H-2-linked Ir gene control of T cell recognition of the Sm nuclear autoantigen and the aberrant response of autoimmune MRL/Mp-+/+ mice

We have examined T cell recognition of the nuclear autoantigen Sm. Rabbit Sm-primed cells from autoimmune MRL/Mp-+/+ (+/+) mice and from all normal strains tested were able to proliferate to rabbit Sm in vitro. In contrast, the reactivity of normal strains to Sm of murine origin was genetically restricted; only H-2f strains B10.M and A.CA, and H-2s strains B10.S and A.SW could recognize mouse Sm, suggesting that responsiveness to mouse Sm was under the control of H-2-linked Ir genes. Although five Iak-bearing normal strains (B10.A, B10.A(2R), B10.BR, A/Sn, and CBA) did not recognize mouse Sm, autoimmune +/+ (Iak) mice were responders. The responsiveness of the +/+ mice to Sm was probably not due to differences in their Iak region, compared with other strains, because the Iak region of normal strains and the autoimmune +/+ strain were indistinguishable by interstrain MLC, immune response gene product function, and recognition by anti-Iak mAb. Inhibition of Sm-induced proliferation by mAb demonstrated that T cells from autoimmune +/+ mice, responder normal strains, and nonresponder normal strains recognized rabbit and mouse Sm in the context of I region-encoded products. The T cell response to Sm antigen in normal mice is therefore Ia region restricted and, for the murine antigen, under Ir gene control. Autoimmune mice that spontaneously make anti-Sm antibodies (+/+) also perceive Sm in an Ia-restricted manner, but their responder status abrogates H-2-linked Ir gene control.     

Role of the Sm antigen in the generation of anti-Sm autoantibodies in the SLE-prone MRL mouse

MRL/Mp-+/+ (+/+) and MRL/Mp-lpr/lpr (lpr) mice spontaneously produce the SLE-specific anti-nuclear antibody, anti-Sm. Previous work on the clonality and specificity of the anti-Sm response has suggested that the Sm antigen itself induces this autoantibody. In the present work, we have directly investigated the immunogenicity of Sm. In short-term cultures, Sm antigen was shown to be important for the de novo generation of anti-Sm PFC in vitro. The addition of purified Sm to cultures of spleen cells from anti-Sm-positive lpr mice augmented the number of anti-Sm PFC on day 4. Also, the addition of Fab anti-Sm to such cultures inhibited the generation of anti-Sm PFC, probably by blocking determinants on endogenous Sm. The ability of the autoantigen to initiate anti-Sm generation in vivo was investigated by hyperimmunizing +/+ mice with Sm from rabbit or mouse sources. Such mice specifically produced antibodies that recognized both rabbit and mouse Sm as determined by ELISA. The IgG subclass distribution of these induced antibodies was similar to that of spontaneous anti-Sm antibodies found in older mice of the same strain. Our data indicate that the Sm antigen can both initiate and augment production of the anti-Sm autoantibody. These findings provide additional evidence that the spontaneous anti-Sm response in SLE is antigen driven.     

Joint meeting of the Belgische Vereniging voor Biofysica Société Belge de Biophysique

Summary The rare earth radionuclides¹⁷⁷Lu and¹⁵³Sm were administered as single i.p. injections in NMRI mice. Lu was deposited principally (up to 60%) in the skeleton if the quantity of stable carrier was low. Increase of stable carrier enhanced deposition in the reticulo-endothelial system. Sm was preferentially deposited in the liver; the liver deposits were further increased by the addition of stable Sm. Liver doses of between 75 and 150 Gy, resulting from a single injection of¹⁵³Sm together with 2 mg/kg stable carrier, led to severe lesions in the liver five months after treatment.Administration of¹⁷⁷Lu resulting in skeletal doses of between 28 and 224 Gy was found to be osteosarcomogenic. Up to 40% osteosarcoma incidence was obtained in animals with 56 and 112 Gy doses in the skeleton. Skeletal doses of this order of magnitude are also known to be osteosarcomogenic when given as⁹⁰Sr injections. The analogous situation with-emitters is discussed.Dedicated to Prof. Dr. Wolfgang Gössner on the occasion of his 60th birthdayIn Association with EURATOM (Contr. Nr. 218-76-1)     

