Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds.

A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon-Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs.

One-year follow-up of transgene expression by integrase-defective lentiviral vectors and their therapeutic potential in spinocerebellar ataxia model mice.

We examined integrase-defective lentiviral vectors (IDLVs) with a mutant (D64V) integrase in terms of their residual integration capability, the levels and duration of transgene expression and their therapeutic potential in comparison to wild-type lentiviral vectors (WTLVs) with a wild-type integrase gene. Compared with WTLVs, the IDLV-mediated proviral integration into host-cell chromosomes was approximately 1/3850 in HeLa cells and approximately 1/111 in mouse cerebellar neurons in vivo. At 2 months, transgene expression by IDLVs in the mouse cerebellum was comparable to that by WTLVs, but then significantly decreased. The mRNA levels at 6 and 12 months after injection in IDLV-infected cerebella were approximately 26% and 5%, respectively, of the mRNA levels in WTLV-injected cerebella. To examine the therapeutic potential, IDLVs or WTLVs expressing a molecule that enhances the ubiquitin-proteasome pathway were injected into the cerebella of spinocerebellar ataxia type 3 model mice (SCA3 mice). IDLV-injected SCA3 mice showed a significantly improved rotarod performance even at 1 year after-injection. Immunohistochemistry at 1 year after injection showed a drastic reduction of mutant aggregates in Purkinje cellsfrom IDLV-injected, as well as WTLV-injected, SCA3 mice. Our results suggest that because of the substantially reduced risk of insertional mutagenesis, IDLVs are safer and potentially effective as gene therapy vectors.

Link between inflammation and aquaporin-5 distribution in submandibular gland in Sjögren's syndrome?

OBJECTIVE: To determine whether a link exists between inflammation and aquaporin-5 distribution in submandibular glands from three animal models for Sjögren's syndrome: IQI/JIC, r1ΔT/r2n and non-obese diabetic mice. METHODS: Mice of different ages were used. Inflammatory infiltrates were quantified using the focus score. Acinar aquaporin-5 subcellular distribution was determined by immunohistochemistry and quantified using labelling indices. RESULTS: Minor inflammatory infiltrates were present in r1f/r2n mice. Massive inflammatory infiltrates and acinar destruction were observed in 24-week-old non-obese diabetic mice, 10-and 13-month-old IQI/JIC mice and some r1ΔT/r2n mice. Aquaporin-5 immunoreactivity was primarily apical in submandibular glands from 8- and 24-week-old Balb/C mice, 8-week-old non-obese diabetic mice, 2-, 4- and 7-month-old IQI/JIC mice and r1f/r2n mice. In contrast, decreased apical aquaporin-5 labelling index with concomitant increased apical-basolateral, apical-cytoplasmic and/or apical-basolateral-cytoplasmic aquaporin-5 labelling indices was observed in 24-week-old non-obese diabetic, 10- and 13-month-old IQI/JIC and r1ΔT/r2n mice with a focus score≥1. CONCLUSIONS: Altered aquaporin-5 distribution in submandibular acinar cells from IQI/JIC, non-obese diabetic and r1ΔT/r2n mice with a focus score≥1 appears to be concomitant to the presence of inflammatory infiltrates and acinar destruction.

Beneficial effects of leukotriene receptor antagonists in the prevention of cedar pollinosis in a community setting.

BACKGROUND: In recent years, many countries have experienced an increase in the prevalence of allergic rhinitis. No effective approach is currently available to prevent the onset of symptoms in allergic individuals. Pranlukast, a leukotriene receptor antagonist with a good safety and efficacy record for the management of allergic inflammation, may be appropriate for early intervention in the management of pollinosis. OBJECTIVE: To investigate the efficacy of pranlukast as an early intervention in the control of cedar pollinosis. METHODS: In a double-blind comparative study, pranlukast (n = 102) or placebo (n = 91) was administered to cedar pollinosis patients immediately before the start of the dispersion season and continued for 4 weeks. Subsequently, pranlukast was administered to all patients for 2 weeks until the end of the cedar pollen dispersion season (mid-March). All patients were carefully monitored for severity of nasal symptoms, symptom scores, medication scores, symptom-medication scores, and quality of life (QOL). RESULTS: Compared with placebo, therapy with pranlukast before and during the dispersion of cedar pollen in these patients significantly improved nasal symptoms (paroxysmal sneezing, rhinorrhea, and nasal congestion), symptom scores, and symptom-medication scores. The drug also significantly reduced deterioration of QOL, and improved nasal symptoms and QOL throughout the dispersion period. CONCLUSION: Administering pranlukast immediately before the beginning of cedar pollen dispersion is effective in reducing symptoms of allergic rhinitis throughout the dispersion period.

