Haplotype tagging for the identification of common disease genes.

Genome-wide linkage disequilibrium (LD) mapping of common disease genes could be more powerful than linkage analysis if the appropriate density of polymorphic markers were known and if the genotyping effort and cost of producing such an LD map could be reduced. Although different metrics that measure the extent of LD have been evaluated, even the most recent studies have not placed significant emphasis on the most informative and cost-effective method of LD mapping-that based on haplotypes. We have scanned 135 kb of DNA from nine genes, genotyped 122 single-nucleotide polymorphisms (SNPs; approximately 184,000 genotypes) and determined the common haplotypes in a minimum of 384 European individuals for each gene. Here we show how knowledge of the common haplotypes and the SNPs that tag them can be used to (i) explain the often complex patterns of LD between adjacent markers, (ii) reduce genotyping significantly (in this case from 122 to 34 SNPs), (iii) scan the common variation of a gene sensitively and comprehensively and (iv) provide key fine-mapping data within regions of strong LD. Our results also indicate that, at least for the genes studied here, the current version of dbSNP would have been of limited utility for LD mapping because many common haplotypes could not be defined. A directed re-sequencing effort of the approximately 10% of the genome in or near genes in the major ethnic groups would aid the systematic evaluation of the common variant model of common disease.

Humoral immunity to respiratory syncytial virus in young and elderly adults.

Respiratory syncytial virus (RSV) has been demonstrated to cause substantial disease in elderly and immunocompromised subjects. The relationship of serum antibody to RSV infection and the risk of infection in elderly subjects is controversial, thus we evaluated the presence of neutralizing antibodies to RSV in healthy people of different age groups and the correlation with viral protection. Baseline blood samples from 197 subjects aged 20-80 years were analysed for the presence of anti-RSV antibodies either by indirect immunofluorescence and microneutralization test. The percentage of people who had neutralizing antibodies to RSV was significantly higher (P=0.001) in the youngest group (92.51%) compared to the frail group (36.21%). The RSV antibody level tends to wane in some older people; this factor could determine proneness to RSV re-infections in the elderly who are at a greater risk of developing severe respiratory disease.

Immunosuppressive monoclonal antibody to CD64 from patients with long-term stable multiple sclerosis.

High intrathecal levels of anti-myelin basic protein (MBP) IgM were previously found to be significantly associated with early favorable course in a cohort of patients with multiple sclerosis (MS). A mAb to MBP 105-120 recognizing the 222-228 epitope of the extracellular domain of high affinity immunoglobulin gamma Fc-receptor I (CD64) was isolated from EBV(+) B cell clones of long-term stable RRMS patients. This mAb exerted immunosuppressive activity on MS-derived T cell lines through induction and release of high amounts of interleukin-10 and decreased levels of interleukin-12 from activated monocytes providing the biological basis for a potential new treatment for MS and other immune-mediated neurological disorders.

Defining origins of malignant B cells: a new circulating normal human IgM(+)D(+) B-cell subset lacking CD27 expression and displaying somatically mutated IGHV genes as a relevant memory population.

In probing the cell of origin in malignant B cells, an imprint of somatic hypermutation (SHM) in immunoglobulin (Ig) variable (V) region genes delineates antigen encounter, and identifying the precise pathway generating SHM in the normal B-cell counterpart becomes relevant. SHM remains the definitive memory imprint in normal human B cells, but CD27 expression also delineates memory. Recently, dye extrusion adenosine triphosphate-binding transporter assays identified circulating isotype-switched memory B cells that lacked CD27, yet exhibited low levels of SHM. To extend findings, we report a pre-switched CD27(-ve) circulating memory B-cell population in normal blood using comparable assays, and isolated CD19(+)IgM(+)D(+)CD27(-ve) cells (>99% purity) for the analysis of IGHV5/IGHV3-IGHM transcripts. Of these (n=334), approximately 78% were germ line and naive B cell derived. Strikingly, 21.9% of the transcripts were mutated. They showed 3-5 mutations (13.5% of sequences) and >5 mutations (8.4% of sequences) per transcript. Accrual of mutations in a subset of CD19(+)IgM(+)D(+)CD27(-ve) cells define a new circulating pre-switched memory B-cell pool, present in substantial numbers in the population harboring naive B cells. These CD19(+)IgM(+)D(+)CD27(-ve) memory B cells may have a distinct lineage and function, and seem relevant to understanding origins of malignant B cells, in particular those of hairy cell leukemia cells, which display mutated V genes yet lack CD27 expression.

Interference with cell cycle progression and induction of apoptosis by dideoxynucleoside analogs.

