Association of platelet response to cilostazol with clinical outcome and CYP genotype in patients with cerebral infarction.

INTRODUCTION: Cilostazol, an anti-platelet drug that inhibits phosphodiesterase 3, is beneficial for patients with atherothrombosis. In contrast to other anti-platelet drugs such as aspirin and thienopyridines, little information is available on the relationship between platelet responses to cilostazol and clinical outcomes. MATERIALS AND METHODS: We conducted a prospective study on patients with cerebral infarction who were treated with cilostazol. The platelet response to cilostazol was assessed with our new assay for the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) subsequent to the pharmacological action of cilostazol. Patients were followed up for 2 years and the relationship between VASP assay results and the recurrence of thrombotic events was examined. We also investigated the effects of CYP3A5 and CYP2C19 genotypes involved in the metabolism of cilostazol on the platelet response to cilostazol. RESULTS: Among the 142 patients enrolled, 130 completed the 2-year follow-up and the recurrence of thrombotic events was noted in 8 (6.2%). VASP phosphorylation levels were significantly lower in patients with than in those without recurrence. The combined genotype of CYP3A5*1/*3 and CYP2C19*1/*1 was associated with a low level of VASP phosphorylation, while either genotype was not. A multivariate analysis showed that high residual platelet reactivity during the cilostazol treatment, which was defined by a low response of platelet VASP phosphorylation to cilostazol, was an independent risk factor for the recurrence of thrombotic events. CONCLUSION: A low platelet response to cilostazol determined by a new platelet assay was associated with the recurrence of thrombotic events in patients with cerebral infarction.

Expansion of the antigenic repertoire of a single T cell receptor upon T cell activation.

Activated T cells and their naive precursors display different functional avidities for peptide/MHC, but are thought to have identical antigenic repertoires. We show that, following activation with a cognate mimotope (NRP), diabetogenic CD8(+) T cells expressing a single TCR (8.3) respond vigorously to numerous peptide analogs of NRP that were unable to elicit any responses from naive 8.3-CD8(+) T cells, even at high concentrations. The NRP-reactive, in vivo activated CD8(+) cells arising in pancreatic islets of nonobese diabetic mice are similarly promiscuous for peptide/MHC, and paradoxically this promiscuity expands as the aviditiy of the T cell population for NRP/MHC increases with age. Thus, activation and avidity maturation of T lymphocyte populations can lead to dramatic expansions in the range of peptides that elicit functional T cell responses.

Aortic atheromatous lesions developed in APA hamsters with streptozotocin induced diabetes: a new animal model for diabetic atherosclerosis. 1. Histopathological studies.

To develop an adequate animal model for atherosclerosis in large vessels of patients with diabetes, i.e. diabetic macroangiopathy, we induced diabetes in APA hamsters with a single injection of streptozotocin (SZ) and examined the aorta histopathologically and immunohistochemically. As a result, hyperglycemia and hyperlipidemia were continuously observed for 26 weeks after the SZ injection (WAI) in APA hamsters. Fatty streaks characterized by a subendothelial accumulation of many foam cells were observed, limited to the aortic arches as early as 6 WAI. In addition to larger fatty streaks developing with the duration of diabetes, fibrous plaques and plaques containing calcium deposits or cholesterol clefts developed at 26 WAI. These lesions are generally similar to the atheromatous lesions developed in humans. Moreover, depositions of apolipoprotein E and advanced glycation end-products immunohistochemically detected in the lesions were very similar to those found in humans. The diabetic APA hamster is therefore considered to be a useful model for studying the formation of atheromatous lesions in diabetic patients.

APA hamster model for diabetic atherosclerosis. 2. Analysis of lipids and lipoproteins.

