in hiv-1 infection , depletion of t cells is caused by productive virus infection and fas - mediated apoptosis of infected and uninfected cells [ 1 , 2 ] . in addition , chronic immune activation , especially of cells of the innate immune system , together with accompanying , counteracting endogenous anti - inflammatory mechanisms , further contributes to t - cell depletion [ 3 , 4 ] . hiv infection of plasmacytoid dendritic cells causes persistent activation , resulting in excessive production of proapoptotic interferon ( ifn)- , as well as immunosuppressive indoleamine-2,3-dioxygenase and transforming growth factor ( tgf)- [ 413 ] . in the case of monocytes / macrophages , translocation of microbial products , especially lipopolysaccharide and dna , across the damaged intestinal epithelium , results in persistent systemic activation of these cells due to interaction with toll - like receptors 4 and 9 , as well as with cytosolic pathogen nucleic acid sensors [ 1423 ] . the resultant production of proinflammatory cytokines , especially tnf- , drives t - cell activation and activation - induced cell death [ 6 , 21 , 22 ] . sustained immune activation is associated with disease progression , aids , and death . while highly active antiretroviral treatment ( haart ) is able to suppress viral replication to levels of < 25 copies / ml plasma and partially restore circulating cd4 t cells , it is unable to normalize immune activation [ 21 , 25 ] . immune activation in hiv infection is associated with the presence of circulating proinflammatory / anti - inflammatory and antiviral cytokines / chemokines , as well as with other biomarkers of immune activation , which vary qualitatively and quantitatively with disease progression [ 2631 ] . however , relatively little is known about the profile of circulating biomarkers of immune activation in the setting of advanced hiv-1 subtype c infection , as well as the usefulness of its measurement , not only in monitoring response to haart , but also as a strategy to detect virologic treatment failure . black , adult ( 18 years ) participants attending the antiretroviral clinic at a district hospital in pretoria , south africa , were included in this study . ethics approval was granted by the research ethics committee , faculty of health sciences , university of pretoria ( ethics committee approval number 46/2011 ) . all participants gave informed consent and whole blood samples were collected in edta vacutainers , processed within 24 hours to separate the plasma component by centrifugation , and stored at 70c for up to 37 months . cd4 t - lymphocyte counts ( cd4 ) ( beckman coulter sa ( pty ) ltd . ) and hiv-1 rna ( vl ) ( nuclisens hiv-1 viral load assay v1.2 or v2.0 ) were measured by standard flow cytometric and pcr - based procedures respectively , according to manufacturer 's instructions . sixty hiv - infected participants were followed from pre - treatment to approximately 6 months on haart as part of a larger study on immune reconstitution inflammatory syndrome ( iris ) . pre - treatment samples were taken prior to the initiation of haart in patients presenting with cd4 counts 200 cells/l blood or who stage 4 disease . twenty patients were randomly selected from those who started haart , were clinically stable , did not develop clinical signs of iris during the first six months of treatment , and were virologically suppressed ( vl < 50 copies / ml plasma ) at approximately 6 months of haart ( suppressed group ) . drug regimens consisted of two nucleos(t)ide reverse transcriptase inhibitors ( nrtis ) ( stavudine ( d4 t ) + lamivudine ( 3tc ) , n = 18 , or tenofovir ( tdf ) + 3tc , n = 2 ) and one nonnucleoside reverse transcriptase inhibitor ( nnrti ) ( efavirenz ( efv ) , n = 14 or nevirapine ( nvp ) ,  n = 4 ) . two patients were started on ritonavir - boosted lopinavir ( lpv / r ) for clinical reasons . a second group consisted of 30 participants failing haart as evidenced by two successive vl results of > 1000 copies / ml plasma at least eight weeks apart despite intensive adherence counselling ( failing group ) . drug regimens consisted of two nrtis ( d4 t + 3tc ,  n = 23 or zidovudine ( azt ) + 3tc , n = 7 ) and one nnrti ( efv , n = 20 or nvp , n = 10 ) . participants had been referred for drug resistance