Marijuana's putative anti-inflammatory properties may benefit HIV-associated comorbidities. How recreational marijuana use affects gene expression in peripheral blood cells (PBC) among youth with ...HIV-1 (YWH) is unknown.
YWH with defined substance use (n = 54) receiving similar antiretroviral therapy (ART) were assigned to one of four analysis groups: YWH with detectable plasma HIV-1 (> 50 RNA copies/ml) who did not use substances (H+V+S-), and YWH with undetectable plasma HIV-1 who did not use substances (H+V-S-), or used marijuana alone (H+V-S+M), or marijuana in combination with tobacco (H+V-S+M/T). Non-substance using youth without HIV infection (H-S-, n = 25) provided a reference group. PBC mRNA was profiled by Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Differentially expressed genes (DEG) within outcome groups were identified by Significance Analysis of Microarrays and used for Hierarchical Clustering, Principal Component Analysis, and Ingenuity Pathways Analysis.
HIV-1 replication resulted in > 3000 DEG involving 27 perturbed pathways. Viral suppression reduced DEG to 313, normalized all 27 pathways, and down-regulated two additional pathways, while marijuana use among virally suppressed YWH resulted in 434 DEG and no perturbed pathways. Relative to H+V-S-, multiple DEG normalized in H+V-S+M. In contrast, H+V-S+M/T had 1140 DEG and 10 dysregulated pathways, including multiple proinflammatory genes and six pathways shared by H+V+S-.
YWH receiving ART display unique transcriptome bioprofiles based on viral replication and substance use. In the context of HIV suppression, marijuana use, alone or combined with tobacco, has opposing effects on inflammatory gene expression.
In
, the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine ...(ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY regulated targets. In this work, we investigated the impact of guanine nucleotides on
physiology and CodY activity by constructing a
null mutant (Δ
).
biosynthesis of guanine monophosphate is abolished due to the
mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out
, activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the Δ
mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, Δ
cells failed to form robust biofilms when limited for guanine or guanosine. RNA-seq analysis of
transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alteration of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that
exhibits a more invasive lifestyle when limited for guanosine. Further, gene-products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections.
infections impose a serious economic burden on healthcare facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence of
in response to environmental cues is critical for developing efficient diagnostics and treatments.
purine biosynthesis is essential for both fitness and virulence in
, since inhibiting production cripples
's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affects
fitness by altering metabolic and virulence gene expression. Characterizing pathways and gene products upregulated in response to guanine limitation can aid in the development of novel adjuvant strategies to combat
infections.
Purpose
Adenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) through an accumulation of toxic metabolites within lymphocytes. Recently, ADA deficiency has been ...successfully treated using lentiviral-transduced autologous CD34+ cells carrying the ADA gene. T and B cell function appears to be fully restored, but in many patients’ B cell numbers remain low, and assessments of the immunoglobulin heavy (IgHV) repertoire following gene therapy are lacking.
Methods
We performed deep sequencing of IgHV repertoire in peripheral blood lymphocytes from a child following lentivirus-based gene therapy for ADA deficiency and compared to the IgHV repertoire in healthy infants and adults.
Results
After gene therapy, Ig diversity increased over time as evidenced by V, D, and J gene usage, N-additions, CDR3 length, extent of somatic hypermutation, and Ig class switching. There was the emergence of predominant IgHM, IgHG, and IgHA CDR3 lengths after gene therapy indicating successful oligoclonal expansion in response to antigens. This provides proof of concept for the feasibility and utility of molecular monitoring in following B cell reconstitution following gene therapy for ADA deficiency.
Conclusion
Based on deep sequencing, gene therapy resulted in an IgHV repertoire with molecular diversity similar to healthy infants.
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