Comparative pathogenicity of auxotrophic mutants of Candida albicans

An induced mutant of Candida albicans with greatly decreased virulence for mice is described. The mutant was one of five auxotrophic mutants obtained by ultraviolet irradiation of a clinical isolate (strain MY 1044). The five mutants included two methionine auxotrophs, one methionine-cysteine auxotroph, one temperature-sensitive serine auxotroph, and one auxotroph with unknown growth requirements. Each of the mutants produced normal mycelium and had a normal profile of susceptibility to four antifungal drugs. The virulence of each mutant was compared with the parent strain by LD50 determination in mice. Four of the five auxotrophs exhibited LD50's that were not significantly different from the parent strain (mean LD50 = 7.5 x 10(5) cells). However, the temperature-sensitive serine auxotroph was significantly less virulent than the parent strain (LD50 greater than 10(7) cells), even though it grew well in vivo and in mouse serum at 37 degrees C in vitro. Use of this mutant in conjunction with its "isogenic" parent should help to elucidate true virulence factors in C. albicans.     

Ribosomal genes in inbred mouse strains: Interstrain and intrastrain variation of copy number and extent of methylation

Quantitative dot hybridization was used to estimate the rDNA copy number in brain tissues of five inbred mouse strains (AKR/JY, NZB/B1OrlY, CBA/CaLacY, 101/HY, and 129/JY), which were obtained from the collection of the Research Center of Biomedical Technologies (Y). In each strain, 9–12 mice aged 1–2 months were examined. The rDNA copy number per diploid genome in strains AKR (range 105–181, mean ± SD 136 ± 27) and NZB (129–169, 148 ± 12) was significantly lower than in strains CBA (172–267, 209 ± 31), 101 (179–270, 217 ± 30), and 129 (215–310, 264 ± 33). Mice of strain NZB were relatively homogeneous in this trait (CV = 8.1%). Strains AKR, CBA, 101, and 129 displayed significant between-group differences, CV varying from 12.5 to 19.9%. The same DNA specimens were digested with MspI or HpaII and used to estimate the extent of methylation of the 28S rDNA region. Regardless of the strain, all mice could be classed into two groups. One group (20 mice) had a methylated fraction accounting for less than 8% of rDNA and included all nine mice of strain NZB, seven out of nine mice of strain 101, and three out of ten mice of strain 129. In the other group (29 mice of strains AKR, CBA, 101, and 109), the methylated fraction varied from 18 to 38%. A possible role of methylation and the genome dosage of ribosomal genes in phenotypic variation (quantitative trait variation) of inbred mouse strains is discussed.     

A murine monoclonal antibody recognizes the 13,000 molecular weight polypeptide of the Sm small nuclear ribonucleoprotein complex

Antibodies to the Sm antigen are closely associated with the rheumatic disease systemic lupus erythematosus (SLE). The Sm antigen exists in the cell as part of a ribonucleoprotein complex containing at least 10 polypeptides and five small nuclear RNA. The major immunoreactive Sm species are three polypeptides of m.w. 27,000, 26,000, and 13,000. By using an MRL/1 mouse, a strain which spontaneously produces a disease with many of the characteristics of human SLE, we have produced an anti-Sm hybridoma specific for the 13,000 m.w. Sm polypeptide. This monoclonal antibody is sufficient to allow for the rapid bulk isolation of the entire class of Sm snRNP, and can be used sequentially with an anti-(U1)RNP monoclonal antibody to subfractionate the Sm snRNP particles.     

