The National NeuroAIDS Tissue Consortium (NNTC) performed a brain gene expression array to elucidate pathophysiologies of Human Immunodeficiency Virus type 1 (HIV-1)-associated neurocognitive ...disorders.
Twenty-four human subjects in four groups were examined A) Uninfected controls; B) HIV-1 infected subjects with no substantial neurocognitive impairment (NCI); C) Infected with substantial NCI without HIV encephalitis (HIVE); D) Infected with substantial NCI and HIVE. RNA from neocortex, white matter, and neostriatum was processed with the Affymetrix® array platform.
With HIVE the HIV-1 RNA load in brain tissue was three log(10) units higher than other groups and over 1,900 gene probes were regulated. Interferon response genes (IFRGs), antigen presentation, complement components and CD163 antigen were strongly upregulated. In frontal neocortex downregulated neuronal pathways strongly dominated in HIVE, including GABA receptors, glutamate signaling, synaptic potentiation, axon guidance, clathrin-mediated endocytosis and 14-3-3 protein. Expression was completely different in neuropsychologically impaired subjects without HIVE. They had low brain HIV-1 loads, weak brain immune responses, lacked neuronally expressed changes in neocortex and exhibited upregulation of endothelial cell type transcripts. HIV-1-infected subjects with normal neuropsychological test results had upregulation of neuronal transcripts involved in synaptic transmission of neostriatal circuits.
Two patterns of brain gene expression suggest that more than one pathophysiological process occurs in HIV-1-associated neurocognitive impairment. Expression in HIVE suggests that lowering brain HIV-1 replication might improve NCI, whereas NCI without HIVE may not respond in kind; array results suggest that modulation of transvascular signaling is a potentially promising approach. Striking brain regional differences highlighted the likely importance of circuit level disturbances in HIV/AIDS. In subjects without impairment regulation of genes that drive neostriatal synaptic plasticity reflects adaptation. The array provides an infusion of public resources including brain samples, clinicopathological data and correlative gene expression data for further exploration (http://www.nntc.org/gene-array-project).
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Well preserved frozen biospecimens are ideal for evaluating the genome, transcriptome, and proteome. While papers reviewing individual aspects of frozen biospecimens are available, we present a ...current overview of experimental data regarding procurement, storage, and quality assurance that can inform the handling of frozen biospecimens. Frozen biospecimen degradation can be influenced by factors independent of the collection methodology including tissue type, premortem agonal changes, and warm ischemia time during surgery. Rapid stabilization of tissues by snap freezing immediately can mitigate artifactually altered gene expression and, less appreciated, protein phosphorylation profiles. Collection protocols may be adjusted for specific tissue types as cellular ischemia tolerance varies widely. If data is not available for a particular tissue type, a practical goal is snap freezing within 20min. Tolerance for freeze–thaw events is also tissue type dependent. Tissue storage at −80°C can preserve DNA and protein for years but RNA can show degradation at 5years. For −80°C freezers, aliquots frozen in RNAlater or similar RNA stabilizing solutions are a consideration. It remains unresolved as to whether storage at −150°C provides significant advantages relative to that at −80°C. Histologic quality assurance of tissue biospecimens is typically performed at the time of surgery but should also be conducted on the aliquot to be distributed because of tissue heterogeneity. Biobanking protocols for blood and its components are highly dependent on intended use and multiple collection tube types may be needed. Additional quality assurance testing should be dictated by the anticipated downstream applications.
