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.

Testicular development in growth-retarded mice.

To assess delayed fertility in male growth-retarded (grt) mice with congenital primary hypothyroidism, their testes were chronologically examined. The testicular weight in grt mice was significantly lower than age-matched normal mice until 8 weeks but was comparable at 13 and 26 weeks. While normal mice had mature sperm cells in both testes and epididymides at 5 weeks, age-matched grt mice did not. The size of the seminiferous tubules in testes of grt mice was smaller than that of normal mice before 13 weeks but was comparable at 26 weeks. These findings suggest that male grt mice might need more than 13 weeks to develop mature testes.

Identification of a quantitative trait locus regulating B cell-dominant infiltration into autoimmune sialitis lesions of the IQI mouse model of primary Sjögren's syndrome.

Sjögren's syndrome (SS) is caused by an autoimmune sialodacryoadenitis, and up to 5% of patients with SS develop malignant B cell growth. The IQI mouse is a spontaneous model of primary SS in which B cells are the dominant cellular subpopulation among mononuclear infiltrates in sialitis lesions. Understanding the genetic control of aberrant B cell growth in IQI mice may help elucidate the genetic mechanisms involved in B-lineage hyperplasia leading to malignant transformation in human SS. B cell-dominant infiltration in the submandibular glands of 6-month-old IQI and C57BL/6 (B6) mice and their F1 and F2 progenies was quantified as B-lymphocytic sialitis score, and a genome-wide scan of 179 (IQI x B6) F2 females was performed to identify a quantitative trait locus (QTL) controlling this phenotype. A QTL significantly associated with variance in B-lymphocytic sialitis score was mapped to the D6Mit138 marker (position of 0.68cM) on proximal chromosome 6, with a logarithm of odds score of 4.3 (p = 0.00005). This QTL, named autoimmune sialitis in IQI mice, associated locus 1 (Asq1), colocalized with Islet cell autoantigen 1 (Ica1), which encodes a target protein of the immune processes that define the pathogenesis of primary SS in humans and in the nonobese diabetic mouse model.

Kinetics of biglycan, decorin and thrombospondin-1 in mercuric chloride-induced renal tubulointerstitial fibrosis.

We investigated the kinetics of decorin, biglycan and thrombospondin-1 in mercuric chloride-treated Brown Norway (BN) rats. BN rats were injected subcutaneously with 1 mg/kg b.w. of mercuric chloride one or three times. The kidney was examined histopathologically and the kinetics of decorin, biglycan and thrombospondin-1 was also examined using immunohistochemistry and real time RT-PCR. As a result, mercuric chloride induced tubular injury and subsequent tubulointerstitial fibrosis. In this lesion, the expression of thrombospondin-1 mRNA was most prominently elevated. The expression of decorin mRNA was next, but biglycan mRNA expression was not elevated. Moreover, decorin and thrombospondin-1 proteins were localized in tubular epithelial cells and peritubular interstitium. Moreover, kinetics of their mRNA expressions was relatively similar to the kinetics of TGF-beta1 mRNA expression previously reported. The present findings suggest that decorin and thrombospondin-1 may participate in the development of tubulointerstitial fibrosis and may have some relation with TGF-beta1 in mercuric chloride-treated BN rats.

Electron microscopic study of subcapsular cell hyperplasia in the adrenal glands of IQI/Jic mice.

Hyperplastic cells in subcapsular cell hyperplasia (SCH) lesion in adrenal glands of female IQI/Jic mice were examined by electron microscopy. These cells were small and polygonal, and had irregular nuclei, elongated mitochondria with lamellar cristae and dense lipid droplets. While these cells showed different features, some of them had desmosomes and basement membranes, and a few round mitochondria with tubular cristae as endocrine cells. These findings suggest that hyperplastic cells in SCH lesions might originate from endocrine blastemic cells.

Effect of lipopolysaccharide (LPS) injection on the immune responses of LPS-sensitive mice.

The effect of lipopolysaccharide (LPS) on humoral and cell-mediated immunity was assessed using LPS-sensitive C3H/HeN mice. A single injection of LPS significantly decreased the anti-sheep red blood cells (SRBC) antibody titers, but not the number of anti-SRBC antibody producing spleen cells. In contrast, double LPS injection did not significantly decrease the anti-SRBC titers and even increased the number of anti-SRBC antibody producing spleen cells. Similarly, single LPS injection significantly suppressed the swelling of the footpad, but double LPS injection caused milder suppression. These results suggest that a difference in the level and timing of exposure to LPS may influence the immune response to infection or vaccination.

Comparative investigation of several sperm analysis methods for evaluation of spermatotoxicity of industrial chemical: 2-bromopropane as an example.

