Mitochondrial genetics cooperate with nuclear genetics to selectively alter immune cell development/trafficking.

The nuclear genome drives differences in immune cell populations and differentiation potentials, in part regulated by changes in metabolism. Despite this connection, the role of mitochondrial DNA (mtDNA) polymorphisms (SNP) in this process has not been examined. Using mitochondrial nuclear exchange (MNX) mice, we and others have shown that mtDNA strongly influences varying aspects of cell biology and disease. Based upon an established connection between mitochondria and immune cell polarization, we hypothesized that mtDNA SNP alter immune cell development, trafficking, and/or differentiation. Innate and adaptive immune cell populations were isolated and characterizated from the peritoneum and spleen. While most differences between mouse strains are regulated by nuclear DNA (nDNA), there are selective changes that are mediated by mtDNA differences (e.g., macrophage (CD11c) differentiation), These findings highlight how nuclear-mitochondrial crosstalk may alter pathology and physiology via regulation of specific components of the immune system.

Immunosuppressive activity of 15-deoxyspergualin on normal and autoimmune peripheral blood mononuclear cells.

Several experimental conditions were used in this study to evaluate the in vitro effects of 15-deoxyspergualin on the function of T lymphocytes, B lymphocytes and monocytes from healthy subjects and patients suffering from systemic lupus erythematosus. Whilst the secretion of polyclonal immunoglobulin (Ig) M and IgG from the B lymphocytes of the healthy subjects was diminished by 15-deoxyspergualin, neither the proliferative response of normal T and B cells to mitogenic stimulation nor the cytokine secretory capacity of these cells (e.g. interleukin-2, -4, -6 and gamma-interferon) and monocytes (e.g. interleukin-1 beta and -6) were affected by the drug. In contrast, on the mononuclear cells obtained from the lupus patients not only did 15-deoxyspergualin inhibit the spontaneous production of polyclonal and anti-DNA IgG antibodies but also suppressed interleukin-1 beta secretion from the monocytes. Other functional responses of T and B cells and monocytes from lupus patients, including mitogenic activation and cytokine secretion, were not altered by the drug. These data suggest that 15-deoxyspergualin possesses a novel mechanism of pharmacological immunosuppression apparently different from that of other immunosuppressants, such as cyclosporin A, FK506 and corticosteroids, that seems to be primarily displayed at the level of autoreactive B cells and monocytes.

Interleukin-10-induced HIV-1 expression is mediated by induction of both membrane-bound tumour necrosis factor (TNF)-alpha and TNF receptor type 1 in a promonocytic cell line.

OBJECTIVE: To investigate whether the upregulatory effect of interleukin (IL)-10 on HIV expression in a model of latent HIV infection is mediated by induction of endogenous tumour necrosis factor (TNF)-alpha and TNF receptors (TNFR). DESIGN: The latently HIV-infected promonocytic cell line U1 was examined, because in this in vitro model IL-10 has been shown to synergize with multiple cytokines, including TNF-alpha, in enhancing HIV production. METHODS: Membrane-bound TNF-alpha, TNFR-1 and TNFR-2 surface expression were determined by flow cytometry. TNF-alpha mRNA was estimated by competitive polymerase chain reaction (PCR), and TNF-alpha, soluble TNFR-1 and soluble TNFR-2 supernatant content by enzyme-linked immunosorbent assay. HIV-1 expression was quantitated by reverse transcriptase assay and p24 antigen release. RESULTS: We demonstrated that IL-10 induces a time and cell-concentration dependent upregulation of HIV expression in U1 cells. This effect is mediated through the endogenous production of TNF-alpha as demonstrated by blocking experiments with anti-TNF-alpha antibodies and by detection of IL-10-induced increase of TNF-alpha mRNA by competitive PCR. More importantly, IL-10 is able to upregulate membrane-bound TNF-alpha and TNFR-1, along with a consistent increase in the shedding of soluble TNFR-1 without inducing detectable TNF-alpha secretion. CONCLUSIONS: IL-10 activates HIV expression through the membrane-bound TNF-alpha/TNFR-1 pathway, suggesting an amplification mechanism of HIV expression that might occur during cell-to-cell interaction. This positive regulatory effect of IL-10 in an in vitro model of chronic HIV infection is consistent with the inexorable progression of disease seen in advanced patients when both IL-10 and TNF-alpha are elevated.

