Resistance to fulminant hepatitis and carcinogenesis conferred by overexpression of retinoblastoma protein in mouse liver.

Previously, retinoblastoma (Rb) transgenic mice were produced under the control of the Rb gene promoter and showed dwarf characteristics. Here, we created transgenic mice, in which the human Rb gene was controlled by the hepatocyte nuclear factor-1 gene promoter/enhancer and was expressed primarily in the liver. The liver of these novel transgenic mice was normally developed. Intriguingly, these mice showed resistance to fulminant hepatitis induced by anti-Fas antibody as well as resistance to chemical carcinogenesis in the liver. These results show that the Rb protein acts as an anti-apoptotic and anti-oncogenic agent in vivo. Our novel construct may be useful as a gene cassette in gene therapy for prevention of fulminant hepatitis and hepatoma.

Oral tolerance.

Oral administration of antigen results in immune tolerance which is mediated by anergy, deletion or the generation of regulatory cells, depending on the dose of antigen administered. Regulatory cells which secrete anti-inflammatory cytokines such as TGF-beta lead to bystander suppression at the target organ of the antigen fed. Oral administration of autoantigens has been shown to suppress autoimmune diseases in several animal models and is being tested as a potential therapy in human autoimmune and other inflammatory diseases.

Suppression of collagen-induced arthritis by oral or nasal administration of type II collagen.

We directly compared the effects of oral and nasal administration of collagen type II (CII) on disease progression, cytokine production and T cell responses in DBA/1 mice. Lymphocytes were assayed for proliferation and cytokine production and cell lines established. T cells from fed or nasally treated groups proliferated significantly less and produced markedly less IFN-gamma than the non-fed immunized group 10 days after immunization and prior to onset of arthritis. T cell lines established from fed or nasally treated mice showed a pattern of cytokine production involving IL-4, IL-10 and TGF-beta, whereas T cell lines from the control group produced more IFN-gamma and IL-2. Suppression of clinical measures of arthritis was equivalent in the nasal and orally treated groups. Animals were then tested for IFN-gamma production 70 days after a booster immunization at a time when disease was apparent. Mucosally treated animals secreted less IFN-gamma as compared to controls, even at this late time point. Suppression of collagen induced arthritis (CIA) by nasal treatment of mice with CII was associated with diminished levels of TNF-alpha and IL-6 mRNA expression in the joints of tolerized mice, two cytokines known to be involved in the inflammatory and pathological process of CIA. These results demonstrate the induction of antigen specific Th2 and TGF-beta secreting regulatory cells following both oral and nasal treatment, which is associated with suppression of local inflammation in the joints and decreased Th1 type responses in the periphery throughout the course of the illness.

Altered expression level of a systemic nuclear autoantigen determines the fate of immune response to self.

One of the hallmarks of systemic autoimmune diseases is immune responses to systemic nuclear autoantigens. We have examined the fate of the immune response against a nuclear autoantigen using human U1 small nuclear ribonucleoprotein-A protein (HuA) transgenic (Tg) mice by adoptive transfer of autoreactive lymphocytes. We obtained two Tg lines that have different expression levels of the transgene. After spleen cells from HuA-immunized wild-type mice were transferred to Tg mice and their non-Tg littermates, these recipients were injected with HuA/IFA to induce a recall memory response. HAB69, which expressed a lower amount of HuA, exhibited a vigorous increase in the autoantibody level and glomerulonephritis. Moreover, the autoreactivity spread to 70K autoantigen. Alternatively, in HAB64, which expressed a higher amount of HuA, the production of autoantibody was markedly suppressed. The immune response to HuA autoantigen was impaired as demonstrated in a both delayed-type hypersensitivity response and proliferation assay. This inhibition was Ag-specific and was mediated by T cells. These data suggest that the expression level of systemic autoantigens influences the outcome of the immune response to self.

IL-4 is a differentiation factor for transforming growth factor-beta secreting Th3 cells and oral administration of IL-4 enhances oral tolerance in experimental allergic encephalomyelitis.

