RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord

ALS is a rapidly progressive, devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression, and molecular insights into pathogenesis and progression are sorely needed. In that context, we used high-depth, next generation RNA sequencing (RNAseq, Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned \u3e50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2, DEseq2, EdgeR) for identification of differentially expressed genes (DEG’s). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples, with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNFα-induced protein 2 (TNFAIP2) as a major network “hub” gene (WGCNA). Using the oPOSSUM algorithm, we analyzed transcription factors (TF) controlling expression of the nine DEG/hub genes in the ALS samples and identified TF’s involved in inflammation (NFkB, REL, NFkB1) and macrophage function (NR1H2::RXRA heterodimer). Transient expression in human iPSC-derived motor neurons of TNFAIP2 (also a DEG identified by all three algorithms) reduced cell viability and induced caspase 3/7 activation. Using high-density RNAseq, multiple algorithms for DEG identification, and an unsupervised gene co-expression network approach, we identified significant elevation of inflammatory processes in ALS spinal cord with TNF as a major regulatory molecule. Overexpression of the DEG TNFAIP2 in human motor neurons, the population most vulnerable to die in ALS, increased cell death and caspase 3/7 activation. We propose that therapies targeted to reduce inflammatory TNFα signaling may be helpful in ALS patients

Plasmacytoid Dendritic Cells Suppress HIV-1 Replication but Contribute to HIV-1 Induced Immunopathogenesis in Humanized Mice

The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment

Unravelling the pluripotency paradox in fetal and placental mesenchymal stem cells: Oct-4 expression and the case of the emperor's new clothes

Mesenchymal stem cells (MSC) from fetal-placental tissues have translational advantages over their adult counterparts, and have variably been reported to express pluripotency markers. OCT- 4 expression in fetal-placental MSC has been documented in some studies, paradoxically without tumourogenicity in vivo. It is possible that OCT- 4 expression is insufficient to induce true "stemness", but this issue is important for the translational safety of fetal-derived MSC. To clarify this, we undertook a systematic literature review on OCT- 4 in fetal or adnexal MSC to show that most studies report OCT- 4 message or protein expression, but no study provides definitive evidence of true OCT- 4A expression. Discrepant findings were attributable not to different culture conditions, tissue sources, or gestational ages but instead to techniques used. In assessing OCT- 4 as a pluripotency marker, we highlight the challenges in detecting the correct OCT- 4 isoform (OCT- 4A) associated with pluripotency. Although specific detection of OCT- 4A mRNA is achievable, it appears unlikely that any antibody can reliably distinguish between OCT- 4A and the pseudogene OCT- 4B. Finally, using five robust techniques we demonstrate that fetal derived-MSC do not express OCT- 4A (or by default OCT- 4B). Reports suggesting OCT- 4 expression in fetal-derived MSC warrant reassessment, paying attention to gene and protein isoforms, pseudogenes, and antibody choice as well as primer design. Critical examination of the OCT- 4 literature leads us to suggest that OCT- 4 expression in fetal MSC may be a case of "The Emperor's New Clothes" with early reports of (false) positive expression amplified in subsequent studies without critical attention to emerging refinements in knowledge and methodology