Comparison of various methods for monitoring hybridoma cell proliferation.

The design of a strategy for the control of large scale cultures of hybridoma cells requires the use of convenient indicators to monitor properly the evolution of the biomass. In order to select appropriate indicators, we have measured in parallel, in bulk cultures of mouse hybridoma cells, the evolution of several metabolic parameters together with those of cell density and viability. We observed that flow cytometry analysis gives an early indication of the proliferative capacity of the cell population. Determination of metabolic rates (i.e. glucose, glutamine, amino acid, consumption, lactic acid or ammonium production) adequately indicates the current metabolic status of the cells. Indeed, a sharp decrease in these metabolic rates occurs rapidly following nutrient deficiency. Finally, measurements of lactate dehydrogenase (LDH) and DNA fragments released into the culture supernatants accurately reflect the kinetics of cell death.

Gene for chronic proximal spinal muscular atrophies maps to chromosome 5q.

Proximal spinal muscular atrophies represent the second most common fatal, autosomal recessive disorder after cystic fibrosis. The childhood form is classically subdivided into three groups: acute Werdnig-Hoffmann (type I), intermediate Werdnig-Hoffmann disease (type II) and Kugelberg-Welander disease (type III). These different clinical forms have previously been attributed to either genetic heterogeneity or variable expression of different mutations at the same locus. Research has been hindered because the underlying biochemical defect is unknown, and there are insufficient large pedigrees with the most common and severe form (type I) available for study. Therefore, we have undertaken a genetic linkage analysis of the chronic forms of the disease (types II and III) as an initial step towards the ultimate goal of characterizing the gene(s) responsible for all three types. We report here the assignment of the locus for the chronic forms to the long arm of chromosome 5 (5q12-q14), with the anonymous DNA marker D5S39, in 24 multiplex families of distinct ethnic origin. Furthermore, no evidence for genetic heterogeneity was found for types II and III in our study, suggesting that these two forms are allelic disorders.

Heterogeneity of HLA genetic factors in IDDM susceptibility.

The association of certain HLA-D alleles with insulin-dependent diabetes mellitus (IDDM) is well known. One hundred and sixty-one non-related diabetic individuals and 142 non-related healthy controls were typed for the HLA DR-DQw-Dw association, using a restriction fragment length polymorphism (RFLP) typing method that combines three probe/enzyme systems: DRB/Taq I, DQB/Taq I, and DQB/Bam HI. Comparison of frequencies in both diabetics and controls confirms previous results in terms of HLA class II and IDDM association. Moreover, we have found that DR3/3 heterozygous individuals are more susceptible to IDDM when they are also Dw25 (associated with B18) than when they are Dw24 (associated with B8). Using oligonucleotide dot-blot hybridizations we analyzed the HLA-DQB1 sequence of DR3,Dw24 and DR3,Dw25 homozygous individuals, and we found no difference at position 57 between these two DR3-carrying haplotypes. This observation points to the heterogeneity of HLA genetic factors in IDDM susceptibility.

Combined immunodeficiency with abnormal expression of MHC class II genes.

The MHC class II CID represents an example of immunodeficiency in which the defect in expression of membrane glycoproteins leads to abnormal cell to cell interactions and thus to abnormal immune responses. It represents an interesting model which confirms the importance of MHC molecules in all immune responses to foreign antigens. It also underlines the complexity of regulatory mechanism which control the expression of MHC class II genes. To elucidate these mechanisms, it is essential to identify and characterize the genes involved in control of MHC class II expression.

[Alpha genes of the T cell receptor: a possible implication in genetic susceptibility to multiple sclerosis]. / Les gènes alpha du récepteur des cellules T: une possible implication dans la susceptibilité génétique à la sclérose en plaques.

