviral activity than did T22 against HIV infection of MT-4 cells, an HTLV-I-bearing T cell line. To clarify the inhibitory mode of T22 and its analogs, we used a single-round replication assay (luciferase assay), in which different envelope-bearing pseudotypes were used to infect CXCR4- or CCR5-bearing U87 cells via CD4. All of the analogs inhibited T cell line-tropic strain HXB-2 (X4) and dual-tropic strain 89.6 (R5X4) HIV infections mediated by CXCR4, but had no effect on macrophage-tropic strain ADA (R5) or 89.6 HIV infections mediated by CCR5. The inhibition by T134 (IC50 of 2.70 nM) and T140 (IC50 of 0.432 nM) was also stronger than that by T22 (IC50 of 5.05 nM). The binding of anti-CXCR4 monoclonal antibody 12G5 to lymphoma-derived T cell line Sup-T1 was more efficiently blocked by T134 and T140 than by T22. Taken together, T22 and its analogs T134 and T140 exerted their inhibition by specific binding to CXCR4. The marked increase in the anti-HIV activity of T134 and T140 was ascribed to an enhancement in their ability to bind to CXCR4.

The relationship between radical intensity and biological activity of cacao husk extracts was investigated. Electron spin resonance (ESR) spectroscopy demonstrated that the radical intensity of hexane, acetone, methanol and 70% methanol extracts increased with water-solubility. Several fractions of these husk extracts, separated by different column chromatographies, significantly inhibited the cytopathic effect of human immunodeficiency virus (HIV) infection in parallel with their radical intensity. However, their cytotoxic activity against human leukemic and carcinoma cell lines is not always correlated with their radical intensity. Water-soluble and lipophilic compounds might induce cytotoxic activity by different mechanisms.

T22, an analog of polyphemusin II (18 amino acid residues), was found to block T-tropic human immunodeficiency virus type 1 (HIV-1) entry into target cells as a CXCR4 inhibitor. We synthesized T134, a small analog (14 amino acid residues) of T22 with reduced positive charges. T134 exhibited highly potent activity and significantly less cytotoxicity in comparison to that of T22. T134 prevents the anti-CXCR4 monoclonal antibody from binding to peripheral blood mononuclear cells but has no effect on the binding of anti-CCR5 monoclonal antibodies. Since T134 inhibits the binding of stromal cell-derived factor-1 (SDF-1) to MT-4 cells, it seems that T134 prevents HIV-1 entry by binding to CXCR4. The bicyclam AMD3100 has also been shown to block HIV-1 entry via CXCR4 but not via CCR5. Both T134 and AMD3100 are CXCR4 antagonists and low-molecular-weight compounds but have different structures. Our results indicate that T134 is active against wild-type T-tropic HIV-1 strains and against AMD3100-resistant strains.

The radical modulation activity of lignins prepared from the cone of Pinus elliottii var. Elliottii was investigated, using ESR spectroscopy. These lignins produced radical(s) under alkaline conditions, and the radical intensity was increased with increasing pH. Lower concentrations of lignins slightly reduced the radical intensity of sodium ascorbate, whereas higher concentrations of lignins enhanced both the radical intensity and cytotoxic activity of sodium ascorbate. Lignins effectively scavenged superoxide anion, produced by hypoxanthine-xanthine oxidase reaction. Elliottii lignins significantly inhibited the human immunodeficiency virus (HIV)-induced cytopathic effect, in similar fashions to other natural, commercial and synthetic lignins. Pretreatment of mice with lignins significantly protected them from the lethal infection with E. coli. Crude alkaline extracts of Elliottii pine cone displayed similar magnitude of activity with lignins. These data further supports the medicinal efficacy of plant extracts.

T22 ([Tyr5,12, Lys7]-polyphemusin II) is an 18-residue peptide amide, which has strong anti-HIV activity. T22 inhibits the T cell line-tropic (T-tropic) HIV-1 infection through its specific binding to a chemokine receptor CXCR4, which serves as a coreceptor for the entry of T-tropic HIV-1 strains. Herein, we report our finding of novel 14-residue CXCR4 inhibitors, T134 and T140, on the basis of the T22 structure. In the assays we examined, T140 showed the highest inhibitory activity against HIV-1 entry and the strongest inhibitory effect on the binding of an anti-CXCR4 monoclonal antibody (12G5) to CXCR4 among all the CXCR4 inhibitors that have been reported up to now.