We have previously shown that following oral administration of myelin basic protein (MBP), regulatory T cells are generated from gut-associated lymphoid tissue and that these cells suppress experimental allergic encephalomyelitis (EAE). These regulatory T cells produce transforming growth factor-beta (TGF-beta) with various amounts of IL-4 and IL-10 and these TGF-beta-secreting T cells have been termed Th3 cells. T cells in lymphoid organs drained by mucosal sites secrete IL-4 as a primary T cell growth factor. In the present study, we examined the role of IL-4 on oral tolerance and in the generation of TGF-beta secreting cells. Treatment of (PLJ x SJL)F1 mice with intraperitoneal (i. p.) IL-4 and low-dose oral MBP (0.5 mg) given three times reduced the severity of EAE, whereas i.p. injection of IL-4 alone or oral MBP alone given in these suboptimal doses, showed no protection. Spleen cells from protected mice produced increased amounts of TGF-beta and reduced IFN-gamma upon stimulation with MBP in vitro. Mucosal MBP-specific IgA production was significantly increased in IL-4 plus MBP fed animals. Moreover, oral administration of IL-4 (1 microg per feeding) also enhanced the suppression of EAE by oral MBP and this protective effect was reversed by administration of anti-TGF-beta antibody in vivo. Reverse transcription-PCR showed enhanced suppression of IFN-gamma in Peyer's patch in animals fed MBP and IL-4 versus those fed MBP alone. We then investigated the role of IL-4 in the generation of TGF-beta-secreting cells using MBP Ac1-11 TCR transgenic animals. Cells were cultured with IL-2, IL-4, or IFN-gamma in the presence of MBP and limiting dilution analysis for cytokine-secreting cells performed. We found that IL-4, but not IL-2 or IFN-gamma, generated TGF-beta-secreting T cells from naive splenic T cells and that these cells provided help for IgA production. These findings demonstrate that IL-4 is a differentiation factor for TGF-beta-secreting Th3 cells and oral IL-4 has a synergistic effect on low-dose oral tolerance that is associated with increased TGF-beta secretion.

Abrogation of autoimmune diabetes in nonobese diabetic mice and protection against effector lymphocytes by transgenic paracrine TGF-beta1.

Paracrine effect of transforming growth factor-beta1 (TGF-beta1) on autoimmune insulitis and diabetes was studied by transgenic production of the active form of porcine TGF-beta1 (pTGF-beta1) in pancreatic islet (islet) alpha cells in nonobese diabetic (NOD) mice under the control of rat glucagon promoter (RGP) (NOD-RGP-TGF-beta1). None of 27 NOD-RGP-TGF- beta1 mice developed diabetes by 45 wk of age, in contrast to 40 and 71% in male and female nontransgenic mice, respectively. None of the NOD-RGP-TGF-beta1 mice developed diabetes after cyclophosphamide (CY) administration. Adoptive transfer of splenocytes of NOD-RGP-TGF-beta1 mice to neonatal NOD mice did not transfer diabetes after CY administration. Adoptive transfer of three types of diabetogenic lymphocytes to NOD-RGP-TGF-beta1 and nontransgenic mice after CY administration led to the lower incidence of diabetes in NOD-RGP-TGF-beta1 mice versus that in nontransgenic mice: 29 vs. 77% for diabetogenic splenocytes, 25 vs. 75% for islet beta cell-specific Th1 clone cells, and 0 vs. 50% for islet beta cell-specific CD8(+) clone cells, respectively. Based on these, it is concluded that autoimmune diabetes in NOD mice is not a systemic disease and it can be completely prevented by the paracrine TGF-beta1 in the islet compartment through protection against CD4(+) and CD8(+) effector lymphocytes.

[Fundamental studies on antibacterial activity of clindamycin against Propionibacterium acnes].

Antibacterial activity of clindamycin against Propionibacterium acnes (P. acnes) was evaluated in comparison with nadifloxacin in vitro. Using a burned-infected mouse model, topical application of 1% gel form of clindamycin phosphate on P. acnes was also evaluated in in vivo. (1) The MIC of clindamycin measured by agar dilution method was 0.02 microgram/ml, and this value was smaller than that of nadifloxacin (0.3 microgram/ml). (2) At concentrations on 1-, 2- and 4- times the MIC clindamycin demonstrated bacteriostatic activity on P. acnes and showed bactericidal activity at 5-times the MIC. Nadifloxacin showed bacteriostatic activity at one half the MIC and bactericidal activity at the MIC. (3) Against acquired resistant strains of P. acnes, the highest concentrations of clindamycin and nadifloxacin that did not inhibit growth of the organism increased 5-fold higher than those against sensitive strain during 25 successive cultures in vitro. Therefore, the resistance of P. acnes was found to be emerged at almost the same ratio against both agents. (4) The chemotherapeutic effects of 1% gel form of clindamycin phosphate and 1% cream of nadifloxacin were evaluated for given subcutaneously to infected P. acnes at the burned site in mice. The topical application of either agents showed a significant reduction of number of bacteria and this result predicted clinical efficacy of topical application of clindamycin.