Onset of autoimmune glomerulonephritis derived from the telomeric region of MRL-chromosome 1 is associated with the male sex hormone in mice.

Female B6.MRLc1(82-100) congenic mice develop more severe autoimmune glomerulonephritis (AGN) than males. We assessed the effects of gonadectomy on the pathogenesis of AGN in these mice. One-month-old male and female mice were divided into sham-operated group (SG) and gonadectomized group (GG), and the pathological changes were investigated at 8 months. SG females showed higher spleen and thymus weights, serum total IgG and autoantibody levels, glomerular damage scores and percent IgG- and CD3-positive glomeruli as compared with SG males. Gonadectomy showed more remarkable effects in males than in females. Spleen and thymus weights, urinary albumin excretion, glomerular damage scores, percent IgG- and CD3-positive glomeruli, and CD3-positive areas in the spleen were significantly higher in GG males than in SG males. CD3-positive cells were observed in both the thymic cortex and medulla in all animals except SG males. The expression ratio of active Fc gamma receptor (Fcgr) 3 to inhibitory Fcgr2b in the kidneys, which we have previously demonstrated to have a great impact on pathogenesis in B6.MRLc1(82-100), was significantly higher in GG males than in SG males. These results suggested that the differences in the pathogenesis of AGN are primarily because of the inhibitory roles of the male sex hormones.

Expression of hepatocyte nuclear factor 4alpha in developing mice.

Hepatocyte nuclear factor (HNF) 4alpha, a transcription factor of the nuclear hormone receptor family, is generally expressed in some endoderm-derived epithelial tissues such as hepatocytes. In mice, an alternative promoter referred to as the P2 promoter is located upstream from the P1 promoter, resulting in the transcription of at least nine isoforms. In this study, we investigated the expression of Hnf4alpha in adult and embryonic mouse tissues, paying special attention to the developing metanephros by using immunohistochemistry and reverse transcriptase-polymerase chain reaction for the detection of P1 and/or P2 promoter-derived products. In adult mouse tissues, the kidney was the only organ expressing Hnf4alpha controlled only by the P1 promoter, and HNF4alpha was detected in the nuclei of epithelial cells in the proximal tubules, but not in other components of the nephron. In the metanephros, HNF4alpha was detected first at the epithelial cell nuclei in part of the comma-shaped body, distributed widely throughout the developing nephron and finally restricted to the proximal tubules. Interestingly, it was noted that Hnf4alpha mRNAs from stomach, pancreas and kidney tissues in embryonic periods were transcribed by both promoters. Immunohistochemistry for HNF4alpha and HNF1alpha revealed that both factors involved the same network of transcription factors, giving the impression that HNF4alpha was upstream of HNF1alpha.

Autoimmune glomerulonephritis induced in congenic mouse strain carrying telomeric region of chromosome 1 derived from MRL/MpJ.

In lupus erythematosus-prone mice, including the BXSB, NZW and NZB strains, telomeric regions of chromosome 1 (Chr.1) contain major glomerulonephritis susceptibility loci such as Bxs3, Sle1, and Nba2. To assess whether strain MRL, a model for lupus erythematosus, had glomerulonephritis susceptibility loci on Chr.1, we created B6.MRLc1(82-100) congenic mice carrying MRL/MpJ Chr.1 (82-100 cM) based on the C57BL/6 background and investigated renal pathology. From 6 months of age, B6.MRLc1 (82-100) showed the onset of diseases such as splenomegaly due to proliferation of CD3- or B220-positive cells, glomerular damage, and an increased serum anti-dsDNA antibody concentration, and these were earlier and severer in females. The score for glomerular damage was higher in B6.MRLc1(82-100) mice over 12 months old than in C57BL/6 or even in wild-type MRL/MpJ. Immune-complex depositions were demonstrated on glomerular basement membrane in B6.MRLc1(82-100) by immunohistochemistry and electron microscopy. For the percentage of IgG1-positive glomeruli, B6.MRLc1 (82-100) had significantly higher values than C57BL/6. In evaluations of clinical parameters, serum levels of blood urea nitrogen and the anti-dsDNA antibody in B6.MRLc1(82-100) were significantly higher than those in C57BL/6. In conclusion, B6.MRLc1(82-100) clearly developed autoimmune-mediated glomerulonephritis, and we demonstrated that MRL Chr.1 contained a novel glomerulonephritis susceptibility locus. We named this locus Mag (MRL autoimmune glomerulonephritis) and it provided new insights into the genetic basis and pathogenesis of lupus nephritis.

