The effect of cellular isolation and cryopreservation on the expression of markers identifying subsets of regulatory T cells.

BACKGROUND: The role of CD4(+) regulatory T cells (Tregs) and their subsets during HIV infection is controversial. Cryopreserved peripheral blood mononuclear cells (PBMC) are an important source for assessing number and function of Tregs. However, it is unknown if PBMC isolation and cryopreservation affect the expression of CD120b and CD39, markers that identify specific subsets of Tregs. METHODS: HIV-uninfected (HIV-) and -infected (HIV+) men were randomly selected from the Multicenter AIDS Cohort Study (MACS). Percentages of CD120b(+) and CD39(+) Tregs measured by flow cytometry in whole blood and in corresponding fresh and cryopreserved PBMC were compared. RESULTS: Percentages of CD120b(+) Tregs were significantly lower in a) fresh PBMC relative to whole blood, and b) freshly thawed frozen PBMC relative to fresh PBMC when the recovery of viable cryopreserved cells was low. When present, low expression of CD120b in frozen PBMC was reversible by 4h of in vitro culture. In contrast, expression of CD39 on Tregs was not affected by isolation and/or cryopreservation of PBMC, or by relative recovery of cryopreserved PBMC. These findings were unaffected by the HIV status of the donor. CONCLUSION: The data suggest that percentages of CD120b(+) Tregs and CD39(+) Tregs can be validly measured in either whole blood or PBMC (fresh and frozen) in HIV- and HIV+ men. However, for measurement of CD120b(+) Tregs one type of sample should be used consistently within a given study, and thawed frozen cells may require in vitro culture if recovery of viable cells is low.

Regulatory T Cells, Frailty, and Immune Activation in Men Who Have Sex With Men in the Multicenter AIDS Cohort Study.

BACKGROUND: Both HIV infection and frailty have been associated with chronic immune activation. One possible explanation for this chronic immune activation could be low levels of CD4(+) T regulatory cells (Tregs), which suppress immune responses. METHODS: HIV-uninfected (HIV-) and HIV-infected (HIV+) men in the Multicenter AIDS Cohort Study (MACS) were classified as frail (or nonfrail) if they expressed (or did not express) the Fried frailty phenotype at two consecutive study visits. Percentages and absolute numbers of total Tregs, and percentages of different subsets of Tregs and of activated T cells were measured by flow cytometry. The function of Tregs was measured by suppression of T-cell proliferation. RESULTS: Percentages of Tregs were higher, rather than lower, in frail men than in nonfrail men, and this difference was significant for HIV- men. Percentages of subsets of Tregs did not differ significantly by frailty status. Among HIV+ men, the suppressive function of Tregs was similar between frail and nonfrail men. Percentages of Tregs and activated T cells were negatively correlated in nonfrail men (HIV- and HIV+) and in frail HIV- men, but this correlation was strongly positive in frail HIV+ men. CONCLUSION: These data suggest that: (a) Tregs were not deficient in frail men; and (b) the immunological pathophysiology of frailty may differ by HIV status.

Sleep disordered breathing, fatigue, and sleepiness in HIV-infected and -uninfected men.

STUDY OBJECTIVES: We investigated the association of HIV infection and highly active antiretroviral therapy (HAART) with sleep disordered breathing (SDB), fatigue, and sleepiness. METHODS: HIV-uninfected men (HIV-; n = 60), HIV-infected men using HAART (HIV+/HAART+; n = 58), and HIV-infected men not using HAART (HIV+/HAART-; n = 41) recruited from two sites of the Multicenter AIDS cohort study (MACS) underwent a nocturnal sleep study, anthropometric assessment, and questionnaires for fatigue and the Epworth Sleepiness Scale. The prevalence of SDB in HIV- men was compared to that in men matched from the Sleep Heart Health Study (SHHS). RESULTS: The prevalence of SDB was unexpectedly high in all groups: 86.7% for HIV-, 70.7% for HIV+/HAART+, and 73.2% for HIV+/HAART-, despite lower body-mass indices (BMI) in HIV+ groups. The higher prevalence in the HIV- men was significant in univariate analyses but not after adjustment for BMI and other variables. SDB was significantly more common in HIV- men in this study than those in SHHS, and was common in participants with BMIs <25 kg/m2. HIV+ men reported fatigue more frequently than HIV- men (25.5% vs. 6.7%; p = 0.003), but self-reported sleepiness did not differ among the three groups. Sleepiness, but not fatigue, was significantly associated with SDB. CONCLUSIONS: SDB was highly prevalent in HIV- and HIV+ men, despite a normal or slightly elevated BMI. The high rate of SDB in men who have sex with men deserves further investigation. Sleepiness, but not fatigue, was related to the presence of SDB. Clinicians caring for HIV-infected patients should distinguish between fatigue and sleepiness when considering those at risk for SDB, especially in non-obese men.

