Immunohistochemical Profile of Ameloblastic Carcinoma Arising from an Amyloid-Producing Odontogenic Tumour in a Miniature Dachshund.

A 13-year-old female miniature dachshund was presented with a centrally-located sublingual mass in the rostral mandibular region. The focally ulcerated growth completely covered the left (305) and right (405) premolar teeth and partially covered the right canine teeth (404). A punch biopsy sample revealed neoplastic proliferation of odontogenic epithelium arranged in irregular cords with frequent comedo-like necrosis. Following the initial diagnosis of ameloblastic carcinoma, a bilateral rostral hemimandibulectomy was performed. Although the detailed examination of the resected mass was consistent with the initial diagnosis, it also contained birefringent congophilic, amelogenin-labelled amyloid deposits similar to an amyloid-producing odontogenic tumour (APOT) in 30-40% of the mass, in continuity with the ameloblastic carcinoma. All neoplastic cells had diffuse moderate expression of cytokeratin (CK) AE1/AE3 and CK5, diffuse mild expression of CK14 and multifocal moderate expression of CK19. Because the APOT-like growth in the mass was histologically benign, the tumour was diagnosed as an ameloblastic carcinoma arising from an APOT.

Local extensive granulomatous inflammation of the neck region and lymphangitis caused by Lichtheimia corymbifera infection in a Japanese Black calf.

A 7-month-old female Japanese Black calf developed elongated, nodular mass measuring 30 × 16 cm extended from the retropharyngeal region to mid lateral neck region. Histological examination revealed granulomatous lymphangitis with non-septate fungal hyphae recognized throughout the lesions. Fungal culture, DNA sequencing and molecular phylogenetic tree analysis confirmed the sequence of Lichtheimia corymbifera. The lymphogenous route was speculated to be the main route of fungal spread leading to the characteristic nodular appearance of this case.

Factors related to allergic transfusion reactions and febrile non-haemolytic transfusion reactions in children.

BACKGROUND AND OBJECTIVES: Allergic transfusion reactions (ATRs) and febrile non-haemolytic transfusion reactions (FNHTRs) are the two major types of transfusion-related adverse reactions (TRARs). Although prestorage leucocyte reduction and diversion of the first aliquot of blood (LR/D) could reduce FNHTRs and bacterial contamination in adult transfusion, ATRs are still problematic. In addition, there is little information about TRARs in paediatric population. MATERIALS AND METHODS: We conducted a single-centre retrospective analysis of all transfusions, except washing products, and TRARs for 153 months to evaluate related factors such as delivery of treatment and the characteristics of recipients. RESULTS: Most TRARs were FNHTRs and/or ATRs in children. In delivering blood products with LR/D, the frequencies of not only FNHTRs but also ATRs were significantly reduced with both platelet concentrates (PCs) and red cell concentrates (RCCs). TRARs of fresh-frozen plasma were infrequent in children. In addition, even after the introduction of LR/D, ATRs were significantly more frequent in patients with primary haematological and malignant diseases who received PCs and RCCs, older patients who received PCs and patients who received frequent RCCs. CONCLUSION: These results suggest that leucocytes or mediators from leucocytes are underlying cause of ATRs in addition to FNHTRs in children. Furthermore, particular characteristics of patients would be other risk factors for ATRs.

Evidence of Spontaneous Recovery of Granulosa-Theca Cell Tumour in a Heifer: A Retrospective Report.