•Frozen biospecimens contain high quality nucleic acids and proteins for testing.•Tissue type and heterogeneity affect biospecimen quality and degradation.•Storage at −80°C preserves DNA and proteins well but RNA can degrade.•Minimize resampling or freeze–thaw events by aliquoting.•Quality assurance approaches can be tailored to the projected biospecimen use.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Elevated plasma lipopolysaccharide (LPS), an indicator of microbial translocation from the gut, is a likely cause of systemic immune activation in chronic HIV infection. LPS induces monocyte ...activation and trafficking into brain, which are key mechanisms in the pathogenesis of HIV-associated dementia (HAD). To determine whether high LPS levels are associated with increased monocyte activation and HAD, we obtained peripheral blood samples from AIDS patients and examined plasma LPS by Limulus amebocyte lysate (LAL) assay, peripheral blood monocytes by FACS, and soluble markers of monocyte activation by ELISA. Purified monocytes were isolated by FACS sorting, and HIV DNA and RNA levels were quantified by real time PCR. Circulating monocytes expressed high levels of the activation markers CD69 and HLA-DR, and harbored low levels of HIV compared to CD4(+) T-cells. High plasma LPS levels were associated with increased plasma sCD14 and LPS-binding protein (LBP) levels, and low endotoxin core antibody levels. LPS levels were higher in HAD patients compared to control groups, and were associated with HAD independently of plasma viral load and CD4 counts. LPS levels were higher in AIDS patients using intravenous heroin and/or ethanol, or with Hepatitis C virus (HCV) co-infection, compared to control groups. These results suggest a role for elevated LPS levels in driving monocyte activation in AIDS, thereby contributing to the pathogenesis of HAD, and provide evidence that cofactors linked to substance abuse and HCV co-infection influence these processes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
OBJECTIVE:To determine whether there is activation of human endogenous retrovirus K (HERV-K) in amyotrophic lateral sclerosis in HIV infection and whether it might respond to treatment with ...antiretroviral drugs.
METHODS:In this case series, we present 5 patients with HIV infection who subsequently developed motor neuron disease involving both upper and lower motor neurons. We monitored HERV-K levels in plasma of 4 of these patients.
RESULTS:Three patients who received antiretroviral therapy had reversal of symptoms within 6 months of onset of neurologic symptoms and the other 2 had slow neurologic progression over several years. Three patients in whom the levels were measured at onset of neurologic symptoms showed elevated HERV-K levels that responded to optimization of antiretroviral therapy for CNS penetration.
CONCLUSIONS:Thus, motor neuron disease in individuals with HIV infection may a treatable entity, but early treatment with CNS-penetrating antiretroviral therapy may be necessary. Monitoring of HERV-K levels may help guide treatment.
Sustainability in Biobanking Abdaljaleel, Maram; Singer, Elyse J; Yong, William H
Methods in molecular biology,
2019, Volume:
1897
Journal Article
Open access
Biobanks are storage places for biospecimens that can be used for current and future scientific research. Biospecimens are exceptional sources of biological data that can be potentially translated ...from molecular and genetic information to clinically relevant treatment modalities. Examples of such biospecimens include, but are not limited to, blood, skin, hair, saliva, stem cells, DNA, and RNA. The volume of biospecimens worldwide continues to grow at an extraordinary rate posing a challenge for biobanks to manage this growth. Due to the vital role of biobanks in research, an understanding of biobanking sustainability is important. Simply starting to collect biospecimens without strategic planning and cost analysis can lead to failure. Components vital to sustainability include fostering public support, cost-effective banking, funding development, standardized protocols, and interoperability.
Mild forms of HIV-associated neurocognitive disorders (HAND) remain prevalent in the era of combination antiretroviral therapy (cART). Although elevated lipopolysaccharide (LPS) and immune activation ...are implicated in HAND pathogenesis, relationships of LPS and inflammatory markers to mild forms of HAND or impairment in specific cognitive domains are unknown. To examine these relationships, we compared plasma soluble CD14 (sCD14), CCL2, and LPS levels with neurocognitive test scores in a cART era cohort.
We analyzed plasma from HIV+ subjects (n = 97) with nadir CD4 counts <300 and high frequency of hepatitis C virus coinfection and illicit drug use for relationships between sCD14, CCL2, and LPS levels and neurocognitive test scores.
