Acute blocking of forearm supination secondary to tearing of the triangular fibrocartilage complex.

We studied the characteristics of acute blocking of supination of the distal radioulnar joint caused by triangular fibrocartilage complex injuries. Twenty-four patients who were treated for acute blocking of supination were retrospectively assessed. Supination was suddenly blocked after minor trauma to the wrist. Active and passive supination was severely restricted with a mean preoperative range of motion (11°), whereas pronation was almost normal. The cause was identified arthroscopically or at open operation. It was found to be a result of avulsion of the dorsal or palmar portion of the radioulnar ligament, which blocked movement of the ulnar head. Blocking was reduced manually in four cases, by arthroscopic surgery in eight cases and by open surgery in 12 cases. After treatment, forearm supination improved to 84° of the mean range of motion. Distal radioulnar joint blocking from a ruptured triangular fibrocartilage complex should be considered in the differential diagnosis of loss of forearm supination.Level of evidence: IV.

Detection of a novel herpesvirus from bats in the Philippines.

Bats are natural hosts of many zoonotic viruses. Monitoring bat viruses is important to detect novel bat-borne infectious diseases. In this study, next generation sequencing techniques and conventional PCR were used to analyze intestine, lung, and blood clot samples collected from wild bats captured at three locations in Davao region, in the Philippines in 2012. Different viral genes belonging to the Retroviridae and Herpesviridae families were identified using next generation sequencing. The existence of herpesvirus in the samples was confirmed by PCR using herpesvirus consensus primers. The nucleotide sequences of the resulting PCR amplicons were 166-bp. Further phylogenetic analysis identified that the virus from which this nucleotide sequence was obtained belonged to the Gammaherpesvirinae subfamily. PCR using primers specific to the nucleotide sequence obtained revealed that the infection rate among the captured bats was 30 %. In this study, we present the partial genome of a novel gammaherpesvirus detected from wild bats. Our observations also indicate that this herpesvirus may be widely distributed in bat populations in Davao region.

Ubiquitin C-terminal hydrolase l1 is expressed in mouse pituitary gonadotropes in vivo and gonadotrope cell lines in vitro.

The ubiquitin-proteasome system (UPS) plays a fundamental role in regulating various biological activities. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme, belonging to the UPS. To date, it has been reported that UCH-L1 is highly and restrictedly expressed in neural and reproductive tissues and plays significant roles in these organs. Although the expression of UCH-L1 in the anterior pituitary gland has been reported, the detailed localization and the role of UCH-L1 remain obscure. In the present study, we detected UCH-L1 protein exclusively in hormone-producing cells, but not non-hormone producing folliculostellate cells in the anterior pituitary lobe. In addition, the cytoplasmic expression of UCH-L1 varied and was limited to gonadotropes and mammotropes. To investigate the role of UCH-L1 in anterior pituitary cells, we performed a comparative analysis using genetically UCH-L1-deficient gad mice. Significant decreases in the numbers of gonadotropes and mammotropes were observed in gad mice, suggesting a close involvement of UCH-L1 in these cells. Moreover, we also determined the expression of UCH-L1 in cultured gonadotropes. Taken together, this is the first report to definitely demonstrate the presence of UCH-L1 in mouse anterior pituitary gland, and our results might provide a novel insight for better understanding the role of UCH-L1 in the hypothalamic-pituitary-gonadal axis and in the reproduction.

Characterization of a variant virus from ascitic fluid of subacute granulomatous serositis in interferon-gamma-deficient C57BL/6 mice persistently infected with murine coronavirus strain JHM.