Ovarian cysts in MRL/MpJ mice originate from rete ovarii.

In MRL mice aged more than 1 year, but not in C57BL/6 mice, ovaries had grossly visible cysts presenting unilaterally or bilaterally. Postnatally, all MRL mice developed ovarian cysts by 8 months of age. Observations by light microscopy, including lectin histochemistry, indicated that the cysts sometimes included papillomatous tissues located at the hilar region and were similar to the rete ovarii system, but not to follicles. Two types of epithelial cells, ciliated and non-ciliated, were arranged on the cysts, in which both cell types had many microvilli projecting in various directions and random ramifications in the cystic lumen. These characteristics suggest that ovarian cysts developing in MRL mice originate mostly from the rete ovarii. Cysts derived from the rete ovarii at 8 months of age were histologically detected in all C3H mice as well as MRL mice, with variable incidence in ICR, AKR, CBA/N and ddY, and none in C57L/6, DBA/2, BALB and A/J mice. However, measurement of the maximum diameters of the ovarian cysts indicated that MRL mice regularly possessed the largest cysts visible to the naked eye. This is the first report of ovarian cysts in this inbred strain, suggesting that ovarian cysts in MRL mice appear with stable incidence and development.

Comparative study of two Japanese rhinoconjunctivitis quality-of-life questionnaires.

CONCLUSION: Two questionnaires were used to assess quality of life (QOL) in allergic rhinitis: the Japanese translation of the Rhino-conjunctivitis Quality of Life Questionnaire (RQLQJ) and an original Japanese QOL questionnaire (JRQLQ). Either questionnaire may be used to assess QOL depending on differences in target domains. OBJECTIVES: Although pollinosis is a common disease which has a major impact on patient QOL, no internationally standardized questionnaire has been available in Japan until now. The aim of this study was to compare two currently available QOL questionnaires for allergic rhinitis in Japan-the RQLQJ and JRQLQ-in terms of their appropriateness for clinical use and their psychometric properties. MATERIAL AND METHODS: A multicenter, inter-group, cross-sectional study was conducted in 187 adult symptomatic patients with Japanese cedar pollinosis in 2003. Patient scores on the two questionnaires were compared in terms of both overall and comparable domains. We also examined the acceptability, construct and reliability of both questionnaires. RESULTS: The questionnaires were highly correlated in terms of both overall and comparable domain scores. In addition, both questionnaires had equal and satisfactory psychometric validity, demonstrating that they are both useful tools for assessing QOL in rhinitis. However, when compared with each other, the JRQLQ focuses mainly on activities of daily life and is simpler, while the RQLQJ focuses mainly on rhinitis-related health and is more responsive.

Proinsulin C-peptide activates vagus efferent output in rats.

The aim of this study was to examine the effect of proinsulin C-peptide on the autonomic nervous systems in rats. Intravenous administration of C-peptide gradually increased electrophysiological activity of the vagus nerves into the stomach and pancreas for at least 90 min. It also slightly increased gastric acid secretion that was suppressed by the treatment with atropine. Intraperitoneal injection of C-peptide did not affect the basal and stress-induced norepinephrine (NE) turnover rate, a biochemical index of sympathetic nerve activity. These results indicate that C-peptide increases parasympathetic nerve activity without affecting sympathetic nerve activity. This could explain, at least in part, the ameliorating effects of C-peptide on impaired cardiac autonomic nerve functions in patients with type 1 diabetes.

Oligoclonal expansion of circulating and tissue-infiltrating CD8+ T cells with killer/effector phenotypes in juvenile dermatomyositis syndrome.

Although triggering by infectious agents and abnormal immune responses may play some role in the pathogenesis of juvenile dermatomyositis syndrome (JDMS), the precise mechanism of muscle destruction and vascular damage is largely unknown. In this study, we tried to elucidate the role of cytotoxic T cells in two patients with JDMS, who were diagnosed based on the characteristic symptoms, laboratory data, MRI findings and electromyographic patterns. Peripheral blood T cell phenotypes were determined by flow cytometry, using mAbs against specific T cell receptor (TCR) Vbetas. Complementarity-determining region3 (CDR3) size analysis was performed by gene scanning of CDR3 polymerase chain reaction (PCR) amplification products specific for each Vbeta. Subsequently, CDR3 nucleotide sequences were obtained after cloning of the predominant products. The distribution of lymphocytes infiltrating the muscle tissue was analysed by immunohistochemistry. In both patients examined, a unique combination of TCR Vbeta repertoires was increased within the CD8+ T cells. These subpopulations expressed a characteristic phenotype, indicating that they are memory/effector T cells with killer functions. At the same time, immunohistological and molecular biological examinations of the biopsied muscle samples revealed that identical CD8+ T cell clones with identical phenotypes/TCR Vbeta infiltrated within the inflammatory tissue, in particular around vessels. These findings indicate that oligoclonal expansion of CD8+ T cells plays a central role in the pathogenesis of muscle injury in the juvenile form of dermatomyositis syndrome and may provide a useful clinical parameter of disease activity and responsiveness to anti-inflammatory therapy.

