SnJ Mice Research Articles

Pegylated Long-Acting Interferon Gamma Treatment Enhances MHC Class II Expression and the Graft-Versus-Leukemia Effect in Preclinical Models of AML Relapse

Relapse after allogeneic hematopoietic stem cell transplantation (HCT)represents the most common type of treatment failure and is thought to result in part from AML cell escape from immune pressure exerted by donor immune cells (the “Graft-versus-leukemia” effect, GvL). We and others have previously reported downregulation of MHC class II (MHC-II) expression in up to half of AML cases relapsing after transplantation, but it is unclear whether a) loss of MHC-II contributes mechanistically to loss of GvL, and b) whether reversal of MHC-II downregulation can restore GvL. We tested the role of MHC-II in mediating GvL in a mouse model of minor-antigen mismatch HCT where C57BL/6 mice (B6) bearing syngeneic AML cells (H-2b) were co-transplanted with or without CD3+ T cells from C3.SW-H2b/SnJ mice (H-2b). Since murine AML cells do not generally express MHC-II at baseline, we transduced MLL-AF9-driven B6 AML cells with a fluorescently-tagged retrovirus expressing CIITA the main transcriptional regulator of MHC-II genes. MHC-II-expressing AML cells were selectively deleted when co-transplanted with donor T cells, in contrast to animals that received no T cells (Figure 1, right) or mice that received AML cells transduced with empty vector (Figure 1, left). To extend these findings to human cells, we used both MHC-II blocking antibodies and CRISPR-mediated disruption of CIITA in human AML cell lines and primary samples. Loss or blocking of MHC-II led to diminished the ability of AML cells to stimulate MHC-mismatched donor T cells in a mixed lymphocyte reaction. Loss of MHC-II expression in AML cells can be reversed in vitro by Interferon gamma (IFNG). We tested the ability of IFNG to enhance the GvL by culturing AML cell lines and primary patient samples with or without IFNG for 48 hours. IFNG-treatment enhanced the ability of AML cells from some-but not all-samples to stimulate third-party T cells. Interestingly, enhanced stimulation was observed in both CD4 and CD8 cells, suggesting that IFNG may also potentiate GvL through MHC-II-independent pathways. Since the use of IFNG in vivo may be limited by its short half-life, we tested a novel long-acting pegylated human interferon gamma (peg-IFNG, Bolder BioTechnology) in a xenograft model of AML. Immunodeficient mice were engrafted with MHC-II-low human AML cells. After engraftment, mice (n=3 each group) received six doses of IFNG, peg-IFNG, or vehicle (15mcg given s.c. M-W-F). After treatment, mice were sacrificed and MHC-II expression was quantified on human AML cells isolated from bone marrow of treated mice. IFNG treatment increased MHC-II expression modestly compared to vehicle (2.7-fold, p=0.04), while peg-IFNG much more robustly stimulated MHC-II stimulation on human AML cells in this xenograft model (12-fold, p=0.02). Finally, we tested whether peg-IFNG can enhance AML cell clearance in a xenograft model. Luciferin-labelled THP1 cells (low MHC-II expression) were injected on day 0 into immundeficient mice (n=8-9 each group). On day 7, mice started treatment with peg-IFNG or vehicle (15mcg given s.c. M-W-F for two weeks). On day 21, baseline tumor burden was measured using bioluminescence, and healthy third-party donor T cells were injected. Mice were followed with weekly CBC and bioluminescence measurments, and moribund mice were sacrificed and banked. Mice treated with peg-IFNG plus T cells had decreased tumor burden during the course of the experiment compared to untreated controls as well as mice treated with T cells plus vehicle only (Figure 2, p<0.01 by ANOVA for both comparisons). In addition, mice treated with peg-IFNG had significantly improved survival compared to control groups (median survival 79 days compared to 63 days and 67 days, p<0.05 by Log-Rank test). Together, these findings suggest that long-acting IFNG analogues may effectively enhance GvL in AML patients after HCT.

Myeloid-Derived Suppressor Cells Restrain Natural Killer Cell Activity in Acute Coxsackievirus B3-Induced Myocarditis.