The in vitro effect of single or combined doses of zidovudine (AZT) and dideoxycytidine (ddC) on PHA-activated human peripheral blood mononuclear cells (PBMC) proliferative response and lymphoblastoid T cell line CEM cell growth was evaluated. Clinically relevant amounts (0.1, 1 and 10 microM) of AZT, ddC and AZT/ddC combination (10 + 10 microM) inhibited 3H TdR uptake in both cell models in a dose-dependent manner. The inhibitory effect on cell growth was confirmed by counting the amount of viable CEM cells recovered after 24, 48 and 72 h exposure to the drugs. On equimolar basis, ddC was considerably more efficient than AZT although the latter potentiates the activity of the former Flow cytometric analysis of PBMC and CEM cells exposed to the dideoxynucleosides revealed a decrease in the rate of DNA synthesis (rate of passage through the S phase of the cell cycle) and a reduced number of cell generations, the latter assessed by measuring the halving of the fluorescent probe 5-6-carboxyfluorescein diacetate succinimidyl ester by flow cytometry. The analysis of CEM cells recovered after exposure to ddC or AZT/ddC combination (10 + 10 microM), showed that in addition to perturbing cell cycle progression, ddC, and most efficiently the AZT/ddC combination, induced cell death by apoptosis. The latter was manifested as enhanced side scatter and decreased, sub-G1, DNA content by flow cytometry, and as DNA breakdown in nucleosomal fragments by gel electrophoresis. Present findings indicate that clinically relevant concentrations of dideoxynucleosides reduce cell growth by hampering DNA replication and inducing apoptosis.

Lack of immunotoxicity of saquinavir (Ro 31-8959) used alone or in double or triple combination with AZT and ddC.

Saquinavir (Ro 31-8959; SQV) has been demonstrated to be a potent inhibitor of human immunodeficiency virus (HIV) proteinases and acts synergistically with dideoxynucleoside analogues. The aim of this study was to investigate the in vitro immunomodulatory effects of SQV on normal human peripheral blood mononuclear cells (PBMC) and on lamina propria mononuclear cells (LPMC). We used the drug either alone or in double and triple combination with AZT and ddC to assess whether SQV enhances the immunomodulatory effects induced by AZT and ddC that we previously observed. We demonstrated that SQV did not induce any modulation of the proliferative response either in PBMC or in LPMC. Similarly, NK cell-mediated cytotoxic activity and cytokine production were not modified by SQV. More importantly, SQV/AZT, SQV/ddC, and SQV/AZT/ddC combinations did not strengthen neither the inhibition of PBMC and LPMC proliferative response or the modulation of cytokine production induced by AZT, ddC, and AZT/ddC. On the other hand, the increased IL-2 production induced by AZT and ddC was not observed adding SQV to the dideoxynucleoside analogues. In conclusion, we demonstrated that SQV used in combination with AZT and ddC did not add any further immunotoxicity.

Oxidized low-density lipoproteins affect natural killer cell activity by impairing cytoskeleton function and altering the cytokine network.

Several lines of evidence indicate that oxidative imbalance can play an important role in determining an impairment of natural killer (NK) cell activity in a variety of human diseases. Because a specific role for oxidized low-density lipoproteins (LDL) as pro-oxidizing agents has been envisaged, we tested the activity of oxidized LDL (ox-LDL) on NK cell-mediated cytotoxicity, cytokine release, and membrane molecule modulation. Native LDL served as control. Treatment with ox-LDL at noncytotoxic concentrations (0.2 mg/ml) during the NK/target cell (TC) interaction markedly reduced NK cytotoxic activity against U937 tumor cells. This inhibitory activity was also noticed when NK cells were pretreated with ox-LDL. Scanning electron microscopy examination of NK-target cell conjugates failed to reveal any morphological cell damage. In addition, the number of conjugates and the expression of some adhesion molecules (CD11a, CD11b, CD18, CD2, and CD62L) were not modified by ox-LDL. These observations argued against a possible interference of ox-LDL with the binding process leading to the formation of NK/TC conjugates. By contrast, immunocytochemical analyses of cytoskeleton components of NK cells exposed to ox-LDL showed a partial depolymerization and a derangement of the microtubular apparatus. These alterations were accompanied by an evident decrease in their intracellular reduced glutathione content. Owing to the important role played by the microtubular network during the killing process, it is possible to infer that a cytoskeleton alteration underlies the inhibitory activity of ox-LDL on NK cell function. In addition, exposure of mitogen-stimulated peripheral blood mononuclear cells to ox-LDL markedly reduced specific mRNA transcription and release of cytokines relevant for NK cell activity (such as tumor necrosis factor-alpha, interferon gamma, and interleukin 12). These data suggest that the impairment of NK cell activity by ox-LDL likely reflects the concomitant dysregulation of some essential mechanisms of NK cell function.

Oxidized low density lipoproteins impair peripheral blood mononuclear cell proliferation and cytokine production.

Oxidized low density lipoproteins (ox-LDL) are known to behave as physiological pro-oxidants leading to the formation of intracellular reactive oxygen species. The presence of these altered lipoproteins in the human plasma has been associated with a number of morbid states, including atherosclerosis and immuno-deficiency. Common features of such pathological conditions seem to be represented by several alterations occurring in the immune system. In this work we analyze the in vitro effects of ox-LDL on both proliferative response and cytokine production of normal human peripheral blood mononuclear cells (PBMC). Our results indicate that ox-LDL significantly inhibit proliferative response and modulate cytokine network interfering both at protein secretion and mRNA synthesis level.