In situ immune response in gut-associated lymphoid tissue (GALT) following oral antigen in TCR-transgenic mice.

Oral administration of Ag results in systemic hyporesponsiveness termed oral tolerance. The regulatory cells induced by oral Ag are effective in the suppression of Th1-type autoimmune diseases. We examined the cytokine microenvironment in gut-associated lymphoid tissue in response to orally administered OVA in OVA TCR-transgenic mice. Mice were fed a low (0.5 mg) or high (500 mg) dose of OVA one time or five times. Immunohistochemical analysis demonstrated increased IL-4, IL-10, and TGF-beta in the gut within 6 h of a low-dose feeding and after five low-dose or high-dose feedings. IFN-gamma and IL-2 either decreased or showed no change, with the exception of a small transient increase in IL-2 at 6 h after a low dose. Increases in IL-4 and IL-10 were found in the dome of the Peyer's patch, and increases in TGF-beta were observed in the interfollicular region and the villi. IL-10 was also substantially increased in the villi. IL-4 and IL-10 were produced predominately by CD4+ T cells. TGF-beta was found predominately in macrophages and CD4+ T cells. Peyer's patches had a marked up-regulation of TGF-beta mRNA as measured by RT-PCR. These results demonstrate the differential activation of cytokine production in discrete regions of gut-associated lymphoid tissue. The induction of cytokines known to inhibit autoimmune disease at the site of Ag absorption indicates an important role for the mucosal immune system in the establishment of oral tolerance.

Reduced T helper 1 responses in IL-12 p40 transgenic mice.

To investigate the antagonistic effect of IL-12 p40 on IL-12 activity in vivo, we generated transgenic (Tg) mice in which p40 gene was regulated by a liver-specific promoter. Three Tg mouse lines were generated, and they expressed the p40 transgene predominantly in liver. Serum p40 level was extremely high, and it consisted of mainly monomer and homodimer and also of higher m.w. complexes. These Tg mice did not show any apparent phenotypic difference from control littermates in lymphoid cells. Enhancement of NK cell lytic activity in spleen by administration of rIL-12 to these mice was greatly diminished. Ag induced cytokine production was impaired: decreased production of IFN-gamma and increased production of IL-4 and IL-10. Delayed-type hypersensitivity response was also significantly reduced. Moreover, these Tg mice showed increased susceptibility to the infection with an intracellular pathogen, blood-stage Plasmodium berghei XAT, which is an irradiation-induced attenuated substrain of P. berghei NK65, presumably due to the decreased IFN-gamma production. These results suggest that p40 functions as an IL-12 antagonist in vivo, and that Th1 responses in p40 Tg mice are significantly reduced. Thus, these Tg mice could be a useful model to evaluate the inhibitory effect of p40 on IL-12-mediated various immune responses in vivo.

Role of cytosolic phospholipase A2 in allergic response and parturition.

Phospholipase A2 (PLA2) comprises a superfamily of enzymes that hydrolyse the ester bond of phospholipids at the sn-2 position. Among the members of this superfamily, cytosolic PLA2 has attracted attention because it preferentially hydrolyses arachidonoyl phospholipids and is activated by submicromolar concentrations of Ca2+ ions and by phosphorylation by mitogen-activated protein kinases (MAP kinases). Here we investigate the function of cytosolic PLA2 in vivo by using homologous recombination to generate mice deficient in this enzyme. These mice showed a marked decrease in their production of eicosanoids and platelet-activating factor in peritoneal macrophages. Their ovalbumin-induced anaphylactic responses were significantly reduced, as was their bronchial reactivity to methacholine. Female mutant mice failed to deliver offspring, but these could be rescued by administration of a progesterone-receptor antagonist to the mother at term. Considered together with previous findings, our results indicate that cytosolic PLA2 plays a non-redundant role in allergic responses and reproductive physiology.

Developmentally ordered V-J recombination in mouse T cell receptor gamma locus is not perturbed by targeted deletion of the Vgamma4 gene.