Murine models of coxsackievirus B3 (CVB3)-induced myocarditis well represent the different outcomes of this inflammatory heart disease. Previously, we found that CVB3-infected A.BY/SnJ mice, susceptible for severe acute and chronic myocarditis, have lower natural killer (NK) cell levels than C57BL/6 mice, with mild acute myocarditis. There is evidence that myeloid-derived suppressor cells (MDSC) may inhibit NK cells, influencing the course of myocarditis. To investigate the MDSC/NK interrelationship in acute myocarditis, we used CVB3-infected A.BY/SnJ mice. Compared to non-infected mice, we found increased cell numbers of MDSC in the spleen and heart of CVB3-infected A.BY/SnJ mice. In parallel, S100A8 and S100A9 were increased in the heart, spleen, and especially in splenic MDSC cells compared to non-infected mice. In vitro experiments provided evidence that MDSC disrupt cytotoxic NK cell function upon co-culturing with MDSC. MDSC-specific depletion by an anti-Ly6G antibody led to a significant reduction in the virus load and injury in hearts of infected animals. The decreased cardiac damage in MDSC-depleted mice was associated with fewer Mac3+ macrophages and CD3+ T lymphocytes and a reduced cardiac expression of S100A8, S100A9, IL-1β, IL-6, and TNF-α. In conclusion, impairment of functional NK cells by MDSC promotes the development of chronic CVB3 myocarditis in A.BY/SnJ mice.

The activating receptor NKG2D of natural killer cells promotes resistance against enterovirus-mediated inflammatory cardiomyopathy.

In enterovirus-induced cardiomyopathy, information regarding the detailed impact of natural killer (NK) cells on the outcome of the disease is limited. We therefore hypothesized that NK cells and certain NK cell receptors determine the different outcome of coxsackievirus B3 (CVB3) myocarditis. Here, we demonstrate in murine models that resistance to chronic CVB3 myocarditis in immunocompetent C57BL/6 mice is characterized by significantly more mature CD11b(high) NK cells, the presence of NKG2D on NK cells, and enhanced NKG2D-dependent cytotoxicity compared to CVB3-susceptible A.BY/SnJ mice. The highly protective role of NKG2D in myocarditis was further proven by in vivo neutralization of NKG2D as well as in NKG2D-deficient mice but was shown to be independent of CD8(+) T-cell-dependent immunity. Moreover, the adoptive transfer of immunocompetent C57BL/6 NK cells pre- (day -1) as well as post-infectionem (day +2) displayed the potential to prevent permissive A.BY/SnJ mice from a progressive outcome of CVB3 myocarditis reflected by significantly improved cardiopathology and heart function. Altogether, our results provide firm evidence for a protective role of NKG2D-activated NK cells in CVB3 myocarditis leading to an effective virus clearance, thus offering novel therapeutic options in the treatment of virus-induced myocarditis.

Proteomic analyses of age related changes in A.BY/SnJ mouse hearts

BackgroundA.BY/SnJ mice are used to study pathological alterations in the heart due to enteroviral infections. Since age is a well-known factor influencing the susceptibility of mice to infection, response to stress and manifestation of cardiovascular diseases, the myocardial proteome of A.BY/SnJ mice aged 1 and 4 months was comparatively studied using two dimensional-differential in-gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS).ResultsComplementary analyses by 2D-DIGE and gel-free LC-MS/MS revealed 96 distinct proteins displaying age associated alterations in their levels. Proteins related to protein transport, and transport chain, lipid metabolism and fatty acid transport showed significant changes in 4 months old mouse hearts compared to juvenile hearts. Proteins involved in lipid metabolism and transport were identified at significantly higher levels in older mice and dysregulation of proteins of the respiratory transport chain were observed.ConclusionThe current proteomics study discloses age dependent changes occurring in the hearts already in young mice of the strain A.BY/SnJ. Besides alterations in protein transport, we provide evidence that a decrease of ATP synthase in murine hearts starts already in the first months of life, leading to well-known low expression levels manifested in old mice thereby raising the possibility of reduced energy supply. In the first few months of murine life this seems to be compensated by an increased lipid metabolism. The functional alterations described should be considered during experimental setups in disease related studies.

