Cardiometabolic and immune factors associated with increased common carotid artery intima-media thickness and cardiovascular disease in patients with systemic lupus erythematosus.

BACKGROUND AND AIM: Patients with systemic lupus erythematosus (SLE) have a higher prevalence of subclinical atherosclerosis and higher risk of cardiovascular (CV) events compared to the general population. The relative contribution of CV-, immune- and disease-related risk factors to accelerated atherogenesis in SLE is unclear. METHODS AND RESULTS: Fifty SLE patients with long-lasting disease (mean age 44 ± 10 years, 86% female) and 50 sex- and age-matched control subjects were studied. Common carotid artery intima-media thickness (CCA-IMT) was used as a surrogate marker of atherosclerosis. We evaluated traditional and immune- and disease-related factors, assessed multiple T-cell subsets by 10-parameter-eight-colour polychromatic flow cytometry and addressed the effect of pharmacological therapies on CCA-IMT. In SLE patients, among several cardiometabolic risk factors, only high-density lipoprotein levels (HDL) and their adenosine triphosphate-binding cassette transporter 1 (ABCA-1)-dependent cholesterol efflux capacity were markedly reduced (p < 0.01), whereas the CCA-IMT was significantly increased (p = 0.03) compared to controls. CCA-IMT correlated with systolic blood pressure, low-density lipoprotein (LDL) cholesterol and body mass index (BMI), but not with disease activity and duration. The activated CD4(+)HLA-DR(+) and CCR5(+) T-cell subsets were expanded in SLE patients. Patients under hydroxychloroquine (HCQ) therapy showed lower CCA-IMT (0.62 ± 0.08 vs. 0.68 ± 0.10 mm; p = 0.03) and better risk-factor profile and presented reduced circulating pro-atherogenic effector memory T-cell subsets and a parallel increased percentage of naïve T-cell subsets. CONCLUSION: HDL represents the main metabolic parameter altered in SLE patients. The increased CCA-IMT in SLE patients may represent the net result of a process in which 'classic' CV risk factors give a continuous contribution, together with immunological factors (CD4(+)HLA-DR(+) T cells) which, on the contrary, could contribute through flares of activity of various degrees over time. Patients under HCQ therapy present a modified metabolic profile, a reduced T-cell activation associated with decreased subclinical atherosclerosis.

Fluorescence microscopic and flow cytometric analysis of bone marrow-derived cells in human epidermis: a search for the human analogue of the murine dendritic Thy-1+ epidermal cell.

Lymphoid cells with an affinity for the epidermis (epidermotropic lymphocytes) have been proposed to play a role in the immune functions of the epidermis. However, antigen-presenting Langerhans cells (LC) and indeterminate cells are presently the only cells in the human epidermis which have been demonstrated to originate in the bone marrow. Recent studies of murine epidermis have identified a population of bone marrow-derived cells which express Thy-1 antigen and which are present in a similar density to, but distinct from, LC. We therefore sought to identify the potential human analogue of the murine Thy-1+ epidermal cell utilizing a battery of antileukocyte reagents in immunohistochemical, flow cytometric, and cell sorting studies. A panel of antibodies failed to detect significant numbers of human Thy-1 antigen-bearing cells, T cells, B cells, monocytes/macrophages (other than LC), and natural killer cells in tissue sections, epidermal sheets, and epidermal cell (EC) suspensions. This was the case using EC suspensions either unfractionated or fractionated on Ficoll-Hypaque to enrich for leukocyte subpopulations. Since the nature of the murine Thy-1+ EC is uncertain, it is possible that antibodies directed against well-defined leukocyte subpopulations may not be of value in the detection of a potential human analogue. We therefore utilized double fluorescence staining with anti-HLe-1, an antibody which identifies all human leukocytes, and anti-HLA-Dr (Dr), which identifies epidermal LC, in order to demonstrate a potential population of HLe-1+ Dr- non-LC, bone marrow-derived cells. The vast majority of HLe-1+ cells were HLA-Dr+ LC; these were present at a density of 608 cells/mm2 in epidermal sheets. A minor population of HLe-1+ cells which did not express HLA-Dr (HLe-1+ Dr-) was observed in tissue sections, epidermal sheets, and EC suspensions. The nondendritic morphology and low density of these HLe-1+ Dr- EC in epidermal sheets (mean density of 4.2 +/- 1.6 cells/mm2) precluded their representing a strict human analogue of the murine Thy-1+ EC, since murine Thy-1+ EC are dendritic and are present in a density similar to that of LC. Purified preparations of the minor HLe-1+ Dr- EC population obtained by electronic cell sorting or panning and examined ultrastructurally were not enriched for any bone marrow-derived cell population. Thus, using currently available markers and sorting technology, we have been unable to identify a human analogue of the murine dendritic Thy-1+ epidermal cell.