Mouse TCR gamma genes in the gamma1 cluster are arranged in the order of Vgamma5, Vgamma2, Vgamma4, Vgamma3, Jgamma1, and Cgamma1 on the chromosome. During thymic ontogeny, each Vgamma gene recombines with the Jgamma1 gene in the order of proximity to Jgamma1. To explore the mechanism of the ordered recombination, we generated Vgamma4-deficient mice by gene targeting and the Cre/loxP system, by deleting the 4.8-kb DNA region between 3' of the Vgamma2 and 3' of the Vgamma4. In semiquantitative PCR analysis, Vgamma2-Jgamma1 recombination was detected frequently in adult thymus, while Vgamma3-Jgamma1 recombination preferentially occurred in fetal thymus of the mutant mice. There was no difference in the frequency of V-J recombinations between control and mutant mice. Southern blot analysis also revealed that recombination of the Vgamma2 gene occurred as frequently as in control mice. In addition, there was no difference in the levels of germline transcripts of Vgamma2 and Vgamma3 genes between control and mutant mice. Therefore, regulation of the Vgamma-Jgamma recombination was not affected by deletion of the Vgamma4 gene. These results suggest that the ordered recombination is controlled by regulatory elements near each Vgamma gene.

Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs.

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool for engineering chromosomal changes in animal cells. Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs. In the present study, we examined the efficacy of this method to remove loxP-flanked DNA sequences in a gene-targeted locus in fertilized eggs. We replaced a part of the T-cell receptor gamma (TCR V gamma) locus with homologous sequences containing a loxP-flanked neogene in mouse embryonic stem (ES) cells by gene-targeting technique. The resulting ES cell clones containing the mutant allele (V gamma LNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild-type female mice. One hundred and seventy-six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS-Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted V gamma locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre-mediated recombination, resulting in a deletion of the loxP-flanked sequences (V gamma delta) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the V gamma delta locus to their offspring. The other 8 pups carried only wild-type alleles. There were no pups carrying the unrecombined V gamma LNL locus. Thus, the frequency of Cre-mediated recombination was 100% (3/3) with this method. In contrast, when closed circular pCAGGS-Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the V gamma LNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes.

The effect of adriamycin against a liver metastatic model by encapsulation in liposomes.

Antitumor activities of liposomes containing adriamycin (L-ADM) and their distribution process into tumour cells were analysed. The lipid composition of the liposomes was dimyristoylphosphatidylglycerol (DMPG)/egg phosphatidylcholine/cholesterol/adriamycin (ADM) in a molar ratio of 11.4:2:12:1.3. Liver-metastasizing murine tumour models, M5076 and L5178Y-ML, were used. In vivo antitumour effect against these tumour models was assessed from increase in life span (ILS). The survival prolongation effect of L-ADM in mice with liver failure caused by M5076 was significantly higher than that of F-ADM. In contrast, significant enhancement of the effects by encapsulation in liposomes was not observed in L5178Y-ML-bearing mice. In vitro cytostatic activities of L-ADM against M5076 cells as well as against other tumour cell lines were lower than those of F-ADM. The in vitro kinetic study on the distribution of L-ADM to the tumour cells revealed that ADM in L-ADM was taken up into the tumour cells mainly after it was released from the liposomes rather than taken up as the liposomal form. Among the cell lines tested, M5076 cells had the highest phagocytic activity and therefore the highest uptake activity of ADM during incubation with L-ADM. These findings suggest that the augmented antitumour activity of L-ADM in M5076-bearing mice was the result of phagocytosis of L-ADM by M5076 cells as well as the reduction of toxicity, prolonged retention of ADM in systemic circulation, and liver accumulation of ADM after administration of L-ADM.

Interleukin 7 receptor-deficient mice lack gammadelta T cells.

The interleukin 7 receptor (IL-7R) plays a crucial role in early B- and T-cell development. It consists of a unique a chain and a common gamma chain [IL-2 receptor gamma chain (IL-2Rgamma)]. Gene inactivation of IL-7, IL-7R, and IL-2Rgamma resulted in severe impairment of B and T lymphopoiesis in mice. In addition, IL-2Rgamma-deficient mice lack gammadelta T cells in the skin and have the impaired development of natural killer (NK) cells and intraepithelial lymphocytes. To explore the role of IL-7/IL-7R system in gammadelta T- and NK-cell development, we have generated and analyzed IL-7R-deficient mice. gammadelta T cells were absent from skin, gut, liver, and spleen in the deficient mice. In contrast, alphabeta T and B cells were detected in reduced, but certain, numbers, and NK cells developed normally. The gammadelta T-cell development in fetal and adult thymus was also completely blocked. These results clearly demonstrate that the signal from IL-7R is indispensable for gammadelta T-cell development in both thymic and extrathymic pathways. On the contrary, it is suggested that NK-cell development requires cytokine(s) other than IL-7.