Comparative immunoproteome analysis of the response of susceptible A.BY/SnJ and resistant C57BL/6 mice to Coxsackievirus B3-infection

Both, innate and cell-mediated immunity contribute to prevention of chronic myocarditis and consecutively, cardiomyopathy. Thus, in resistant C57BL/6 mice myocarditis induced by Coxsackievirus B3 (CVB3)-infection is abrogated by immune-mediated mechanisms. However, susceptible A.BY/SnJ mice develop dilated cardiomyopathy (DCM) due to chronic myocarditis. Cardiac auto-antibodies have been shown to play a pivotal role in the initiation and/or progression of inWammatory DCM. In order to investigate differences in the autoimmune response of susceptible and resistant mice to infection with CVB3, the patterns of autoantibodies reacting with heart proteins in A.BY/SnJ and C57BL/6 mice were profiled by 2-D Western blot analysis during the acute and chronic phases of myocarditis up to three months, when the pathophysiological phenotype in the susceptible mice has progressed to DCM. In the early phase of infection both mouse strains displayed similar autoantibody patterns. In contrast, at later time points compared to the resistant C57BL/6 strain susceptible A.BY/SnJ mice displayed a much stronger autoimmune response against proteins associated with cell structure, protein transport as well as primary metabolic processes such as energy production. During chronic myocarditis strong antibody responses against myosin heavy chain 6, mitochondrial and heat shock proteins were observed in A.BY/SnJ mice. Antibodies directed against alpha-enolase, serotransferrin, radixin and two processed myosin protein species accumulated late and only in A.BY/SnJ mice suffering from inWammatory DCM. Functional assignment of the target proteins of cardiac autoantibodies indicates that these might be directly involved in cardiac dysfunction.

Cyclophilin A affects inflammation, virus elimination and myocardial fibrosis in coxsackievirus B3-induced myocarditis

Extracellular cyclophilin A (CyPA) and its receptor Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147) modulate inflammatory processes beyond metalloproteinase (MMP) activity. Recently, we have shown that CyPA and CD147 are upregulated in patients with inflammatory cardiomyopathy. Here we investigate the role of CyPA and CD147 in murine coxsackievirus B3 (CVB3)-induced myocarditis. CVB3-infected CyPA(-/-) mice (129S6/SvEv) revealed a significantly reduced T-cell and macrophage recruitment at 8 days p.i. compared to wild-type mice. In A.BY/SnJ mice, treatment with the cyclophilin-inhibitor NIM811 was associated with a reduction of inflammatory lesions and MMP-9 expression but with enhanced virus replication 8 days p.i. At 28 days p.i. the extent of lesion areas was not affected bei NIM811, whereas the collagen content was reduced. Initiation of NIM811-treatment on day 12 (after an effective virus defense) resulted in an even more pronounced reduction of myocardial fibrosis. In conclusion, in CVB3-induced myocarditis CyPA is important for macrophage and T cell recruitment and effective virus defense and may represent a pharmacological target to modulate myocardial remodeling in myocarditis.

The mitochondrial respiratory chain has a critical role in the antiviral process in Coxsackievirus B3-induced myocarditis

Well-established differences in Coxsackievirus B3 (CVB3) elimination in resistant C57BL/6 and permissive A.SW/SnJ mice provide suitable models for studying the significance of the link between mitochondrial respiratory chain (RC), antioxidative stress components and mitochondrion-related apoptosis in the context of myocardial virus elimination. Distinct myocardial CVB3 titer in C57BL/6 (2.5±1.4 × 104 plaque-forming units (p.f.u.)/g tissue) and A.SW/SnJ mice (1.4±0.8 × 107 p.f.u./g) were associated with differences in the cardiac mitochondrial function 8 days post infection (p.i.). Infected C57BL/6 mouse hearts disclosed increased complex I (CI) and CIII activity, but restricted CII and normal CIV activity of RC. Reduced expression of the antioxidative catalase was accompanied by elevated lipid peroxidation (LPO), indicating oxidative stress. Intrinsic apoptosis was activated demonstrated by elevated levels of Bax, Bcl-2, caspase 3 and DNA degradation. In contrast, all myocardial RC complex activities were restricted in CVB3-infected A.SW/SnJ mice. The antioxidative system provided sufficient protection against oxidative stress shown by an elevated catalase expression and unaltered LPO. Bax and Bcl-2 levels were unchanged in CVB3-infected A.SW/SnJ mice, while caspase 3 was moderately increased but no DNA degradation was detectable. Correlation analyses including data from the two mouse strains revealed that reduced CVB3 titer correlated with increased CI and CIII activity, oxidative stress as well as active apoptosis during acute myocarditis (MC). C57BL/6 mice completely eliminated CVB3 and inflammation and normalized all intracellular parameters, while A.SW/SnJ mice showed permanently restricted CI activity in chronic MC 90 days p.i., at which time the replicating virus was no longer detectable but immunological processes were still active. Consequently, the regulation of energy metabolism appears crucial for an effective virus elimination and may be of prognostic and therapeutic significance for patients with virus-induced MC.

