Patent Application: US-45186408-A

Abstract:
the present invention relates to the use of monocyte markers for diagnostic , prognostic or theranostic applications during diseases and syndromes caused by hiv infection . more specifically , it relates to a method comprising isolation of monocytes and determining gene expression , preferably pbef1 gene expression . the method is useful to determine the evolution of the disease or can be used to evaluate the efficacy of a treatment .

Description:
fifty ml blood samples were collected in edta - tubes from therapy - naïve hiv - 1 - seropositive patients from the hiv clinic of the institute of tropical medicine in antwerp , belgium . patient details are shown in tables 1 and 6 . peripheral blood mononuclear cells ( pbmcs ) were separated via a ficoll gradient and plasma was concomitantly aspirated and stored at − 80 ° c . monocytes were purified from the pbmc fraction using the negative selection - based monocyte isolation kit ii from miltenyi - biotec ( bergisch gladbach , germany ), according to the manufacturer &# 39 ; s instructions . yields were minimally 5 million monocytes with a purity & gt ; 85 %, as verified through flow cytometry . hiv bal was either inactivated with aldrithiol - 2 ( at - 2 , 200 μm in dmso at 37 ° c . for one hour ) or mock - inactivated with dmso alone . virus was subsequently enriched by filtration over a 100 kda cut - off membrane , aliquotted and stored at − 80 ° c . until use . monocytes were purified via counterflow elutriation and subsequent e - rosetting from buffy coats of healthy blood donors from the blood transfusion centre of antwerp . cells were cultured at 3 × 10 & lt ; 6 & gt ; cells / ml in rpmi medium supplemented with 10 % fetal bovine serum and were treated with infectious and at - 2 - inactivated hiv bal for the indicated times at a concentration corresponding to 50 ng / ml of p24 . for rna extraction , monocytes isolated from patients or treated in vitro with virus were immediately lysed in trizol ( invitrogen , carlsbad , calif ., usa ), and trizol pellets were stored at − 80 ° c . total rna was prepared from the trizol pellets by chloroform extraction , as per the manufacturer &# 39 ; s recommendations . ten randomly selected samples were checked for integrity on a bioanalyzer ( biorad , hercules , calif ., usa ). no protein contamination or degradation of rna was detected . selected rna samples were prepared and hybridized to codelink hwg bioarrays according to the manufacturer &# 39 ; s instructions ( amersham biosciences , freiberg , germany ). codelink datasets were analyzed using the genemaths xt software package ( applied maths , st . martens - latem , belgium ). after background correction , genes that were called as “ absent ” in more than four arrays were eliminated from the datasets . subsequently , array normalization was performed ; both quantile and simple mean normalization were performed , without significant differences in the datasets . in this fashion , a normalized dataset containing only genes with a present call in a minimum of eight arrays was constructed . normalized “ present ” datasets were further analyzed for over - representation of specific processes / pathways . for this type of analysis , two different software applications were used : the freeware program genmapp / mappfinder ( doniger et al ., 2003 ; on the world - wide web at genmapp . org ) and the commercially available package genego ( on the world - wide web at genego . com ). genmapp / mappfinder clusters genes together in common pathways / processes using the associated gene ontology ( go ; ashburner et al ., 2000 ) annotations ; additionally , users can contribute pathways ( mapps ) for which over - representation can also be assessed . genego uses a system of manually curated pathways , which are publicly available on the world - wide web at invitrogen . com / ipath . the macrophage activation state ( mas ) array was developed as a focused and flexible tool for the analysis of gene expression patterns in monocytes / macrophages . a collection of 700 genes associated with different macrophage activation states was compiled , using a combination of literature data - mining and human “ translation ” of murine models of macrophage activation available in our laboratory . subsequently , gene - specific primers were designed for the genes in this collection and fragments were amplified from total cdna pools of monocytes under various in vitro and in vivo conditions . these fragments were applied in duplicate on 7 × 10 cm nylon membranes and were cross - linked to the membranes using uv - exposure . rna samples from all patients were selected for analysis on this mas array . a reverse transcription was performed on 1 μg total rna using oligo - dt and superscript ii reverse transcriptase ( invitrogen ) in the presence of & lt ; 33 & gt ; p - dctp ( amersham biosciences ), and the labeled cdna was then hybridized to the membranes for 20 hours at 42 ° c . in northernmax hybridization buffer ( ambion , austin , tex ., usa ). membranes were subsequently washed with sds - containing buffer at 68 ° c . and were exposed to a phosphorscreen to reveal bound radioactivity . phosphorscreens were then scanned in a phospho - imager ( biorad ). spot recognition and quantification , background correction and array normalization were all performed using custom - designed software based on the program imagej ( image processing and analysis in java , sun microsystems , santa clara , calif ., usa ). expression of individual genes was examined using real - time semi - quantitative pcr ( rtqpcr ). cdna was prepared from 1 μg total rna using oligo - dt and superscript ii reverse transcriptase ( invitrogen ) and gene - specific primers for the gene of interest ( pbef1 ) and a housekeeping gene ( gapdh ) were designed : pcr reactions were performed in duplicate in a biorad mycycler , with biorad iq sybr green supermix ; each pcr cycle consisted of 60 - second denaturation at 94 ° c ., 45 - second annealing at 55 ° c ., and 60 - second extension at 72 ° c . gene expression was normalized using the gene gapdh , coding for the enzyme glyceraldehyde - 3 - phosphate dehydrogenase , as a housekeeping gene . for in vitro infection experiments , monocytes were obtained from buffy coats of healthy donors of the blood transfusion center of antwerp ( rode kruis vlaanderen , belgium ) by counterflow elutriation , as described previously ( van herrewege et al ., 2002 ). these cells were then differentiated to monocyte - derived macrophages ( mdm ) during seven days in rpmi 1640 medium ( bio - whittaker , verviers , belgium ) supplemented with 10 % bovine fetal calf serum ( biochrom , berlin , germany ), penicillin ( 100 u / ml ) and streptomycin ( 100 μg / ml ) ( roche diagnostics , mannheim , germany ) and 40 ng / ml m - csf ( peprotech , london , united kingdom ) at 37 ° c . and 5 . 0 % co 2 . half the medium was replaced after four days . cells were then harvested and used for experiments in the same medium ( without m - csf ). recombinant visfatin was obtained from peprotech and alexis ( zandhoven , belgium ). as both batches gave similar results in preliminary studies , all further experiments were performed using recombinant protein from peprotech . the recombinant protein batches contained & lt ; 0 . 01 ng / μg lps , as assessed by quantitative chromogenic limulus amoebocyte lysate assay ( qlal ) ( bio - whittaker ). for infection experiments , mdm were plated in 96 - well plates at 7 . 5 × 10 5 cells / ml and pre - treated with recombinant visfatin ( 200 ng / ml ) for 1 hour at 37 ° c . and 5 . 0 % co 2 . then , virus was added in six - fold and incubated for 2 hours , again at 37 ° c . and 5 . 0 % co 2 . cells were then washed 3 × to remove unbound virus and incubated for 14 days . productive infection was monitored via an in - house - developed p24 antigen elisa , as described elsewhere ( beirnaert et al ., 1998 ). plasma separated from patient blood samples by lymphoprep separation was stored at − 80 ° c . until use . one ml samples were added to 5 × 10 6 phytohemagglutinin ( pha )/ interleukin - 2 ( il2 ) stimulated pbmcs obtained from buffy coats of healthy donors of the blood transfusion center of antwerp and were cultured in rpmi 1640 medium ( bio - whittaker ) supplemented with 10 % bovine fetal calf serum ( biochrom ), penicillin ( 100 u / ml ) and streptomycin ( 100 μg / ml ) ( roche ), pha ( 0 . 5 μg / ml ) ( murex biotech ltd ., dartford , united kingdom ) and il2 ( 5 ng / ml ) ( roche ). medium was refreshed twice weekly , and supernatants were monitored using p24 antigen elisa . additional pbmc were added on an ad hoc basis when cells became depleted . cultures were followed until p24 levels in the supernatants reached overflow values in elisa . then viruses were harvested , aliquoted and stored at − 80 ° c . for co - receptor usage - determination assays , plasma viruses were serially diluted and added to u87 . r5 or u87 . x4 cells in quadruplicate . unbound virus was washed away after 2 hours incubation at 37 ° c . and 5 . 0 % co 2 , and productive infection was monitored by p24 elisa after 7 and 14 days . productive infection of either u87 . ccr5 or u87 . cxcr4 , signifying , respectively , r5 and x4 usage , was clear - cut in all cases . hiv bal and hiv iiib viruses were assayed in parallel as positive controls for , respectively , r5 and x4 virus . for codelink hwg bioarray data , an uncorrected t - test ( p - value & lt ; 0 . 01 significant ) and a benjamini - hochberg - corrected t - test ( p - value & lt ; 0 . 05 significant ) to control the false positive rate ( benjamini & amp ; hochberg , 1995 ) were used ; for the mas data , only an uncorrected t - test ( p - value & lt ; 0 . 