Previously, we showed that intraperitoneal infection with murine coronavirus strain JHM (JHMV) established a persistent infection with subacute granulomatous serositis in interferon-gamma-deficient C57BL/6 (B6-GKO) mice. Herein, we characterize a variant virus from B6-GKO mice persistently infected with JHMV. Viruses were isolated from ascites at 25 d post-infection and cloned by limiting dilution on DBT cells; one variant was named 25V16G. To compare pathogenicity in vivo, we inoculated 25V16G and JHMV intraperitoneally into 8- to 12-week-old B6-GKO mice. Whereas nearly all of the B6-GKO mice infected with JHMV survived over 14 d, all of those infected with 25V16G died by 9 d post-infection. Histopathological examination revealed that 25V16G induced acute fulminant hepatitis in B6-GKO mice, whereas JHMV caused severe but focal hepatitis. The virus titer of 25V16G in the liver was 50- and 250-fold higher than that of JHMV at 5 and 7 d post-infection, respectively. However, there was no significant difference in viral growth between 25V16G and JHMV in cell lines cultured in vitro. Nucleotide sequencing of the S gene of 25V16G and JHMV revealed a deletion of 29 amino acids encompassing S(511-539), which covers a major cytotoxic T lymphocyte (CTL) epitope in C57BL/6 mice, and two point mutations resulting in amino acid changes in the S protein of 25V16G. One explanation for the greater pathogenicity of 25V16G is that 25V16G escapes CTL-mediated protection in B6-GKO mice. This experimental model may be used to assess the role of IFN-gamma in viral persistence in vivo.

Aortic ER stress in streptozotocin-induced diabetes mellitus in APA hamsters.

Atherosclerosis is thought to be associated with endoplasmic reticulum (ER) dysfunction and the accumulation of unfolded proteins. In this study, we examined the relationship between atherosclerosis and ER stress and the effect of sodium 4-phenylbutyrate (4-PBA), a kind of chemical chaperone, on atherosclerosis in streptozotocin-induced diabetic APA hamsters. Male, 8-week-old, APA hamsters were injected with streptozotocin (30 mg/kg body weight) to induce diabetes mellitus, and ER stress was evaluated immunohistochemically or by semi-quantitative RT-PCR analysis using ER stress markers such as calreticulin and GPR78. Control hamsters were injected with citrate buffer and were similarly analyzed. In the aorta of control animals, a weak ER stress was detected, and 4-PBA treatment decreased the calreticulin- and GRP78-positive areas and also reduced the mRNA levels of calreticulin and GRP78. On the other hand, strong ER stress was detected at the lesser curvature of the aortic arch of streptozotocin-induced diabetic APA hamsters. However, 4-PBA treatment failed to lessen the ER stress in the aorta and had no effect on improvement of the atherosclerotic lesions. These results may provide an explanation for the complex etiology of atherosclerosis accompanied by diabetes mellitus and various other clinical phenotypes of atherosclerosis.

Molecular cloning and sequencing of the cDNAs encoding the bat interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12p40, and tumor necrosis factor-alpha.

This is the first report on the cDNA sequences of bat interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12 p40, and tumor necrosis factor (TNF)-alpha. The cDNAs of bat IL-2, IL-4, IL-6, IL-10, IL-12 p40, and TNF-alpha comprise 459, 405, 624, 537, 990, and 699 base pairs respectively. Moreover, each of the cDNAs of bat IL-2, IL-4, IL-6, IL-10, IL-12 p40, and TNF-alpha contain a single open reading frames encoding 152, 134, 207, 178, 329, and 232 amino acids, respectively. The comparison of bat cytokines with Perrissodactyla (horse), Carnivora (dog and cat), and Cetartiodactyla (cattle and pig) orthologs revealed a high degree of homology. Although the N-terminal amino acids and cysteine residues are highly conserved in each mature cytokine, the deduced N-linked glycosylation sites vary across species.

Ubiquitin C-terminal hydrolase L1 deficiency decreases bone mineralization.

Ubiquitin C-terminal hydrolase L1 is a component of the ubiquitin proteasome system, which evidences unique biological activities. In this study, we report the pattern of UCH-L1 expression, and show that it regulates bone mineralization in osteogenesis. UCH-L1 was expressed in osteoblasts, osteoclasts, and hematopoietic precursor cells of bone marrow in the metaphysis and diaphysis of the femora. To further assess the involvement of UCH-L1 in the regulation of bone mineralization, we evaluated the bone mineral density (BMD) rate of gad mice, using the Latheta computed tomography system. Male gad mice evidenced a significantly decreased BMD rate in the metaphysis and diaphysis of the femora. These findings of decreased BMD rate in the bones of gad mice may suggest that UCH-L1 function regulates bone mineralization during osteogenesis.