Antigen‐presentation capacity of dendritic cells is impaired in ongoing enterovirus myocarditis

Coxsackievirus B3 (CVB3)-infection is a frequent cause of acute myocarditis, which may result in chronic myocarditis and virus persistence. Investigation of the initial immune responses to CVB3 may shed light on the mechanisms that contribute to ongoing disease. DCs, as key professional APCs, were investigated in two MHC-matched hosts: while C57BL/6 mice are resistant to chronic CVB3-myocarditis, the A.BY/SnJ mouse strain exhibits susceptibility. DC maturation and activation were critically impaired in A.BY/SnJ mice, as reflected by the failure of DCs to induce co-stimulatory molecules and cytokine/chemokine responses. MHC class I-restricted antigen presentation via the cross-presentation pathway was also affected in DCs from A.BY/SnJ mice. DC maturation involves the accumulation of DC aggresome-like induced structures (DALISs) and the transient storage of defective ribosomal products (DRiPs). DCs from A.BY/SnJ mice showed permanent DALIS accumulation; the detection of poly-ubiquitinylated CVB3 proteins in these DALISs suggested a limitation in the MHC class I antigenic supply in this host. In conclusion, ongoing chronic disease in A.BY/SnJ mice due to a failure to clear the virus may be attributed to defects in DC maturation/activation and DC MHC class I antigen processing.

Cardioprotective effect of NO‐metoprolol in murine coxsackievirus B3‐induced myocarditis

The effect of NO-metoprolol, that is, 3-nitrooxypivaloyl metoprolol-amide, a novel NO-releasing derivative of the β1-blocking drug metoprolol was investigated in A.CA/SnJ mice infected with coxsackievirus B3 (CVB3) and compared to metoprolol and placebo. Daily treatment of mice with the respective drug started immediately (experiment A) or 3 days after virus infection (experiment B) and was continued until day 13 post-infection (p.i.). Two doses of NO-metoprolol were administered. Body mass differences, viral load, and histopathological signs of myocarditis were compared between the several groups. As a result, NO-metoprolol diminished significantly the body weight loss, the viral load and the histopathology, whereas metoprolol treatment led solely to a significant attenuation of myocardial damage. In experiment A, low dose NO-metoprolol decreased significantly enteroviral copy numbers. Both doses of NO-metoprolol had a significant effect on reduction of myocardial infiltrates and fibrosis. The data suggest that delayed drug administration might more advantageous. Both doses of NO-metoprolol reduced significantly the scores of four tested parameters compared to placebo. Body weight loss, virus titers, plus-strand as well as minus-strand enteroviral RNA levels, infiltration and fibrosis scores were diminished significantly when NO-metoprolol was given 3 days p.i. In addition, a significant difference regarding the enteroviral copy numbers was observed between low dose NO-metoprolol- and metoprolol-treated mice. Treatment with metoprolol reduced insignificantly the viral load and body weight loss (experiment A and B) but led to a significant reduction of myocardial histopathology in experiment A. The results indicate that NO-metoprolol treatment has a greater therapeutic benefit than metoprolol.

T Cell Epitope Specificity and Pathogenesis of Mouse Hepatitis Virus-1–Induced Disease in Susceptible and Resistant Hosts