A 14-month-old Japanese Black heifer was evaluated on Day 0 (D 0) for enlargement of the right ovary (RO). Transrectal ultrasonography (TRUS) revealed that the RO was markedly enlarged and multicystic, while the left ovary (LO) was small and inactive. The presumptive diagnosis was granulosa-theca cell tumour (GTCT), which was confirmed by markedly elevated plasma anti-Müllerian hormone (AMH) of 4.42 ng/ml. Therefore, ovariectomy of the RO was the treatment of choice. The heifer was checked by TRUS and blood sampling on D 42, D 63 and immediately before ovariectomy on D 85. On D 42, TRUS did not show marked changes in either ovary in comparison with D 0. However, on D 63, the RO had transformed into a single cyst, and on D 85, the LO had resumed cyclic activity. The RO was extracted on D 85 by hand-assisted laparoscopic ovariectomy to allow better control. Unexpectedly, histopathology revealed the lesion to be a fluid-filled cystic structure, with no neoplastic proliferation of follicular epithelium that would indicate GTCT. The wall of the cystic structure consisted of collagen fibres and a few degenerated granulosa cells. The retrospective hormonal analysis revealed that the AMH concentrations had markedly dropped on D 63 and 85, which coincided with resumption of cyclicity in the LO. These findings suggest that the GTCT had self-cured and transformed into a cyst-like structure. The heifer then received an oestrous synchronization regime on D 105, was artificially inseminated on D 115 and became pregnant.

Expression analysis of stemness genes in a rat thyroid cell line FRTL5.

Detection and analysis of a small subpopulation of cells such as stem cells or cancer stem cells are recognized to be a key technique in a recent regeneration and cancer science. However, in the thyroid, no marker that identifies stem cells has been established yet. We previously established a novel method to analyze cells collected by fluorescence-activated cell sorting (FACS), named mRNA quantification after FACS (FACS-mQ). By using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. In this study, we analyzed the expression of stemness genes in a rat thyroid cell lines FRTL5 using FACS-mQ. 3 stemness genes, NANOG, ABCG2 and GATA4, were expressed in FRTL5. In FRTL5 cells, varied expression of thyroglobulin (TG) among cells was observed by flow cytometry. Cell populations with high or low TG expression were analyzed by FACS-mQ. The cell population with low TG expression showed increased expression of the stemness genes. Furthermore, Ki67-positive cells showed increased expression of TG, which suggested that cells with high TG proliferated rapidly. These results indicated that FRTL5 contains a cell population with high stemness gene expression and less differentiated features, resembling stem cells. These cells might regulate proliferation in FRTL5.

The functional polymorphisms of VDR, GC and CYP2R1 are involved in the pathogenesis of autoimmune thyroid diseases.

Vitamin D is a multi-functional immune regulator, and a low serum concentration of vitamin D promotes autoimmune inflammation. In this study, we evaluate the association between the prognosis of autoimmune thyroid disease (AITD) and the functional polymorphisms of genes that regulate vitamin D metabolism. For 139 Graves' disease (GD) patients, 116 Hashimoto's disease (HD) patients and 76 control subjects, we genotyped the following polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP): vitamin D receptor (VDR): rs731236, rs7975232, rs2228570 and rs1544410; group-specific component (GC): rs7041 and rs4588; and CYP2R1: rs10741657. The frequency of the TT genotype for the rs731236 polymorphism was higher in GD patients than in HD patients (P = 0·0147). The frequency of the C allele for the rs7975232 polymorphism was higher in GD patients than in control subjects (P = 0·0349). The proportion of GD patients whose anti-thyrotrophin receptor antibody (TRAb) level was >51% was higher in those with the CC genotype than in those with the CA+AA genotypes (P = 0·0065). The frequency of the CC genotype for the rs2228570 polymorphism was higher in HD patients than in control subjects (P = 0·0174) and GD patients (P = 0·0149). The frequency of the Gc1Gc1 genotype for the GC polymorphism and the AG genotype for the CYP2R1 polymorphism were lower in intractable GD than in GD in remission (P = 0·0093 and 0·0268, respectively). In conclusion, genetic differences in the VDR gene may be involved in the development of AITD and the activity of GD, whereas the genetic differences in the GC and CYP2R1 genes may be involved with the intractability of GD.

Associations of single nucleotide polymorphisms in precursor-microRNA (miR)-125a and the expression of mature miR-125a with the development and prognosis of autoimmune thyroid diseases.

