Lack of K1b9 light chains in Basilea rabbits is probably due to a mutation in an acceptor site for mRNA splicing.

Rabbits of the Basilea strain do not produce normal K1b9 light chains but continue to produce immunoglobulins with light chains of the rare K2 isotype and of lambda type. To understand the molecular basis for this unusual expression of kappa light chains in Basilea rabbits, we undertook an analysis of their kappa genes. We isolated and sequenced the mutant kappa 1b9 gene and found a substitution of A for G in the highly conserved AG dinucleotide of the 3' acceptor splice site. Although we cannot rule out additional alterations of portions of the gene we did not sequence, this spontaneous change of the G found in the normal gene provides a likely molecular explanation for the loss of K1 light chain expression in Basilea rabbits.

Sequence of a cDNA encoding Basilea kappa light chains (K2 isotype) suggests a possible relationship of protein structure to limited expression.

We present the complete sequence of a cDNA encoding rabbit immunoglobulin kappa light chains of the Basilea isotype (K2). Although all rabbits seem to possess a K2 constant region gene, expression of this gene in most rabbits is minimal if present at all. Even in Basilea rabbits the majority of expressed immunoglobulins are of lambda type. We find that the sequence of our Basilea cDNA constant region and the sequence of a "silent" K2 gene from b4 rabbits (bas-N4) are almost identical. The bas (K2) isotype lacks cysteine at position 171 in the constant region that is present in all K1 constant regions and usually forms an interdomain disulfide bond, with a cysteine at position 80 of the variable region. We postulate that one factor contributing to the low expression of the bas (K2) isotype could be a paucity of V kappa regions lacking cysteine at position 80. If a typical rabbit V kappa encoding Cys at position 80 is rearranged and expressed with th K2 isotype. B cells with mRNAs encoding light chains with free sulfhydryl groups would result. These cells may fail to form functional immunoglobulin receptors. Only a small subset of rabbit variable regions that lack the cysteine at position 80 would rearrange and encode K2 light chains lacking a free sulfhydryl group.

Structural studies on induced antibodies with defined idiotypic specificities. IX. Framework differences in the heavy- and light-chain-variable regions of monoclonal anti-p-azophenylarsonate antibodies from A/J mice differing with respect to a cross-reactive idiotype.

Amino terminal amino acid sequence analyses have been performed on the heavy and light chains of induced monoclonal antibodies with specificity for the hapten p-azophenylarsonate. Four of the eight antibodies react with conventional antisera to the previously described A/J anti-arsonate cross-reactive idiotype (CRI). Of the 16 chains analyzed, all but one contain sequence differences in their first framework segment (residues 1-30) that distinguish them from the heavy- and light-chain sequences found in anti-arsonate antibodies isolated from A/J serum or ascites fluid. The presence of such framework differences appears to be independent of whether or not the hybridoma antibodies bear the CRI. In spite of the framework substitutions, all four of the CRI-positive hybridoma antibodies have variable (V)-region frameworks that are very similar to each other and to the CRI-positive molecules found in A/J serum. Two of the four CRI-negative molecules are also structurally similar to the serum antibodies. Two others, however, are strikingly different from any serum anti-arsonate antibody thus far described and appear to reflect a completely separate repertoire of anti-arsonate antibodies in the A/J MOUSE. In addition, serological analyses with an anti-idiotypic antiserum generated against a CRI-positive hybridoma product suggest that each monoclonal antibody may possess individual antigenic specificities different from the determinant(s) detected with the conventional rabbit anti-CRI. The consistent appearance of framework substitutions in what has been thought to be a homogeneous antibody population has important implications for our understanding of the generation of antibody diversity and for the precise chemical definition of an idiotype.

Presence of highly conserved idiotypic determinants in a family of antibodies that constitute an intrastrain cross-reactive idiotype.

It has been shown that A/J anti-p-azophenylarsonate antibodies that share a major cross-reactive idiotype (CRI) comprise a family of closely related, but nonidentical, molecules. Our results demonstrate that 12 of 14 monoclonal hybridoma products that express the CRI have in common at least one highly conserved idiotypic determinant. It is proposed that this reflects conservation of a portion of the amino acid sequence, presumably in hypervariable regions. That the conserved determinant(s0 are located in the region of the hapten-binding site is indicated by the ability of haptens to inhibit idiotype-anti-idiotype interactions involving the conserved, or public determinants.

