Human keratinocyte differentiation requires translational control by the eIF2α kinase GCN2

Appropriate and sequential differentiation of keratinocytes is essential for all functions of the human epidermis. While transcriptional regulation has proven to be important for keratinocyte differentiation, little is known about the role of translational control. A key mechanism for modulating translation is through phosphorylation of the α subunit of eIF2. A family of different eIF2α kinases function in the integrative stress response to inhibit general protein synthesis coincident with preferential translation of select mRNAs that participate in stress alleviation. Here we demonstrate that translational control through eIF2α phosphorylation is required for normal keratinocyte differentiation. Analyses of polysome profiles revealed that key differentiation genes, including involucrin, are bound to heavy polysomes during differentiation, despite decreased general protein synthesis. Induced eIF2α phosphorylation by the GCN2 protein kinase facilitated translational control and differentiation-specific protein expression during keratinocyte differentiation. Furthermore, loss of GCN2 thwarted translational control, normal epidermal differentiation, and differentiation gene expression in organotypic skin culture. These findings underscore a previously unknown function for GCN2 phosphorylation of eIF2α and translational control in the formation of an intact human epidermis

Translational Repression Protects Human Keratinocytes from UVB-Induced Apoptosis through a Discordant eIF2 Kinase Stress Response

This study delineates the mechanisms by which UVB regulates protein synthesis in human keratinocytes and the importance of translational control in cell survival. Translation initiation is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2-P) that causes decreased global protein synthesis coincident with enhanced translation of selected stress-related transcripts, such as activating transcription factor 4 (ATF4). ATF4 is a transcriptional activator of the integrated stress response (ISR) that has cytoprotective functions as well as apoptotic signals through the downstream transcriptional regulator C/EBP homologous protein (CHOP; GADD153/DDIT3). We determined that UVB irradiation is a potent inducer of eIF2-P in keratinocytes, leading to decreased levels of translation initiation. However, expression of ATF4 or CHOP was not induced by UVB as compared with traditional ISR activators. The rationale for this discordant response is that ATF4 mRNA is reduced by UVB, and despite its ability to be preferentially translated, there are diminished levels of available transcript. Forced expression of ATF4 and CHOP protein before UVB irradiation significantly enhanced apoptosis, suggesting that this portion of the ISR is deleterious in keratinocytes following UVB. Inhibition of eIF2-P and translational control reduced viability following UVB that was alleviated by cycloheximide (CHX), indicating that translation repression through eIF2-P is central to keratinocyte survival

Resistance Mutations to Zidovudine and Saquinavir in Patients Receiving Zidovudine plus Saquinavir or Zidovudine and Zalcitabine plus Saquinavir in AIDS Clinical Trials Group 229

The relationships among treatment regimens, plasma human immunodeficiency virus (HIV) RNA levels, and resistance mutations to saquinavir (codons 48 and 90) and zidovudine (codon 215) were examined in a cohort of 144 patients from the AIDS Clinical Trials Group 229 study. After 24-40 weeks of therapy, no patients who had received the two-drug combination (zidovudine plus saquinavir) had only codon 48 mutations, 45.8% had only codon 90 mutations, and 8.3% had both codon 48 and 90 mutations. Mutations developed by patients who had received the three-drug combination (zidovudine and zalcitabine plus saquinavir) were codon 48 alone in 1.4%, codon 90 alone in 33.3%, and both codons 48 and 90 in 4.2%. The difference between the groups showed a trend toward reduced mutations with three versus two drugs but did not reach significance (p = .11, two-sided χ2). Higher baseline HIV RNA levels correlated with the development of protease mutations. Mutations at codon 215 were present in 82% of all patients at baseline and in 87% after therap

Acceptability of Cell and Gene Therapy for Curing HIV Infection among People Living with HIV in the Northwestern United States: A Qualitative Study

