In this study, we examined changes in gene expression profiles in peripheral blood induced by methcathinone abuse. All but one of the subjects had a clinically relevant extrapyramidal syndrome. RNA expression patterns of drug users clearly differed from those of healthy controls. Also, RT-PCR results confirmed differences between these two groups. The overlap between controls and HIV negative methcathinone users in the middle of the heatmap (Figure ) suggested that most of the hierarchical clustering was driven by HIV infection status. During recent years several microarray studies of HIV induced alterations in host cell gene expression have been performed using in vitro
infected primary cells, cell lines, or tissue samples (Giri et al., 2006
). However, few in vivo
studies in HIV infected humans have evaluated expression profiles of tissue samples (Masliah et al., 2004
; Everall et al., 2005
) or peripheral blood mononuclear cells (Motomura et al., 2004
; Kottilil et al., 2009
; Monaco et al., 2009
). Several of the same apoptosis, antigen presentation, and immune response genes (IFIT3, IFI27, IFI44, IFNG, NFAT, STAT1, TCR, CCR5, ERK, IFITM3, LY6E, IFNG, NFkB, OAS1, PLSCR1) described in previous HIV microarray studies were upregulated in our analysis. Thus peripheral blood can be used for finding gene expression alterations in HIV infected humans. Besides cell death and antigen presentation the genetic network with the highest score was also related to neurological disease, consistent with known associations of HIV infection and neurodegeneration (Clifford et al., 2005
Further minor subgroup analysis of HIV negative users who were all positive for HCV indicated that genes related to cell cycle, cellular growth and proliferation, cancer, and cellular development were upregulated compared to healthy controls. The same pathways have been shown to be involved in HCV induced hepatic changes like cirrhosis, dysplasia, or hepatocellular carcinoma (Wurmbach et al., 2007
; De Giorgi et al., 2009
As the aim of our study was to analyze the drug abuse effect, we decided to stratify our group of subjects. We analyzed only HIV and HCV positive subjects according to their injection status (past versus current) and a clinical measure of the severity of the neurological syndrome UPDRS. The most significantly enriched network after comparing HIV positive current and past users included genes annotated with functions in Genetic Disorder, Immunological Disease, or Cellular Movement categories (25 genes, enrichment score 46). This network confirms the involvement of the immune system in drug abuse induced pathologies. However, as both comparison groups had neurological symptoms we can not draw any conclusions on the potential causes of the extrapyramidal syndrome. On the other hand, injection status seems to influence immune system and this is one factor involved in the development of a neurodegenerative syndrome. Psychostimulant abuse has been shown to compromise immunological status (Everall et al., 2005
). Chronic Mn exposure has similar effects on the immune system (Sengupta et al., 2007
). Thus, both of these factors have potential to induce neurodegeneration.
The UPDRS score reflects the clinical severity of the extrapyramidal syndrome. No significant correlations were found between the duration of Mn–methcathinone use and UPDRS or between blood RNA profiles and UPDRS.
There are several limitations in our study. The main problem is related to the sample organization. Our sample contains many confounding factors – HIV and HCV infection, different duration of methcathinone use, concomitant consumption of other drugs, alcohol, and tobacco. We have not analyzed the clinical status of the HIV and HCV infections. In addition, several drug abusers were undernourished which makes identification of a relevant control group even more complicated. To overcome these limitations, we have applied complex bioinformatics analytical tools in relatively large samples (N
in both groups) that allowed us to perform some cohort stratification. For further studies within methcathinone users more balanced control samples are needed. We have tried to collect similar balanced sample as a control group, but due to the specific and complex nature of our study group it is difficult and time consuming. Ideal controls would be either drug-free HIV and HCV positive subjects or addicts abusing other drugs. We fully understand the limitations caused by the complexity of the problem we analyze. On the other hand, the situation of a non-balanced control sample is quite common in clinical research.