Plasma sCD14 levels were higher in subjects with test scores indicating global impairment (P = 0.007), particularly in attention and learning domains (P = 0.015 and P = 0.03, respectively), regardless of HAND diagnosis. Plasma sCD14 levels correlated inversely with global, attention, and learning T scores (P = 0.036, 0.047, and 0.007, respectively) and yielded higher area under receiver operating characteristic values for predicting impaired scores than single-marker models based on plasma or cerebrospinal fluid viral load or CD4 count (area under receiver operating characteristic values = 0.71, 0.81, and 0.71, respectively) and in 4-marker models based on plasma sCD14 and 3 conventional markers compared with the 3-marker models.
Plasma sCD14 is a biomarker associated with impaired neurocognitive testing in attention and learning domains in HIV-infected individuals with advanced disease, suggesting involvement of cortical and limbic pathways by inflammatory processes in the cART era. Plasma sCD14 is a potential biomarker to monitor HAND progression and therapeutic responses.
Combined antiretroviral therapy (cART) does not eradicate HIV, which persists for years and can re-establish replication if treatment is stopped. The current challenge is identifying those tissues ...harboring virus through cART. Here, we used HIV env-nef single genome sequencing and HIV gag droplet digital PCR (ddPCR) to survey 50 tissues from five subjects on cART with no detectable plasma viral load at death. The spleen most consistently contained multiple proviral and expressed sequences (4/5 participants). Spleen-derived HIV demonstrated two distinct phylogenetic patterns: multiple identical sequences, often from different tissues, as well as diverse viral sequences on long terminal branches. Our results suggested that ddPCR may overestimate the size of the tissue-based viral reservoir. The spleen, a lymphatic organ at the intersection of the immune and circulatory systems, may play a key role in viral persistence.
BACKGROUND:Activated monocytes/macrophages play a role in severe forms of HIV-associated neurocognitive disorders (HAND), but little is known about the mechanisms driving milder forms that are ...prevalent despite combination antiretroviral therapy (cART). To examine relationships of monocyte activation markers to HAND of varying severity, we compared plasma and cerebrospinal fluid (CSF) biomarker levels with neurocognitive test scores in HIV+ subjects.
METHODS:Plasma and CSF soluble CD14 (sCD14), CCL2, and interleukin (IL) 6 were measured by enzyme-linked immunosorbent assay in 67 HIV+ subjects with nadir CD4 <300, and CSF inflammatory biomarkers were measured by multiplex assay in 14 subjects on suppressive cART.
RESULTS:Eighty-two percent were on cART, with 31% having undetectable plasma viral load (VL). CSF sCD14 was increased in subjects with impaired neurocognitive testing (P = 0.02), correlated inversely with global T scores in subjects with detectable but not undetectable plasma VL (P = 0.02), and yielded higher area under the receiver operating characteristic curve values for predicting impaired T scores (0.659) than plasma or CSF VL and current or nadir CD4 counts in single-marker and multivariate models. CSF sCD14, IL-6, IL-8, CCL2, CCL3, CXCL10, and interferon (IFN) gamma were increased in subjects on suppressive cART regardless of cognitive status and predicted patient class in unsupervised analyses, with IL-8, CCL2, and IFNγ explaining most of the variance.
CONCLUSIONS:CSF sCD14 is associated with impaired neurocognitive testing in patients with HIV on nonsuppressive cART, suggesting potential utility as a biomarker to monitor HAND progression. CSF sCD14, IL-6, IL-8, CCL2, CCL3, CXCL10, and IFNγ remain elevated in patients on suppressive cART regardless of cognitive status, implying ongoing intrathecal inflammation even in the absence of clinical manifestations.