Association between systemic inflammation and obstructive sleep apnea in men with or at risk for HIV infection.

BACKGROUND: We sought to determine whether markers of systemic inflammation are associated with the presence of moderate/severe obstructive sleep apnea (OSA) and whether this association differs based on HIV and HIV treatment status. METHODS: HIV-uninfected men (HIV-; n=60), HIV-infected men receiving HAART (HIV+/HAART; n=58) and HIV-infected men not receiving HAART (HIV+/no HAART; n=41) underwent polysomnograpy and measurement of plasma levels of tumour necrosis factor (TNF)-α, soluble TNF-α receptors I and II (sTNFRI and sTNFRII) and interleukin (IL)-6. The relationship between moderate/severe OSA (respiratory disturbance index ≥15 apnea/hypopnea events/h) and inflammatory markers was assessed with multivariable regression models. RESULTS: Compared with the HIV- men, HIV+/HAART men and HIV+/no HAART men had higher levels of TNF-α, sTNFRI and sTNFRII, independent of age, race, smoking status, obstructive lung disease (OLD) and body mass index (BMI). Moderate/severe OSA was present in 48% of the sample (HIV- 57%; HIV+/HAART 41%; HIV+/no HAART 44%). Among the HIV+/no HAART men, but not in the other groups, TNF-α, sTNFRII and IL-6 levels were higher in those with moderate/severe OSA compared to men with no/mild OSA after adjustment for age, race, smoking status, OLD and BMI. Within this group, the association of high TNF-α concentrations with moderate/severe OSA was also independent of CD4(+) T-cell count and plasma HIV RNA concentration. CONCLUSIONS: Compared with HIV+/HAART men and HIV- men, markers of systemic inflammation were higher in HIV+/no HAART men. In these men, TNF-α was significantly related to OSA, independent of HIV-related covariates.

Anthropometry in the prediction of sleep disordered breathing in HIV-positive and HIV-negative men.

BACKGROUND: Body mass index (BMI), waist circumference (WC) and neck circumference (NC) are important screening tools for sleep disordered breathing (SDB); however, the utility of anthropometry for this purpose has not been evaluated among HIV-positive patients. METHODS: HIV-negative men (n=60), HIV-positive men receiving highly active antiretroviral therapy (HIV-positive/HAART; n=58) and HIV-positive men not receiving HAART (HIV-positive/no HAART; n=41) from the Multicenter AIDS Cohort Study underwent a nocturnal sleep study and anthropomorphic assessment. Moderate-severe SDB was defined as an apnea/hypopnea event rate > or =15 episodes/h. Receiver operating characteristic (ROC) curves were used to compare the ability of different anthropometric measurements to predict SDB within each group. RESULTS: Moderate-severe SDB was found in 48% of men (HIV-negative [57%], HIV-positive/HAART [41%] and HIV-positive/no HAART [44%]). The performance of BMI, WC and NC to predict SDB was excellent among the HIV-negative men (ROC areas under the curve [AUCs] 0.83, 0.88 and 0.88, respectively) and fair among the HIV-positive/HAART group (AUC 0.71, 0.77 and 0.77, respectively). By contrast, these measurements had no predictive value in the HIV-positive/no HAART group (AUC 0.43, 0.41 and 0.45, respectively). Moreover, in the HIV-positive/no HAART group, moderate-severe SDB was independently associated with serum C-reactive protein > or =3.0 mg/l (odds ratio 6.9; P=0.04) and HIV RNA>10,000 copies/ml (odds ratio 7.1; P=0.05). CONCLUSIONS: BMI, WC and NC had a better predictive value for moderate-severe SDB in HIV-positive men compared with HIV-positive [corrected] men, and had no value among HIV-positive/no HAART men. Among this latter group, systemic inflammation might contribute to the pathogenesis of SDB.