Molecular characterization of a supratypic cross-reactive idiotype associated with IgM autoantibodies.

Neoplastic B cells from patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphomas (SLL) frequently express surface Ig reactive with the mouse mAb, Lc1. Raised against a human monoclonal IgM with rheumatoid factor activity, Lc1 detects a major cross-reactive Id (CRI) present on the H chain of many monoclonal IgM autoantibodies. In contrast to other major autoantibody-CRI investigated to date, we note that the Lc1-CRI is expressed by subpopulation of cells in the germinal centers, as well as in the mantle zones, of secondary human B cell follicles. To examine the molecular basis for Lc1 expression, we used the polymerase chain reaction to isolate the functionally rearranged Ig VH genes of monoclonal Lc1-reactive B cell populations from six unrelated patients with CLL or SLL. Although the neoplastic B cells from most patients with CLL or SLL express the CD5 surface differentiation Ag, the lymphoma cells from one patient with SLL were CD5-negative. We find that the Lc1-reactive cells from each cell population have Ig rearrangements involving a VH gene of the VH4 subgroup. However, the VH4 genes rearranged in different Lc1-reactive tumor populations may originate from at least two disparate germ-line VH4 genes. Also, in contrast to the CD5-positive tumor populations, we find evidence for intraclonal diversity in the functionally rearranged VH4 genes of the CD5-negative SLL. Collectively, this study discerns a degeneracy in the VH4 genes that can encode the Lc1 CRI, indicating the term "supratypic cross-reactive idiotype" may best describe the specificity of the Lc1 mAb. Also, this study suggests that expression of CD5 may delineate categories of B cell SLL that differ in their relative rates of constitutive Ig V gene somatic mutation.

Autoantibody-encoding kappa L chain genes frequently rearranged in lambda L chain-expressing chronic lymphocytic leukemia.

Patients with kappa L chain expressing chronic lymphocytic leukemia (CLL) frequently have leukemia cells reactive with a murine mAb, designated 17.109. Raised against a monoclonal IgM rheumatoid factor autoantibody, this mAb recognizes a major kappa-L chain-associated cross reactive Id, designated 17.109-CRI. Molecular studies reveal that the 17.109-CRI in CLL is a serologic marker for expression of a conserved kappa L chain V region gene (V Kappa gene) of the V Kappa 3 subgroup, designated Humkv325. We isolated an upstream gene fragment of Humkv325 to examine for Ig gene rearrangements of this and other closely related V Kappa 3 genes by Southern analyses. Consistent with Humkv325 encoding the 17.109-CRI, we find that the genomic DNA from all 17.109-reactive leukemia cell populations have gene rearrangements that are detected using this probe. In addition, we observe V Kappa 3 gene rearrangements frequently in the genomic DNA of lambda L chain-expressing leukemia cells. Of the genomic DNA from 33 lambda-L chain-expressing CLL samples, 8 (24%) had additional nongerm-line bands detected with the Humkv325 probe. Consistent with these bands representing Ig gene rearrangements, the additional band in each but one sample also hybridized with probes specific for the J Kappa region and/or the kappa-deleting element. Using the polymerase chain reaction (PCR), we examined the genomic DNA from all lambda L chain-expressing CLL for V Kappa 3 gene rearrangements to J Kappa and/or Kde. PCR on each DNA sample with V Kappa 3 gene rearrangements detected by Southern analysis generated gene fragments that hybridized specifically with oligonucleotides corresponding to framework or CDR of the Humkv325 gene. Nucleic acid sequence analyses of representative samples confirmed that these DNA contained abortive Humkv325 gene rearrangements. PCR for rearranged V Kappa 3 genes in the DNA of other lambda-L chain-expressing CLL either did not generate any PCR product or produced fragments that failed to hybridize with all Humkv325 oligonucleotide probes. Nucleic acid sequence analyses of the latter demonstrated that these represent abortive V Kappa gene rearrangements involving another conserved V Kappa 3 gene, designated Vg. These studies indicate that Humkv325 and Vg frequently may undergo Ig gene rearrangement independent of their expression. As such, the frequent use of Humkv325 in CLL may be secondary, in part, to an enhanced propensity of this V Kappa 3 gene to undergo genetic rearrangement during B cell ontogeny.

Developmentally restricted immunoglobulin heavy chain variable region gene expressed at high frequency in chronic lymphocytic leukemia.

During fetal development, murine and human B-lineage cells rearrange and express a highly restricted set of immunoglobulin heavy chain variable region genes (VH genes). We noted that a VH gene of the restricted human fetal repertoire, designated 51p1, potentially could encode the VH region of two human IgM rheumatoid factor proteins. These rheumatoid factors share a cross-reactive idiotype (CRI) defined by reactivity with G6, a murine monoclonal antibody that recognizes an antibody heavy chain determinant present on many human IgM autoantibodies, particularly rheumatoid factors. Recently, we found that the G6 CRI also is expressed frequently by neoplastic CD5 (Leu1) B cells from patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma. However, neoplastic CD5-negative B cells from patients with lymphomas of follicular center cell origin rarely express this CRI. Here, we report that G6-reactive leukemic cells from two unrelated CLL patients express a VH gene that shares greater than 99% homology with a rearranged VH gene previously isolated from the leukemic cell DNA of another CLL patient and that is identical to VH 51p1. Using the polymerase chain reaction, we find that this VH gene is rearranged, and presumably expressed, in the genomic DNA of all examined cases of G6-reactive CLL or small lymphocytic lymphoma. Thus these data indicate that the autoantibody-associated G6 CRI is a serologic marker for a conserved and developmentally restricted VH1 gene that is expressed at high frequency in CD5 B-cell malignancies and early B-cell ontogeny.