Syrian hamsters of the APA strain (APA hamsters) have recently been shown to have atheromatous lesions in the aortic arches under diabetic condition induced by a single injection of streptozotocin (SZ). In that model, fatty streaks, which are the initial lesions of atherogenesis, develop by 6 weeks after the injection (WAI). In this study, we evaluated plasma lipid concentrations and lipoprotein profiles in diabetic APA hamsters at 6 WAI to reveal the early stage of atherogenesis clinicopathologically. As a result, by biochemical analysis, hyperglycemic APA hamsters showed signs of hypercholesterolemia and hypertriglyceridemia. Low-density lipoprotein (LDL) cholesterol significantly increased, but high-density lipoprotein (HDL) cholesterol significantly decreased. Agarose gel electrophoresis showed an obvious increase in the fractions of chylomicron, LDL and abnormal lipoprotein. Plasma LDL in diabetic animals was in a state more susceptible to oxidization. In addition, a significant increase in glycated LDL was also found in the diabetic animals by enzyme linked immunosorbent assay (ELISA). Moreover, lipid peroxidation product (4-hydroxynonenal (4 HNE))-adducted proteins and advanced glycation end-products (AGE) were immunohistochemically detected in the foam cells of the fatty streaks. These results revealed that diabetic APA hamsters had hyperlipidemia characterized by increases in chylomicron, LDL and abnormal lipoprotein, and suggested that oxidized LDL and/or glycated LDL might be actively uptaken by macrophages and play an important role in the initial stage of atherogenesis.

Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin.

Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions. In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization. Of these isoforms, only type III was detected in epidermis and hair follicles. Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase. In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription-polymerase chain reaction and by rapid amplification of cDNA ends methods. This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770). To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria. The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III. The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively. An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles. Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone. The enzyme was also expressed in the cuticle layer of hair. On the other hand, expression of the enzyme in the epidermis was very low. These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation.

[Hemophagocytic syndrome in a patient with rheumatoid arthritis].

A 62-year-old man with 16 year-history of rheumatoid arthritis (RA) was admitted due to progressive pancytopenia, general fatigue, and high fever. He was treated with 5 mg methotrexate weekly in RA. His bone marrow examination revealed a decreased nuclear cell count (2.1 x 10(4)/microliter), megakaryocyte count (16/microliter), and macrophages phagocytizing blood cells (13.2%), indicating the presence of hemophagocytic syndrome. No infections agent was detected in cultures derived from his blood or other sources. The serological tests for several viruses revealed no obvious viral etiology. The systemic lymphonodes were not swelling. Administration of 40 mg prednisolone daily improved his abnormal hematological findings. This is a case of RA accompanied by hemophagocytic syndrome, which is a rare complication of RA.

[Acute mitral regurgitation caused by malignant lymphoma of the posterior mediastinum].

An 82-year-old man was admitted to our hospital in September 1996 due to dysphagia and cardiomegaly. Physical examination detected the fourth heart sound and a Levine III/VI systolic murmur in the cardiac apex. Surface lymph nodes were not palpable. LDH 662 IU/I was detected by laboratory examinations, and ultrasound cardiography showed grade 3 mitral regurgitation. Computed tomography revealed a huge mass in the posterior mediastinum, pressing the heart from the posterior direction. Thereafter, a left pleural effusion developed and aspiration was performed. Cytological examination of the fluid showed clusters of lymphoid cells with a positive immunophenotype for CD10, CD19 and HLA-DR. Chromosome analysis revealed complex abnormal karyotypes including t(8;14) (q24;32). A diagnosis of B cell lymphoma was made, and combination chemotherapy consisting of cyclophosphamide, THP-adriamycin, vincristine, and prednisolone was initiated. The patient's mass disappeared promptly, and his mitral reguration subsided. We reported this case because malignant lymphoma of the posterior mediastinum is rare, and because we are unaware of any previous reports of malignant lymphoma causing acute mitral regurgitation.

[A case of Sjögren's syndrome associated with advanced hemolytic anemia caused by worsening autoimmune cholangiopathy].

We report a rate case of autoimmune cholangiopathy (AIC) and autoimmune hemolytic anemia (AIHA) in a patient with Sjögren's syndrome. A 59-year-old woman was admitted to Matsuyama Red Cross Hospital in September 1996 because of worsening liver dysfunction. She had suffered from keratoconjunctivitis sicca and xerostomia and had been diagnosed as having Sjögren's syndrome in February 1994, based on histological examination of the minor salivary gland and sialography, and positivity for SS-A and SS-B antibody. Liver dysfunction had first become evident in September 1995. Histological examination of a liver biopsy specimen obtained by laparoscopy showed that the structure of the hepatic lobules was mostly preserved, whereas most of the biliary ducts were transformed, being consistent with AIC. On admission, the patient was given 40 mg of prednisolone. Although the serum transaminase level decreased, jaundice persisted and hemolytic anemia developed. Further administration of 60 mg of prednisolone and plasmapheresis ameliorated the hemolytic anemia and cured the jaundice. We consider that an increased immunological response caused by the worsening AIC might have played a role in the development of AIHA in the present case.

