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HIV-associated dementia tied to proviral DNA levels
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Note from Jules Levin: As I've reported previously, as a large population with HIV age several issues will be key to follow: heart disease, lipids, diabetes, cancers, and mental health.
"......Neither being treated with HAART, nor achieving undetectable VL, excludes the diagnosis of HAD, as shown by us and others....
.......In the current study, data are presented that suggest circulating HIV DNA may be an important link in the pathogenesis of HAD. Furthermore, an exploratory evaluation suggests that the greatest burden may be among the CD14/CD16 subsets. It is particularly interesting that the correlation of HAD with circulating HIV DNA remained significant among individuals with undetectable VL, further underscoring that circulating HIV DNA may be involved in pathways leading to HAD.....
........In summary, these data indicate that circulating proviral DNA may be significantly elevated in HAD when compared to HIV-1-seropositive patients with normal cognition, even after controlling for plasma HIV-1 RNA loads. Our future work will determine if proviral DNA represents a sensitive marker to identifying HIV-1-infected individuals at risk for developing HAD or HAD progression; these findings could have therapeutic or prognostic implications......"
Reuters report: By David Douglas
NEW YORK (Reuters Health) - Levels of circulating proviral HIV DNA appear to be elevated in patients with HIV-associated dementia (HAD), researchers report in the January 3rd issue of AIDS.
Lead investigator Dr. Bruce Shiramizu told Reuters Health that "HAD and milder forms of neurocognitive disorders continue to be seen despite effective treatment which lowers HIV-1 RNA levels. Because monocytes/macrophages are cells that continue to harbor the virus, in spite of effective therapy, our group was interested to see if proviral DNA in these cells is associated with HAD."
Dr. Shiramizu of the University of Hawaii, Honolulu, and colleagues compared HIV DNA levels within circulating leukocytes in 27 HIV patients with HAD and 22 HIV patients with normal cognition.
Those with HAD had considerably higher levels of circulating proviral DNA. Analysis of a subset of 11 HAD patients and 13 with normal cognition, all of whom had undetectable viral load, showed that the odds ratio for HAD attributable to HIV DNA copy number was 2.76.
Findings in a subset of 5 patients indicated that activated macrophages/monocytes were the primary source of HIV DNA.
"Further studies," Dr. Shiramizu continued, "are being conducted to confirm that more virus is found in macrophages/monocytes. If confirmed, then this may have implications in developing strategies to target macrophages/monocytes with HIV DNA to prevent HAD."
AIDS 2005;19:45-52.
Circulating proviral HIV DNA and HIV-associated dementia
AIDS: Volume 19(1) 3 January 2005 p 45-52
Shiramizu, Brucea; Gartner, Suzanneb; Williams, Andrewa; Shikuma, Ceciliaa; Ratto-Kim, Silviaa; Watters, Michaela; Aguon, Joleena; Valcour, Victora
aFrom the Hawaii AIDS Clinical Research Program, University of Hawaii, Honolulu, Hawaii
bDivision of Neurology, Johns Hopkins University, Baltimore, Maryland, USA
Abstract
Objective: Individuals continue to develop HIV-1-associated dementia (HAD) despite treatment with highly active antiretroviral therapy (HAART). Monocytes/macrophages (M/M) can harbor proviral DNA that is not eradicated by HAART. To determine if HAD is associated with the level of HIV-1 infection within circulating leukocytes, we quantified HIV-1 DNA copy number in peripheral blood mononuclear cells (PBMC), and in PBMC subsets.
Design: Cross-sectional analysis within the Hawaii Aging with HIV Cohort comparing participants with HAD to those with normal cognition (NC).
Methods: Real-time PCR assays assessing HIV DNA copy number/1 Å~ 106 cells were performed on PBMC and subsets.
Results. Individuals with HAD (n = 27) had a median (interquartile range) of 9.11 (37.20) HIV DNA per 1 x 106 PBMC compared to 0.49 (0.89) HIV DNA per 1 Å~ 106 PBMC in individuals with NC (n = 22). Using a univariate analysis in the subset of individuals with undetectable viral load (HAD, n = 11; NC, n = 13), the odds of HAD attributable to HIV DNA copy number was 2.76 (1.28-5.94), P < 0.01. Preliminary analysis of a small subset of patients (n = 5) suggested that the primary source of HIV DNA may be the activated M/MÉ>= (CD14/CD16) subset.
Conclusions. These findings suggest a potentially important association between circulating provirus and HAD.