Idiotypic analysis of polyclonal and monoclonal anti-p-azophenylarsonate antibodies of BALB/c mice expressing the major cross-reactive idiotype of the A/J strain

The idiotypic cascade allows the induction of silent idiotypes, and as such, the immune system can be reprogrammed towards predetermined goals. To understand the genetic origin of silent idiotypes, we have used a system in which detailed structural and genetic information is available. The major cross-reactive idiotype (CRIA) of A/J mice (positive strain) immunized with arsonate coupled to a carrier can be regularly induced in BALB/c mice (negative strain) by anti-idiotypic treatment with or without subsequent antigen immunization. By using a panel of monoclonal anti-idiotypic antibodies, we have found that the germline-encoded CRIA displays a mosaic of at least five idiotopes. Polyclonal and monoclonal anti-arsonate antibodies prepared from idiotypically manipulated BALB/c mice have been studied. Four germline idiotopes are shared between the CRIA of the A/J strain and the CRIA-like idiotype induced in BALB/c mice. Furthermore, CRIA-like antibodies can appear "spontaneously" in some BALB/c mice immunized with antigen only. The data suggest that anti-idiotypic treatment in BALB/c mice selects a preexisting subset of antibodies. From the serological analysis, it is predicted that CRIA molecules from A/J and CRIA-like molecules from BALB/c employ different VH subgroups but share some components of the hypervariable regions. These predictions are tested in a forthcoming paper that describes the amino acid sequences of BALB/c monoclonal antibodies displaying the major cross-reactive idiotype of the A/J strain.     

Mouse mammary tumor virus proviral sequences congenital to C3H/Sm mice are differentially hypomethylated in chemically induced, virus-induced, and spontaneous mammary tumors

C3H/Sm mice have lost the exogenous milk-borne mouse mammary tumor virus (MMTV) characteristic of the C3H strain and have a very low (1.5%) incidence of spontaneous mammary tumors, yet they are highly susceptible to mammary carcinogenesis by either chemical carcinogens or infection with the milk-borne virus. We have analyzed the MMTV proviral DNA content of normal tissues and of spontaneous, virus-induced, and chemically induced mammary tumors by restriction endonuclease digestion and Southern blot analysis. Although the results clearly showed additional MMTV sequences in the virus-induced tumor which are not present in normal liver DNA, none of the spontaneous or chemically induced tumors could be shown to contain either newly acquired exogenous or amplified endogenous MMTV sequences. Interestingly, mammary tumors arising in C3H/Sm mice treated simultaneously with infectious MMTV (C3H) and dimethylbenz[a]anthracene (DMBA) possessed new exogenous MMTV DNA even though no quantitative change in tumor production was observed when these mice were compared with C3H/Sm mice treated with DMBA alone (Smith et al., Int. J. Cancer 26:373-379, 1980). Our data indicate that the endogenous MMTV proviral units are extensively methylated in normal tissues, such as livers and normal nonlactating mammary glands. In the absence of MMTV (C3H), we found that in the rare, spontaneously occurring C3H/Sm mammary tumors, certain endogenous MMTV sequences were specifically hypomethylated. Hypomethylation of endogenous MMTV sequences was also noted in the chemically induced mammary tumors, even though radioimmune competition assays for MMTV gp52 and p28 are negative (Smith et al., Int. J. Cancer 27:81-86, 1981). Our results support the conclusion that amplification of endogenous MMTV sequences is not intrinsic to C3H/Sm mouse mammary tumors arising spontaneously or after induction by chemicals. On the other hand, integration of exogenous MMTV DNA into the genome was a constant feature of mammary tumors developing in MMTV (C3H)-infected C3H/Sm mice, even when DMBA was used as the carcinogen. Hypomethylation of some endogenous MMTV sequences is characteristic of C3H/Sm mammary tumors, whether spontaneous or induced by chemicals, which suggests that these sequences are located in actively transcribing regions of the tumor cell genome.     