Reproductive toxicity of 2-bromopropane (2BP), a substitute for ozone layer-depleting chloro-fluorocarbon, was found among the workers in an electronics factory in Korea in 1995. Furthermore the importance of testicular toxicity has been realized since the problem of endocrine disruptors arose all over the world, but manual methods must rely on subjective assessment. Recently, computer-assisted sperm analysis (CASA) was proposed but this system requires vast investment. We then investigated the applicability of the MTT method with a microplate and sperm quality analyzer (SQA) as simple, rapid, and economic instrumental methods for the examination of sperm quality in rats, comparing it with the manual microscopic method and CASA. Epididymal fluid derived from male F344/N Slc (Fischer) rats intraperitoneally injected with 2BP in the dose range of 125-1,000 mg/kg/d twice a week (total 8 times) were examined by these methods as a model experiment. Sperm count measured by the manual method and CASA in the epididymal fluid, absorbance by the MTT method and sperm motility index value by the SQA method were significantly lower in the 2BP 1,000 mg/kg administered group than in the control group. This result suggests that the MTT method can detect oligospermia. With the microplate and microplate reader, the efficiency of detection becomes much better. Sperm analyses by the MTT method with the microplate reader and the SQA method are available for reproductive toxicity study in rats.

Comparative study on picryl chloride (PCL)-induced contact dermatitis in female IQI/Jic and BALB/c mice.

Ear skin responses to picryl chloride (PCL)-induced contact dermatitis were compared in detail between IQI/Jic mice developed in Japan and BALB/c mice often used for the investigation of contact dermatitis. PCL was applied to the left ear of each mouse 4 (1st), 11 (2nd), 18 (3rd) and 25 days (4th) after sensitization of the abdominal skin with PCL. Time course examinations were carried out on the ear swelling responses, total IgE levels, skin histology and immunohistochemistry for infiltrated cells after the 1st and 4th application. In IQI mice, the peak time of the ear swelling responses tended to shift from 24 h to 9 h with marked elevation of total IgE levels and marked increase of mast cells showing degranulation after the 4th application when CD8(+) cells as well as CD4(+) cells also prominently increased. In BALB/c mice, except for the total IgE levels and the number of mast cells, the degrees of ear swelling responses, histological changes and increase of CD4(+) and CD8(+) cells were much less severe. Female IQI mice are considered to be a useful mouse strain for further investigations on the role of CD4(+) and CD8(+) T cells in the pathogenesis of contact dermatitis.

Presence of B7-2+ dendritic cells and expression of Th1 cytokines in the early development of sialodacryoadenitis in the IqI/Jic mouse model of primary Sjörgren's syndrome.

Subpopulations of infiltrating lymphocytes, professional antigen-presenting cells (APCs), and Th1/Th2 cytokines that could initiate an autoimmune sialodacryoadenitis were studied in the IQI/Jic mouse model of primary Sjögren's syndrome. Although lymphocytic infiltrations were first seen in submandibular glands (SMGs) of females and in lacrimal glands (LGs) of males at 8 weeks of age, clusters of MHC class II+, CD11c+, B7-2+ dendritic cells (DCs) were already localized in these tissues at 4 weeks. At 8 weeks, the infiltrating lymphocytes consisted of almost equal numbers of B cells and CD4+ T cells. In the inflammatory foci, MHC class II+, CD11c+, B7-2+ DCs formed network-like structures. Duct cells in the lesions showed immunoreactivities for MHC class II and ALCAM (a costimulatory adhesion molecule). IL-12 and IFN-gamma transcripts were detected by RT-PCR in SMGs of females and in LGs of males at 8-12 weeks. These results suggest that the clustered DCs might play an important role in the initiation of the adenitis, and further suggest that the DCs and epithelial cells may participate in the activation of CD4+ T cells. It is also likely that Th1 cytokines mediate the functional interactions between the APCs and CD4+ T cells in the early lesions.

Ultrastructural features of mast cells in picryl chloride (PCL)-induced contact dermatitis in IQI/Jic mice.

Mast cells are one of the major effector cells in the pathogenesis of allergic diseases such as contact dermatitis. In the present study, ultrastructural features of mast cells in contact dermatitis were examined. Namely, the ear of IQI/Jic mice was topically applied with picryl chloride (PCL) at 4 (1st), 11 (2nd), 18 (3rd) and 25 days (4th) after the sensitization with PCL to the abdominal skin. The changes in the ear swelling responses, total serum IgE levels and histology including mast cell numbers were similar to those of previous reports by our research group (Ikeda et al. 2000; Jung et al. 2001). Ultrastructurally, after the 1st application, a close spacial relationship between mast cells and neutrophils and phagocytosis of mast cell granules by neutrophils were observed. Mast cells generally contained non-fused swollen granules filled with altered contents with low electron density and showed an extrusion of membrane-free granules through membrane pores. In addition, interestingly, a few mast cells secreted membrane-bound granules into the dermis without leaving cell membrane damage. After the 4th application when the number of mast cells prominently increased and the total serum IgE level was greatly elevated, in addition to mast cells showing typical anaphylactic degranulation, many mast cells probably in the recovery process from degranulation and several immature mast cells characterized by well-developed Golgi apparatus, many ribosomes and a few electron-dense secretory granules in the peripheral cytoplasm were also observed at the same time. The present results clarified the ultrastructural features of mast cells in the course of PCL-induced contact dermatitis in IQI/Jic mice.