Multiple sclerosis (MS) is a neurological disease in which 60% of patients are DR2 (versus 20% in controls). Restriction fragment length polymorphism (RFLP) associated with T cell receptor alpha-chain and beta-chain genes have been analysed in a sample of 46 MS patients and compared with those of 142 controls. The alpha-chain gene polymorphism is localized to the V-J region and consists of 3 Bgl II alleles (alpha a = 3.2 kb; alpha b = 2.9 kb; alpha c = 2.8 kb). A significant difference was found in the distribution of these three alleles since 97% of DR2 patients versus 60% in DR2 non-MS individuals were found to be homozygotes alpha a/alpha a. These results suggest the influence of T cell antigen receptor germ line repertoire on the etiopathology of this disease.

New methods for detection of HLA genes polymorphism useful for associated diseases studies.

We have studied in 22 informative families typed for HLA the segregation of DNA restriction fragments obtained with five restriction enzymes and hybridized with one class I and three class II probes. Most of the fragments correlate with serologically defined specificities. Many fragments can be grouped in clusters, whose genetic significance is discussed. The RFLP distribution in patients with insulin-dependent diabetes mellitus, multiple sclerosis or narcolepsy, three diseases known to be associated with some HLA-DR specificities, has been also studied. Many fragments allow to distinguish between patients and HLA-DR matched controls. Hybridization of genomic DNA with synthetic oligomers will refine moreover the understanding of the HLA system polymorphism.

Exuberant restriction fragment length polymorphism associated with the DQ alpha-chain gene and the DX alpha-chain gene.

Cellular DNAs from individuals of 23 families were digested with five restriction endonucleases (Pvu II, EcoRI, HindIII, BamHI, and EcoRV) and then probed with a DX alpha-chain gene probe. Seventeen allogenotopes were observed, each of which could be assigned to a serologically defined haplotype by noting its segregation in families. Six sets of allogenotopes forming allelic series were noted. In comparison with restriction maps of the DQ alpha and the DX alpha regions, each of these series has been assigned to the DQ alpha or the DX alpha locus. Allogenotopes of the four DQ alpha series constitute three clusters correlating with the supertypic groups of class II histocompatibility antigens DQw1 (DR1, DR2, and DRw6), DRw53 (DR4, DR7, and DR9), and DR3 plus DR5 plus DR8. These 13 DQ alpha fragments constitute 22 different patterns. The two DX alpha series constitute two clusters, one of which is not found to be correlated strongly with DR specificities, whereas the other is correlated loosely (r = 0.45) with DR5 and DR7. This absence of strong linkage disequilibrium between the DX alpha series and the DR series contrasts with the DQ alpha series and suggests a recombination point between DQ alpha and DX alpha loci.

Genotyping with DNA probes in combined immunodeficiency syndrome with defective expression of HLA.

We performed HLA genotyping by using restriction-enzyme fragments hybridized with specific HLA probes instead of traditional immunologic methods in two patients whose lymphocytes expressed so few HLA antigens on the cell surface that serologic methods failed. Segregation of restriction-fragment-length polymorphism permitted identification of the genotypes. In addition, known correlations between serologically determined antigens and restriction-fragment-length polymorphism were confirmed. We applied this approach in making therapeutic decisions regarding bone marrow transplantation.

Clusters of HLA class II beta restriction fragments describe allelic series.

Eighty-eight HLA haplotypes have been investigated for the presence or absence of 52 restriction fragments generated by four restriction enzymes (EcoRI, EcoRV, HindIII, BamHI), and detected by a DQ beta cDNA probe. Correlation analysis showed several sets of positively associated fragments forming 11 clusters. They constitute three different allelic series. The first coincides with DR alleles, the second with DQ alleles, and within the third, one cluster coincides with DRw53 (MT). As shown by comparative hybridization, most fragments belonging to the DR- as well as the DQ-related series correspond to DQ beta genes. In contrast, the MT-related series corresponds to DR beta genes. The evolutionary significance of these restriction fragment clusters is discussed.

Class II HLA-DC beta-chain DNA restriction fragments differentiate among HLA-DR2 individuals in insulin-dependent diabetes and multiple sclerosis.