Idiotype profile of an immune response. II. Reversal of the relative dominance of major and minor cross-reactive idiotypes in arsonate-specific T-independent responses.

Two different cross-reactive idiotype (CRI) groups are distinguishable in the Ab response of A/J mice to the p-azobenzenearsonate (ABA) hapten: CRIA and CRIm. These two groups showed distinct patterns of relative dominance in the ensuing response depending on whether the inducing Ag was a T cell-dependent (TD) form of ABA, such as ABA-KLH or ABA-CGG, or a T-independent type 1 (TI-1) form, such as ABA-Brucella abortus or ABA-lipopolysaccharide (LPS), and on whether the response was elicited in vivo or in vitro. The CRI+ component of primary in vivo plaque-forming cell (PFC) responses to TD ABA Ags was largely (greater than 90%) CRIA+ as was, to a slightly lesser extent (greater than 75%) the CRI+ portion of secondary or hyperimmune serum Ab or PFC responses to the same Ags. In contrast, in vivo primary and hyperimmune PFC responses to ABA-Bru or ABA-LPS showed a significantly lower CRIA/CRI ratio, averaging 0.5-0.6, with some individual mice giving figures as low as 0.2, indicating predominance of CRIm over CRIA. Serological analysis of hyperimmune anti-ABA Abs from a group of 5 A/J mice immunized with ABA-Bru gave a figure of less than 0.5 for the CRIA/CRI ratio. The most striking disparity from the TD pattern was seen in primary in vitro PFC responses to the TI ABA Ags; here ratios of less than 0.2 were generally seen. Since T cell removal did not alter the Id pattern in the TI responses, CRIA-specific Ts cells do not account for the weak expression of CRIA in such responses. We propose a model that explains these results on the basis of differential expression of IdX dominance by two distinct B cell subpopulations--equatable to the Lyb-5+ and Lyb-5- B cell subsets--along with differential relative activation of these subsets in different types of responses. Examination of anti-ABA PFC responses of F1 progeny of CBA/N and A/J mice to ABA-Bru lends support to this hypothesis since CRIA expression was significantly lower in mice with the xid defect.

In vitro cell fusion between CD4(+) and HIV-1 Env(+) T cells generates a diversity of syncytia varying in total number, size and cellular content.

Syncytia formation in HIV infections is driven by the virus fusion-active molecules (Env) interacting with membrane components of hosts cells. HIV-syncytia are usually interpreted as pathogenic entities and although they may potentially vary in size, numbers and types of constituent cells, little is known about the extent and significance of their diversity. Here, we describe numerically the cell population dynamics and the diversity of syncytia produced in the in vitro cell-fusion between two Jurkat T cell lines, one CD4(+) and the other Env(+). Cell-fusion partners were differentially stained with the lipophilic DiI and DiO, or with the cytoplasmic CMFDA and CMTMR tracers and syncytia showing double fluorescence were counted in a flow cytometer. The total number of syncytia formed, their size, cellular complexity and ratio of CD4(+)/Env(+) cells recruited, varied significantly in relation with time of reaction and initial proportions of fusion partners. The considerable structural diversity of syncytia formed, in so limited an in vitro cell fusion reaction, suggests that a greater heterogeneity may be formed in the natural course of disease. Identification of the main determinants of syncytia diversity allows for a detailed study of the relation between the syncytia structure and function.

Preliminary crystallographic study of the Fab fragment of a monoclonal anti-phenylarsonate antibody.

Preliminary crystallographic data are given for the Fab fragment of a monoclonal anti-p-phenylarsonate antibody. This crystalline Fab fragment was found by screening a number of monoclonal anti-arsonate antibodies obtained from hybrids of A/J immune spleen cells with a non-secreting mouse myeloma line. The protein crystallizes in the monoclinic space group P21 with a = 86.2 +/- 0.1 A, b = 80.4 +/- 0.2 A, c = 75.8 +/- 0.1 A, beta = 90.3 +/- 0.1 degrees. Precession photographs show X-ray reflections extending to a resolution of 3 A.