testing and study samples were taken at the time of referral . they had been on haart for a median time of 30 months ( range 997 months ) and had been failing treatment for a median of 15.5 months ( range 538 months ) . five patients ( 17% ) had been referred from peripheral clinics and the duration of treatment failure could not be determined . three patients ( 10% ) had experienced treatment interruptions at some time before treatment failure and 13 ( 43% ) never had a suppressed vl while on haart . all patients with cd4 200 cells/l ( n = 21 ) were on cotrimoxazole or dapsone prophylaxis . a third group ( n = 8) of black , hiv - uninfected , healthy control subjects was also included in the study . the median ages of the control , suppressed , and failing groups were 29 ( range 2449 ) , 41.5 ( 2563 ) , and 40.5 ( 2755 ) years , respectively , and the corresponding male  :  female ratios were 1  :  0.6 , 1  :  4 , and 1  :  4 . these were selected on the basis of being largely representative of t - cell , monocyte / macrophage , dendritic cell , and natural killer cell activation . circulating cytokines / chemokines were measured using ( i ) the bioplex suspension bead array system ( bio - rad laboratories inc . , hercules , ca , usa ) ( il-6 , il-10 , ifn- , tnf- , ccl2/mcp-1 , ccl3/mip-1 , ccl4/mip-1 , and cxcl10/ip-10 ) or ( ii ) conventional elisa , namely , ifn- ( ebioscience inc . , san diego , ca , usa ) ; tgf-1 total ( biolegend , san diego , ca , usa ) ; cxcl9/mig and stnf - r1 ( raybiotech inc . , norcross , ga , usa ) ; and scd14 ( abcam , cambridge , ma , usa ) . c - reactive protein ( crp ) and 2-microglobulin ( 2 m ) were assayed by nephelometry ( siemens healthcare diagnostics , bn prospec nephelometer , newark , usa ) . previously published ranges for each of these parameters together with supporting references are shown as supplementary data ( see supplementary material available online at http://dx.doi.org/10.1155/2014/198413 ) . as participant groups consisted of 30 individuals , data were considered to be nonparametric and distribution - free statistical tests implemented in stata v11.2 ( statacorp ) . median concentrations of each parameter were compared between cohorts using the wilcoxon mann - whitney test for independent groups and wilcoxon signed rank sum test for matched groups . correlations between parameters were determined using the spearman correlation test for the hiv - infected pre - haart group ( n = 20 ) , as well as for this group combined with the group failing haart ( n = 50 ) . statistical significance was set at p  0.05 . cd4counts , hiv-1 vl , and levels of inflammatory biomarkers are shown in table 1 . as expected , plasma vl decreased from a median of 53,000 to < 50 rna copies / ml plasma and there was a significant increase in the circulating cd4 count ( 83 to 208 cells/l ;  p < 0.0001 ) in the suppressed group . with respect to the circulating biomarkers of immune activation , cxcl9 , cxcl10 , tgf-1 , stnf - r1 , 2  m , and scd14 were significantly elevated ( p < 0.03 ) and ccl4 significantly decreased ( p = 0.04 ) in the pre - haart group relative to the control group , while ifn- was moderately increased but not significantly so ( p = 0.07 ) . following 6 months of haart , cxcl9 , cxcl10 , 2  m , ifn- , il-6 , tnf- , and stnf - r1 were significantly decreased ( p < 0.01 ) , ccl4 increased ( p < 0.001 ) , while tgf-1 and scd14 also remained elevated despite undetectable plasma vl . it is difficult to attribute major significance to the decreases in tnf- and il-6 as the pretherapy values for both were low . no difference was observed in ifn- , ccl3 , and crp either between the hiv - infected and uninfected control group or the virologically suppressed group pre- and post - haart . in the failing group , the same 5 biomarkers ( cxcl9 , cxcl10 , tgf-1 , 2 m , and scd14 ) were also significantly elevated compared with the control group ( p < 0.02 ) , the values for cxcl10 and 2 m being somewhat lower than those of the pre - haart group ( p < 0.03 ) , while those of cxcl9 , tgf-1 , and scd14 were essentially comparable ( p > 0.5 ) . although the value for ccl2 was significantly lower and that of il-10 higher than the corresponding values of the control group , interpretation is difficult as these values were low