In vitro type-1 and type-2 cytokine production in systemic lupus erythematosus: lack of relationship with clinical disease activity.

OBJECTIVE: To investigate the relationship between disease activity and in vitro cytokine, soluble(s)CD23 and polyclonal and anti-DNA antibody production by PBMC from patients with active systemic lupus erythematosus (SLE). METHODS: Cytokines, sCD23 and immunoglobulins were estimated by ELISA in unstimulated and polyclonal mitogen-stimulated culture supernatants. RESULTS: PHA-induced IL-2 and IFN-gamma production were decreased, whereas spontaneous and PHA-induced IL-6 and IL-10 production were increased in cultures of SLE lymphocytes. Conversely, spontaneous and PHA-stimulated IL-4 and sCD23 production was comparable between patients and controls. Finally, we found an increase in in vitro spontaneous polyclonal and anti-DNA IgG secretion. CONCLUSIONS: We demonstrated an expanded type-2 cytokine profile with no correlation with parameters of disease activity.

In vitro production of type 1 and type 2 cytokines by peripheral blood mononuclear cells from high-risk HIV-negative intravenous drug users.

OBJECTIVE: To study type 1 and type 2 cytokine patterns in HIV-negative high-risk intravenous drug users (IVDU). DESIGN: We investigated interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-6 and IL-10 production by phytohaemagglutinin (PHA)-stimulated and unstimulated peripheral blood mononuclear cell (PBMC) cultures from HIV-negative high-risk IVDU, HIV-negative controls and HIV-positive subjects. METHODS: Cytokine production was measured in supernatants using enzyme-linked immunosorbent assay (ELISA) in 10 HIV-negative high-risk IVDU, 25 HIV-negative controls, and 12 HIV-positive IVDU. We also determined spontaneous in vitro immunoglobulin (Ig) G and IgM production. RESULTS: HIV-negative high-risk IVDU showed increased IFN-gamma and decreased IL-4, IL-10 and IL-2, although the latter was not significant compared with HIV-negative controls. Further, HIV-negative high-risk IVDU had reduced IgG production and impaired IgM-IgG switch. CONCLUSIONS: The reduced IL-2 and IL-4 production suggest an impaired CD4+ T-cell function in HIV-negative high-risk IVDU. The increased IFN-gamma production along with the decreased type 2 cytokine profile is consistent with the hypothesis that protective immunity against HIV may reside in type 1 responses and cell-mediated immunity.

TH1 and TH2 cytokine production by peripheral blood mononuclear cells from HIV-infected patients.

OBJECTIVE: To study the TH1-->TH2 cytokine switch, thought to occur during the progression of HIV infection. DESIGN: We investigated interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-6 and IL-10 production by phytohaemagglutinin (PHA)-stimulated and unstimulated peripheral blood mononuclear cell (PBMC) cultures from HIV-negative controls and HIV-positive subjects, stratified according to the Centers for Disease Control and Prevention (CDC) criteria. We correlated the above parameters with markers of disease progression. METHODS: Cytokine production was measured in supernatants using enzyme-linked immunosorbent assay (ELISA) in 40 patients and 17 controls. To evaluate disease progression, we also determined CD4+ cell counts, PHA-induced proliferative response, p24 release and spontaneous immunoglobulin (Ig) G and IgM production. RESULTS: In agreement with the TH1-->TH2 switch hypothesis, we found that in the course of HIV disease mitogen-stimulated IL-2 production decreased, spontaneous and stimulated IL-6 production and spontaneous IL-10 secretion increased; IL-4 showed an increasing trend, although it was reduced in HIV-positive subjects. Finally, immunoglobulin production increased over time. In contrast, mitogen-stimulated IFN-gamma and IL-10 production did not change among the CDC categories, although the former decreased and the latter increased in comparison with HIV-negative controls. CONCLUSIONS: Our data partially agree with the TH1-->TH2 switch hypothesis. Since IL-6 and IL-10 are produced by different cell types, whose proportions and functional features vary in the course of the disease, further experiments with purified lymphocyte subsets and monocytes are required. Nevertheless, as already suggested, we believe that a switch from a type 1 to a type 2 response occurs in HIV infection.

Beta-endorphin content in HIV-infected HuT78 cell line and in peripheral lymphocytes from HIV-positive subjects.