Preparation of F(ab')2 fragments from mouse IgG of various subclasses.

We have investigated the effects of peptic digestion on mouse monoclonal immunoglobulins of each subclass of IgG. F(ab')2 fragments were obtained in good yield from IgG1, IgG2a and IgG3 proteins. The relative rates of digestion were IgG3 greater than IgG2a greater than IgG1. Variations in rate of digestion were noted for individual monoclonal IgG3 proteins. Five different IgG2b proteins were degraded very rapidly without the detectable formation of antigen-binding F(ab')2 fragments. One IgG1 protein out of four tested was also rapidly degraded.

Carbohydrate epitopes of Entamoeba histolytica cell surface glycoproteins are major targets of the human humoral response.

The antigens of Entamoeba histolytica recognized by antibodies in 11 individual sera from patients treated for amebic liver abscess were determined both by immunoprecipitation of metabolically-radiolabeled whole trophozoite proteins and by immunoblotting.Collectively, twenty-s even antigens ranging from 167 to 21 kDa were detected in immunoblots of whole trophozoite extracts; eight of these were recognized by all tested patient sera. Immunoprecipitation studies also revealed a complex amebic antigenic profile. Of a total of twenty immunoprecipitated polypeptides (from 200 to 24 kDa), seventeen were uniquely recognized by the patient sera. Eight of these seventeen antigens were immunoprecipitated by most immune sera. The cellular localization of trophozoite antigens was determined by analyzing plasma membrane and soluble cytosol fractions. Plasma membranes contained virtually as many antigenic moieties as the total trophozoite extract; in contrast, the soluble fraction was antigenically less complex. Mild periodate oxidation of plasma membrane antigens indicated that surface glycoproteins are highly immunogenic for the human host and that antibodies to their carbohydrate epitopes are a major component of the total response of most patients.

Effect of oral zinc supplementation upon Taenia crassiceps murine cysticercosis.

The effect of zinc supplementation on Taenia crassiceps murine cysticercosis was studied in susceptible BALB/cAnN mice. Female offspring of mice supplemented with high zinc throughout gestation and lactation were intraperitoneally infected with T. crassiceps cysticerci. Offspring from nonsupplemented mothers were used as controls. Significantly fewer parasites were recovered from zinc-supplemented mice (Zsm) 30 days after infection. Increased resistance was not related to the IgG antibody response. At early stages of infection, T cells from Zsm proliferated to T. crassiceps antigens, whereas cells from control mice did not respond. Infection caused in both groups a decrease in CD3+ cell percentages, which was more pronounced in the controls, and paralleled by a decrease in CD8+ cells; CD3+ and CD8+ percentages returned to normal levels at later stages of infection. In contrast, the CD4+ subpopulation only decreased in control mice. Intracellular cytokine determinations indicate that zinc supplementation favored a stronger and persistent type-1 T cell response in cysticerci-infected mice, which probably participates in the observed increased resistance.

Human immunodeficiency virus 1 (HIV-1) envelope-dependent cell-cell fusion modulation by HIV-positive sera is related to disease progression.

Fusion of CD4+ cells by HIV-1 envelope proteins (Env) is a mechanism of virus spread and cell damage. Production of antibodies able to influence cell-cell fusion in vivo may affect the course of the infection. The effect of sera from 49 HIV-1-positive patients was tested on an in vitro fusion assay using Env-expressing and normal Jurkat T cells labelled with DiI and DiO dyes, and flow cytometry for quantification of cell-cell fusion. Sera varied in their activity on fusion: 69.4 % inhibited, 24.5 % had no effect and 6.1 % enhanced cell fusion. Fusion activity correlated positively with the CD4+ T-cell count and inversely with the viral load. Removal of IgG or IgM from sera reduced or eliminated inhibition and enhancing activities, respectively. Antibodies with inhibitory activity predominate in early and intermediate stages of infection, whereas loss of inhibition or enhancement of fusion correlates with progression to AIDS.