Intranasal mouse hepatitis virus-1 (MHV-1) infection of susceptible mouse strains mimics some important pathologic features observed in the lungs of severe acute respiratory syndrome (SARS)-coronavirus-infected humans. The pathogenesis of SARS remains poorly understood, although increasing evidence suggests that immunopathology could play an important role. We previously reported that the adaptive immune response plays an important protective role in MHV-1-infected resistant B6 mice and that both CD4 and CD8 T cells play a significant role in the development of morbidity and lung pathology following intranasal MHV-1 infection of susceptible C3H/HeJ and A/J mice. In this study, we have identified novel CD4 and CD8 epitopes in MHV-1-infected susceptible and resistant strains of mice. Susceptible C3H/HeJ mice mount robust and broad MHV-1-specific CD4 T cell responses, whereas in resistant B6 mice, Ag-specific CD8 T cell responses dominate. We also show that previously immunized susceptible C3H/HeJ mice do not develop any morbidity and are completely protected following a lethal-dose MHV-1 challenge despite mounting only a modest secondary T cell response. Finally, we demonstrate that the resistance displayed by B6 mice is not solely accounted for by the elaboration of a broad and vigorous MHV-1-specific CD8 T cell response, as MHV-1 infection of C3.SW-H2(b)/SnJ mice, which mount an equally robust CD8 T cell response of the same specificity, is still associated with significant morbidity. Thus, identification of novel CD4 and CD8 T cell epitopes for MHV-1 permitted high-resolution analyses of pulmonary T cell responses in a mouse model of SARS.

Osteopontin

The characteristics of dilated cardiomyopathy (DCM) resulting from chronic viral myocarditis are remodeling processes of the extracellular matrix. Based on our findings of enhanced osteopontin (OPN) expression in inflamed human hearts, we further investigated in the murine model of acute and chronic coxsackievirus (CV)B3-myocarditis the role of OPN regarding its involvement in resolution of cardiac virus infection and fibrosis. In hearts of A.BY/SnJ mice susceptible to chronic CVB3-myocarditis, a pronounced increase of OPN expression levels was detected by microarray analysis and quantitative RT-PCR during acute stages of myocarditis. Combined immunohistochemistry and in situ hybridization identified infiltrating macrophages as main OPN producers. In contrast to resistant C57BL/6 and OPN gene-deficient mice, transcription levels of matrix metalloproteinase-3, TIMP1 (tissue inhibitor of metalloproteinases-1), uPA (urokinase-type plasminogen activator), and transforming growth factor beta1 were elevated in susceptible mice, and as a consequence, procollagen-1alpha mRNA expression and fibrosis was considerably enhanced. Treatment of infected susceptible mice with the vitamin D analog ZK 191784 led to decreased myocardial expression levels of OPN, metalloproteinase-3, TIMP1, uPA, and procollagen-1alpha and subsequently to reduced fibrosis. Concurrently, the fibrosis-relevant signaling molecules pERK (phosphorylated extracellular signal-regulated kinase) and pAkt (phosphorylated Akt), increased in A.BY/SnJ mice, were diminished in ZK 191784-treated mice. Here, we show that high expression levels of OPN in acute myocarditis are associated with consecutive development of extensive fibrosis that can be reduced by treatment with a vitamin D analog. Thus, OPN may serve as a diagnostic tool as well as a potential therapeutic target to limit cardiac remodeling in chronic myocarditis.

Effective Chemokine Secretion by Dendritic Cells and Expansion of Cross-Presenting CD4−/CD8+Dendritic Cells Define a Protective Phenotype in the Mouse Model of Coxsackievirus Myocarditis

Enteroviruses such as coxsackievirus B3 (CVB3) are able to induce lethal acute and chronic myocarditis. In resistant C57BL/6 mice, CVB3 myocarditis is abrogated by T-cell-dependent mechanisms, whereas major histocompatibility complex (MHC)-matched permissive A.BY/SnJ mice develop chronic myocarditis based on virus persistence. To define the role of T-cell-priming dendritic cells (DCs) in the outcome of CVB3 myocarditis, DCs were analyzed in this animal model in the course of CVB3 infection. In both mouse strains, DCs were found to be infectible with CVB3; however, formation of infectious virions was impaired. In DCs derived from C57BL/6 mice, significantly higher quantities of interleukin-10 (IL-10) and the proinflammatory cytokines IL-6 and tumor necrosis factor alpha were measured compared to those from A.BY/SnJ mice. Additionally, the chemokines interferon-inducible protein 10 (IP-10) and RANTES were secreted by DCs from resistant C57BL/6 mice earlier in infection and at significantly higher levels. The protective role of IP-10 in CVB3 myocarditis was confirmed in IP-10(-/-) mice, which had increased myocardial injury compared to the immunocompetent control animals. Also, major differences in resistant and permissive mice were found in DC subsets, with C57BL/6 mice harboring more cross-priming CD4(-) CD8(+) DCs. As CD4(-) CD8(+) DCs are known to express 10 times more Toll-like receptor 3 (TLR3) than other DC subsets, we followed the course of CVB3 infection in TLR3(-/-) mice. These mice developed a fulminant acute myocarditis and secreted sustained low amounts of type I interferons; secretion of IP-10 and RANTES was nearly abrogated in DCs. We conclude that MHC-independent genetic factors involving DC-related IP-10 secretion and TLR3 expression are beneficial in the prevention of chronic coxsackievirus myocarditis.