Comparison of the Pathogenicity for Mice of Mycobacterium fortuitum and Mycobacterium abscessus

The pathogenicity for mice of 12 strains of Mycobacterium abscessus was compared with that for 8 strains of M. fortuitum. Both species caused lesions in kidneys and produced "spinning disease" resulting from inner ear infections. No major differences in pathogenicity of these two species were demonstrated. Strain to strain variation was marked, especially with M. abscessus. For example, 1.6 x 10(6) organisms of strain 11188 of M. abscessus produced death in four of five animals within 42 days, whereas strain 380 of M. abscessus failed to produce any deaths within 42 days. In the case of M. fortuitum, the greatest mortality observed was one of five animals, yet the incidence of spinning disease and kidney disease occurred earlier postinfection than in mice infected with M. abscessus. Histologically, abscess formation by a strain of M. abscessus was greater than by a strain of M. fortuitum, but this difference cannot be interpreted as a species difference.     

Leakage of 5′-Guanosine Monophosphate from Candida lipolytica (IFO, 0746) and Its Mutants

5′-Guanosine monophosphate (5′-GMP) was found to leak from Candida lipolytica (IFO, 0746) and its mutant Y and Sm.

Mutant Sm obtained from the parent strain by treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and ultraviolet irradiation was found to leak 5′-GMP at 5 times the amount of the parent strain. The properties of mutant Y and Sm were compared with the parent strain, and differences were present in growth, amount of 5′-GMP leakage and ribonuclease activity.     

R-Plasmids in Pasteurella multocida.

Multiple drug-resistant strains of Pasteurella multocida were associated with a high incidence of fatal pneumonia in feedlot cattle. A representative strain, CAH160, resistant to tetracycline (Tc), streptomycin (Sm), and sulfonamide (Su) was studied. The minimal inhibitory concentration (MIC) of Tc was 32 μg/ml while Sm had an MIC of 256 μg/ml. Plasmid DNA was isolated from CAH160 by cesium chloride-ethidium bromide centrifugation. Agarose gel electrophoresis showed that at least three distinct species of plasmid DNA were present. DNA isolated from CAH160 was used to transform Escherichia coli K12 strain C600 r_k^?m_k^?. Transformants resistant to Tc; to Sm, Su; and to Tc, Sm, Su were obtained. Contour length measurements of plasmid DNA isolated from transformant cells showed that Tc resistance was associated with a 3-Mdal plasmid (pSR10), while Sm, Su resistance resided on a 2.7-Mdal molecule (pSR11). More than 20% of the transformants were resistant to Tc, Sm, Su and contained both plasmid species. In E. coli the MIC of Tc was 256 μg/ml and that of Sm was 64 μg/ml. The buoyant density of pSR10 was 1.699 g/cm³, while the density of pSR11 was 1.709 g/cm³.     

Animal models in Q fever: pathological responses of inbred mice to phase I Coxiella burnetii

The susceptibility of inbred strains of mice to infection by phase I Coxiella burnetii, the aetiological agent of Q fever, was investigated by evaluating morbidity, mortality, antibody production and in vitro proliferative responses of splenic lymphocytes. Among the 47 strains of mice tested for morbidity and mortality to C. burnetii infection, 33 were resistant, 10 were of intermediate sensitivity, and four were sensitive. A/J mice exhibited the highest mortality, and surviving mice of this strain yielded high concentrations of viable rickettsiae from essentially all organs for more than 3 weeks after inoculation. However, A/J mice developed a protective immune response after vaccination with inactivated C. burnetii cells. Induction of gross pathological responses and antibody production were similar in sensitive mice (strain A/J) and resistant mice (strain C57BL/6J). The LD50 of phase I C. burnetii for A/J mice was about 1000-fold lower than that for the more resistant C57BL/6J mice. Mice of both strains developed antibody titres against phase I cells, phase II cells, and phase I lipopolysaccharide after the injection of one or more viable phase I organisms of C. burnetii; five or more rickettsiae caused splenomegaly that was almost proportional to the infecting dose. Suppression of in vitro proliferative responses of splenic lymphocytes to concanavalin A, a T-cell mitogen, was apparent after infection of sensitive A/J mice with as few as one to five phase I micro-organisms. However, suppression of proliferation of splenic lymphocytes from resistant C57BL/6J mice required 10(7) phase I C. burnetii.     