To elucidate the cellular activation mechanisms of lipoteichoic acid (LTA) compared with those of lipopolysaccharide (LPS), a quantitatively major LTA fraction, QM-1M, was prepared from hot phenol-water extracts of Enterococcus hirae (ATCC 9790) by hydrophobic octyl-Sepharose chromatography and by ion-exchange membrane (QMA-Mem Sep 1010) chromatography as a 60% 1-propanol- and 1 M NaCl-eluted fraction. Unlike the reference Escherichia coli LPS, QM-1M did not demonstrate any ability to induce cytokines in a human whole blood culture system in this study, whereas QM-1M induced a few cytokines such as interleukin (IL)-8 and tumor necrosis factor-alpha in human monocytic THP-1 cell and human peripheral mononuclear cell (PBMC) cultures in the absence of serum. Fetal calf and human sera decreased the above cytokine induction by QM-1M in THP-1 and PBMC cultures, whereas sera increased activities of the reference LPS. IL-8 induction in the absence of serum in response to QM-1M was demonstrated to proceed through a CD14-independent pathway unlike the reference LPS.

We have previously found that T22 ([Tyr(5,12), Lys7]-polyphemusin II) has strong anti-human immunodeficiency virus (HIV) activity, and that T22 inhibits T cell-line-tropic HIV-1 infection mediated by CXCR4/fusin. T22 is an 18-residue peptide amide, which takes an antiparallel beta-sheet structure that is maintained by two disulfide bridges. Structure-activity relationship (SAR) studies on T22 have disclosed the contributions of each region of T22 to activity or cytotoxicity, and have provided the following useful information to develop new CXCR4 antagonists: The number of Arg residues in the N-terminal and C-terminal regions of T22 is closely related to anti-HIV activity. Addition of a variety of functional groups at the N-terminal end results in increases in activity. Disulfide rings, especially the major disulfide loop, are indispensable for anti-HIV activity and maintenance of the beta-sheet structure. Trp3 can be replaced by other aromatic residues (Tyr, Phe and L-2-naphthylalanine). Between two repeats of Tyr-Arg-Lys, which are a characteristic structure in T22, Tyr-Arg-Lys in the N-terminal portion is more closely associated with anti-HIV activity and maintenance of the beta-sheet structure. A positive charge in the side chain at the (i + 1) position of the beta-turn region is necessary for strong activity. Through these studies, we have found several compounds having higher selectivity indexes (50% cytotoxic concentration/50% effective concentration) than that of T22.

The radical modulation activity of hot water and alkaline extracts from leaf of Ceriops decandra, a mangrove plant, was investigated using ESR spectroscopy. IR and NMR analyses demonstrate that the leaf extracts have a lignin-like polyphenolic structure. All these extracts produced radical(s) under alkaline conditions. The radical intensity of sodium ascorbate was slightly reduced at lower concentrations of the extracts, but it was synergistically enhanced at higher concentrations. All the extracts effectively scavenged superoxide anion, produced by hypoxanthine-xanthine oxidase reaction. Pretreatment of mice with the extracts significantly protected them from the lethal infection by E. coli. Similar activity was found in lignins from pine seed shell of Pinus parviflora Sieb. et Zucc. These data further support the medicinal efficacy of plant extracts.