It is important to search the biomarker to predict the development and prognosis of autoimmune thyroid diseases (AITDs) such as Hashimoto's disease (HD) and Graves' disease (GD). MicroRNA (miR) bind directly to the 3' untranslated region of specific target mRNAs to suppress the expression of proteins, promote the degradation of target mRNAs and regulate immune response. miR-125a is known to be a negative regulator of regulated upon activation normal T cell expressed and secreted (RANTES), interleukin (IL)-6 and transforming growth factor (TGF)-ß; however, its association with AITDs remains unknown. To clarify the association between AITDs and miR-125a, we genotyped the rs12976445 C/T, rs10404453 A/G and rs12975333 G/T polymorphisms in the MIR125A gene, which encodes miR-125a, using direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods in 155 patients with GD, 151 patients with HD and 118 healthy volunteers. We also examined the expression of miR-125a in peripheral blood mononuclear cells (PBMCs) from 55 patients with GD, 79 patients with HD and 38 healthy volunteers using quantitative real-time PCR methods. We determined that the CC genotype and C allele of the rs12976445 C/T polymorphism were significantly more frequent in patients with HD compared with control subjects (P < 0·05) and in intractable GD compared with GD in remission (P < 0·05). The expression of miR-125a was correlated negatively with age (P = 0·0010) and down-regulated in patients with GD compared with control subjects (P = 0.0249). In conclusion, miR-125a expression in PBMCs and the rs12976445 C/T polymorphism were associated with AITD development and prognosis.

Association of polymorphisms in DNMT1, DNMT3A, DNMT3B, MTHFR and MTRR genes with global DNA methylation levels and prognosis of autoimmune thyroid disease.

To clarify the association between factors regulating DNA methylation and the prognosis of autoimmune thyroid diseases (AITDs), we genotyped single nucleotide polymorphisms in genes encoding DNA methyltransferase 1 (DNMT1), DNMT3A, DNMT3B, methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), which are enzymes essential for DNA methylation. Subjects for this study included 125 patients with Hashimoto's disease (HD), including 48 patients with severe HD and 49 patients with mild HD; 176 patients with Graves' disease (GD), including 79 patients with intractable GD and 47 patients with GD in remission; and 83 healthy volunteers (control subjects). The DNMT1+32204GG genotype was more frequent in patients with intractable GD than in patients with GD in remission. Genomic DNA showed significantly lower levels of global methylation in individuals with the DNMT1+32204GG genotype than in those with the AA genotype. The MTRR+66AA genotype was observed to be more frequent in patients with severe HD than in those with mild HD. The DNMT1+14395A/G, DNMT3B-579G/T, MTHFR+677C/T and +1298A/C polymorphisms were not correlated with the development or prognosis of AITD. Our study indicates that the DNMT1+32204GG genotype correlates with DNA hypomethylation and with the intractability of GD, and that the MTRR+66AA genotype may correlate with the severity of HD.

In vitro regulatory effect of epididymal serpin CRES on protease activity of proprotein convertase PC4/PCSK4.

PC4 or PCSK4 belongs to the 9-member superfamily of mammalian subtilases collectively called Proprotein Convertases or Proprotein Convertase Subtilisin/Kexins that convert inactive precursor proteins into their active mature forms by endoproteolytic cleavage. PC4-activity plays a crucial role in mammalian fertilization via activation of sperm surface proteins. PC4 knockout mice exhibit severely impaired male fertility due to premature sperm acrosome reaction. Regulation of sperm-PC4 activity during its storage and transport through epididymis is an important determinant for ultimate egg-binding and fertilizing capacities of sperms. Herein we show that epididymal serpin CRES (cystatin related epididymal spermatogenic) recombinant protein inhibits PC4 activity in vitro in a differential manner when measured against the fluorogenic substrate Boc- RVRR-MCA depending on its oligomeric state. Thus while CRES-dimer exhibits K(i) ∼8 µM, the corresponding monomer showed K(i) > 100 µM. Both forms also blocked PC4-mediated processing of human proIGF-2 in human placenta tropoblast cell line with dimer being more efficient. Using specific inhibitors and substrates, we also demonstrated the presence of PC4-like activity and CRES protein in varying levels in the fluids of various epididymal compartments. Our observations suggest a potential function of CRES as a regulator of PC4 in sperm-egg interaction and fertilization.