Spontaneous development of multiple glandular and extraglandular lesions in aged IQI/Jic mice: a model for primary Sjogren's syndrome.

OBJECTIVE: In primary Sjögren's syndrome (SS), systemic exocrine and non-exocrine organs are frequently affected, in addition to the major target tissues of the lacrimal and salivary glands. This study aimed to examine whether the IQI/Jic mouse, an animal model of SS whose autoimmune dacryoadenitis and sialoadenitis have been documented, develops inflammatory lesions in multiple organs as in primary SS. METHODS: Systemic histopathological analysis was performed on IQI/Jic mice at various ages. Phenotypes of infiltrated lymphocytes were determined using immunohistochemical techniques. RESULTS: Inflammatory lesions were observed not only in the lacrimal and salivary glands, but also in multiple organs, including the lung, pancreas and kidney at advanced ages, and were mainly composed of CD4(+) T cells and B cells. The incidence and severity of the inflammatory lesions increased with age in all these organs. The histological appearance and spreading of lesions were similar to those in human primary SS. CONCLUSIONS: IQI/Jic mice spontaneously develop inflammatory cellular infiltrates in multiple exocrine and non-exocrine organs. This characteristic distinguishes IQI/Jic mice from other murine models, making them favourable for studies on the pathogenesis of systemic involvement in primary SS.

Scavenger receptor cysteine-rich domains 9 and 11 of WC1 are receptors for the WC1 counter receptor.

Workshop cluster 1 (WC1) is a member of the scavenger receptor cysteine-rich (SRCR) superfamily that includes CD5, CD6, CD163, and M160. Bovine WC1 consists of 11 SRCR domains, a unique domain 1, and two homologous 5 SRCR domain cassettes, WC1 domains 2-6 and 7-11. The porcine orthologue of WC1 contains five SRCR domains with a different domain arrangement. Although the function of WC1 is unknown, WC1 is proposed to be an accessory or homing molecule. Thus, identification of cells that express the counter receptor for WC1 (WC1-CR) is critical to understanding the function of WC1. For this reason, we constructed WC1-human immunoglobulin G1 fusion proteins to identify the binding domain of WC1 and cells that express the WC1-CR. Immunohistochemical analysis revealed WC1 domains 9 and 11 bind cells with macrophage and dendritic cell morphology and cells in ellipsoids in the spleen. These results and the finding of conserved signaling motifs in the cytoplasmic tail suggest WC1 may be an accessory molecule.

Effect of formaldehyde on the expression of adhesion molecules in nasal microvascular endothelial cells: the role of formaldehyde in the pathogenesis of sick building syndrome.

BACKGROUND: Formaldehyde is associated with sick building syndrome (SBS), a set of diffuse and irritative symptoms predominantly involving the eyes and the respiratory tract. However, its pathophysiological mechanism in SBS has not yet been clarified. OBJECTIVE: In this study we investigated the effect of formaldehyde on the expression of adhesion molecules on human mucosal microvascular endothelial cells (HMMECs). Furthermore, we investigated the effect of formaldehyde on adhesiveness of HMMECs to eosinophils. MATERIALS AND METHODS: HMMECs were incubated with various concentrations of formaldehyde (1 ng/mL-1 microg/mL) for 24 h, and the expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) on HMMECs were evaluated by flow cytometry. The change in the expression of ICAM-1 and VCAM-1 mRNA was then evaluated by reverse transcriptase polymerase chain reaction. To understand the role of formaldehyde in eosinophilic inflammation of the nasal mucosa, we examined the effects of formaldehyde on the adhesiveness between HMMECs and eosinophils by eosinophil adhesion assay. RESULTS: Formaldehyde increased the surface expressions of ICAM-1 and VCAM-1 on HMMECs. Formaldehyde also induced ICAM-1 and VCAM-1 mRNA. In addition, the adhesiveness between HMMECs and eosinophils was also increased by formaldehyde. CONCLUSION: These in vitro studies suggest that formaldehyde may play a role as the irritant of the nasal mucosa by increasing the expressions of adhesion molecules on HMMECs and by enhancing the adhesiveness between HMMECs and eosinophils.