Fas and Fas ligand mutations inhibit autoantibody production in pristane-induced lupus

Mutations of Fas (lpr) or Fas ligand (gld) cause a limited lupus-like syndrome in B6 mice by interfering with the deletion of autoreactive B and/or T cells. A more generalized lupus syndrome reminiscent of that of MRL mice can be induced in nonautoimmune strains by pristane, which causes a nonspecific inflammatory response in the peritoneal cavity. We hypothesized that, as in MRL mice, the lpr and gld mutations might accelerate lupus in pristane-treated mice. Pristane-treated B6 mice developed anti-nRNP/Sm, Su, and ribosomal P Abs, but little anti-ssDNA or chromatin. In contrast, B6/lpr and B6/gld mice spontaneously developed anti-ssDNA/chromatin Abs, but not anti-nRNP/Sm/Su/ribosomal P. Unexpectedly, B6/lpr and B6/gld mice were highly resistant to the induction by pristane of IgM anti-ssDNA (2 wk) and IgG anti-nRNP/Sm/Su/ribosomal P autoantibodies (6 mo), suggesting that intact Fas signaling is necessary. Interestingly, pristane did not enhance IgG chromatin Ab production in B6/lpr or B6/gld mice, suggesting that it did not influence the production of autoantibodies that develop spontaneously in the setting of Fas deficiency. Pristane treatment also decreased lymphoproliferation in B6/lpr mice. Increased production of IL-12 was associated consistently with the production of anti-nRNP/Sm/Su/ribosomal P as well as anti-DNA/chromatin. In contrast, production of anti-DNA/chromatin Abs was associated with IL-6 overproduction in pristane-treated mice, but not in lpr mice. The data strongly support the idea that different subsets of autoantibodies are regulated differentially by cytokine stimulation and/or Fas signaling.     

Nodulation competitiveness between contrasting phage phenotypes of pigeonpea rhizobial strains

Competitiveness between (I) lysogenic vs. phage-indicator strains, (II) phage-resistant vs phage-sensitive strains, and (III) large plaque vs. small plaque developing strains was examined under laboratory and field conditions in order to study the involvement of these crucial phage sensitivity patterns in the competition for nodule occupancy of pigeonpea rhizobia. The phage-indicator strain (A039) exhibited higher competitiveness over the lysogenic strain (A025 Sm(r)); the phage sensitive strain (IHP-195) over the phage resistant strain (IHP 195 Sm(r)V(r)); and the large plaque developing strain (A059) over the small plaque developing strain (IHP195 Sm(r)) in association with pigeonpea cv. bahar both under laboratory and field conditions. Dual inoculation of A025 Sm(r) + A039 and A059 + IHP195 Sm(r) (mixed in equal proportion just before treatment) improved the nodule occupancy by inoculant strains against native rhizobia and resulted into higher plant dry weight and yield as compared to their application as single inoculum. The phage-resistant mutant IHP195 Sm(r)V(r) showed reduced competitiveness against native rhizobia, compared to its parental strain. The dual inoculation of parental strain and phage-resistant mutant gave the same result as the inoculation of parental strain alone.     

Two isoforms of Serratia marcescens nuclease. Crystallization and preliminary X-ray investigation of the enzyme.

Two isoforms of an extracellular endonuclease, nucleases Sm1 and Sm2, were purified from culture fluid of Serratia marcescens strain BIO MI by ligand-exchange chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI-values for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The nuclease Sm1 has been crystallized from ammonium sulfate solution by the vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell constants a = 69.0, b = 106.7, c = 74.8 A, contain two molecules in an asymmetric unit, packing density Vm = 2.3 A/Da, and diffract to at least 1.5 A resolution. The Pt- and UO2-derivatives of the protein were obtained. Preliminary X-ray investigation of nuclease Sm2 crystals was carried out.     

