Different allelic distribution of a single SNP between sexes in humans.

We searched for a difference in allele distribution between males and females of a single nucleotide polymorphism located in the human beta T-cell receptor, in 500 subjects (200 males and 300 females). Genotype analysis gave the following results: among the males, 114 (57%) were heterozygous for the T/C polymorphism, 52 (26%) were homozygous (T/T), and 34 (17%) were homozygous (C/C). Among the females, 142 (47.3%) were heterozygous, 73 (24.3%) were homozygous (T/T), and 85 (28.3%) were homozygous (C/C). The allele frequency was significantly different between sexes (chi2 = 8.799, P = 0.012).

A possible mechanism for non-replication of allelic association between a single nucleotide polymorphism of the human beta T-cell receptor and autoimmune diseases.

Gene polymorphisms, in particular single nucleotide polymorphisms (SNPs), have been associated to multifactorial diseases such as cancer, inflammation and autoimmunity. Indeed for some autoimmune diseases, it has been possible to identify critical residues that play a major role in susceptibility to diseases. The association of a common T/C polymorphism in the promoter region of the beta 2 constant chain of the T-cell receptor with autoimmune diseases, such as insulin-dependent diabetes, autoimmune hepatitis, IgA nephropathy, membranous nephropathy, Graves' disease and Hashimoto's thyroiditis, was described in the 1990 s. These reports have not been confirmed in the last few years. We also failed in a previous study to detect any difference between 70 normal subjects and 70 patients with primary biliary cirrhosis; however, we found a difference in allelic frequency between males and females. This finding led us to make an allele frequency study of this single nucleotide polymorphism between sexes in a new series of patients. We studied 165 subjects, 80 males and 85 females, and we found a significant difference between sexes especially for the CC homozygous genotype: 34% of females vs. 14% of males (P = 0.008). If the higher frequency of CC homozygous genotype (that is associated with an increased risk of autoimmune diseases) in females would be confirmed in normal population, this could be an explanation of the controversial results obtained by association studies made between this SNP and autoimmune diseases.

Chromosome 7 monosomy and deletions in myeloproliferative diseases.

We studied deletion and monosomy of chromosome 7 in 150 patients with myeloproliferative diseases. We found 8/150 patients with monosomy 7 by cytogenetics and 4/150 with deletions of the long arm of chromosome 7 by restriction fragment length polymorphism (RFLP) analysis performed with Southern and polymerase chain reaction. To overcome limitation of RFLP analysis, we restricted loss of heterozygosity study with microsatellites to 45 patients, observing deletion 7q31.1 in 7/45 patients. In all patients with molecular alterations the deletion was observed only in myeloid cells, while the monosomy was detected in both myeloid precursor and lymphocytes. This finding suggests a CD34-totipotent stem cell origin for the monosomy and a colony forming unit - granulocyte, erythrocyte, monocyte, megakaryocytes (CFU-GEMM) stem cell origin for the deletions.

A common T/C polymorphism in the promoter region of the beta T-cell receptor gene.

Polymorphisms in the T-cell receptor genes can provide important information for the study of the immune response system, particularly for autoimmune diseases. This report characterizes a common T to C polymorphism in the promoter of the beta 2 constant chain of the T-cell receptor, which abolishes a recognition site for BglII restriction endonuclease.

Ceftriaxone/Amikacin vs Ceftazidime/Amikacin as Empirical Therapy for Fever in Patients with Haematological Malignancy and Severe Granulocytopenia: Clinical and Economic Outcomes.

