First recognized community outbreak of haemorrhagic colitis due to verotoxin-producing Escherichia coli O 157.H7 in the UK.

The first recognized outbreak of haemorrhagic colitis due to Escherichia coli O 157.H7 in the United Kingdom affected at least 24 persons living in East Anglia over a 2-week period. The illnesses were characterized by severe abdominal pain and bloody diarrhoea of short duration. Eleven patients were admitted to hospital and there was one death. Patients were mainly adult women who had not eaten out of the home in the 2 weeks before onset. Unlike previously reported outbreaks hamburgers were not the vehicle of infection, and a case-control study suggested that handling vegetables, and particularly potatoes, was the important risk factor.

Implication of the H-2L locus in hybrid histocompatibility (Hh-1).

The Hh-1 (hybrid histocompatibility) effect in which F1 hybrids with heterozygosity at the "D end" of the H-2 complex can reject parental grafts of the H-2b haplotype was examined in four H-2 mutants wherein the mutation had affected the H-2Db, H-2Dd, or H-2Ld genes. In bone marrow grafting experiments it was shown that two separate mutations affecting the H-2Db locus did not affect the Hh-1 gene, suggesting that H-2Db and Hh-1 are probably two different genes. By contrast, experiments with two mutants, one affecting the H-2Dd and H-2Ld loci (B10.D2-H-2dm1) and the other a loss mutation at the H-2Ld locus (BALB/c-H-2dm2), demonstrate an alteration in the hybrid histocompatibility phenomenon and the presumption is made that the Hh-1 and H-2Ld locus are identical. This presumption was supported by studies of Hh-1 using the EL4 tumor grafting model where marked differences in growth were noted in the (B10 X BALB/c)F1 and (B10 X BALB/c-H-2dm2)F1 hybrids. By contrast, the functionally related natural killer (NK) phenomenon appeared to be the same in the BALB/c parent and dm2 mutant. Hybrid histocompatibility is a complex phenomenon but at this time we conclude that the H-2L locus is related to, if not identical to the Hh-1 gene but the H-2L locus is distinct from genes affecting the NK phenomenon.

Ir gene testing in H-2 mutants.

Four different C57BL/6 mutants, each with a mutation presumably in the H-2Kb locus, were studied to determine whether the mutations had affected Ir gene function. When tested with the antigens, (TG)-A-L, ovalbumin (OA), and lactic dehydrogenase (LDHB), there was no difference in the antibody response of the mutants when compared to the parental C57BL/6 strain and there was also no difference in the response to the histocompatibility H-Y antigen. The responses to these antigens have been mapped, wholly or partially in the K-IA, IA or IB subregions and therefore the identical response of parent and mutants may provisionally exclude H-2Kb as a site of Ir gene function for the four antigens tested. In addition, the BALB/c-dm2 mutant, a mutation in the H-2Ld locus, gave identical responses to the parental BALB/cKh strain.

Cell membrane alloantigenic determinants of several unusual mouse strains.

Several interesting and clinically important strains of Mus musculus were serotyped with alloantisera recognizing the different alleles of the Thy-1, Ly-1, Ly-2, Ly-3, Ly-4, Ly-5, Ly-6 and Ly-7 loci. The strains were the Biozzi high and low responder strains, or importance in immune responsiveness studies, and the strains BXSB, MRL and MRL--lpr/lpr--strains which spontaneously develop immune complex disease. In addition the related species Mus musculus casteneus was typed with the same reagents. The knowledge of the cell surface phenotype should prove useful in various functional studies involving these interesting strains.

Murine lymphocyte alloantigens. II. The Ly-7 locus.

The murine Ly-7 locus was identified by the reactivity of alloantisera prepared as B6,C-H-2d x CXBG)F1 anti-CXBK. Most strains, except C57BL/6 and related strains, were Ly-7.2+. Absorption analysis demonstrated that the Ly-7 specificity was only found on lymphocytes and was absent from liver, kidney, brain, and red blood cells. Direct testing by cytotoxicity and protein A rosetting showed that Ly-7 is expressed in very low amounts on thymocytes, but is readily detectable on more mature T and B lymphocytes (including antibody-forming cells), with the greatest expression on B cells. Typing backcross mice with Ly-7.2 Ly-4.2, H-2.33 antisera showed that Ly-7 is not linked to the Ly-4 or H-2 loci. In raising the antisera by hyperimmunization, it was found that the antibody response to the Ly-7.2 specificity was influenced by an H-2-linked immune response gene.