Inter-laboratory relative fluorescence intensity measurements using FlowCal 575 calibration beads: a baseline study.

Twenty-one laboratories participated in a baseline study of their ability to agree on the measurement of fluorescence intensities of a stable multi-peak reference material (FlowCal 575) and of stained and fixed CD4 lymphocytes. Relative fluorescence intensities were calculated as ratios to the most fluorescent bead. The good correlation between laboratories suggests that this simple approach may be useful in multi-laboratory studies. The data also provide a baseline for the evaluation of any improvement of inter-laboratory agreement gained by more rigorous and demanding approaches.

Flow cytometric analysis of whole blood lysis, three anticoagulants, and five cell preparations.

We studied the effects of anticoagulants and cell preparation methods on lymphocyte forward-angle scatter (FSC), autofluorescence, and immunofluorescent staining for CD45, CD14, and CD13. Blood samples collected in ethylenediaminetetracetic acid (EDTA), heparin, and acid citrate dextrose (ACD) were processed by using conventional Hypaque-Ficoll (HF) separation and four whole blood (WB) lysis techniques: Immuno-lyse, Q-Prep, FACS Lyse, and Gen Trak Lysis. Lymphocytes prepared by using three of the four whole blood methods gave FCS values comparable to those isolated by HF, while one method (FACS Lyse) gave consistently lower values. Autofluorescence values were comparable by all methods except Immuno-lyse, which showed consistently higher values in blood stored for 24 h with any anticoagulant. Immunofluorescent values for CD45-stained cells were quite consistent across all methods, and among the whole blood methods, FACS Lyse and Q-Prep uniformly gave the highest purity of CD45-positive cells in the lymphocyte light scatter gates. Additionally, propidium iodide (PI) analyses of CD45-stained whole blood, and analyzed without lysis, confirmed that ACD and heparin were superior to EDTA for maintaining viable leucocytes overnight. Future studies should focus on other commonly used reagents, a wide variety of abnormal samples, and cell viability.

9-Nitrocamptothecin inhibits HIV-1 replication in human peripheral blood lymphocytes: a potential alternative for HIV-infection/AIDS therapy.

The ability of the anti-cancer drug, 9-Nitrocamptothecin (9NC), to inhibit replication of HIV-1 in clinically relevant primary lymphocytic cells was studied. Primary peripheral blood lymphocytes (PBLs) from a non-infected donor were freshly infected with HIV-1 and treated with 9NC by using three different treatment schedules. Cells were monitored for cytotoxicity by the XTT metabolic cell proliferation assay and a sensitive flow cytometric assay that was capable of measuring cell cycle changes and apoptosis. 9NC inhibited replication of HIV-1 in PBLs by greater than 95% in a dose-dependent manner as measured by the level of extracellular HIV-1 p24 release. Similar results were observed, whether 9NC was applied in a single, double, or triple dose regimen. Minimal cytotoxicity was observed for both non-infected and infected PBLs, as determined by the XTT assay. Moreover, 9NC induced apoptosis within 24 hours of drug treatment in freshly infected, but not non-infected, PBLs. The data showed that 9NC reduced replication of HIV-1 in primary human lymphocytes; thus, it indicates the potential clinical utility of this drug as an alternative or adjunct therapy for HIV-infection/AIDS.