A cluster of rabbit T-cell beta-chain variable region genes.

A genomic Eco RI DNA fragment of 4291 base pairs contains three V beta genes (V44T0, V44T1, and V44T2), each represented as only a single copy in the haploid genome of the rabbit. These V beta do not appear to be pseudogenes; all three can be found on approximately 1.3 kb (mature size) transcripts in thymus RNA, although they do not contain classical upstream promoter sequences. V beta T0 and T1 exhibit high DNA sequence homologies with mouse V beta 10 and V beta 1 genes, respectively (approximately 81 and 73%). In the mouse, the two genes are also closely linked. Rabbit V beta gene T2 resembles mouse V beta 11 and human YT35 DNA sequences but the homologies are lower (approximately 63%). The deduced protein sequences of the rabbit and mouse gene pairs are also conserved and ratios of replacement changes to silent base changes are low, suggesting that there has been selection for conservation of certain portions of the protein sequences. In contrast, the DNA and deduced protein sequences of the adjacent closely linked rabbit V beta genes are remarkably different from each other (approximately 54-65% DNA; approximately 33-55% protein homologies). These facts suggest early duplication of V beta genes and evolutionary conservation of certain members.

Genomic sequence and organization of rabbit Tcrb constant region genes.

We previously reported that some rabbits have three different copies of T-cell receptor b (Tcrb) constant region genes unlike man, mice, and rats who generally have two copies. Two of these C beta genes were found on an approximately 14 kilobase (kb) and one on an approximately 6 kb Eco RI fragment. The gene on the 6 kb fragment is of beta 2 type. A previously described portion of the 14 kb fragment appeared to have sequences characteristic of C beta 1. We have now shown that the 6 kb fragment is adjacent to and 3' of the 14 kb fragment. Furthermore, the second linked sequence of C beta gene present on the 14 kb fragment resembles to a large extent the C beta 2 gene present on the 6 kb fragment. Moreover, this second C beta gene has a 5' cluster of J beta sequences resembling J beta 2 of other species. However, exon 4 and the 3' untranslated region (3'UT) are of the beta 1 type. Mapping studies using Southern analyses of both genomic DNA and the 14 kb clone have identified another cluster of J beta 2 sequences 5' of the third tandem C beta 2 gene present on the 6 kb Eco RI fragment. Thus, the second gene on the 14 kb fragment appears to be a chimeric genomic Tcrb gene that may have arisen by an unequal crossing-over event analogous to that which may have deleted C beta 1, D beta 2, and J beta 2 in NZW mice.

Allotypes of the constant region of the rabbit T cell receptor beta-2 chain.

Our laboratory previously reported that there was restriction fragment length polymorphism of TCR C beta genes in rabbits. EcoRI digests of DNA from different rabbits gave fragments of 9 and 6 kb (C beta a) or 14 and 6 kb (C beta b) that hybridized to a C beta cDNA probe. We also reported that the 9- and 14-kb types segregated as Mendelian traits and that there were allotypic differences in the first exon of the C beta 1 genes of C beta a and C beta b animals. Here we report the DNA sequence of the C beta 2 gene present in the 6-kb EcoRI fragment from a C beta b animal and compare the exon sequences with that of a cDNA from a C beta a animal. We find replacement changes in the first and third exons that probably represent allotypic forms of the rabbit C beta 2 gene. The genomic DNA 5' of exon 1 of both beta 1 and beta 2 contain alternating purine/pyrimidine repeat sequences. The genomic C beta 2 has an open reading frame of 69 amino acids in frame with exon 1 similar to a longer one previously found 5' of exon 1 of C beta 1. Further 5' of this region, rabbit C beta 1 and C beta 2 DNA sequences are only about 66% similar. Both the C beta 1 and C beta 2 sequences have two chi sequences; one in exon 1 with a perfect match and one in the intron downstream of exon 1 with one mismatch. Alternating purine/pyrimidine repeats and chi sequences found in rabbit C beta 1 and C beta 2 genes may have contributed to process(es) of gene duplication and/or conversion.

Relationship of idiotypes of the anti-p-azophenylarsonate antibodies of A/J and BALB/c mice.