To assess the economic outcomes produced when a conventional antibiotic treatment regimen requiring three administrations per day was replaced with a treatment regimen requiring only one daily administration, the efficacy, tolerability and cost of ceftazidime was compared with that of ceftriaxone (both drugs in combination with amikacin) for the empirical treatment of febrile granulocytopenic patients with haematological malignancy. 102 febrile patient-episodes were randomly assigned to receive ceftazidime (6g in three divided doses) or ceftriaxone (2g as a single daily dose), both in combination with amikacin. The response was evaluable in 94 patients (47 in each group). 75 (80%) patients had an absolute granulocyte count lower than 100/mm(3) at the onset of fever or during the first week of antibiotic therapy. 61 (64.9%) were affected by acute leukaemia. Multiple daily ceftazidime plus amikacin was effective in 33 of 47 (70.2%) patients, and single daily ceftriaxone plus amikacin in 31 of 47 (66%) patients (p > 0.2). Among patients successfully treated, median time to defervescence was 3.3 days (range 1 to 11) for ceftazidime plus amikacin and 4.5 days for ceftriaxone plus amikacin (range 1 to 15) [p = 0.14]; study drugs were continued for 12 (range 7 to 26) and 12.3 days (range 7 to 28), respectively. Our study demonstrated that single daily administration of ceftriaxone was as effective and well tolerated as multiple daily administration of ceftazidime when both were administered in combination with amikacin. Cost analysis showed that compared with the thrice daily regimen, administration of single daily doses of ceftriaxone for a 12-day treatment period would result in a net cost saving of $US392 (626 940 Italian lire).

[Multiple myeloma. Role of prognostic factors and staging in a therapeutic program]. / Il mieloma multiplo. Ruolo dei fattori prognostici e della stadiazione in un programma terapeutico.

Patients affected with multiple myeloma constitute an heterogeneous population with very different clinical patterns, varying from asymptomatic to very compromised patients with severe and uncontrolled disease. Most common clinical and biological staging systems have been in use for many years. Recently new prognostic factors have been identified; among them, serum levels of beta-2 microglobulin, C-reactive protein and interleukin-6 employed with already known parameters have been useful in the new staging system, permitting a more focalized therapy. As today is not yet possible to define the best treatment schedule, as the most common treatments are incapable to eradicate myeloma neoplastic clone even in responsive patients. Nevertheless extensive use of biologic response modifiers in the last years, as alpha interferon, have added new powerful and hopeful therapeutic tools even if the results need to be confirmed in future trials. It is important to remind the primary role of bone marrow transplantation associated with high dose polychemotherapy even if just a minority of patients is eligible for this therapeutic chance.

Susceptibility of human-mouse T cell hybrids to HIV-productive infection.

Interspecies human x mouse cell hybrids were used to investigate the genetic basis of human permissivity to HTLV-IIIB infection. T cell hybrids between the mouse BW 51.47 T lymphoma line and normal, PHA-IL-2 activated, human peripheral mononuclear cells (PBMCs) were generated. These hybrids preferentially segregated human chromosomes, as assessed by phenotype and karyotype analysis. Viral integration occurred only in those hybrids expressing CD4+ at the cell surface. However, infectious progeny production was demonstrated only in two of the three CD4+ hybrids tested. By segregation analysis, we could correlate the absence of human chromosomes 1, 3, and 9 with the lack of infectious viral progeny.

Detection of human chromosomes in somatic cell hybrids by PCR analysis.

The detection of human chromosomes in somatic cell hybrids is usually made by chromosomal analysis, Southern blot analysis with human probes, and starch-gel electrophoresis of isoenzymes. We describe here a new, quick, and very efficient method to detect human chromosomes in somatic cell hybrids between human and rodent (rat and mouse) cells. The method is based on the polymerase chain reaction to promote amplification of human DNA, using primers derived from localized genes or DNA fragments from each human chromosome.

Assignment of the urokinase-type plasminogen activator receptor gene (PLAUR) to chromosome 19q13.1-q13.2.