Murine lymphocyte alloantigens. I. The Ly-6 locus.

The Ly-6 locus and the Ly-6.2 specificity have been previously described, and we now further define this locus and the allelic specificities, Ly-6.1 and Ly-6.2. Back-cross studies and examination of several recombinant inbred (RI) lines demonstrate that Ly-6 is distinct from other loci determining CMAD, except for ALA-1 and evidence is presented for the identity of Ly-6 and ALA-1. The Ly-6 congenic strain C3H.B6-Ly-6b is described and was used to prepare antisera in combinations congenic for Ly-6. The allelism of the Ly-6.1 and Ly-6.2 specificities was confirmed by the reactivity of these antisera with a segregating F2 generation. Antisera can be prepared between the Ly-6 congenic strains, although the magnitude of the response is under the control of one or more genes in A strain, not linked to H-2 or Ly-4 loci. Absorption analysis using separated T and B lymphocyte populations demonstrate that Ly-6.2 does not have exclusive peripheral T cell representation as originally reported, but is also present on B cells in amounts one-fourth to one-eighth of that found on T cells and is found in greater quantities on T blast cells.

The definition of a new Ia antigenic specificity using the B6.C-H-2bml2 I-region mutant strain.

A new Ia specificity has been defined using the IA-subregion mutant B6.C-H-2bm12. The immunization to produce the antiserum was bml2 antiA.BY, as all other immunizations, such as bml2 anti-C57BL/6, failed to produce antibody. By selecting strains of C57BL origin for testing, it was shown that, (a) the serum was only weakly cytotoxic but gave substantial reactions using a rosetting assay; (b) the antibody reacted with B cells and not T cells; (c) strains of the b,d,p and q H-2 haplotypes were positive, whereas f,k,r and s were negative; (d) absorption studies demonstrated only a single specificity to be present and by testing recombinant strains, the reaction mapped to the IA subregion; (e) SDS-PAGE demonstrated that the antiserum reacted with a molecule of MW approximately 33,000. Preliminary studies indicate this new specificity, present on C57BL/6 and lost from bml2, is present on the same molecule as other I-A specificities.

Studies of H-2Kb mutant mice. I. Description and serological studies of B6.C-H-2bm9, -H-2bm10, and -H-2bm11.

Three new H-2b mutant strains, B6.C-H-2bm9, B6.C-H-2bm10 and B6.C-H-2bm11, are described. The three mutant strains are of the gain and loss type as they reject skin grafts reciprocally with the parental C57BL/6Kh. The mutations, which arose independently, are all allelic at the same locus as 11 other mutant strains already described. By complementation and other studies the mutated gene has been shown to be H-2Kb. The strains were typed directly and by absorption with antisera specific for H-2Kb and H-2Db private and public specificities and for Iab specificities. Each strain typed differently with these sera. The strin B6.C-H-2bm9 was found to be serologically identical with C57BL/6. The strains B6.C-H-2bm10 and B6.C-H-2bm11 were found to have alterations in the private H-2Kb specificity, H-2.33, and in the public specificity, H-2.5, but to a different extent. B6.C-H-2bm10 had a marked decrease in the amount of H-2.33 expressed on the splenic cell surface as compared to C57BL/6 and also has a marked decrease in the expression of H-2.5 on both spleen and red blood cells. In comparison, B6.-H-2bm11 has a decrease in the expression of H-2.33 but an increase in the expression of H-2.5 on both splenic and red blood cells. The other H-2b specificities appeared to be unaltered as compared with C57BL/6.

Studies of H-2Kb mutant mice. II. Definition of new H-2 specificities (H-2.68, 69, 71, 72) using the H-2Kb mutant, B6.C-H-2bm10.