Sustained Nitric Oxide Synthesis Contributes to Immunopathology in Ongoing Myocarditis Attributable to Interleukin-10 Disorders

Ongoing coxsackievirus B3 (CVB3) myocarditis is characterized by persistence of viral RNA and chronic inflammation primarily mediated by macrophages and T cells. Activated macrophages produce anti-viral effector molecules comprising reactive nitrogen intermediates; however, reactive nitrogen intermediates also contribute to host tissue damage. Controlled activation of macrophages depends on interferon (IFN)-gamma and interleukin (IL)-10. To evaluate mechanisms involved in CVB3-induced pathogenesis of myocarditis, we determined the relationship of inducible nitric-oxide synthase (iNOS) mRNA expression with IFN-gamma and IL-10 secretion during CVB3 infection in different mouse strains. We found in susceptible A.BY/SnJ mice that develop ongoing myocarditis, a low and delayed IFN-gamma secretion and highly diminished IL-10 production compared with resistant C57BL/6 mice. Consequently, iNOS mRNA synthesis was delayed but clearly prolonged in susceptible mice. IL-10 gene-deficient mice confirmed the regulatory role of IL-10 in the outcome of CVB3 myocarditis. These mice did not establish a persistent cardiac infection and revealed IFN-gamma secretion kinetics similar to resistant mice but showed a slightly elongated cardiac iNOS mRNA expression resulting in extended myocarditis. We conclude that coordinated secretion of IFN-gamma and IL-10 is crucial for the effective resolution of CVB3 myocarditis. Moreover, lack of regulatory IL-10 leads to uncontrolled iNOS mRNA production, thus contributing to ongoing myocardial injury.

The Major Histocompatibility Complex Haplotype Affects T-Cell Recognition of Mycobacterial Antigens but Not Resistance to Mycobacterium tuberculosis in C3H Mice

Both innate and adaptive immunity play an important role in host resistance to Mycobacterium tuberculosis infection. Although several studies have suggested that the major histocompatibility complex (MHC) haplotype affects susceptibility to infection, it remains unclear whether the modulation of T-cell immunity by the MHC locus determines the host's susceptibility to tuberculosis. To determine whether allelic differences in the MHC locus affect the T-cell immune response after M. tuberculosis infection, we infected inbred and H-2 congenic mouse strains by the respiratory route. The H-2 locus has a profound effect on the antigen-specific CD4+-T-cell response after M. tuberculosis infection. CD4+ T cells from infected mice of the H-2(b) haplotype produced more gamma interferon (IFN-gamma) after in vitro stimulation with mycobacterial antigens than mice of the H-2(k) haplotype. A higher level of IFN-gamma was also detected in bronchoalveolar lavage fluid from infected mice of the H-2(b) haplotype. Furthermore, C3.SW-H2(b)/SnJ mice generate and recruit activated T cells to the lung after infection. Despite a robust immune response, C3.SW-H2(b)/SnJ mice succumbed to infection early and were similarly susceptible to infection as other C3H (H-2(k)) substrains. These results suggest that although the MHC haplotype has a profound impact on the T-cell recognition of M. tuberculosis antigens, the susceptibility of C3H mice to infection is MHC independent.

