PDF《LPS and HIV gp120 modulate》

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1938 LPS and HIV gp120 modulate monocyte/macrophage CYP27B1 and CYP24A1

1939 The following parameters were extracted from medical records: patient demographics, HCV and/or HBV co-infection, body mass index (BMI) and HIV-related parameters, including time since HIV diagnosis and initiation of HAART, route of infection, CDC stage, HAART regimen. Information on smoking habit was ob- tained by subject interview. The following bio- chemical and viro-immunological parameters were measured: serum glucose, total cholesterol, high-density lipoprotein (HDL) cholesterol, in- sulin, creatinine, D-dimer, hs-CRP, parathyroid hormone (PTH), CD4+ and CD8+ T-cell count, HIV RNA (Cobas Amplicor HIV-1 monitor test, minimal detection threshold of

20 copies/ml). 25OHD levels were measured using the DiaSorin radioimmunoassay (Stillwater, MN, USA)46 . The level of detection for 25OHD was <

4 ng/ml. The reference range is 32-100 ng/ml. According to EuroSida indications, 25OHD concentration was defined normal when >

30 ng/ml, insufficient when ranging between

10 and

30 ng/ml, deficient when <

10 ng/ml47 . Cell Cultures Human monocytes were isolated from fresh buffy coats of healthy volunteers as described in details elsewhere32 . Peripheral blood mononu- clear cells (PBMCs) were diluted with phos- phate-buffered saline (PBS) supplemented with 2.5 mM EDTA and plated at a density of

1 *

106 cells/well in 6-well plates (Becton Dickinson, Franklin Lakes, NJ, USA). Monocytes were al- lowed to attach to the wells for

2 hours (h) at 37°C in 5% CO2, after which non-adherent cells were removed by washing three times with PBS. Monocytes were cultured in Iscove'

s Modified Dulbecco'

s Medium supplemented with rHuman Macrophage-Colony Stimulating Factor (M- CSF)

5 ng/ml (PeproTech, BDA, Segrate, Milan, Italy), 10% fetal bovine serum (FBS),

2 mM glu- tamine and 1% of penicillin/streptomycin (Invit- rogen, Milan, Italy). Mature macrophages were obtained from monocytes after

7 days of culture without changing the medium. To determine the effects of glycoprotein

120 (gp120) on Vitamin D (1,25-dihydroxyvitamin D3) receptor (VDR), CYP24A1, CYP27B1 and IL-6 expression on freshly isolated monocytes, cells were treated immediately after adhesion with gp120 (1 ?g/ml, ImmunoDiagnostics, Boston, MA, USA) or left untreated, samples were evaluated at 3h and 24h. Analogously, to evaluate the effects of LPS on VDR, CYP24A1, creased prevalence of metabolic and cardiovas- cular disease, malignancies, bone and renal dis- ease observed in HIV-infected subjects13-28 . In fact, direct or indirect markers of inflammation, such as highly sensitive (hs)-C reactive protein (CRP) and Interleukin (IL)-6, and coagulation, such as D-dimer, and circulating levels of bacter- ial lipopolysaccharide (LPS), have been associat- ed with increased morbidity and mortality in the setting of HIV29,30 . Vitamin D is involved not only in bone home- ostasis, but it also has non-skeletal functions, in- cluding immune regulation31,32 . Vitamin D defi- ciency has been associated with increased levels of pro-inflammatory cytokines and increased systemic inflammation33-35 . In recent years a growing number of studies have reported high prevalence rates of vitamin D deficiency among HIV-infected patients36-38 . Hy- povitaminosis D has a multifactorial origin, in- cluding non-HIV-related risk factors, such as fe- male sex, low dietary intake and low sun expo- sure, and HIV-related factors, i.e. immune acti- vation and antiretroviral adverse effects39,40 . Sev- eral reports have identified vitamin D deficiency as an independent risk factor not only for osteo- porosis and fragility fractures41,42 , but also for cardiovascular and metabolic disorders among HIV-positive subjects43-45 . In the present study, we cross-sectionally eval- uated the prevalence of vitamin D deficiency in a cohort of HIV-infected subjects and the risk fac- tors associated with hypovitaminosis D, includ- ing inflammation and coagulopathic markers. Then, we studied in vitro the expression of CYP27B1 and CYP24A1, the enzymes involved in vitamin D metabolism and catabolism, respec- tively, and vitamin D receptor (VDR), upon monocyte and macrophage stimulation with LPS or the HIV envelope protein gp120. We also measured vitamin D levels in the supernatant of monocytes stimulated with LPS, LPS plus 25-hy- droxyvitamin D (25OHD) or 25OHD alone. Patients and Methods Study Population From March to May 2011,