Author Discussion
The value of proviral DNA levels to assess HIV-1 treatment status has been previously reported, but its utility as a prognostic marker for HAD has not been studied [35,39-42]. While HAART does decrease HIV DNA, the rate of decline is generally lower than its effect on plasma VL [43,44]. HIV DNA plays a role in M/M viral persistence and the levels observed in M/M are generally higher than those found in CD4 lymphocytes [40,45]. In the current study, data are presented that suggest circulating HIV DNA may be an important link in the pathogenesis of HAD. Furthermore, an exploratory evaluation suggests that the greatest burden may be among the CD14/CD16 subsets. It is particularly interesting that the correlation of HAD with circulating HIV DNA remained significant among individuals with undetectable VL, further underscoring that circulating HIV DNA may be involved in pathways leading to HAD.
Neither being treated with HAART, nor achieving undetectable VL, excludes the diagnosis of HAD, as shown by us and others [5,12,24]. While previous studies have shown a variable relationship between HIV DNA level and progression of HIV disease status, we believe the current study is the first to report a link between circulating proviral DNA and HAD [41,46-49]. Our findings are consistent with a model in which M/M harboring HIV DNA are involved in the pathogenesis of HAD. Several mechanisms have been proposed by which M/M might synergistically impact HAD including monocyte trafficking, involvement of perivascular macrophages, and secretion of neurotoxic substances by M/M [2,5,50-56]. This may be partially explained by presence of HIV DNA in M/MÉ>=. We acknowledge several limitations to the current study, including the inability to confirm that these results are specific to M/MÉ>= in all individuals with HAD. While an intriguing association was observed suggesting an association between HIV DNA and activated M/M, we were not able to definitively confirm this relationship due to limited specimens. Additionally, the data were obtained by extrapolating and normalizing HIV DNA copy number per 1 Å~ 106 million cells. Therefore, future analyses could benefit by using a more sensitive assay. In the limited subset of patients analyzed, the highest HIV DNA copy number was found within the CD14/CD16 cells and not within the T-cell compartment.
We recognize that while these patients have higher HIV DNA copy numbers in M/M compared to CD4 T cells, the total CD4 counts are also high, therefore the total HIV DNA burden may be significant in these cells. While we have not conclusively shown that the high HIV DNA copy numbers are primarily due to CD14/CD16 monocytes, we feel that our results are highly suggestive and deserve further evaluation. In-depth quantitative studies on suspected target cells would provide insight on the role that proviral DNA plays in the pathogenesis of HAD.
In summary, these data indicate that circulating proviral DNA may be significantly elevated in HAD when compared to HIV-1-seropositive patients with normal cognition, even after controlling for plasma HIV-1 RNA loads. Our future work will determine if proviral DNA represents a sensitive marker to identifying HIV-1-infected individuals at risk for developing HAD or HAD progression; these findings could have therapeutic or prognostic implications.
Results
The demographic constitution of the cohort used in this study is summarized in Table 1. The quality and quantity of DNA were adequate from all of the specimens. The distributions of HIV DNA were skewed, as reflected in the standard deviations and differences between mean and median HIV DNA values (Fig. 1, Table 2).
Individuals with HAD had a median [interquartile range (IQR)] of 9.11 (37.20) HIV DNA per 1 Å~ 106 PBMC compared to 0.49 (0.89) HIV DNA per 1 x 106 PBMC in individuals with NC (Table 2). In an unadjusted logistic regression model, the association of HIV DNA with HAD resulted in an odds ratio (OR) of 2.83 (IQR, 1.57-5.08; P < 0.001). The analyses were repeated after controlling for VL, CD4 cell count, HAART (71% of the participants were on HAART), duration of infection, education, age, and ethnicity which continued to show significance (P < 0.01). To evaluate the possibility that these findings were driven primarily by a few participants with very high HIV DNA values, we excluded the participants with the five highest values. This did not substantially change our results. We repeated the univariate analysis in the subset of individuals who had undetectable VL (HAD, 11; NC, 13) to determine whether the effect we observed was attributable to plasma VL. This also did not substantially change the results (OR, 2.76; IQR, 1.28-5.94; P < 0.01) (Table 2 and Fig. 1).