Coronavirus disease 2019 (COVID-19) is emerging as the greatest public health crisis in the early 21
century. Its causative agent, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), is an ...enveloped single stranded positive-sense ribonucleic acid virus that enters cells via the angiotensin converting enzyme 2 receptor or several other receptors. While COVID-19 primarily affects the respiratory system, other organs including the brain can be involved. In Western clinical studies, relatively mild neurological dysfunction such as anosmia and dysgeusia is frequent (~70-84%) while severe neurologic disorders such as stroke (~1-6%) and meningoencephalitis are less common. It is unclear how much SARS-CoV-2 infection contributes to the incidence of stroke given co-morbidities in the affected patient population. Rarely, clinically-defined cases of acute disseminated encephalomyelitis, Guillain-Barré syndrome and acute necrotizing encephalopathy have been reported in COVID-19 patients. Common neuropathological findings in the 184 patients reviewed include microglial activation (42.9%) with microglial nodules in a subset (33.3%), lymphoid inflammation (37.5%), acute hypoxic-ischemic changes (29.9%), astrogliosis (27.7%), acute/subacute brain infarcts (21.2%), spontaneous hemorrhage (15.8%), and microthrombi (15.2%). In our institutional cases, we also note occasional anterior pituitary infarcts. COVID-19 coagulopathy, sepsis, and acute respiratory distress likely contribute to a number of these findings. When present, central nervous system lymphoid inflammation is often minimal to mild, is detected best by immunohistochemistry and, in one study, indistinguishable from control sepsis cases. Some cases evince microglial nodules or neuronophagy, strongly supporting viral meningoencephalitis, with a proclivity for involvement of the medulla oblongata. The virus is detectable by reverse transcriptase polymerase chain reaction, immunohistochemistry, or electron microscopy in human cerebrum, cerebellum, cranial nerves, olfactory bulb, as well as in the olfactory epithelium; neurons and endothelium can also be infected. Review of the extant cases has limitations including selection bias and limited clinical information in some cases. Much remains to be learned about the effects of direct viral infection of brain cells and whether SARS-CoV-2 persists long-term contributing to chronic symptomatology.
HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma HIV RNA. While combined antiretroviral therapy (cART) can reduce plasma viral load (pVL) to ...undetectable levels, the degree that HIV is eliminated from other anatomical sites remains unclear. We investigated the HIV DNA levels in 229 varied autopsy tissues from 20 HIV-positive (HIV(+)) cART-treated study participants with low or undetectable plasma VL and cerebrospinal fluid (CSF) VL prior to death who were enrolled in the National Neurological AIDS Bank (NNAB) longitudinal study and autopsy cohort. Extensive medical histories were obtained for each participant. Autopsy specimens, including at least six brain and nonbrain tissues per participant, were reviewed by study pathologists. HIV DNA, measured in tissues by quantitative and droplet digital PCR, was identified in 48/87 brain tissues and 82/142 nonbrain tissues at levels >200 HIV copies/million cell equivalents. No participant was found to be completely free of tissue HIV. Parallel sequencing studies from some tissues recovered intact HIV DNA and RNA. Abnormal histological findings were identified in all participants, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. All brain tissues demonstrated some degree of pathology. Ninety-five percent of participants had some degree of atherosclerosis, and 75% of participants died with cancer. This study assists in characterizing the anatomical locations of HIV, in particular, macrophage-rich tissues, such as the central nervous system (CNS) and testis. Additional studies are needed to determine if the HIV recovered from tissues promotes the pathogenesis of inflammatory diseases, such as HIV-associated neurocognitive disorders, cancer, and atherosclerosis.
It is well-known that combined antiretroviral therapy (cART) can reduce plasma HIV to undetectable levels; however, cART cannot completely clear HIV infection. An ongoing question is, "Where is HIV hiding?" A well-studied HIV reservoir is "resting" T cells, which can be isolated from blood products and succumb to cART once activated. Less-studied reservoirs are anatomical tissue samples, which have unknown cART penetration, contain a comparably diverse spectrum of potentially HIV-infected immune cells, and are important since <2% of body lymphocytes actually reside in blood. We examined 229 varied autopsy specimens from 20 HIV(+) participants who died while on cART and identified that >50% of tissues were HIV infected. Additionally, we identified considerable pathology in participants' tissues, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. This study substantiates that tissue-associated HIV is present despite cART and can inform future studies into HIV persistence.
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