The urokinase-type plasminogen activator receptor (uPAR) is a key molecule in the regulation of cell-surface plasminogen activation and, as such, plays an important role in many normal as well as pathological processes. We applied a cDNA probe from the corresponding gene (PLAUR) in a location analysis using a panel of human/rodent cell hybrids and in a multipoint linkage analysis of 40 CEPH families. These two independent studies both found PLAUR to be located on chromosome 19. The cell hybrid study suggested that PLAUR is located at chromosome 19q13-qter, and the multipoint analysis indicated that PLAUR is located at chromosome 19q13.1-q13.2 and surrounded by DNA markers in the following way (with distances given in recombination fractions): D19S27-.11-CYP2A-.06-PLAUR-.03-D19S8-.04-APOC 2-.24-PRKCG. Further, a ligand-binding study performed on cell hybrids verified the species specificity of the uPAR and confirmed the chromosome assignment.

A rapid method for monitoring DNA labelling reactions with haptens.

A rapid and simple method for evaluating the efficiency of DNA labelling reactions with haptens is described. The method, called the Flow-Through Hapten-DNA Assay (FT-HDA), relies on binding of anti-hapten antibodies/alkaline phosphatase conjugates to hapten-DNA, immobilized on disposable capillary absorbent filters, and visual detection of blue-grey coloured spots appearing on the filter after chromogenic reaction with enzyme substrates. FT-HDA of hapten-DNA is markedly faster and simpler than conventional diffusion assays on membranes.

Increased amplification of abl oncogene in K562 cells after passage in nude mice.

Human chronic myelogenous leukemic cells K562, carrying the Ph+ chromosome and an amplified abl oncogene, were injected subcutaneously in nude mice and gave rise to myeloid solid tumors after 4-5 weeks. DNA from the cells of the solid tumors was analyzed by Southern blotting using v-abl and actin probes. Increased amplification of human abl oncogene, but not of the actin gene was found. This suggests a clonal selection of a cell population with more copies of the abl oncogene. This in vivo selection appears to increase the tumorigenicity of K562 cells since, in a second transplantation in nude mice, we observed a shorter period of latency before tumor appearance (3 weeks) when compared to the initial transplantation.

Two mechanisms for the extinction of gene expression in hybrid cells.

When two different mammalian cell types are fused to generate a stable hybrid cell line, genes that are active in only one of the parents are frequently shut off, a phenomenon called extinction. In this study two distinct, complementary mechanisms for such extinction of growth hormone gene expression were identified. In hybrids formed by fusing fibroblasts to pituitary cells, pituitary-specific proteins that bind to the growth hormone promoter were absent. In addition, a negative regulatory element located near the rat growth hormone promoter was specifically activated.

Inhibition of DNA restriction enzyme digestion by anthracyclines.

The inhibition of restriction enzyme digestion of lambda phage DNA by anthracyclines (i.e., adriamycin, daunomycin, epirubicin, idarubicin and esorubicin) commonly used in the treatment of human leukemia and cancer has been studied in vitro. The anthracyclines used inhibit DNA digestion by SmaI, AvaII, HaeIII, HhaI and HpaII, which cut DNA at guanine-cytosine (G-C) sequence sites, and by EcoRI, which cut DNA at adenine adenine-thymine thymine (AATT) sequence sites. Adriamycin completely inhibits DNA restriction by the indicated enzymes, daunomycin, epirubicin and idarubicin determine only a partial inhibition, while esorubicin does not inhibit DNA restriction at all. An attempt is made to correlate the extent of the in vitro interaction between anthracyclines and DNA to in vivo cardiotoxicity.

Translocation of oncogene c-sis from chromosome 22 to chromosome 17 in a human myelogenous leukemia cell line.

By combining somatic cell genetics, in situ hybridization and Southern hybridization, we found that the c-sis oncogene in the human myelogenous leukemia cell line ML3 is translocated from the long arm (q11----qter) of chromosome 22 to the long arm (mid-portion or q21 region) of chromosome 17. This translocation does not result in rearrangement of the c-sis oncogene.

Human histone genes map to multiple chromosomes.