Association of functional GITR gene polymorphisms related to expression of glucocorticoid-induced tumour necrosis factor-receptor (GITR) molecules with prognosis of autoimmune thyroid disease.

The glucocorticoid-induced tumour necrosis factor (TNF)-receptor (GITR) affects the functions of regulatory T (T(reg)) and effector T (T(eff)) cells, but the significance of this phenomenon is still unclear. To examine the association of single nucleotide polymorphisms (SNPs) in the GITR gene with the expression of GITR molecules on T cells and with the pathological conditions in patients with autoimmune thyroid disease (AITD), we examined the frequencies of four candidate SNPs in AITD patients and healthy volunteers by restriction enzyme analysis and direct sequence analyses. We also analysed the GITR expression on peripheral T(reg) and T(eff) cells in AITD patients by three-colour flow cytometry. The CC genotype in the rs3753348 C/G SNP was significantly more frequent in patients with mild Hashimoto's disease (HD) than in those with severe HD [P = 0·0117, odds ratio (OR) = 3·13]. The AA genotype in the rs2298213 A/G SNP was significantly more frequent in patients with mild HD than in patients with severe HD (P = 0·010, OR = 4·43). All patients and healthy individuals had the GG genotype in rs60038293 A/G and rs11466696 A/G SNPs. The proportions of GITR(+) cells in T(reg) and T(eff) cells were significantly higher in AITD patients with the CC genotype of the rs3753348 SNP than in those with the GG genotype (P = 0·004 and P = 0·011, respectively). In conclusion, the rs3753348 C/G SNP in the GITR is associated with HD prognosis and expression on T(reg) and T(eff) cells.

Association of functional polymorphisms in promoter regions of IL5, IL6 and IL13 genes with development and prognosis of autoimmune thyroid diseases.

To clarify the association of genetic producibility of interleukin (IL)-5, IL-6 and IL-13, which are secreted by T helper type 2 (Th2), with the development and prognosis of autoimmune thyroid disease (AITD), we genotyped IL5-746C/T, IL6-572C/G and IL13-1112C/T polymorphisms, which are functional polymorphisms in the promoter regions of the genes regulating these cytokines. Fifty-seven patients with intractable Graves' disease (GD), 52 with GD in remission, 52 with severe Hashimoto's disease (HD), 56 with mild HD and 91 healthy controls were examined in this study. The IL13-1112T allele, which correlates with higher producibility of IL-13, was more frequent in patients with GD in remission than in those with intractable GD [P=0·009, odds ratio (OR)=3·52]. The IL5-746T allele, which may correlate with lower levels of IL-5, was more frequent in patients with GD in remission than controls (P=0·029, OR=2·00). The IL6-572G allele carriers (CG and GG genotypes), which have higher producibility of IL-6, were more frequent in AITD patients (P=0·033, OR=1·75), especially in GD in remission (P=0·031, OR=2·16) and severe HD (P=0·031, OR=2·16) than in controls. Interestingly, both allele and genotype frequencies of Th2 cytokine genes were similar between GD and HD patients. In conclusion, functional polymorphisms in the genes encoding Th2 cytokines are associated differently with the development and prognosis of AITD from each other.

A multiple shock tube and chemical kinetic modeling study of diethyl ether pyrolysis and oxidation.

The pyrolysis and oxidation of diethyl ether (DEE) has been studied at pressures from 1 to 4 atm and temperatures of 900-1900 K behind reflected shock waves. A variety of spectroscopic diagnostics have been used, including time-resolved infrared absorption at 3.39 mum and time-resolved ultraviolet emission at 431 nm and absorption at 306.7 nm. In addition, a single-pulse shock tube was used to measure reactant, intermediate, and product species profiles by GC samplings at different reaction times varying from 1.2 to 1.8 ms. A detailed chemical kinetic model comprising 751 reactions involving 148 species was assembled and tested against the experiments with generally good agreement. In the early stages of reaction the unimolecular decomposition and hydrogen atom abstraction of DEE and the decomposition of the ethoxy radical have the largest influence. In separate experiments at 1.9 atm and 1340 K, it is shown that DEE inhibits the reactivity of an equimolar mixture of hydrogen and oxygen (1% of each).