Of particular interest were two individuals with HAD who had relatively high HIV DNA and undetectable VL: 114 and 618 HIV DNA per 1 x 106 PBMC. In an attempt to understand potential differences in these two individuals, analyses were performed on these two, and three other specimens, to ascertain HIV DNA in subsets of PBMC. PBMC from the five individuals (three with HAD, including the two with highest HIV DNA per 1 x 106 PBMC, and two individuals with NC) were obtained and separated into CD14/CD16 and CD14-negative subsets. A representative flowcytometer plot is shown in Fig. 2 where 11-15% of the monocytes demonstrate CD14/CD16 phenotype. The CD14+/CD16- subset was not recovered. HIV DNA was measured from each of the retained subsets (Table 3). From the two individuals with relatively high HIV DNA in PBMC, the HIV DNA per 1 x 106 PBMC was the highest in the CD14/CD16 subset (activated M/MÉ>=) compared to the CD14-negative subset. By flow cytometry, the CD14-negative subset included CD4 T cells. The number of cells lysed from the isolated subsets used to obtain DNA varied depending on the total number of cells originally obtained, ranging from 30 000 to 500 000. Therefore, the HIV DNA per 1 x 106 PBMC was extrapolated from the number of cells used to isolate DNA for each of the specimens. For low levels of HIV DNA (below 1 copy number per million cells), such as the CD14-negative subsets in Table 3, the values were extrapolated based on the standard curves generated by the iCycler IQ Software Program, version 3.0 (BioRad). Representative graphs of the É¿-globin and HIV-1 PCR standard curves and unknown samples are shown in Fig. 3, as described previously [32]. From the β-globin and HIV-1 PCR standard curves, the equivalent cell number and HIV-1 copy number, respectively, are calculated for each unknown specimen. The ratio of HIV DNA copy number per 1 x 106 cells is then acquired. At the lower limits of the standard curves, if limited amount of a specimen is available, the IQ Software Program extrapolated the HIV DNA copy number per 1 x 106 cells, which assumes a linear pattern.
Introduction
HIV-1-associated dementia (HAD) persists among HIV-1-infected individuals despite highly active antiretroviral therapy (HAART) [1-3]. In the pre-HAART era, approximately 15% of patients with low CD4 cell counts progressed to HAD [4]. While the incidence of HAD currently appears to be decreasing, the prevalence has risen from 6.6/100 person years in 1994 to 10.1/100 person years in 2000 [4-6]. A determination of the factors associated with prevalent HAD despite effective suppression of peripheral HIV-1-infection is needed.
In the era of HAART, the utility of circulating plasma HIV-1 virus (viral load, VL) measurement as a marker of HAD has decreased [7-10]. We propose that detectable plasma VL is neither necessary nor sufficient for the presence of HAD, and that other factors are involved in the pathogenesis of HAD [5,9,11-14].
Monocytes/macrophages (M/MÉ>=) are important in the pathogenesis of HAD. Individuals with HAD have higher percentages of circulating activated M/MÉ>= [14]; these cells are probably important in the neuropathogenesis of HIV-1 possibly by increased virus trafficking into the central nervous system [1,14,15]. As important HIV-1 reservoirs, M/MÉ>= could harbor proviral DNA [16-18], suggesting a mechanism of increased risk among individuals with undetectable plasma VL [19,20]. In recent experiments, productive HIV-1 infection of macrophages in SCID mice suggest a role in neurological disease [16]. We were therefore interested in assessing the correlation of proviral DNA with HAD by quantitatively assessing HIV-1 DNA copy number (HIV DNA) in PBMC, and further, to determine the association with activated M/M.
Materials and methods
Cohort and clinical data
The participants for this study were selected from the Hawaii Aging with HIV Cohort (HAHC) following informed consent [21]. This longitudinal cohort was established to examine HAD and other neurologic complications in older (>=50 years old) compared to younger (20-39 years old) HIV-1-seropositive individuals living in Hawaii. Less than 5% of the participants in the HAHC identify injecting drug use as a risk factor for HIV-1 infection [6,22-24]. Individuals were excluded for the following reasons: (i) a major psychiatric or neurologic disorder; (ii) a history of head injury with loss of consciousness greater than 1 h; (iii) current or past opportunistic infection with brain involvement; (iv) a diagnosed learning disability; or (v) delirium due to medications at the time of examination. Evaluations included the macro-neurological examination, a medical history questionnaire, a medication/adherence history, a DSM-IV-based substance abuse/dependence inventory, immunologic and virologic laboratory tests, and neuropsychological testing. Research-based neurocognitive diagnoses were established for each participant using the American Academy of Neurology 1991 criteria [25]. Full details of the diagnostic approach within HAHC are provided elsewhere [21,26,27].
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