[A case of the active intestinal tuberculosis detected during the examination of the right pleural effusion].

A 76-year-old female was admitted to our hospital because of fever and the right pleural effusion. On the analysis of pleural effusion, the total cell count was 6720/microliter with 95% lymphocytes, and ADA was 38.1 U/l. The culture of pleural effusion was negative, and the smear and PCR for Mycobacterium were also negative. For examinations, we performed eterography that showed cicatricial strictures of intestine. X-ray examination of the colonated colonoscopy showed ulcers (circular type), shortening of the colon, Bauhin's value insufficiency and diverticulum-like deformity. Then, she was diagnosed as intestinal tuberculosis. The smear and PCR of biopsy specimens from the lesion were positive, and antituberculotic therapy was effective. Finally, the culture of pleural effusion for Mycobacterium tuberculosis was positive after 8 weeks. We thought intestinal examination may be useful for the diagnosis of tuberculosis, when lymphocyte-rich exudative pleural effusion of unknown etiology is seen.

[Neoplastic angioendotheliosis with Roth's spots].

A 81-year-old woman was admitted with fever, anemia and an elevation of serum LDH on September, 1995. She had anasarca and various abnormalities of the ocular fundus including Roth's spots. Skin eruption and psycho-neurological abnormalities were not seen. Laboratory data exhibited Hb 7.6g/dl, Plt 9.3 x 10(4)/microliters, WBC 6,300/microliters and LDH 1932 IU/l. Antibiotics, antituberculotic drugs and steroids were not effective and she died on October, 1995 with heart failure. An autopsy revealed that the small vessels of lungs, kidneys and the mucosa of the bladder and the gastrointestinal tract were filled with large mononuclear cells. Immunohistochemically these cells were positive in LCA and L26 stains, and negative in UCHL-1 stains. There fore this case was finally diagnosed as neoplastic angioendotheliosis (NAE). Although there has been no case report of NAE with Roth's spots previously, there is the possibility that Roth's spots appeared as a sign of vascular occlusion due to NAE. Roth's spots may be noteworthy as a sign of NAE that has few clinical features.

Morphologic changes in hepatocyte nuclei of streptozotocin (SZ)-induced diabetic mice.

Morphological examinations were carried out on hepatocyte nuclei of streptozotocin (SZ)-induced diabetic mice. The area of hepatocyte nuclei in diabetic mice was about two times larger than that in control mice, and the incidence of hepatocytes with intranuclear inclusions was 3.4 +/- 0.2% in diabetic mice and 0% in control mice, respectively. Although the incidence of binuclear hepatocytes was not significantly different between diabetic (14.5 +/- 4.6%) and control mice (16.4 +/- 4.4%), the morphology of the nuclei of binuclear hepatocytes was apparently different between diabetic and control mice. Namely, the nuclei of binuclear hepatocytes of control mice were round and identical in ultrastructural appearance, and they did not differ from those of mononuclear diploid hepatocytes. On the other hand, the nuclei of binuclear hepatocytes of diabetic mice were not identical in distribution pattern of chromatin granules, and they frequently varied in size and showed irregular contours.

Modification of spontaneous renal lesion of APA hamsters by streptozotocin-induced diabetes.

Syrian hamsters of the APA strain (APA hamsters) develop spontaneous mesangial thickening in the renal glomeruli from an early age. They also develop focal and segmental glomerulosclerosis (FSG) at and after 6 months of age. In this study, histopathological, immunohistochemical and lectin histochemical examinations were conducted to clarify the modification of the spontaneous renal lesions of APA hamsters by streptozotocin(SZ)-induced diabetes. Histopathological analysis revealed that the expansion of the mesangial region was more prominent and the thickening of the glomerular basement membrane (GBM) was weaker in SZ-treated animals than in non-treated ones. Immunohistochemical analysis suggested that type IV collagen and laminin were involved in the expansion of the mesangial region and thickening of the GBM. In lectin histochemical analysis, podocytes, capillary endothelial cells, GBM and a part of mesangial region of SZ-treated animals were positive for RCA120 and GSL-I with neuraminidase-pretreatment although they were negative for these lectins in non-treated animals. These results suggest that the spontaneous glomerular lesion of APA hamsters is modified qualitatively and quantitatively by SZ-induced diabetes.