Gelatinous transformation of the bone marrow in systemic lupus erythematosus.

Gelatinous transformation of the bone marrow is a rare disease characterised by a focal marrow hypoplasia, fat atrophy and accumulation of extracellular mucopolysaccharides abundant in hyaluronic acid, which is often associated with extreme malnutrition and weight loss. There are only two reports describing its association with systemic lupus erythematosus (SLE). One described underlying diseases in 155 cases of gelatinous transformation of the bone marrow and found one case with clinical diagnosis of SLE, but no clinical details were provided. The other described three SLE patients with gelatinous transformation of the bone marrow; however, two of these were cachectic and one was diagnosed with concomitant tuberculosis. We describe one active SLE patient without other comorbidities whose pancytopaenia was histologically confirmed as gelatinous transformation. The combination of high-dose steroid, intravenous immunoglobulin and mycophenolate mofetil improved the peripheral blood cytopaenia and reversed the bone marrow abnormalities.

Increased expression levels of tensin3 mRNA in thyroid functional adenomas as compared to non-functioning adenomas.

Tensin3 is a member of tensin family which is localized in focal adhesion. In our previous study, a high level of tensin3 mRNA expression was observed in the thyroid but not in other tissues, thus, tensin3 gene was regarded as a novel thyroid-specific gene. The high expression level of tensin3 mRNA in normal thyroid tissue suggests some fundamental roles in thyroid functions. In fact, the expression level of tensin3 mRNA was low in most thyroid carcinomas and non-functioning thyroid follicular adenomas which do not produce thyroid hormone. In the present study, we measured the expression levels of tensin3 mRNA in twelve functional adenomas and compared the results with those in normal thyroid tissues, adenomatous goiters and non-functioning thyroid follicular adenomas. Tensin3 mRNA was expressed abundantly in all twelve functional adenomas at almost the same level as in normal thyroid tissues and adenomatous goiters, while its expression was significantly lower in non-functioning follicular adenomas. Considering these facts, an abundant expression of tensin3 mRNA is observed in tissues that produce thyroid hormone, which suggests some fundamental roles in basic thyroid functions.

Development of liver dysfunction after delivery is possibly due to postpartum autoimmune hepatitis. A report of three cases.

Autoimmune diseases, especially autoimmune thyroid disease, frequently develop after delivery due to the immune rebound mechanism. Most cases involve transient dysfunction of affected organs. We examined three patients who developed liver dysfunction after delivery. They were all diagnosed with definite or probable autoimmune hepatitis using the scoring system of the International Autoimmune Hepatitis Group. Moreover, all of them had anti-CYP2D6 antibodies detected by a sensitive radioligand assay. Our findings strongly suggest that liver dysfunction is induced by postpartum autoimmune hepatitis, and clinicians should be aware of this disease.

Characteristics of experimental autoimmune hypophysitis in rats: major antigens are growth hormone, thyrotropin, and luteinizing hormone in this model.

We produced experimental autoimmune hypophysitis (EAH) in rats and investigated its characteristics. Female Lewis rats were immunized by two injections with homologous pituitary homogenate and complete Freund's adjuvant. Blood was collected serially from the rats, and serum antibodies to pituitary antigens were examined. The rats were sacrificed 2 or 4 weeks after the final immunization, and histological examinations of the endocrine organs were carried out. Histological examination revealed slight, focal infiltration of mononuclear cells in the pituitary gland only in the rats immunized with the pituitary homogenate. Infiltration of mononuclear cells was not observed in the thyroid gland, pancreas, adrenal gland, or ovary. In the serological examination, antibodies to both cytosolic antigens and cytoplasmic particle antigens from the pituitary gland were detected by enzyme-linked immunosorbent assay (ELISA), and these antibody levels increased with time. Western blotting using the serum antibodies identified an immunoreactive protein of approximately 21.5 kDa among these antigens, and we confirmed that this protein was rat growth hormone (GH). Furthermore, antibodies to GH, thyrotropin (TSH), and luteinizing hormone (LH) were detected by ELISA. Antibodies to follicule stimulating hormone, prolactin, or adrenocorticotropin were not detected. These data suggest that several antigens from the pituitary gland are involved in EAH in rats, and that GH, TSH, and LH are major antigens among the pituitary antigens in this model.