Histone genes were mapped to at least three human chromosomes by Southern blot analysis of DNAs from a series of mouse-human somatic cell hybrids (using 32P-labeled cloned human histone DNA as probes). Chromosome assignment was confirmed by in situ hybridization of radiolabeled histone gene probes (3H-labeled) to metaphase chromosomes. One human histone gene cluster (lambda HHG41) containing an H3 and H4 gene resides only on chromosome 1, whereas other clusters containing core (H3, H4, H2A, and H2B) alone (lambda HHG17) or core together with H1 histone genes (lambda HHG415) have been assigned to chromosomes 1, 6, and 12. These results suggest that the multigene family of histone coding sequences that reside in a series of clusters may be derived from a single cluster containing one each of the genes for the five principal classes of histone proteins. During the course of evolution, a set of events, probably involving reduplication, sequence modification, and recombination, resulted in the present pattern of human histone gene distribution among several chromosomes.

Tissue-type plasminogen activator gene is on chromosome 8.

Tissue plasminogen activator is one of the two plasminogen activators, both serine proteases, that catalyze the conversion of inactive plasminogen to plasmin, which then degrades the fibrin network of blood clots. By combining somatic cell genetics, in situ hybridization, and Southern blot hybridization, we localized the human tissue plasminogen activator gene to the pericentromeric region of chromosome 8.

Human urokinase gene is located on the long arm of chromosome 10.

Urokinase is one of the two plasminogen activators that catalyze the conversion of inactive plasminogen to plasmin. By combining somatic cell genetics, in situ hybridization, and Southern hybridization, we localized the human urokinase gene on the distal third of the long arm (q24-qter) of chromosome 10.

Specific immunoglobulin production and enhanced tumorigenicity following ascites growth of human hybridomas.

Human X human hybridomas constructed with the B6 lymphoblastoid clone, which produces antitetanus toxoid (TT) antibody, and the lymphoblastoid cell line KR-4 or human hybrid myeloma KR-12, were adapted to growth as ascites in pristane-treated BALB/c nude mice by a single prior passage as a solid subcutaneous (s.c.) tumor in irradiated nude mice followed by in vitro culture. Both B6 X KR-4 and B6 X KR-12 hybrids produced anti-TT antibody and phenotypically resembled the lymphoblastoid KR-4, or the hybrid myeloma KR-12 parent, respectively. Growth as ascites increased the tumorigenicity of both hybrids in nude mice as measured by tumor incidence and rate of tumor growth. The observed increase in tumorigenicity of these hybrid cells after ascites growth was associated with a substantial loss of chromosomes. Passage of the B6 X KR-4 lymphoblastoid hybrid resulted in several reversible morphological changes characteristic of myeloma cells. These changes correlated with increased human Ig production. These observations provide a system for greatly amplifying human monoclonal antibody production.

A human hybrid myeloma for production of human monoclonal antibodies.

We produced somatic cell hybrids between human myeloma cells and a lymphoblastoid cell line that is hypoxanthine phosphoribosyl transferase-deficient and ouabain-resistant. These hybrids were phenotypically similar to the human myeloma parental cells and grew as well as the human lymphoblastoid parental cells. After counterselection in 6-thioguanine, mutants that were 6-thioguanine-and ouabain-resistant were obtained, one of which was used as a fusion partner with lymphoblastoid B cells that produce anti-tetanus toxoid (TT) antibodies. These hybrids secreted human anti-TT monoclonal antibodies in much larger amounts than the parental lymphoblastoid cells, and were stable for a period of over 10 mo until the present time. Thus, by hybridizing plasmacytomas with lymphoblastoid cells, we constructed a fusion partner that secretes large amounts of immunoglobulin (Ig), grows at a fast rate, has a high fusion frequency, and supports the production of monoclonal antibodies over long periods of time. Moreover, anti-TT antibody-producing hybrids have been grown as solid tumors in irradiated BALB/c nude mice and then adopted to ascites growth, producing 1 to 8 mg of human immunoglobulin per 1 ml of ascites fluid.