HLA-DR2 allele is negatively associated with insulin-dependent diabetes and positively associated with multiple sclerosis (MS). A 2.2-kilobase-pair EcoRI DNA restriction fragment detected with a beta-chain HLA-DC cDNA probe was found to be strongly correlated with HLA-DR2 in the normal population, but was absent in HLA-DR2 insulin-dependent diabetic patients. This fragment was found in HLA-DR2 multiple sclerosis patients with the same frequency as in controls. A beta-chain HLA-DC 12-kilobase-pair BamHI fragment might differentiate multiple sclerosis patients from healthy individuals.

Analysis of HLA class I genes with restriction endonuclease fragments: implications for polymorphism of the human major histocompatibility complex.

Cellular DNA from HLA-typed individuals was digested with the restriction endonucleases HindIII, EcoRV, and EcoRI. The separated restriction endonuclease fragments were hybridized with a HLA class I cDNA probe by using the Southern transfer technique. Digestion of cellular DNA with HindIII generated 22 restriction endonuclease fragments, 11 of which showed polymorphism for presence or absence in a population sample. With EcoRV, 13 fragments were identified; 6 showed polymorphism. EcoRI generated 11 fragments, of which 1 was polymorphic. Of these 18 polymorphic fragments generated by the three restriction endonucleases, each of 5 was found to be positively associated with one allele of the HLA-A or -B allelic series (HLA-Aw24, -B8, -B15, -Bw35, and -B40). One fragment was positively associated with two HLA-A series alleles (HLA-A1 and -A11). Another fragment was positively associated with five HLA-B series alleles (HLA-B5, -B7, -B14, -Bw16, and -Bw35) and one fragment was positively associated with alleles at two loci (HLA-B14 and -Cw5). The serologically defined allele HLA-Aw24 was associated with two polymorphic fragments, one association showing a positive correlation and the other a negative correlation. Each informative family studied thus far has shown segregation of the restriction fragment with the associated serologically defined allele. The fragments associated with serologically defined alleles occurred in the population sample studied at low or moderate frequencies. The remaining polymorphic fragments occur at high frequency, suggesting that class I genes not serologically detected show less polymorphism than serologically defined class I genes.

Polymorphic restriction endonuclease fragment segregates and correlates with the gene for HLA-B8.

Cellular DNA from HLA-typed individuals was digested with the restriction endonuclease EcoRV. After electrophoresis and transfer to a hybridization membrane, the restriction endonuclease fragments were probed with cDNA carrying the nucleotide sequence encoding a class 1 HLA gene. Polymorphism for presence or absence of various EcoRV fragments was noted in a panel of unrelated HLA-typed individuals. A polymorphic 8.6-kilobase pair EcoRV fragment was found which correlated in the panel with the serologically defined gene HLA-B8. A family study revealed that this fragment segregated with the haplotype carrying the HLA-B8 gene. This fragment may carry the gene for HLA-B8 or it may represent another class 1 gene (or pseudogene) in linkage disequilibrium with HLA-B8.

[Polymorphism of HLA genes: I. Demonstration of a close correlation between DNA fragments determined by BglI restriction enzyme and HLA class I antigens]. / Polymorphisme des gènes HLA: I. Mise en évidence d'une étroite corrélation entre des fragments d'ADN déterminés par l'enzyme de restriction BglI et des antigènes HLA de classe I.

Description of DNA fragments associated to HLA class I gene is possible by using restriction enzymes which determine these fragments and specific DNA probes which permit their detection. In one family, with a child presenting a recombination between HLA-A and C, six fragments determined by the enzyme BglI were found to be polymorphic. The informative fragments segregate with HLA, either with a whole haplotype or with one of the two recombinant segments of the HLA complex. In a small sample of population they correlate with one (A11) or with a group of known cross-reactive antigens serologically defined (A3 and A11; A25 and A26. Another fragment is associated with unknown cross-reactive antigens (A2 and A29).