Distinct immunohistochemical localization in Kuru plaques using novel anti-prion protein antibodies.

By immunizing Prnp-knockout mice with synthetic polypeptides, a panel of mAbs directed to bovine PrP(C) was obtained. The mAb panel was characterized by the ELISA method, where synthetic polypeptides were used for epitope mapping. Different reactivity patterns were identified. The ability of these mAbs to detect abnormal PrP(Sc) in CJD cases was studied by immunohistochemistry. All mAbs were tested for PrP(Sc) in murine, bovine, monkey and human brain tissues. Three mAbs recognized the fragmented PrP epitope in our ELISA. Antibody 1D12 was strongly reactive to ovine and squirrel monkey tissues infected with a scrapie agent, although non-reactive to scrapie-infected mouse tissues. Antibody 2D8 was clearly reactive to type-2 but not type-1 CJD human tissues. Of particular interest was the reactivity of mAb 6C4 with the inner structure of Kuru plaques (peripheral pattern) in a type-2 CJD case and mAb T2, 1D12, 2B11, 2D8, 4B5 and 6G3-2 with the central area (central pattern). The fact that different anti-PrP mAbs possess distinct staining properties suggests that the PrP(c) to PrP(Sc) conversion might involve a multiple-step process.

Discrimination of antibody to herpes B virus from antibody to herpes simplex virus types 1 and 2 in human and macaque sera.

The antigenic cross-reactive characteristics of herpes B virus and herpes simplex virus (HSV) type 1 (HSV-1) and HSV-2 are responsible for false-positive diagnoses by serological assays in humans and macaques. In the present study, we developed a fluorometric indirect enzyme-linked immunosorbent assay (ELISA) with recombinant herpes B virus glycoprotein D (gD) and HSV-1 and HSV-2 gG (gG-1 and gG-2, respectively) to discriminate between the three primate herpesvirus infections. The secreted form of gD, gDdTM, was used to detect antibody to herpes B virus gD. Sera positive for herpes B virus, HSV-1, and HSV-2 showed specific reactions to gD, gG-1, and gG-2, respectively. Sera collected from humans and rhesus macaques were investigated for the presence of antibodies to the recombinant proteins of the three herpesviruses. The results suggested that the approach is able to discriminate between herpes B virus and HSV infections. The ELISA was also found to be able to detect infections with multiple primate herpesviruses and may have the potential to identify a subsequent infection in individuals that have already been infected with another herpesvirus. In addition, we found evidence of a greater cross-reactivity of herpes B virus with HSV-1 than with HSV-2. It is suggested that the ELISA with the recombinant antigens is useful not only for the serodiagnosis of primate herpesvirus infections but also for elucidation of the seroprevalence of herpesviruses in humans and primates.

Abeta upregulates and colocalizes with LGI3 in cultured rat astrocytes.

1. The leucine-rich glioma inactivated (LGI) family of genes encodes a leucine-rich repeat (LRR) protein, proteins that are thought to be specifically involved in protein-protein and protein-matrix interactions. Since amyloid beta peptide (Abeta) has been previously shown to induce the expression of another LRR-encoding gene in neural cells, we assessed how Abeta affects LGI gene expression in rat primary cerebral cortical cultures and astrocyte cultures. Both RT-PCR and Western Blotting analyses revealed that Abeta robustly induced the expression of LGI3 in rat astrocyte cultures.2. Western Blotting analyses also showed that both glial fibrillary acidic protein (GFAP) and apolipoprotein E (ApoE) significantly increased coincidentally with the Abeta-induced upregulation of LGI3. Immunocytochemistry showed that LGI3 colocalized with Abeta at plasma membranes and also with internalized Abeta in astrocytes. These findings suggest that activated LGI3 may be involved in the astroglial response against Abeta.

Roles of the small intestine for induction of toll-like receptor 4-mediated innate resistance in naturally acquired murine toxoplasmosis.