Multiple strategies to cure HIV infection are under investigation, including cell and gene therapy (C&GT) approaches. Research, and ultimately treatment, with these novel strategies will require patients' willingness to participate. To elicit the perspectives of people living with HIV specific to these novel approaches, we conducted 4 focus group discussions with a diverse group of 19 English-speaking men and women living with HIV in care at a large academic HIV clinic in the northwestern United States. Thematic analysis indicated participants expressed initial fear about C&GT research. They articulated specific concerns about risks, including analytical treatment interruptions, and thought only a person in desperate straits would participate. They voiced significant mistrust of research in general and believed there was already a cure from HIV that was being withheld from the poor. Overall, they were satisfied with their health and quality of life on antiretroviral therapy. These findings suggest the importance of community engagement and educational efforts about C&GT for HIV cure to ensure optimal collaborative partnerships

Effects of traumatic brain injury on cognitive functioning and cerebral metabolites in HIV-infected individuals.

We explored the possible augmenting effect of traumatic brain injury (TBI) history on HIV (human immunodeficiency virus) associated neurocognitive complications. HIV-infected participants with self-reported history of definite TBI were compared to HIV patients without TBI history. Groups were equated for relevant demographic and HIV-associated characteristics. The TBI group evidenced significantly greater deficits in executive functioning and working memory. N-acetylaspartate, a putative marker of neuronal integrity, was significantly lower in the frontal gray matter and basal ganglia brain regions of the TBI group. Together, these results suggest an additional brain impact of TBI over that from HIV alone. One clinical implication is that HIV patients with TBI history may need to be monitored more closely for increased risk of HIV-associated neurocognitive disorder signs or symptoms

Raltegravir in second-line antiretroviral therapy in resource-limited settings (SELECT): a randomised, phase 3, non-inferiority study

For second-line antiretroviral therapy, WHO recommends a boosted protease inhibitor plus nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs). However, concerns about toxicity and cross-resistance motivated a search for regimens that do not contain NRTIs. We aimed to assess whether boosted lopinavir plus raltegravir would be non-inferior to boosted lopinavir plus NRTIs for virological suppression in resource-limited settings

Interpreting cerebrospinal fluid pleocytosis in HIV in the era of potent antiretroviral therapy

Background: Cerebrospinal fluid (CSF) pleocytosis may be seen in asymptomatic HIV-infected individuals. This finding complicates interpretation of CSF abnormalities when such individuals are evaluated for other central nervous system infections. The goal of this study was to determine the relationship between CSF pleocytosis, central nervous system (CNS) antiretroviral penetration, adherence to antiretroviral medication regimens, neurological symptoms and performance on neuropsychological tests. Methods: Clinically stable HIV-infected individuals at any peripheral blood CD4+ T cell count or any plasma viral load were asked to attend study visits at entry and every 6 months thereafter for at least one year. At each visit, they underwent a standardized neurological and medication history; neurological examination; a brief neuropsychological test battery: venipuncture; lumbar puncture; and assessment of medication adherence. Generalized estimating equations (GEE) were used to assess the relationships between CSF pleocytosis and other variables. Results: CSF pleocytosis was independently and significantly related to lack of current antiretroviral use (OR 5.9, 95% CI 1.8-18.6, p = 0.003), CD4 count >200/ul (OR 23.4, 95% CI 3.1-177.3, p = 0.002) and detectable plasma HIV RNA (OR 3.3, 95% CI 1.1-9.4, p = 0.03). At visits where antiretrovirals were used, and taking into account detectable plasma HIV RNA, an antiretroviral regimen that contained two or more agents with good CNS penetration conferred a trend toward lower odds of CSF pleocytosis (OR 0.45, 95% CI 0.18-1.12, p = 0.087). Conclusion: CSF pleocytosis is a characteristic of HIV disease that varies significantly with easily identifiable clinical and laboratory features. Use of antiretroviral agents decreases the odds of pleocytosis. This association may be stronger when the regimen contains two or more agents with good CNS penetration.This work was supported by National Institutes of Health grant U54 NS 39406 (AI and CMM)