05 significant ) was used . significance of rt - qpcr data was assessed via a nonparametric mann - whitney test . eight hiv patient samples with a broad range of cd4 + t lymphocyte counts and four healthy control samples were selected for analysis on codelink hwg microarrays ( p01 - p08 and c01 - c04 ; table 1 ) and were processed as described . a principal components analysis ( pca ) was performed on the normalized datasets of “ present ” genes . this allowed a segmentation of the data into a cluster of hiv - samples , a cluster of control samples and one outlier control sample ( fig1 ), suggesting that monocyte function is distinctly modulated during in vivo hiv infection . samples were grouped according to serostatus , i . e ., no stratifications according to cd4 + t lymphocyte count or viral load were performed , and gene expression values were compared between the hiv - positive and hiv - negative groups . two different types of meaningful information can be extracted from datasets of this magnitude . on the one hand , by performing over - representational analysis on a broad group of genes for which expression is significantly different ( i . e ., p - value & lt ; 0 . 01 as only criterion ), it is possible to identify specific pathways and / or functional groups of genes that are influenced as a whole . on the other hand , by using more stringent criteria ( i . e ., p - value & lt ; 0 . 01 and fold change & gt ; 1 . 5 ), individual genes that may play pivotal roles in the model at hand or that may be candidate molecular markers for certain conditions can be identified . in the context of this study , over - representational analysis using the two described software applications ( genego and genmapp ) revealed several cellular pathways / processes involved in apoptosis that were significantly modulated in monocytes of hiv patients ( table 2 ), confirming our notion that the cellular apoptotic machinery is disturbed on a molecular level during hiv infection . another class of processes that appears to be targeted in monocytes by hiv infection is a group of pathways involved in lipid metabolism and / or insulin signaling ( table 3 ). to the best of our knowledge , no study to date has focused on these processes in monocytes during hiv infection . an analysis of the datasets aimed at the identification of individual genes with an interesting expression pattern yields several candidates . one of the genes that pass the set criteria ( p - value & lt ; 0 . 01 , fold change & gt ; 1 . 5 ) is the novel cytokine / adipokine pbef1 ( pre - b - cell colony - enhancing factor 1 or visfatin ) ( fig2 ), which possesses a documented involvement in both lipid metabolism and apoptosis . all hiv patient ( n = 29 ) and healthy control ( n = 8 ) samples ( table 1 ) were analyzed on our custom mas array as described . patients were grouped together according to their cd4 + t lymphocyte count : t4 & lt ; 200 cells / mm & lt ; 3 & gt ; ( group 1 ), 200 & lt ; t4 & lt ; 500 cells / mm & lt ; 3 & gt ; ( group 2 ) and t4 & gt ; 500 cells / mm & lt ; 3 & gt ; ( group 3 ). as this smaller - scaled array is less geared towards pathway analyses and more towards identification of individual genes of interest , over - representation / pathway analysis was not performed on this dataset . statistical analysis , however , again with an additional fold change cut - off of 1 . 5 , revealed a list of genes to be significantly up - regulated or to be significantly down - regulated in at least one of these patient groups ( table 4 ). within this list , a cluster of apoptosis - associated genes ( n = 6 ) could be compiled ( table 5 ), using the gene ontology ( go ) annotations in combination with a thorough screening of the available literature . several of these genes possess previously documented hiv - associated properties in cells of monocyte lineage . an example is stat1 , which was previously found to be induced in monocytes and monocyte - derived macrophages by in vitro treatment with hiv - 1 nef or infectious hiv ( federico et al ., 2001 ) and in immature dendritic cells by hiv - 1 tat expression or in vitro hiv infection ( izmailova et al ., 2003 ). additionally , as an interferon - γ - associated transcription factor , stat1 is involved in many inflammatory pathways and has been implicated in hiv - associated pathogenesis in a multitude of studies ( e . g ., abbate et al ., 2000 ; asensio et al ., 2001 ; roberts et al ., 2003 ). pbef1 , on the other hand , was , to the best of our knowledge , never associated with monocyte dysfunction during hiv infection and was only very recently linked with hiv infection in general , in the context of haart - treated hiv patients ( schindler et al ., 2006 ). rt - qpcr verification of pbef1 expression in monocyte samples from hiv - infected patients versus healthy controls gene expression was analyzed in selected patient samples ( p03 - p19 and c01 - c06 ; table 1 ) using gene - specific primers for pbef1 . gene expression data were normalized using the housekeeping gene gapdh and were compared between hiv patients and healthy controls ( fig3 ). when patients were stratified according to cd4 + t lymphocyte count , only group 2 ( 200 & lt ; t4 & lt ; 500 cells / mm & lt ; 3 & gt ;) appeared to display a significant ( mann - whitney , p & lt ; 0 . 