We investigated beta-endorphin (BE) content in an HIV-infected cell line and in peripheral blood mononuclear cells (PBM) from HIV-positive subjects. HIV infection increased BE content in HuT78 cell line compared to uninfected cells. Accordingly, BE content was greater in HIV-positive subjects than in healthy controls, both in fresh PBM and in mitogen-stimulated or unstimulated cultured cells. Further, in PHA-stimulated cultures, BE increase was correlated with disease progression. Opioids are known to decrease immune responsiveness in vivo, and it may be that the increased BE concentrations contribute to HIV-associated immune deficiency. In HIV-positive subjects, but not in healthy controls, intracellular BE concentration was positively correlated with PHA-induced PBM proliferation. The latter data suggest an alternative explanation: that the increased BE content represents a paradoxical response of the host in an attempt to balance virus-induced immunodepression. Thus, BE may be important in fine-tuning of the immune response with its up- and downregulation dependent upon differences in immune status.

Autoantibodies against beta 2-microglobulin-free HLA antigens in AIDS patients.

Serum samples from 88 human immunodeficiency virus (HIV)-positive drug addicts have been investigated for the presence of antibodies to both beta 2-microglobulin (beta 2m)-free and beta 2m-associated HLA class I molecules. Using HIV-negative drug addicts as background control, we found that none of the Centers for Disease Control (CDC) stage II, 9.1% of CDC III, 36.4% of CDC IV A, and 45.5% of CDC IV C1 patients had significant levels of autoantibodies competing with the binding of the monoclonal antibody specific for beta 2m-free HLA I (L31 mAb). Using the mAb 01.65, recognizing the beta 2m-associated form of HLA class I molecules, a similar percentage of positive samples was found in the CDC II, CDC III, and CDC IV A patient groups; conversely, the percentage of positive serum samples was lower in the CDC IV C1 group. A lower number of systemic lupus erythematosus serum samples and none of the specimens from healthy adult subjects or patients suffering from recurrent Epstein-Barr virus infections were positive in both assays. Our data demonstrate the existence of an ongoing HLA class I-specific autoimmune response during AIDS disease development, which probably reflects a molecular mimicry between autologous histocompatibility antigens and HIV components. The relationship between the prevalence of autoantibodies against beta 2m-free HLA class I and disease progression suggests a possible pathogenetic role of these antibodies in the induction of the HIV-associated immune deficiency.

Prevention of cyclophosphamide-induced diabetes in the NOD/WEHI mouse with deoxyspergualin.

Ten out of 20 (50%) 17-week-old female NOD/WEHI mice developed an acute form of autoimmune diabetes when injected with two large doses of cyclophosphamide (CY), given at 14-day intervals. If these mice were treated under a prophylactic regimen with 2.5 mg/kg body weight per day of the novel immunosuppressant deoxyspergualin (DSP) the onset of diabetes was completely prevented. Moreover, DSP-treated animals showed reduced signs of pancreatic insulitis, had lower percentages of splenic lymphoid cells (SLC) expressing IL-2 receptors and Ly-6C antigens on their surfaces, and these cells released lower amounts of interferon-gamma (IFN) when stimulated in vitro. These data, providing evidence for the capacity of DSP to protect NOD/WEHI mice from experimental autoimmune diabetes and to modulate histo-immunological pathogenic pathways, indicate DSP as a drug of potential interest in the treatment of human insulin-dependent diabetes mellitus.

In vitro and ex vivo effect of tiaprofenic acid on human peripheral blood mononuclear cells.

The effect of the non-steroidal anti-inflammatory drug (NSAID) tiaprofenic acid on different human immune parameters was investigated in vitro or following in vivo administration in healthy adult volunteers. Results from the in vitro study demonstrated an increased mitogen-induced blastogenesis and interleukin 2 (IL-2) production together with a reduced polyclonal immunoglobulin (Ig) secretion in the presence of the drug. Results from the ex vivo study showed that treatment with tiaprofenic acid had no significant effects on the immune parameters investigated, i.e. unstimulated and mitogen-induced proliferation and IL-2 production, spontaneous and stimulated Ig synthesis, lymphocyte subpopulations, serum Ig and complement levels.

Enrichment of IgG anti-DNA-producing lymphoblastoid cell lines by antigen-coated immunomagnetic beads.