in both groups . correlations between cd4 , vl , and the various biomarkers in the pre - haart group are shown in table 2 . cd4 counts correlated negatively and significantly with vl and with stnf - r1 and ccl2 . significant positive correlations were also observed between several of the biomarkers including , but not limited to , ifn- , cxcl10 , ccl2 , ccl3 , and tnf-. although not shown , correlations for the composite group ( consisting of the pre - haart and failing groups ) were generally comparable , albeit weaker , with the exception of cd4 count with vl ( r = 0.64 , p < 0.001 ) , while the following modest correlations were found : ( i ) ccl4 with cxcl9 , il6 , ccl3 , and ifn- ( r = 0.30 , 0.43 , resp . ; p < 0.03 ,  p < 0.001 ) ; and ( ii ) 2 m with cd4 counts , il-6 , and ifn- ( r = 0.28 ,  0.43 , resp . ; p < 0.05 ,  p < 0.02 ) . our findings in patients infected with hiv-1 subtype c are consistent with the coexistence of distinct mechanisms of immune activation , which appear to be differentially affected by successful haart [ 21 , 25 ] . although only moderately elevated pre - haart , it is likely that ifn- , probably originating from cd4 and cd8 t cells , underpins the increases in cxcl9 and 10 , a contention supported by the strong , positive intercorrelation between ifn- and ccl10 , as well as that of ccl9 with ccl10 . other cell types such as dendritic cells and monocytes may also contribute to the increases in these cytokines pre - haart following exposure of the cells to alternative activators such as ifn-1 [ 11 , 32 , 33 ] . the unexpectedly low level of ifn- , as well as those of ccl2 and 3 , may be due to advanced immunosuppression in the setting of high levels of tgf-1 in this group of patients . in the case of 2 m , cxcl 9 and 10 , and tnf - r1 ( a surrogate for tnf ) , haart - associated decreases most likely reflect efficient viral suppression and consequent decreased turnover and reactivity of both cd4 and cd8 t cells . the absence of effects of haart on plasma scd14 , as previously reported by us and others [ 17 , 21 ] , as well as the increase in ccl4 , is consistent with ongoing chronic inflammation due to sustained activation of monocytes / macrophages , even in the face of virally suppressive therapy , and may persist for several years [ 21 , 35 ] . in this setting , the persistent activation of monocytes / macrophages , predominantly the subtype which coexpresess cd14 and cd16 , is most likely driven by the process of microbial translocation [ 21 , 24 , 36 ] . the consequence is sustained generation of proinflammatory mediators and cytokine - driven t - cell death pathways . interestingly , sandler et al . recently reported significant positive correlations between plasma scd14 , il-6 , crp , serum amyloid a , and d - dimer in patients infected with hiv-1 subtype b . subjects with the highest quartile of plasma scd14 concentrations had a 6-fold higher risk of death than those in the lowest quartile . in addition , supported by the findings of the current study , endogenous , monocyte / macrophage - targeted , anti - inflammatory mechanisms are also likely to contribute to ongoing immunosuppression with tgf-1 appearing to play a pivotal role . notwithstanding platelets , plasmacytoid dendritic cells , macrophages of the m2 phenotype , and immunoregulatory cd8 t cells , immunosuppressive and profibrotic tgf-1 is likely to originate predominantly from regulatory t cells [ 38 , 39 ] . in this context it is noteworthy that extensive fibrosis of the t - cell zone of lymphoid tissue appears to be a significant factor in the failure of t - cell reconstitution following successful haart . persistently elevated plasma levels of tgf-1 and scd14 , even in the setting of ostensibly successful haart , may therefore identify a subset of patients at highest risk of a poor outcome . in the group of patients failing haart , the circulating concentrations of cxcl9 , cxcl10 , and 2 m were also significantly higher than those of the control group and , with the exception of cxcl9 , significantly lower than the pre - haart values for the suppressed group . the circulating concentrations of scd14 and tgf-1 in the failing group were comparable to those of the suppressed group both before and after therapy . persistent elevations , or a rebound