Major Locus on Mouse Chromosome 17 and Minor Locus on Chromosome 9 are Linked with Alopecia Areata in C3H/HeJ Mice

Alopecia areata is an autoimmune disease that targets actively growing (anagen) hair follicles in humans, mice, rats, dogs, horses, and cattle. C3H/HeJ mice spontaneously develop alopecia areata from 5 mo of age and older in females and later in males. Frequency of disease approached 20% in a colony by 18 mo of age. C57BL/6J mice do not develop alopecia areata. A segregating F2 population of female mice (n=1096) was generated from crossing these two strains. Alopecia areata (n=138) and clinically normal (n=214) mice were genotyped at 12 mo of age using 211 microsatellite probes. The peak logarithm of odds ratio score on mouse chromosome 17 (10.9) was around marker D17Mit134 at 16.9 cM from the centromere. The mouse histocompatibility locus, H2, the mouse equivalent of human leukocyte antigen in humans, was a likely candidate. Twelve-month-old C3H.SW-H2b/SnJ mice (C3H/HeJ congenic mice in which the H2k purported susceptibility locus was replaced with the H2b purported resistance locus) did not develop alopecia areata, supporting this locus as being important in alopecia areata. A suggestive linkage was also found on mouse Chromosome 9 (logarithm of odds ratio score 2.0) around D9Mit206, 20 cM from the centromere. The interval on mouse Chromosome 17 contains several orthologous genes potentially associated with human alopecia areata.

CD8+ T cells inhibit immunoglobulin E synthesis in low responder SJL/J mice.

In an effort to examine the basis for low IgE responsiveness of SJL/J strain mice, we analysed the profiles of cytokines, such as interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma), in SJL/J and A.SW/SnJ mice following immunization. Splenocytes of ovalbumin (OVA)-immunized SJL/J mice, secreted significantly higher levels of IL-4 and lower levels of IFN-gamma than those of A.SW/SnJ mice. A time-course analysis of cytokine expression in in vitro cultures of spleen cells indicated that the levels of IL-4 and IL-2 remained persistently high throughout in the cultures of SJL/J splenocytes as opposed to those of A.SW/SnJ. Depletion of CD4+ T cells in vivo suppressed the production of IL-2, IL-4 and IFN-gamma suggesting that CD4 T cells are the producers of most cytokines in both SJL/J and A.SW/SnJ mice. Depletion of CD8+ T cells in vivo not only induced productive epsilon transcript but also enhanced IgE production in SJL/J mice. Moreover, CD8 depletion in SJL/J mice led to decreased production of IFN-gamma, resulting in a net decrease in the ratio of IFN-gamma to IL-4. A similar shift in the IFN-gamma/IL-4 ratio was found in splenocytes of SJL/J mice following irradiation, which is known to enhance IgE synthesis in these mice. Taken together, it is concluded that low IgE responsiveness in SJL/J mice is not due to a defect in IL-4 production per se. Increased IFN-gamma production by the CD8+ T cells inhibits class switch and suppresses IgE antibody production in SJL/J mice.

The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa--a review.

Pseudomonas aeruginosa (Pa) produces several surface-associated adherence factors or adhesins which promote attachment to epithelial cells and contribute to the virulence of this pathogen. Among them, the type-4 pilus accounts for about 90% of the adherence capability of Pa to human lung pneumocyte A549 cells. Furthermore, it is responsible for more than 90% of the virulence in AB.Y/SnJ mice. Pa type-4 pili display a tip-base differentiation with the adherence function located at the tip of the pilus. All Pa pili prototypes characterized so far contain an intrachain disulfide loop (DSL) of 12 to 17 semi-conserved amino acid residues at the C-terminus of pilin. In Pa, this DSL comprises the epithelial cell-binding domain. Despite little sequence homology, DSL-containing peptides of different pilin prototypes seemingly reveal striking structural similarities. Two beta-turns within the loop and the disulfide bridge impose significant structural rigidity on the DSL pilin peptide, suggesting a conformationally conserved binding domain. Insertions of C-terminal pilin peptides with disrupted DSL displayed on the surface of bacterial S-layer mediate the same receptor binding characteristics as pili, indicating that a DSL is not essential in maintaining the functionality of the binding domain. Pa pili bind specifically to the carbohydrate moiety of the glycosphingolipids (GSL) asialo-G(M1) and asialo-G(M2) and, to a much weaker extent, to lactosyl ceramide and ceramide trihexoside. The disaccharide sequence GalNAc beta(1-4)Gal, common in both asialo-G(M1) and asialo-G(M2), likely represents the minimal structural receptor motif recognized by the pili. Pa pili also bind to surface-localized proteins of human epithelial cells and other cell types, suggesting that non-sialylated GSL and (glyco)proteins function as receptors of pili. In addition to the major pilus adhesin, exoenzyme S and, as recent studies indicate, flagella, are further protein adhesins of Pa with GSL receptor binding specificities similar to those of pili.