Clonal prevalence of T cells infiltrating into the pancreas of prediabetic non-obese diabetic mice.

The non-obese diabetic (NOD) mouse spontaneously develops T-cell-mediated autoimmune insulitis. We analyzed the clonotypes of T cell infiltrates of the NOD mouse islets using a new method we have developed recently, which consists of RT-PCR amplification of the CDR3 region of the TCR beta chain mRNA and subsequent single-strand conformation polymorphism (SSCP) analysis. NOD mice of 10-32 weeks of age were shown to accumulate oligoclonal T cells in the pancreas. To examine whether each T cell clone stays in a small area of the pancreas or spreads over the whole pancreas, a pancreas was divided into two pieces, which were then subsequently analyzed in a pair by the above PCR-SSCP method. When a pair produces common bands with the same mobility in SSCP gel, they are likely to represent the presence of the same T cell clones between these two parts of the pancreas. Aged mice (24-32 weeks old) with severe insulitis obviously produced more common bands for most of the Vbeta subfamilies than younger mice (10 weeks old) with only periinsulitis. DNA sequencing verified that these common bands have the same TCR junctional sequences, suggesting that they were derived from the same T cell clones. These results suggest that clonal prevalence of T cells infiltrating into the pancreas occurs in the late stage of insulitis development and that a limited number of T cell clones finally predominate over the whole pancreas.

Heterogeneity of N insertion capacity in fetal hematopoietic stem cells.

TCR gene rearrangement is strictly regulated during mouse ontogeny. The V-(D)-J junctions of alphabeta and gammadelta TCR transcripts expressed in the adult thymus are more highly diverse than those in the fetal thymus. We previously showed that adult hematopoietic stem cells (HSC) have a higher capacity to insert N nucleotides into Vgamma4 TCR transcripts than fetal HSC and that the level of N nucleotide insertion is determined, at least in part, at the level of HSC. To analyze this developmental change of HSC at the single cell level, we investigated N nucleotide insertions in three TCR transcripts (Vgamma4, Vgamma2 and Vbeta8) derived from limiting numbers of fetal liver HSC by fetal thymic organ culture. Eight day-14 fetal liver HSC clones showed various levels of N nucleotide insertions in Vgamma transcripts (0-78%). On the other hand, the level of N insertions was similarly regulated in Vgamma4, Vgamma2, and Vbeta8 TCR transcripts in a clone-specific way. These results suggested that the level of N insertion is programmed at the level of single HSC and that fetal liver contains a heterogeneous population of HSC in terms of N insertion capacity. After 3 weeks culture with a stromal cell line, fetal HSC showed higher levels of N insertion capacity than before culture. This result and the presence of HSC with intermediate N insertion capacity support the hypothesis that the developmental potential of individual HSC gradually changes from fetal to adult type in one stem cell lineage.

[Production and characterization of a monoclonal antibody specific for Salmonella O19-antigen].

A monoclonal antibody, SBY1 (IgM, kappa), against the Salmonella O-antigen was generated by using the myeloma cell line Sp2/O-Ag14 as a fusion partner with spleen cells from BALB/c mice immunized with S. senftenberg 963 K. SBY1 was characterized by the slide agglutination and absorption test. SBY1 was believed to show the specificity to O1-, O3- or O19-antigens of Salmonella because S. Senftenberg 963 K (O1, 3, 19) was used as the antigen for immunization. The slide agglutination test with the Salmonella serovars indicated the responsiveness of SBY1. SBY1 was reactive only with strains that possessed O19-antigen. The agglutinating ability of SBY1 was absorbed completely with bacilli possessing O19-antigen. These finding indicates that SBY1 is specific for O19-antigen. Polyclonal factor sera for he serotyping of the O3, 10 group of Salmonella cross-reacted with Salmonella group O1, 3, 19 in the slide agglutination test. In contrast, SBY1 did not cross-react with serovars from several other Salmonella groups. These data suggest the usefulness of SBY1 as a serodiagnostic tool for serotyping of Salmonella.