Peroral infection of Toxoplasma gondii is thought to reflect the typical infection route of naturally acquired toxoplasmosis in humans. We have investigated possible differential roles of toll-like receptor 2 (TLR2) and TLR4 in host defense against naturally acquired murine toxoplasmosis. After peroral inoculation of T. gondii ME49 cysts, TLR4-deficient C3H/HeJ mice were more susceptible to infection than wild-type (WT) C3H/HeN mice, as shown by increased cyst number and low production of cytokines, which are the key factors in protective immunity. When mice were inoculated by intra-peritoneal inoculation of T. gondii, there were no significant differences in the number of brain cysts and cytokine productions between C3H/HeJ and C3H/HeN mice. Histopathologic examination revealed severe inflammation in the small intestine of C3H/HeJ (TLR4-deficient) mice, while an increased number of TLR4-positive mononuclear cells was found in C3H/HeN (WT) mice. To confirm these phenomena, TLR2(-/-) or TLR4(-/-) mice were infected perorally with T. gondii cysts. TLR4(-/-) mice were more susceptible to infection compared with TLR2(-/-) and C57BL/6 mice. Nuclear factor-kappa B activation through TLR4 agonistic activity of T. gondii ME49 was demonstrated by luciferase assay using stably expressing mouse (m) TLR2 or mTLR4/mMD-2 transfectants. We demonstrate here for the first time that innate immune recognition by TLR4 is involved in protective mechanisms against peroral infection with T. gondii ME49. These results suggest that the small intestine plays an important role in the induction of innate immunity in naturally acquired toxoplasmosis.

Gene expression profiling of mouse embryonic stem cell progeny differentiated by Lumelsky's protocol.

Successful conversion of embryonic stem (ES) cells into insulin-producing cells has been reported by Lumelsky et al. (Science 2001;292:1389-1394); however, it remains controversial. In this study, we investigated the properties of ES cell progeny-induced differentiation according to Lumelsky's protocol by immunocytochemistry, oligonucleotide microarray and real-time RT-PCR. Insulin-positive cells were observed at stages 3, 4 and 5. Microarray analysis demonstrated upregulation and appearance of some genes involved in pancreatic development but not beta-cell-specific functional genes in cells at stage 5. Similarly, real-time RT-PCR revealed that expression of beta-cell-specific functional genes such as islet amyloid polypeptide, insulin I and II was not increased in cells at stage 5. These results suggest that terminal differentiation of ES cell progeny toward functional pancreatic beta-cell is insufficient. This study also demonstrates the usefulness of multiple time-course expression profiles for validating differentiation fates of ES cell progeny.

Molecular cloning and sequencing of the cDNA encoding the bat CD4.

Chiroptera is thought to be a vector or a natural reservoir of various pathogenic microbes. However, there are few basic studies on the subject of chiroptera immune systems. This is the first report to determine the sequence of bat CD4 cDNA. Comparison with other animals' CD4 and phylogenetic analysis have shown that bat CD4 had a higher homology to cat and dog CD4 than to human and mouse CD4. Moreover, from the analysis of the structure of the CD4 Ig-like C-type 1 region, in bat CD4 there was an insertion of 18 extra amino acids. In addition, bat CD4 lacked cystein, which suggested that the disulfide bond could not be formed. Human, monkey and mouse CD4 have the cystein and the disulfide bond, but pig, cat, whale and dog CD4, like that of the bat, lacked the cystein. We conducted the present study in order to help elucidate the infectious diseases derived from the bat as well as bat immune systems.

Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermatogenesis in transgenic mice.

Ubiquitin carboxyl-terminal hydrolase 1 (UCH-L1) can be detected in mouse testicular germ cells, mainly spermatogonia and somatic Sertoli cells, but its physiological role is unknown. We show that transgenic (Tg) mice overexpressing EF1alpha promoter-driven UCH-L1 in the testis are sterile due to a block during spermatogenesis at an early stage (pachytene) of meiosis. Interestingly, almost all spermatogonia and Sertoli cells expressing excess UCH-L1, but little PCNA (proliferating cell nuclear antigen), showed no morphological signs of apoptosis or TUNEL-positive staining. Rather, germ cell apoptosis was mainly detected in primary spermatocytes having weak or negative UCH-L1 expression but strong PCNA expression. These data suggest that overexpression of UCH-L1 affects spermatogenesis during meiosis and, in particular, induces apoptosis in primary spermatocytes. In addition to results of caspases-3 upregulation and Bcl-2 downregulation, excess UCH-L1 influenced the distribution of PCNA, suggesting a specific role for UCH-L1 in the processes of mitotic proliferation and differentiation of spermatogonial stem cells during spermatogenesis.