Using the H-2Kb mutant strain, B6.C-H-2bm10, antibodies were produced which define four new H-2 specificities. This is in contrast to other H-2Kb mutant strains in which attempts to produce antibodies against the mutation have failed with the exception of H-2bm3 in which a new specificity, H-2.62, was described by absorption analysis of an H-2.33 antiserum. With the mutant strain as immunization donor in the combination (C57BL/10 x 129)F1 anti B6.C-H-2bm10, two new H-2 specificities, H-2.68 and H-2.69, were produced. H-2.68 is found only on B6.C-H-2bm10, while H-2.69 is present on B6.C-H-2bm10 and on all cells of the H-2k haplotype. By testing the appropriate H-2 recombinant strains, the reaction with the H-2k haplotype could be mapped to the H-2D locus, whereas in B6.C-H-2bm10 it must have occurred in the H-2Kb gene. The reciprocal immunization of congenic strains failed, but with B6.C-H-2bm10 anti-A.BY, the specificities H-2.71 and H-2.72 were produced: H-2.71 was present on cells of the b,d, and k haplotypes, while H-2.72 was found only on the b haplotype. Genetic mapping using H-2 recombinant strains showed the H-2.72 reaction to be associated with the H-2Kb, H-2Dd and H-2Kk genes. The specificities described were shown to be of the H-2 type by their reactivity with H-2 congenic and recombinant strains, their segregation in a backcross and their ability to precipitate a molecule of 45,000 daltons molecular weight on SDS-PAGE. Therefore, the mutation carried by B6.C-H-2bm10, which resulted in a marked alteration of the H-2Kb specificities H-2.33 and H-2.5, is such that specific antibodies were able to be produced against both the gain and loss alterations produced by the mutation.

Skin graft enhancement studies with antigenic differences arising from the H-2K, H-2D, and H-2L loci.

Enhancement of skin grafts was previously shown to be possible when there were antigenic differences arising from the @K end" but not the @D end" of the H-2 complex. Subsequent studies demonstrated that anti-Ia antibodies in the anti-K end sera were responsible for graft prolongation and not the anti-H-2K antibodies and that D end antisera were not enhancing as they lacked Ia antibody activity. The studies reported herein have extended these findings by two separate approaches. First, by using appropriate congenic and recombinant strains, enhancement by D end antigenic differences could be observed, provided that Ia antibodies were also present. In this way, H-2D and H-2K alloantigens are similar and both require additional I region differences to enable skin graft enhancement to be observed. Second, H-2Kb, H-2Dd, and H-2Ld mutants were examined and putative antisera used to attempt to prolong graft survival in these unique coisogenic strains. In no case was graft survival significantly prolonged, even in the presence of demonstrable antibodies. Therefore, in two different approaches, wherein single gene products were examined (derived by mutation and by recombination), enhancement of skin allografts could not be observed, unless additional Ia alloantigenic differences were also present.

High titres of antibody to murine leukaemia virus in lymphocyte (Ly) alloantisera.

The radioimmune precipitation (RIP) assay was used to examine the antibody titres against endogenous AKR murine leukaemia virus (MuLV) in a number of antisera to lymphocyte (Ly) alloantigens. The sera from normal donor and unimmunized recipient mice used in raising the alloantisera were also examined for anti-MuLV activity. It was found that all the antisera had high anti-MuLV titres and that in all but one case alloantigen immunization augmented the anti-viral titres. The degree of augmentation did not appear to be related to the anti-MuLV titre in the donor strain sera. Three I-region antisera were also examined for anti-MuLV antibodies and were found to have lower anti-viral titres than the Ly antisera even though immunization to I-region products greatly augmented the anti-viral titre. These results caution against the use of Ly antisera in characterizing the phenotype of lymphoid tumour cells without prior virus absorption.

Studies of two H-2Db mutants: B6. C-H-2bm13 and B6.C-H-2bm14.

Two new C57BL/6 H-2 mutants, B6.C-H-2bm13 and B6.C-H-2bm14 are described. They arose independently in C57BL/6 as spontaneous mutations of the gain and loss type. Complementation studies map the mutations in both bm13 and bm14 to the H-2Db gene. However, these two mutant strains are not identical, but occurred as independent mutations at the same locus, as shown by reciprocal graft rejection and by the inability of the (bm13 X bm14)F1 hybrid to accept C57BL/6 grafts. Serological studies by direct testing (cytotoxicity and hemagglutination) and by quantitative absorption demonstrated a decrease in the H-2Db private specificity H-2.2 in both bm13 and bm14 when compared to C57BL/6. This was confirmed by SDS-PAGE analysis using antisera detecting the H-2.2 specificity. Attempts to produce antibodies to either the gained or lost specificities of the two mutant strains failed.