Ultrastructure of atheromatous lesions experimentally induced in Syrian hamsters of the APA strain.

In order to examine whether diabetes enhances, primary aortic lesions up to atherosclerotic ones, mild primary lesions were induced in aorta of APA hamsters by an administration of vitamin D2 (VD) and/or stop-and-reflow (SR)-operation, a modification of renal artery clamping. At 2 months after the treatment with the combination of VD-administration and SR-operation, atheromatous lesions, characterized by an appearance of many foam cells in the intima, were observed in the abdominal aorta, the site of SR-operation, in streptozotocin (SZ)-induced diabetic APA hamsters. Foam cells in the atheromatous lesions were originated from smooth muscle cells and monocyte/ macrophages. On the other hand, neither VD-administration alone nor SR-operation alone developed atheromatous lesions in SZ-induced diabetic APA hamsters. In conclusion, we succeeded in a rapid induction of atherosclerotic lesions in abdominal aorta of SZ-induced diabetic APA hamsters by the combination of VD-administration and SR-operation.

Age-related histochemical and ultrastructural changes in renal glomerular mesangium of APA hamsters.

Syrian hamsters of the APA strain (APA hamsters) develop spontaneous mesangial thickening in the renal glomeruli from an early age. They also develop focal and segmental glomerulosclerosis (FSG) at and after 6 months of age. In this study, histopathological, histochemical and electron microscopical examinations were conducted to clarify the age-related renal glomerular changes in spontaneous FSG of APA hamsters. Histopathological analysis revealed that expansion of the mesangial region and thickening of the glomerular basement membrane (GBM) in the glomeruli of APA hamsters progressed with age. These age-related changes appeared earlier in male animals. Immunohistochemical analysis suggested that type IV collagen was responsible for the expansion of the mesangial region and thickening of the GBM. In lectin histochemical analysis, positive sites for WGA with and without neuraminidase pretreatment and PNA with neuraminidase pretreatment were detected mainly in podocytes and were expanded with age. Ultrastructurally, the increase in basement membrane-like materials in the mesangial matrix, development of intracellular organella of mesangial cells and migration of a part of the mesangial cell cytoplasm into the GBM were observed at and after 6 months of age. These features suggested that mesangial cells played an important role in the age-related expansion of the mesangial region and thickening of the GBM.

Age-related changes of aorta in Syrian hamsters of APA strain.

Age related changes in thoracic aorta (TA) and abdominal aorta (AA) of male APA hamsters from 3 to 12 months of age were examined morphometrically and ultrastructurally. The nuclear density of smooth muscle cells (SMCs) was larger in AA than in TA, and it decreased with advancing age. In contrast, the collagen fibre density was larger in TA than in AA, and it increased correlatively with aging, especially in TA. Electron microscopic examinations revealed that subendothelial cystic spaces and aggregations of fragments of elastic and collagen fibres were found at 3 months of age and progressed with advancing age in TA, while they were not evident in AA even at 12 months of age. Irregularity of medial SMC contours and an amount of SMC-associated collagen fibres were more prominent in TA than in AA throughout the experimental period. Degenerative changes of endothelial cells and medial SMCs progressed with aging in both TA and AA, and degenerated SMCs were characterized by aggregations of swollen mitochondria.

Ultrastructure of spontaneous glomerular lesions in syrian hamsters of APA strain.

Electron microscopic observations were carried out on the spontaneous glomerular lesions in male APA hamsters from 3 to 12 months of age. Until 6 months of age, focal expansion of mesangial region due to an increase of matrix material and mesangial cells was characteristic, and segmental thickening of capillary basement membrane and partial effacement of foot processes of podocytes were also sometimes observed. At 12 months of age, although all of these changes became more severe, the most prominent alteration was found in podocytes, which showed various degenerative changes. No deposition of amyloid fibrils was detected in any portion of the glomerulus.