In this paper we describe the establishment of four anti-DNA-producing lymphoblastoid cell lines (LCL) by Epstein-Barr virus (EBV) infection of peripheral blood B-cells from a patient with systemic lupus erythematosus. The LCL showed a heterogeneous cell composition: the frequencies of cells in active proliferation, cells secreting IgG or IgM, and cells effectively producing IgG or IgM anti-DNA were estimated by limiting dilution analysis. The cells producing anti-DNA antibodies were a small fraction of the whole cell population constituting the LCL. In order to enrich the fraction of cells producing the desired antibody we performed a positive selection by DNA-coated immunomagnetic beads. Results show that in two out of three IgG anti-DNA-secreting LCL the frequency of DNA-producing cells increased after incubation with DNA-coated beads. At variance, IgM anti-DNA-secreting cells were completely lost after immunomagnetic separation. This approach could represent a further tool to obtain better fusion partners to construct stable hybrids secreting human monoclonal antibodies. The advantages of the presented technique would be: (a) removing of the fraction of low-affinity IgM-producing cells, which is often prevalent in EBV-induced LCL; and (b) the enrichment of IgG anti-DNA producing cells, which better represent the antibodies involved in the pathogenesis of the disease.

Antiphospholipid antibodies cross-reacting with erythrocyte membranes. A case report.

We describe a patient with primary antiphospholipid syndrome (PAPS) and haemolytic anemia with cryoagglutinins, in whom antibodies eluted from red blood cells (RBC) displayed anti-cardiolipin (CL) binding activity. This activity was confined to the IgG isotype and was directed to the negatively-charged phospholipids only. In addition, the patient's IgG fraction displayed a higher binding to RBC in comparison to normal control IgG and this reactivity was inhibited after absorption of the anti-CL activity with CL-micelles. These findings further sustain the association between antiphospholipid antibodies (APA) and Coombs' positivity with or without haemolytic anemia and suggest that APA could be at least in part responsible for anti-RBC activity.

Immunopharmacological activity of cefodizime in young and elderly subjects: in vitro and ex vivo studies.

The biological response modifying activities of cefodizime (CDZ), a new third-generation cephalosporin, were investigated in vitro and ex vivo. In vitro investigations using cells isolated from the blood of young healthy donors showed no stimulating activity of CDZ on peripheral blood lymphocytes, natural killer cell activity, IL-1 production by adherent mononuclear cells, PMN chemiluminescence or PMN chemotaxis. A slight but statistically insignificant increase in PMN phagocytosis and phagocytic index was observed in the same population. IL-1 production was increased in three subjects with low resting state values. In a controlled ex vivo study, 20 healthy elderly subjects selected on the basis of depressed phagocytic function were treated with CDZ 1 g i.m. b.i.d. or placebo for eight days. PMN function was determined at baseline and on the day after the last dose. In the CDZ group a significant increase in both phagocytosis and phagocytic index was found, while there were no changes in the placebo group. In conclusion, CDZ restored depressed PMN phagocytic function in a population of elderly subjects. Patients with impaired PMN function who require antibiotic treatment may benefit from this activity of CDZ.

Combination of mutant EL-4 thymoma cells and EBV-infection in B lymphocyte activation a) EBV-infection of EL-4-activated lymphocytes. b) Use of EL-4 cells as feeders for lymphoblastoid cell lines.

The combination of mutant EL-4 thymoma cells and Epstein-Barr virus (EBV)-infection in the activation of human B lymphocytes was studied with two approaches: a) limiting numbers of B cells were first activated by EL-4 contact in order to expand the B cell population and then infected with EBV. Results show that EBV could induce further Ig synthesis, although was unable to determine proliferation or to generate immortalized lines from EL-4 activated cells. b) EL-4 cells were compared to conventional PBM as feeders for cultures of established lymphoblastoid cell lines (LCL) at low cell densities. EL-4 feeder activity was strictly dependent on the presence of phorbol-myristate-acetate (PMA) and supernatant from stimulated T-lymphocytes (T-SN). EL-4 feeders induced earlier proliferation peak and greater Ig synthesis by LCL. The latter effect could be also mediated by the addition of PMA and T-SN, even if a cooperating cell-to-cell signal by PMA and T-SN-sensitized-EL-4 cells could not be excluded. Altogether results indicate that EL-4 cells do not represent a clear advantage over classic PBM as feeders for cultures of established LCL at low cell densities.