following an earlier decrease , in plasma cxcl9 , cxcl10 , and 2 m appear to be associated with a poor response to haart , suggesting that serial measurement of these biomarkers may be a useful adjunctive strategy . with respect to previous studies , our findings are generally in agreement with a recent study by kamat et al . in which elevated circulating concentrations of cxcl9 , cxcl10 , scd14 , and soluble il-2 receptor ( sil-2r ) represented a profile which distinguished viremic and aviremic subjects infected with hiv-1 subtype b from uninfected , healthy control subjects . in agreement with the report of kamat et al . , we also detected a significant , negative correlation between numbers of circulating cd4 t cells and vl but failed to show a correlation between these disease markers and cxcl10 in the pre - haart group . however , this correlation was detected when the pre - haart and failing groups were combined , most likely due to increased statistical power . we also detected a significant positive correlation between cxcl9 and cxcl10 , while in contrast to these authors , a significant , positive correlation between cxcl10 and ifn- was evident as can be expected in conditions of chronic inflammation . notwithstanding the different viral types investigated , several other important differences underscore the strengths of the current study . most importantly , the profile of biomarkers of immune activation measured by kamat et al . , which did not include 2 m or tgf-1 , was not measured serially in a single cohort of patients pre- and post - haart as done in the current study , which may account for the observed lack of effect of haart on ifn- in the former study . limitations , however , are ( i ) small sample sizes ; ( ii ) measurement of circulating biomarkers at a single time point ( 6 months ) following initiation of haart in the suppressed group ; and ( iii ) no pretherapy measurement of circulating biomarkers prior to initiation of therapy in the failing group . nonetheless , the general agreement with previous studies , predominantly in the setting of hiv-1 subtype b infection , supports the reliability of our findings . in conclusion , successful administration of haart to patients with hiv-1 subtype c infection is accompanied by significant decreases in circulating biomarkers associated with t - cell activation and turnover ( ifn- , cxcl9 , cxcl10 , stnf - r1 , and 2 m ) . serial measurement of 3 of these ( cxcl9 , cxcl10 , and 2 m ) may represent a useful adjunct to measurement of viral loads in monitoring responses to haart . in addition , persistently elevated levels of scd14 and tgf-1 , despite successful haart , are consistent with chronic activation of monocytes / macrophages and possible risk of a poor outcome , underscoring the adjunctive therapeutic potential of monocyte / macrophage - targeted anti - inflammatory chemotherapy in patients with advanced hiv infection .
few studies have examined immune activation profiles in patients with advanced hiv-1 subtype c infection or assessed their potential to predict responsiveness to haart . 
 bioplex , elisa , and nephelometric procedures were used to measure plasma levels of inflammatory biomarkers in hiv-1 subtype c - infected patients sampled before and after 6 months of successful haart ( n = 20 ) ; in patients failing haart ( n = 30 ) ; and in uninfected controls ( n = 8) . prior to haart , cxcl9 , cxcl10 , 2 m , stnf - r1 , tgf-1 , ifn- , il-6 , tnf , and scd14 were significantly elevated in hiv-1-infected patients compared to controls ( p < 0.01 ) . 
 all of these markers , with the exception of stnf - r1 , were also elevated in patients failing haart ( p < 0.05 ) . 
 the persistently elevated levels of cxcl9 , cxcl10 , and 2 m in patients failing therapy in the setting of a marked reduction in these markers in patients on successful haart suggest that they may be useful not only to monitor immune activation during haart , but also to distinguish between good and poor responders . in the case of scd14 and tgf-1 
 , the levels of these biomarkers remained persistently elevated despite haart - induced virological suppression , a finding that is consistent with ongoing monocyte - macrophage activation , underscoring a potential role for adjuvant anti - inflammatory therapy .