Hyperinsulinemia has been associated with cardiovascular disease (CVD), but whether this relation is independent of other CVD risk factors is uncertain. Most studies have focused on coronary heart disease (CHD), but few have included peripheral vascular disease (PVD) and stroke. Moreover, evidence in elderly and minority populations is limited. Between 1991 and 1993, 3562 elderly (71 to 93 years) Japanese-American men from the Honolulu Heart Program were examined and had fasting insulin levels measured. Hyperinsulinemia, defined as a fasting insulin > or =95th percentile among nonobese men with normal glucose tolerance and no diabetic history or medication use, was observed in 22% of the population. Subjects with hyperinsulinemia had a more adverse CVD risk factor profile and had higher age-adjusted prevalences of CHD, angina, PVD, thromboembolic stroke, and hemorrhagic stroke compared with those without hyperinsulinemia. Age-adjusted fasting insulin levels but not 2-hour levels were also significantly elevated (P<.01) in those with prevalent CVD compared with those without. In logistic regression analyses, adjustment for multiple CVD risk factors attenuated the relations of hyperinsulinemia with CHD, angina, and PVD to nonsignificant levels, whereas those involving thromboembolic and hemorrhagic stroke were strengthened and remained significant (odds ratios=2.27 and 7.53, 95% confidence intervals=1.25 to 4.13 and 1.65 to 34.25, respectively). When multivariate analyses were restricted to nondiabetic subjects, associations were slightly weaker and in general nonsignificant. Nondiabetic men with thromboembolic stroke were twice as likely to have hyperinsulinemia as those who were stroke-free, although this association was of borderline significance (odds ratio= 1.99, 95% confidence interval=0.95 to 4.17, P=.069). In subjects with elevated total cholesterol levels, somewhat stronger associations were observed for PVD and stroke but not for CHD. Although further prospective studies are indicated, particularly for PVD and stroke, these cross-sectional results are consistent with an indirect role for insulin in CVD, wherein hyperinsulinemia or an underlying insulin-resistant state may adversely affect other CVD risk factors or serve as a marker for an atherogenic or thrombogenic state.

The pattern of expression of HNF-3beta in organizing centers and axial structures during early vertebrate development suggests an important role for this protein in the establishment of the vertebrate body plan. To establish whether the pattern of expression during embryogenesis is species specific, a comparative immunohistochemical study of two mammalian species, the house shrew, insectivore, and the mouse was carried out; it is difficult to obtain accurate morphological differences from the study of remotely related animals. The earliest expression of HNF-3beta appeared in the node and hypoblast (or endoderm) in both species, where the presumptive foregut endoderm showed intense immunoreactivity prior to the formation of the axial mesoderm, suggesting a role different from that in axial formation. The anterior extension of immunopositive axial mesoderm and the median band of the neural plate varied between the two species, and was delayed in the house shrew. HNF-3beta in the anterior end of the foregut disappeared transiently in the house shrew but persisted in the mouse embryo. An asymmetric pattern of distribution in the primitive streak was also observed in the mouse but not in the house shrew. The present immunohistochemical study elucidated that the distribution of HNF-3beta is conserved initially but soon manifests species specificities in development even between mammalian species.

The topographic patterns of peripheral receptors and effectors seem to contribute to the construction of the neuronal circuit in the central nervous system (CNS) in mammals. Many patterns replicating those of the periphery have been found in the CNS, and fasciculation has been regarded as having a central role in the pattern replication. The house shrew, Suncus murinus, is an excellent species in which to study this topic because it has a vibrissae system arranged in a single ordered fashion and extraordinarily well-developed trigeminal spinal tracts. Using immunostaining and retrograde-tracing techniques, we examined the developmental pattern of the maxillary nervous system in the house shrew. The results indicate that the basic pattern of axonal extension reiterates with a parallel arrangement throughout the course of development except at a site in the brainstem where the central processes bifurcate into ascending and descending branches. Dorsoventral inversion of the peripheral pattern in the spinal tract occurs with this dual-leveled bifurcation in association with the mediolaterally ordered entry of the central processes into the brainstem. The basic pattern of the central processes is established prior to the appearance of the vibrissae, indicating that the basic topographic pattern of the maxillary nerve is not related to the vibrissae system. The fasciculation pattern does not correspond to the overall layout of the arrays of vibrissae, and there are frequent exchanges of axons between fascicles both in the periphery and centrally. The parallel organization of the majority of the processes, together with the free exchange of processes between fascicles, suggests that these processes have an important role in the formation of the fasciculation and somatotopic patterns.