Expression of C-C chemokine TARC in human nasal mucosa and its regulation by cytokines.

BACKGROUND: Although interleukin (IL)-4 and IL-5 have been demonstrated to play a critical role in the pathophysiology of allergic diseases such as allergic rhinitis, the mechanism that causes the predominance of Th2 lymphocytes has yet to be clarified. Thymus and activation-regulated chemokine (TARC) has been known to facilitate the recruitment, activation and development of Th2 polarized cells, leading investigators to suggest a role for TARC in the development of Th2 responses. OBJECTIVE: To gain a better understanding of the role of TARC in the pathogenesis of allergic rhinitis we investigated the cellular sources of this chemokine in nasal mucosa. In addition, the effect of cytokines on TARC production has been investigated. METHODS: The expression of TARC in human nasal mucosa was assessed by immunohistochemistry. To study the effect of cytokines on TARC production, epithelial cells, endothelial cells and fibroblasts, isolated from inferior nasal mucosa samples, were stimulated by a variety of cytokines including IL-4, IL-13, tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma. RESULTS: Epithelial cells in nasal mucosa in subjects with allergic rhinitis expressed higher signal level than those in non-allergy patients. Combined stimulation with IL-4 and TNF-alpha, as well as IL-13 and TNF-alpha, synergistically induced TARC expression in epithelial cells. Furthermore, the amount of TARC induced by these cytokines was higher in epithelial cells obtained from patients with allergic rhinitis than in those from non-allergic patients. CONCLUSION: These results demonstrate a crucial role of nasal epithelial cells in the expression of TARC, and that Th2 cytokine IL-4 and IL-13 may promote Th2 responses by inducing TARC production from epithelial cells.

Association between nasal allergy and a coding variant of the Fc epsilon RI beta gene Glu237Gly in a Japanese population.

The gene for the beta-chain of the high-affinity receptor for IgE (Fc epsilon RI beta) has been proposed as a candidate gene for atopy. A coding variant Glu237Gly has been studied in various populations with asthma and atopy, and the results were controversial for association of the variant with atopy/asthma. Because nasal allergy is a more common atopic disease and shows less remission than asthma, we analyzed whether the Glu237Gly variant is correlated with nasal allergy. The study enrolled 233 patients with nasal allergy and 100 control subjects. Further, three subgroups were selected: patients with perennial nasal allergy (n=149), Japanese cedar pollinosis (n=189), and allergy to multiple allergens (n=45). The allele frequency of Gly237 in the controls and patients was 0.14 and 0.20, and the frequency of Gly237-positive subjects was 0.23 and 0.356, respectively. There was a significant association between Gly237-positivity and nasal allergy, perennial nasal allergy, Japanese cedar pollinosis, and allergy to multiple allergens. Among all 333 subjects we observed a significant relationship between Gly237 and elevated levels of serum total IgE (>250 IU/ml) and very high IgE (>1000 IU/ml). Among patients positive for a specific IgE, Gly237 was significantly associated with high IgE for house dust, mite, and Japanese cedar pollen. These results suggest that the Glu237Gly variant of the Fc epsilon RI beta gene is involved in the development of nasal allergy through the process for the production of both specific and nonspecific IgE antibodies.

Pathogenesis of laryngeal narrowing in patients with multiple system atrophy.

1. We do not fully understand the pathogenesis of nocturnal laryngeal stridor in patients with multiple system atrophy (MSA). Recent studies suggest that inspiratory thyroarytenoid (TA) muscle activation has a role in the development of the stridor. 2. The breathing pattern and firing timing of TA muscle activation were determined in ten MSA patients, anaesthetized with propofol and breathing through the laryngeal mask airway, while the behaviour of the laryngeal aperture was being observed endoscopically. 3. Two distinct breathing patterns, i.e. no inspiratory flow limitation (no-IFL) and IFL, were identified during the measurements. During IFL, significant laryngeal narrowing was observed leading to an increase in laryngeal resistance and end-tidal carbon dioxide concentration. Development of IFL was significantly associated with the presence of phasic inspiratory activation of TA muscle. Application of continuous positive airway pressure suppressed the TA muscle activation. 4. The results indicate that contraction of laryngeal adductors during inspiration narrows the larynx leading to development of inspiratory flow limitation accompanied by stridor in patients with MSA under general anaesthesia.