05 ) up - regulation of pbef1 ( fig3 , panel a ), and no significant correlation was found between lymphocyte counts and pbef1 expression levels . however , according to the lymphocyte counts , this grouping of patients may not be the best strategy , as the aberrant gene expression profile in monocytes may , in the first place , be a direct result of the circulating virus , and correlation between viral load and cd4 + t lymphocyte count is not always strong . therefore , the gene expression was also plotted versus the viral loads of the patients ( fig3 , panel b ). linear regression analysis reveals that pbef1 expression is significantly correlated with the viral load ( p = 0 . 001 ). rt - qpcr analysis of the effect of haart on pbef1 expression in monocyte samples from hiv - infected patients blood samples were collected from therapy - naïve and haart - treated hiv patients ( table 6 ). body mass index was between 20 and 25 for all patients . inclusion criteria for therapy - naïve patients were never to have received therapy and to have a viral load of more than four log copies / ml . for haart patients , inclusion criteria were to have been on therapy for at least one year and to have an undetectable viral load . samples from healthy seronegative donors with matching age and nationality were collected as negative controls . monocytes were isolated from these blood samples and pbef1 expression was analyzed through rt - qpcr analysis using gene specific primers for pbef1 . also in this analysis ; therapy - naïve hiv patients displayed significantly higher pbef1 expression than healthy controls . however , pbef1 expression in patients on haart was reduced to the levels also found in healthy controls ( fig4 ). this observation confirms our previous findings and is in accordance with the role of visfatin as an inflammatory product ( the viral load in patients on haart is reduced to undetectable levels , resulting in a decreased inflammatory phenotype ). however , it also suggests that circulating monocytes play no role in the increase in plasma visfatin described to occur by schindler et al in patients undergoing haart . rt - qpcr analysis of the effect of hiv treatment on pbef1 expression in monocyte samples from non - infected individuals monocytes were isolated from buffy coat from healthy control volunteers by counterflow elutriation . these cells were seeded in six - well plates and were treated with infective hiv bal virus or with virus that had been treated with aldrithiol - 2 ( at2 ), which is reported to covalently modify essential zinc fingers in the nucleocapsid of hiv , rendering it incapable of productive infection while conserving its structure and binding properties ( rossio et al ., j . virol . 1998 , 72 : 7992 - 8001 ). both infective and inactivated viruses were added at a concentration corresponding to 50 ng / ml of p24 . rt - qpcr analysis revealed that pbef1 induction appeared to be an early event in monocyte cultures treated both with active and inactive virus ( fig5 ), suggesting that simple interaction of the virus with the cell is enough to induce significant transcriptional changes in the monocyte . addition of recombinant visfatin decreases the viral infectivity of the m - tropic r5 hiv labstrain bal as these expression analyses of visfatin showed interesting tendencies , we initiated a series of experiments aimed at elucidating the functional role of visfatin during hiv infection . experiments concerning the effect on productive infection with hiv proved to be very promising . addition of recombinant visfatin to activated pbmc or monocyte - derived macrophage ( mdm ) cultures prior to in vitro infection with the mtropic r5 hiv lab - strain bal decreases the viral infectivity for these cultures ( fig6 ). this viral infectivity can be quantified by calculating the tcid50 ( tissue culture infectious dose 50 %, i . e ., the dose of virus that has a 50 % chance of infecting a cell culture ) values of the viral stocks . addition of visfatin reduces the tcid50 values of bal by approximately one log in both pbmc and mdm cultures , signifying a 90 % reduction in viral infectivity ( fig7 , panel a ). interestingly , visfatin does not mediate this effect on the t - tropic x4 hiv lab - strain iiib (= h × b2 ). this signifies that by selectively inhibiting r5 and not x4 hiv , visfatin contributes to the hiv co - receptor switch seen in ca . 