It has previously been shown that A/J anti-Ar antibodies contain 2 different families of cross-reactive idiotypes, referred to as the major and minor idiotypes populations. The present report shows that the minor A/J idiotype is related to a major idiotype of BALB/c anti-Ar antibodies. Anti-idiotype directed against the minor A/J idiotype binds 5 to 10% of A/J anti-Ar but an average of about 40% of BALB/c anti-Ar. This BALB/c population corresponds to the major BALB/c anti-Ar idiotype. For individual BALB/c anti-Ar preparations the maximum percentages of antibody bound by anti-id directed to A/J or BALB/c anti-Ar are very similar. Anti-id reactive with the minor A/J idiotypic population suppressed the formation of the BALB/c major idiotype when injected into BALB/c mice. Adsorption experiments showed that only about one-third of the minor A/J population is related to the BALB/c idiotype and that the expression of this idiotype is highly variable in individual A/J sera. Several types of evidence, obtained with hybridoma products expressing the major A/J idiotype, revealed no detectable relationship between the major A/J and BALB/c anti-Ar idiotypes.

Regulation of expression of a family of cross-reactive idiotypes.

It has recently been shown that anti-p-azophenylarsonate antibodies of A/J mice, which share a common idiotype, are not homogeneous but actually comprise a family of idiotypically related molecules. To obtain additional insight into the degree of relatedness of antibodies within this family, we have determined whether inoculation of any idiotype-positive hybridoma product, conjugated to thymocytes, can activate a regulatory mechanism capable of controlling the entire cross-reactive idiotypic response. In addition anti-idiotypic antibodies were prepared against individual hybridoma products, and their suppressive effects on the idiotypic component of the humoral response were determined. Inoculation of each of the idiotype-positive hybridoma products or anti-idiotypic antisera caused virtually complete suppression of the idiotypic component of the immune response. Furthermore, it was shown that the state of suppression induced by the inoculation of a hybridoma products could be adoptively transferred with T cells to mildly irradiated syngeneic recipients. The results indicate that there are sufficient structural similarities among the family of antibodies comprising the major cross-reactive idiotype to permit their recognition by the same set of elements involved in idiotypic regulation.

Genomic DNA encoding rabbit T cell receptor beta-chains: isotypes and allotypes of C beta.

We have discovered sequence differences in DNA encoding the first exon of rabbit T cell receptor beta-chains from unrelated rabbits that probably reflect allelic C beta 1 allotypes. Rabbit I was from a colony bred to maintain the K1-expression mutation Basilea, and rabbit II was from a colony bred to maintain the K1b9 allotype. Genomic DNA from rabbits I and II also exhibit restriction fragment length polymorphism of C beta on Southern blots. In addition, several different restriction enzyme digests of DNA from rabbit I give three bands, whereas DNA from rabbit II gives two when probed with C beta. An approximately 14-kb cloned genomic DNA fragment from rabbit I has two copies of C beta exon 1 and a 6-kb fragment has a third copy, suggesting that rabbit I has three different C beta genes. The DNA sequence of a germ-line genomic DNA fragment encoding the first exon of the beta-chain constant region from rabbit I also has an open reading frame encoding 140 amino acids immediately 5' of the C beta sequence. A corresponding sequence had previously been found in a cDNA clone from the second rabbit (rabbit II).

Naegleria actin elicits species-specific antibodies.

Actin, the major protein of amebae of Naegleria gruberi, proved to be strongly immunogenic in rabbits. The resulting precipitating antibodies are specific to actin of Naegleria. In a competitive solid-phase radioimmunoassay, these antibodies bound similarly to Naegleria G- and F-actin. Actins from amebae of Acanthamoeba and Dictyostelium, plasmodia of Physarum, sea urchin eggs, and vertebrate muscles gave no competition in the radioimmunoassay. Estimates of the amount of actin in Naegleria amebae ranged from a minimum of 5% of the total cell protein by radioimmunoassay to a maximum of 16% by electrophoresis. The unusual species specificity of these antibodies indicates that Naegleria actin, although conserved in many properties, is different enough to have unique antigenic determinants.