Spontaneous central nervous system remyelination in strain A mice after infection with the Daniel's (DA) strain of Theiler's virus.

Intracerebral infection of susceptible SJL/J (H-2s) mice with the Daniel's strain of Theiler's murine encephalomyelitis virus produces chronic, progressive, inflammatory central nervous system demyelination, with minimal spontaneous remyelination. To assess the role of host genetic factors in spontaneous myelin repair following chronic infection with the Daniel's strain of Theiler's virus, we examined demyelination and spontaneous remyelination in strain A mice after infection with Theiler's virus. We found that A.BY/SnJ (H-2a), and A.SW/SnJ (H-2s) mice all developed chronic demyelination with substantial spontaneous remyelination 90 days after infection. In the spinal cords of both A/J and A/WySnJ mice, one quarter of the total lesion area showed spontaneous remyelination, whereas in A.SW/SnJ mice, the extent of remyelination increased to two thirds of the total lesion area. The spontaneous remyelination seen in strain A mice was consistent with myelin repair by oligodendrocytes and Schwann cells, and occurred despite the presence of persistent virus antigen. These results indicate that host-pathogen interactions play an important role in myelin regeneration after virus-induced demyelination, and suggest that host genetic factors influence spontaneous remyelination.

Myogenic Expression of Mesenchymal Stem Cells within Myotubes ofmdxMicein Vitroandin Vivo

The myogenic potential of bone marrow- and periosteum-derived mesenchymal stem cells (MSCs) was studied in vitro by coculture of MSCs of snj mice with myoblasts of newborn snj mice or 3-week-old mdx mice. MSCs were labeled with [(3)H]thymidine and cocultured with muscle precursor cells. At 5 different time points, the cocultures were harvested and prepared for autoradiography. Cocultures of MSCs and mdx mouse-derived myoblasts were immunostained for dystrophin before autoradiography. Autoradiographic grains were detected over isolated nuclei in myotubes, which stained positively with antidystrophin antibody. In vivo myogenic potential of MSCs was tested by direct injection into growing muscle of mdx mice. Equal numbers of nonmutant bone marrow-derived MSCs or myoblasts were injected separately into the tibialis anterior muscles of mdx mice. Muscle samples were harvested at 6, 8, and 10 weeks after injection, weighed, and stained with antidystrophin antibody. A small yet significant increase in muscle mass was observed in both the myoblast-injected (11% increase) and MSC-injected muscles (3%), as compared to controls. Muscle injected with myoblasts showed a remarkable conversion from dystrophin-negative to dystrophin-positive fibers (30-40%) in mdx mice injected with normal myoblasts, as previously reported by others. The frequency of dystrophin-positive fibers in mdx mouse muscle injected with marrow-derived MSCs was lower than that of the muscles injected with myoblasts, but was significantly higher than control muscles injected with medium. These results suggest that within the population of MSCs there are cells that are able to differentiate into skeletal muscle.

Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice

The disulfide loop domain of Pseudomonas aeruginosa PAO pilin was altered by insertion of a chloramphenicol acetyltransferase gene into the pilin gene so that the C-terminal nine amino acids were replaced with 11 new amino acids. The altered pilin gene was transferred into wild-type PAO by recombination, where it did not affect normal piliation as observed by transmission electron microscopy or change of sensitivity to f116, PO4, B9, and Pf1 pilus-specific bacteriophages. However, the binding to human pneumocyte A549 cells was markedly reduced when tested in an in vitro binding assay (2 to 6 bacteria bound per A549 cell for the mutant bacteria compared with 50 bacteria per A549 cell for the wild-type bacteria). Additionally, when susceptible A.BY/SnJ mice were challenged with wild-type P. aeruginosa PAO and with P. aeruginosa PAO-MP (altered pilin gene), a 50% lethal dose of 3 x 10(6) bacteria per mouse was observed for PAO-MP compared with 7 x 10(4) bacteria per mouse for PAO. Approximately 90 of the adherence capability of P. aeruginosa PAO is seemingly attributable to the C-terminal disulfide loop adherence domain of pili. The pilus adherence function contributes significantly to the virulence of P. aeruginosa PAO in the A.BY/SnJ mouse infection model.