50 % of all patients in which the co - receptor usage of the dominant quasispecies within the patient switches from the moderately virulent r5 to the more aggressive x4 , leading to increased progression to aids . because the bal and iiib strains differ significantly in their genetic background and not only in their co - receptor usage , we repeated the same experiments using clinical isolates in lieu of lab strains . r5 and x4 strains of the 968 viral clone were isolated from one patient , and differ only in co - receptor usage . for the r5 strain of the 968 clone ( 968 - 3 ), the results were similar to those of the r5 lab strain bal . for the x4 strain ( 986 - 1 ), visfatin even increased the viral titer in mdm ( fig7 , panel b ). while this strain is incapable of infecting untreated mdm , addition of visfatin allowed the 968 - 1 clone to establish a productive infection . in order to rule out lps contamination ( which also mediates effects on viral infectivity in mdm and pbmc ) as a possible contributing factor to this effect , the experiments concerning viral infectivity were reproduced using nicotinamide mononucleotide ( nmn ), the endproduct of the enzymatic function of visfatin . simple addition of nmn to pbmc cultures also reduces the infectivity of hivbal ( fig8 ), proving that it is , in the first place , the enzymatic function of visfatin that is mediating the described effects , rather than possible lps contamination of the recombinant protein . additionally , these results prove that it is specifically the enzymatic function of visfatin that is responsible for these effects , rather than , e . g ., the published cytokine - like or insulin - mimetic properties of this molecule . in order to identify correlates for visfatin expression in hiv patients other than the viral load , several other parameters were examined . one such parameter was the co - receptor , usage of primary viruses isolated from patient plasma samples . co - receptor usage of these isolates was evaluated through infection experiments of ccr5 - or cxcr4 - expressing u87 cells ( u87 . r5 and u87 . x4 ). when patients were grouped according to the co - receptor usage of their corresponding viral isolates ( two individuals with r5 virus and three with x4 virus ), significant differences were found between the groups at the level of visfatin protein expression . high visfatin expression appeared to correlate with the presence of x4 virus in the clinical isolates , while low visfatin expression was associated with r5 viruses ( fig9 ). this correlation , combined with our observation that visfatin is induced late in infection in patients with high viral loads , supports the application of visfatin as a biomarker for the co - receptor switch , and fits with the contribution of visfatin to that switch . table 2 . processes , pathways and molecular functions associated with apoptosis , identified by the software applications genmapp and genego as over - represented in codelink hwg datasets . ppf : name of the identified process , pathway or function ; either the gene ontology ( go )/ contributed term ( genmapp ) or the name of the curated pathway ( genego ; on the world - wide web at invitrogen . com / ipath ). p - val : p - value of over - represented pathway , as calculated by software application ; % changed : percentage of the genes in the pathway that were called as significant ; author : author of contributed genmapp ( on the world - wide web at genmapp . org /). table 3 . processes , pathways and molecular functions associated with lipid metabolism / insulin resistance , identified by the software applications genmapp and genego as over - represented in codelink hwg datasets . ppf : name of the identified process , pathway or function ; either the gene ontology ( go )/ contributed term ( genmapp ) or the name of the curated pathway ( genego ; on the world - wide web at invitrogen . com / ipath ). p - val : p - value of over - represented pathway , as calculated by software application ; % changed : percentage of the genes in the pathway that were called as significant ; author : author of contributed genmapp ( on the world - wide web at genmapp . org /). table 4 . genes identified from the mas analysis as differentially expressed between samples from hiv patients and controls . t4 : cd4 + t lymphocyte count ( cells / mm & lt ; 3 & gt ;). diff groups : groups of patients ( defined by t4 counts ) in which the genes are differentially expressed . tr : type of regulation : i = induction , s = suppression . table 5 . apoptosis - associated genes differentially expressed between monocytes of hiv patients and of healthy controls , as assessed by custom mas array analysis . group : patient group , based on cd4 + t lymphocyte count ( cells / mm & lt ; 3 & gt ;); evidence : evidence for assigning the gene to the cluster “ apoptosis - associated genes ”; go : gene ontology annotation ; fc : fold change ; p - val : p - value , determined via uncorrected student &# 39 ; s t - test . table 6 . clinical details of patients enrolled in the study for pbef1 levels in the monocytes of therapy - naïve patients and patients on haart . t4 : cd4 + t lymphocyte count ( cells / mm & lt ; 3 & gt ;); na : not applicable . abbate i ., f . dianzani , and m . r . capobianchi ( 2000 ). activation of signal transduction and apoptosis in healthy lymphocytes exposed to bystander hiv - 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