B6.C-H-2bm12. A new H-2 mutation in the I region in the mouse.

The B6.C-H-2bm12 mutant is described and evidence is presented for the mutational site occurring in the IA subregion. The mutant is of the gain and loss type as bm12 in equilibrium or formed from C57BL/6 grafts are rejected in 14-16 d. Mapping studies by the gene-complementation method using H-2 recombinant strains place the mutation in the K or IA regions of the H-2 complex and furthermore, the use of this test and the use of other H-2 mutants indicate that H-2Kb is not the site of the mutation, making the IA region the most likely site. Serological analysis with a battery of H-2b, Iab, and other Ia sera, both by cytotoxicity, rosetting, and also by absorption analysis, indicated no alteration in H-2 specificities, particularly in H-2.K33. By contrast, all of the Iab specificities coded for by the IA subregion (Ia.3, 8, 9, 15, and possibly 20) are extensively altered and are either absent or greatly reduced in amount indicating an extensive alteration in the Ia-bearing molecule. The bm12 mutant strongly stimulates the parental C57BL/6 strain in an mixed lymphocyte reaction (MLR), and the reciprocal also occurs, the degree of stimulation being similar to that obtained with K + IA differences originating in another H-2 haplotype and points to the mutation effecting the Lad-1 locus. The presence of an extensive histocompatibility change, a marked alteration in the serologically detected Ia specificities, and a strong MLR, all produced by the one mutation, provides strong evidence for the identity of the Ia-1, Lad-1, and H-2(IA) loci in the IA subregion. The bm12 mutant should be of value in determining the relationship of Ia specificities, Ir genes, and other phenomena effected by the I region.

Detection of mouse alloantibodies by rosetting with protein A-coated sheep red blood cells.

Staphylococcal protein A has an affinity for the Fc portion of the IgG molecule of different species and can therefore be used to detect cell-bound immunoglobulin. Using this property, protein A coupled to sheep red blood cells via chromic chloride can detect alloantibodies to mouse H-2, Thy-1, Ly-1, 2, 4, 5, 6, and 7, and Ia antigenic specificities bound to the surface of lymphocytes by the formation of rosettes. In comparison with other rosetting and cytotoxicity assays, the protein A assay shows a greater sensitivity than does cytotoxicity using spleen cells as the target, as does the sheep anti-mouse Ig rosetting assay, whereas cytotoxicity shows greater sensitivity with some antisera on thymocytes. The major advantages of the protein A assay are that constant low reproducible backgrounds are obtained, there is no need to remove surface Ig by capping prior to antiserum treatment, and that viable cells can be recovered.

The relationship between theH-2 loss mutations ofH-2(da) andH-2 (db) in the mouse.

The mouse strain B10.D2-H-2(da) carries the mutantH-2(da) allele, derived after chemical induction, and this has been shown to be a gain and loss mutation involving theH-2D(d) locus.BALB/c- H-2(db), derived spontaneously, is a loss mutation only, and appears not to involve theH-2D(d), but rather theH-2L(d) locus. The two mutations effectboth graft rejection and serologically detected H-2 specificities (Type II mutation). In the experiments described in this study, theloss mutations in theH-2(da) andH-2(db) mutants have been compared by skin grafting, and by direct and absorption serological techniques: (1) By skin grafting, using the well established complementation method, it has been shown thatH-2(da) andH-2(db) do not complement each other, i.e., the mutation in both occurred at the same 'locus.' However, by appropriate selection of donor and recipient, it has become clear thatH-2(da) had a greater loss than didH-2(db), althoughH-2(da) includes the loss found inH-2(db). (2) Serological studies have demonstrated that H-2D.4 was altered inH-2(da), but not inH-2(db); 'H-2.28' (detected by D-28b and D-29) was decreased or lost in both mutants;H-2(db) anti-BALB/c failed to react withH-2(da); both mutants reacted similarly with D-28 sera. In addition, sera made usingH-2(da) as donor did not contain an anti-H 2.28 antibody. The loss mutation involvingH-2(da) therefore appears to have led also to the loss of H-2.28 as found inH-2(db). We conclude that theH-2(da) strain arose after a complex mutation or recombination event which involvedboth theH-2D(d) locus and the closely linkedH-2L(d) locus, whereasH-2(db) affects only theH-2L locus.