Identification of an acidic ribosomal protein reactive with anti-Sm autoantibody

Autoantibody to Sm Ag is a highly specific marker for the diagnosis of SLE. The Sm Ag exists in the cell nucleus as part of a ribonucleoprotein complex containing five small nuclear RNA. The major immunoreactive Sm species have been reported to be three polypeptides of m.w. 28,000/29,000 (B/B') and 16,000 (D). We report here that a m.w. 21,000 peptide is another major target of anti-Sm antibody. This peptide was originally identified by Western blotting as an acidic ribosomal protein (RP21) reactive with IgG from some SLE patients. Anti-RP21 is distinct from anti-ribosomal P protein antibody (anti-P) which has been previously identified as a lupus-specific autoantibody. Cell fractionation experiments showed that RP21 existed only in the ribosomal fraction and was never detected in other cellular compartments including nuclei. However, when nuclear extracts were used as Ag sources in immunoblotting, affinity-purified anti-RP21 was found to react with m.w. 28,000 and 16,000 peptides, suggesting that anti-RP21 reactivity might be due to the cross-reaction of anti-Sm. This was further confirmed by the evidence that two kinds of murine anti-Sm mAb independently derived from MRL/lpr mouse recognized RP21. These results indicate that anti-Sm antibodies in SLE are reactive with both nuclear and ribosomal ribonucleoproteins. Previous reports have described certain similarities, i.e., antibody subclass restriction and incidence, of anti-Sm and anti-P in both humans and autoimmune mice. Our present study demonstrated a close physical association of target molecules reactive with anti-Sm and anti-P, and might, therefore, provide some clue to the origin of these two types of lupus-specific autoantibodies.     

Mycobacterial codon optimization of the gene encoding the Sm14 antigen of Schistosoma mansoni in recombinant Mycobacterium bovis Bacille Calmette-Guérin enhances protein expression but not protection against cercarial challenge in mice

A mycobacterial codon-optimized gene encoding the Sm14 antigen of Schistosoma mansoni was generated using oligonucleotide assembly. This synthetic gene enhanced approximately fourfold the protein expression level in recombinant Mycobacterium bovis Bacille Calmette-Guérin (rBCG) when compared to that obtained using the native gene in the same expression vector. Immunization of mice with rBCG expressing Sm14 via the synthetic gene induced specific cellular Th1-predominant immune responses, as determined by interferon-gamma production of Sm14-stimulated splenocytes, which were comparable to those recorded in animals immunized with an rBCG strain expressing the native gene. Administration of a single dose of the rBCG-Sm14 construct carrying the synthetic gene conferred protection against cercarial challenge in outbred Swiss mice, at a level equivalent to those provided by either a single dose of rBCG expressing the native gene or three doses of Escherichia coli-derived recombinant Sm14. Our data demonstrated that despite improving the level of antigen expression, the codon optimization strategy did not result in enhanced immunity or protection against cercarial S. mansoni challenge.     

A few characteristics of mouse having high sensibility to anaphylatic shock separated from El mouse (author's transl)]

During an experiment of shaking in El mice, a strain which was less susceptible to convulsion was found out. In this strain (ASK), the mortality to anaphylactic shock and susceptibility to audiogenic seizure were compared with five other strains of mice. Six strains of mice (ASK, EL, IDT, JCC-ICR, dd, C57BL/6J) were sensitized by two subcutaneous injections with 2 mg/head of crystalline egg albumin at five and six weeks of age, and then challenged by the intravenous injection of 0.125, 1, 8, 64, 512 and 4096 mug/head at seven weeks of age. In both sexes of ASK strain, the mortality was the maximum (75--95%) after the challenge of 8--4096 mug egg albumin. The mortality of the female El and IDT and the both sexes of JCL-ICR strains was middle (55--70%) after the challenge with 64 mug egg albumin, while that of other strains (dd and C57BL/6J) was very low (0--20%). The susceptibility of 3-weeks-old mice of ASK, El, IDT, JCL-ICR and dd strains was low (1.9--19.0%), whereas mice of DBA/2 strain showed a very high susceptibility (80%) to 110 phons.