Safe and efficient collection of cytokine-mobilized peripheral blood cells from cynomolgus monkeys (Macaca fascicularis) with human newborn-equivalent body weights.

Hematopoietic stem cells in bone marrow can be mobilized into peripheral blood by cytokine administration. Cytokine-mobilized peripheral blood stem cells are of great use in clinical applications. We previously established a modified procedure for the collection of cytokine-mobilized peripheral blood cells from rhesus monkeys (Macaca mulata) using a commercially available apparatus originally developed for human subjects. In this study, we examined the efficacy and safety of this method with even smaller macaques, cynomolgus monkeys (Macaca fascicularis), which are equivalent to human newborns in body weight (mean = 3.3 kg). Using the manufacturer's unmodified protocol (n=6), one monkey died of cardiac failure and three developed severe anemia. In contrast, using our modified procedure (n=6), no such complication was observed in any animal. In addition, the harvested nuclear cell, mononuclear cell and CD34(+) cell counts were significantly higher with the modified method. The modified method should allow safe and efficient collection of cytokine-mobilized peripheral blood cells from non-human primates as small as human newborns in a non-invasive manner.

Histopathological studies of aortic dissection in streptozotocin-induced diabetic APA hamsters.

Syrian hamsters of the APA strain (APA hamsters) are known to show continuous diabetes accompanied by its complications, such as glomerulosclerosis and atherosclerosis, following a single injection of streptozotocin (SZ). Recently, we observed Stanford type B aortic dissection in three diabetic APA hamsters and histopathological analysis was performed. The histopathologic observations in the false lumen, such as proliferation of granulation tissues, neointima and pseudoneointima, corresponded to the non-thrombosed type of human aortic dissection, and blood clots of the thrombosed type were similar to the remodeling structures of aortic dissection found in human cases. Thus, this model may be useful for investigating the etiology and pathogenesis of aortic dissection accompanying diabetes mellitus in humans.

Ubiquitin C-terminal hydrolase L-1 is essential for the early apoptotic wave of germinal cells and for sperm quality control during spermatogenesis.

Ubiquitination is required throughout all developmental stages of mammalian spermatogenesis. Ubiquitin C-terminal hydrolase (UCH) L1 is thought to associate with monoubiquitin to control ubiquitin levels. Previously, we found that UCHL1-deficient testes of gad mice have reduced ubiquitin levels and are resistant to cryptorchid stress-related injury. Here, we analyzed the function of UCHL1 during the first round of spermatogenesis and during sperm maturation, both of which are known to require ubiquitin-mediated proteolysis. Testicular germ cells in the immature testes of gad mice were resistant to the early apoptotic wave that occurs during the first round of spermatogenesis. TUNEL staining and cell quantitation demonstrated decreased germ cell apoptosis and increased numbers of premeiotic germ cells in gad mice between Postnatal Days 7 and 14. Expression of the apoptotic proteins TRP53, Bax, and caspase-3 was also significantly lower in the immature testes of gad mice. In adult gad mice, cauda epididymidis weight, sperm number in the epididymis, and sperm motility were reduced. Moreover, the number of defective spermatozoa was significantly increased; however, complete infertility was not detected. These data indicate that UCHL1 is required for normal spermatogenesis and sperm quality control and demonstrate the importance of UCHL1-dependent apoptosis in spermatogonial cell and sperm maturation.

Amelioration of the beta-cell dysfunction in diabetic APA hamsters by antioxidants and AGE inhibitor treatments.