Ig V region gene expression in small lymphocytic lymphoma with little or no somatic hypermutation.

Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V kappa gene) rearrangement in small lymphocytic non-Hodgkin's lymphomas that express Ig bearing a major kappa-L chain associated cross-reactive Id, designated 17.109. Previously, we identified the 17.109-cross-reactive Id in chronic lymphocytic leukemia as a serologic marker for expression of a highly conserved V kappa gene, designated Humkv325. Using sense-strand oligonucleotides specific for the 5'-end of this V kappa gene and antisense oligonucleotide specific for a J kappa region consensus sequence, we could amplify specifically Humkv325 when juxtaposed with J kappa through Ig gene rearrangement. This allowed us to amplify rearranged V kappa genes from DNA isolated from minute amounts of lymphoma biopsy material for molecular analyses. Our studies demonstrate that 17.109-reactive SL NHL, with or without associated CLL, rearrange, and presumably express, Humkv325 without substantial somatic diversification. Our data suggest that malignant B cells in SL NHL, in contrast to NHL of follicular center cell origin, may express immunoglobulin variable region genes with little or no somatic hypermutation.

Suppression of pentylenetetrazol-elicited seizure activity by intraosseous lorazepam in pigs.

Use of the intraosseous (i.o.) route as an alternative venous access for drug administration has increased. This study examined the efficacy of i.o. lorazepam (LZP) in suppressing pentylenetetrazol (PTZ)-induced seizure activity in pigs. Domestic swine (13-20 kg) were prepared for recordings of arterial blood pressure, EEG, and electrocortical activity. Seizure activity was induced by PTZ (100 mg/kg i.v.). Sixty seconds after onset of seizure activity, animals either received no drug (control) or LZP (1.0 mg/kg) administered i.v. or i.o. via an 18- or 13-gauge spinal needle inserted in the right proximal tibia. Both i.o. and i.v. LZP significantly suppressed the duration of seizure activity (DSA) (s/min interval) within 1 min following drug administration: DSA control, 46.2 +/- 3.6; i.v. LZP, 25.0 +/- 5.1; i.o. (18-gauge) LZP, 27.6 +/- 6.0; i.o. (13-gauge) LZP, 24.0 +/- 2.4. Seizure activity was essentially abolished at 1 min following LZP infusion. In addition, both i.v. and i.o. LZP did not have significant effects on the basal heart rate and mean arterial blood pressure. The data demonstrate that in swine (1) the i.o. route is an effective alternative venous access for LZP administration, and (2) the size of spinal needles does not affect the antiepileptic efficacy of i.o. infusion of LZP.

A survey of the alpha-tubulin gene family in chicken: unexpected sequence heterogeneity in the polypeptides encoded by five expressed genes.

To characterize the alpha-tubulin gene family in chicken, we have isolated five chicken alpha-tubulin genes and determined the majority of the sequences of the encoded polypeptides. Three of these (c alpha 3, c alpha 5/6 and c alpha 8) encode novel, expressed alpha-tubulins that have not previously been analyzed, whereas one gene segment is a pseudogene and another appears capable of encoding a functional subunit (although we were unable to document its expression in a survey of chicken tissues). Together with two additional expressed, functional alpha-tubulins reported earlier, we conclude that the chicken alpha-tubulin family is comprised of at least five functional genes whose polypeptide products are substantially more heterogeneous than found in preceding analyses of vertebrate alpha-tubulins. Comparison of the amino acid sequences reveals that the five polypeptides are between 96 and 83% identical, with the extreme carboxy-terminal residues representing a highly heterogeneous variable domain. Since some alpha-tubulins undergo cyclic post-translational removal and readdition of a carboxy-terminal tyrosine, the notable sequence divergence in this domain suggests that individual tubulins probably participate to different extents in this modification cycle.

A divergent testis-specific alpha-tubulin isotype that does not contain a coded C-terminal tyrosine.

On the basis of analysis of cDNA clones of alpha-tubulin RNAs expressed during spermiogenesis in chickens, we report the identification of a novel alpha-tubulin which is expressed exclusively in chicken testes. Comparison of its sequence with those previously determined not only demonstrates that the encoded polypeptide is significantly divergent from other alpha-tubulins but also supports the hypothesis that alpha-tubulin isotypes are distinguished by a carboxy-terminal variable region sequence and, to a lesser extent, by a domain near the amino terminus. Since essentially all previously known alpha-tubulins undergo a unique cycle of removal and posttranslational readdition of a tyrosine residue at the extreme carboxy terminus, the presence in this testes alpha-tubulin of a very divergent carboxy terminus that does not contain an encoded tyrosine raises the possibility that this polypeptide does not participate in the usual cycle of tyrosination/detyrosination.