Accumulation of somatic mutants in the B cell compartment after primary immunization with a T cell-dependent antigen.

The accumulation of somatic mutants in splenic B lymphocytes early after primary immunization with the hapten (4-hydroxy-3-nitro-phenyl)acetyl (NP) coupled to chicken gamma globulin (CG) was determined. Rearranged V186.2 heavy chain genes were amplified by the polymerase chain reaction from genomic DNA and subjected to nucleotide sequence analysis. Somatic antibody mutants become detectable on day 6 after immunization, and most of the somatic mutations accumulating in the memory compartment are introduced until day 14. At this time strong selection for mutants expressing high binding affinity for NP is apparent. Extrapolation from the mutation frequency increases between day 6 and day 14 to the previously determined mutation frequency at week 6 (Weiss. U. and Rajewsky, K., J. Exp. Med. 1990, 172: 1681) leads to the prediction that the process of mutant generation ceases to operate around day 22 after primary immunization.     

Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development.

Hodgkin disease (HD) is characterized by a small number of putative malignant cells [Hodgkin and Reed-Sternberg (HRS) cells] among a background of lymphocytes and histiocytes. The lineage of HRS cells is still elusive and a clonal origin of these rare cells has not formally been demonstrated. We isolated HRS cells by micromanipulation from histological sections of three cases of Hodgkin lymphoma (each representing a distinct subtype of the disease) and analyzed individual cells for immunoglobulin variable (V) gene rearrangements by PCR. In each of the three cases a single heavy-chain V (VH) (and in one case, in addition, a kappa light-chain) gene rearrangement was amplified from the HRS cells, identifying these cells as members of a single clone. A potentially functional VH rearrangement was obtained from a case of nodular sclerosis HD. Somatic mutations and intraclonal diversity in the VH genes indicate a germinal center B-cell origin of the HRS cells in a case of lymphocyte-predominant HD, whereas in a case of mixed-cellularity HD the sequence analysis revealed only nonfunctional V gene rearrangements, suggesting a pre-B-cell origin. This indicates that HRS cells can originate from B-lineage cells at various stages of development.     

Development and proliferation of lymphocytes in mice deficient for both interleukins-2 and -4

Interleukin (IL)-2 and IL-4 are considered as important regulators of growth and differentiation of lymphocytes. We report that in mice made deficient for both IL-2 and IL-4 by gene targeting all major T cell subsets and B cells were normal, indicating that IL-2 and IL-4 are not essential for development of the immune system. Paradoxically, proliferation of T cells was increased in both IL-2- and IL-4-deficient homozygous mice.     

Increased drug cytotoxicity at reduced pH counteracts cyclophosphamide resistance in cultured rat mammary carcinoma cells

The sensitivity of a cyclophosphamide (CP)-resistant MIR rat mammary carcinoma cell variant (MIRCPr) in monolayer culture towards the cytotoxic effect of mafosfamide (an analogue of "activated" CP) was measured as a function of extracellular pH (pHe). An inverse correlation was found between cell survival and the H+ ion concentration in the culture medium. At pHe 7.4, the fraction of clonogenic MIRCPr cells exposed to mafosfamide (7.5 micrograms/ml) for 24 hr was 1 X 10(-1) in relation to untreated control cells. At pHe 6.2, however, this value was reduced to 3 X 10(-4), i.e., a value equal to that for the CP-sensitive parental MIR cells exposed to the same concentration of mafosfamide at pHe 7.4. Our data indicate complete compensation of CP resistance in MIRCPr cells at pHe 6.2. MIRCPr cells were not resistant to the cytotoxic effect of nornitrogen mustard. This suggests that resistance to CP in MIRCPr cells is due to enzymatic inactivation of the primary intermediates in CP bioactivation. The alkylating activity of nornitrogen mustard (and less so that of phosphoramide mustard) is strongly enhanced at low pH. In MIRCPr cells shifted to an acidic environment, therefore, a (putative) decrease in the intracellular concentration of alkylating CP metabolites may be counteracted by an enhancement of their alkylating activity (on a molar basis). By parenteral administration of glucose, the pH in malignant tumors of both animal and human origin can be lowered to values between 5.6-6.6. Our data suggest that an upshift of H+ ion concentration in malignant tissues may at least partially counteract CP resistance in cancer cells in vivo.     

Predominance of null mutations in ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a Pl 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the Pl 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.