BACKGROUND: In our recent report, probucol treatment ameliorated glucose intolerance and increased the insulin-positive area in the pancreas of streptozotocin (SZ)-induced diabetic APA hamsters. The data suggested that the beneficial effects of probucol treatment on beta-cell function might result from its additive effect as an antioxidant. Here, we examined the antioxidant effects on the beta-cell function in SZ-induced diabetic APA hamsters treated with three different agents, N-acetyl-L-cysteine (NAC), aminoguanidine (AG) and pyridoxamine (PM). METHODS: The control (CB group) and diabetic (SZ group) hamsters were treated with NAC, AG or PM for four weeks from several days after SZ injection. RESULTS: Non-fasting plasma glucose and glycoalbumin levels were significantly reduced in SZ animals by NAC or PM treatment. Glucose tolerance test revealed that fasting plasma glucose levels of SZNAC and SZPM animals were low, similar to the corresponding control animals. Thirty minutes after glucose injection, amelioration in the plasma glucose levels of SZNAC and SZPM animals was observed. Immunohistochemistry revealed that the pancreata of SZNAC, SZAG and SZPM animals showed significantly higher percentages of insulin-positive area than those of non-treated SZ animals. The plasma 8OHdG and malondialdehyde plus 4-hydroxy-2-nonenal (4HNE) levels were significantly decreased especially in SZNAC and SZPM animals. 4HNE-positive cells stained by anti-4HNE antibody were reduced in the islets of each agent-treated animal. SZNAC and SZPM animals showed significantly increased beta-cell proliferation determined by insulin and BrdU double staining. All SZ groups treated with NAC, AG or PM had the high expression levels of Reg and INGAP, which are known to be expressed in regenerating islets. CONCLUSIONS: These data suggest that NAC and PM treatment of SZ-injected diabetic hamsters reduces oxidative stress and restores beta-cell function, but that AG treatment has little beneficial effect on the diabetic conditions of SZ-injected hamsters.

Inhibition of staurosporine-induced neuronal cell death by bisphenol A and nonylphenol in primary cultured rat hippocampal and cortical neurons.

We examined whether bisphenol A (BPA) and 4-nonylphenol (NP) influenced staurosporine-induced neuronal cell death in primary cultured rat hippocampal and cortical neurons. In hippocampal neurons, 17beta-estradiol (E2) (1 nM and 10 microM) and BPA (10 microM) significantly inhibited the staurosporine-induced release of lactate dehydrogenase (LDH). In cortical neurons, BPA significantly inhibited the LDH release, while E2 did not. In hippocampal neurons, E2 and BPA significantly inhibited the staurosporine-induced increase in caspase-3 activity. In cortical neurons, BPA and NP significantly inhibited the increase in caspase-3 activity, while E2 did not. Furthermore, low-dose BPA (10 nM) also significantly inhibited the increase in caspase-3 activity in both hippocampal and cortical neurons. BPA and NP might impede normal brain development by inhibiting even desirable neuronal cell death, interfering with caspase-3 activation.

Spontaneous T-cell-rich B-cell lymphoma in a cynomolgus monkey (Macaca fascicularis).

A spontaneous T-cell-rich B-cell lymphoma (TCRBCL) occurred as a subcutaneous mass in the buccal region and enlarged submandibular lymph node in a 6-year-old female cynomolgus monkey (Macaca fascicularis). The constituent cells were examined by histology, immunohistochemistry and the double labeled-immunofluorescence method (dl-IF). Further, in situ hybridization (ISH) was employed to detect the gene expression of Epstein Barr virus (EBV). Histologically, the mass was comprised mainly of neoplastic large lymphoid cells and reactive small mononuclear cells. Immunohistochemically, the neoplastic large lymphoid cells were positive for CD20, CD79 alpha, MHC class II, and either IgG, IgM, or IgA. Polyclonal Ig production by the neoplastic large lymphoid cells was demonstrated by dl-IF, although IgG-positive ones predominated in number. On the other hand, most of the small mononuclear cells were positive for CD3 and were regarded as reactive T lymphocytes, while the remaining cells appeared to be histocytes or reactive B-cells. Transcripts of EBV gene were not demonstrated in these neoplastic or reactive cells by ISH. This is the first reported case of spontaneous TCRBCL in the cynomolgus monkey.