Molecular cloning and characterization of a new human histamine receptor, HH4R.

A new histamine receptor, HH4R, was cloned from human leukocyte cDNA. The deduced amino acid sequence showed about 40% identity to that of the human histamine H3 receptor, HH3R. HH4R-expressing cells responded to histamine, inhibiting forskolin-induced cAMP accumulation. An H3 agonist, N-alpha-methylhistamine (NAMHA), bound specifically to HH4R, while another H3 agonist, R(-)-alpha-methylhistamine (RAMHA), and the H3 antagonist, thioperamide, competed with this binding. RAMHA, NAMHA, and imetit inhibited forskolin-induced cAMP accumulation in HH4R-expressing cells. However, the binding affinities and agonistic activities of H3 agonists to HH4R were weaker than those to HH3R. Low expression of HH4R was detected in a wide variety of peripheral tissues by RT-PCR; however, in contrast with HH3R, expression was not detected in the brain. These observations indicate that the clone is a distinct histamine receptor from HH3R, and thus is named HH4R.

Enzyme-linked immunosorbent assay for anti-tropomyosin antibodies and its clinical application to various heart diseases.

Tropomyosin is one of the key proteins for muscle contraction. We developed an enzyme-linked immunosorbent assay for antibodies to porcine muscle tropomyosin and measured serum anti-tropomyosin antibodies in patients with heart diseases and in normal controls. The mean values of absorbance in the ELISA assay of patients with ischemic heart disease (n=36, P<0.001), dilated cardiomyopathy (n=28, P<0.005), valvular heart disease (n=27, P<0.05), and collagen disease (n=38, P<0.05) were significantly higher than those of normal controls (n=53), but the value in patients with hypertrophic cardiomyopathy (n=19) was not significantly different from that of normal controls. When the cut-off value was fixed at the mean+2 SD of absorbance in normal controls, positive reactions were found in 19.4%, 7.1%, 18.5% and 15.8% of patients with ischemic heart disease, dilated cardiomyopathy, valvular heart disease, and collagen disease, respectively. An inhibition study revealed that anti-tropomyosin antibodies were different from anti-myosin antibodies, but there was a partial cross-reactivity between the two. Thus, there was a weak correlation of the titers of the two types of antibody within the group of heart diseases. These data indicate that measurement of anti-tropomyosin antibodies by ELISA is helpful for detecting autoimmune abnormalities in various heart diseases.

Dual function of the propeptide of prouroguanylin in the folding of the mature peptide: disulfide-coupled folding and dimerization.

Guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, is produced via the processing of the precursor protein (prepro-GCAP-II). We have previously shown that the propeptide in pro-GCAP-II functions as an intramolecular chaperone in the proper folding of the mature peptide, GCAP-II (Hidaka, Y., Ohno, M., Hemmasi, B., Hill, O., Forssmann, W.-G., and Shimonishi, Y. (1998) Biochemistry 37, 8498-8507). Here, we report an essential region in pro-GCAP-II for the correct disulfide pairing of the mature peptide, GCAP-II. Five mutant proteins, in which amino acid residues were sequentially deleted from the N terminus, and three mutant proteins of pro-GCAP-II, in which N-terminal 6, 11, or 17 amino acid residues were deleted, were overproduced using Escherichia coli or human kidney 293T cells, respectively. Detailed analysis of in vivo or in vitro folding of these mutant proteins revealed that one or two amino acid residues at the N terminus of pro-GCAP-II are critical, not only for the chaperone function in the folding but also for the net stabilization of pro-GCAP-II. In addition, size exclusion chromatography revealed that pro-GCAP-II exists as a dimer in solution. These data indicate that the propeptide has two roles in proper folding: the disulfide-coupled folding of the mature region and the dimerization of pro-GCAP-II.