Raman-Enhanced Spectroscopy (RESpect) Probe for Childhood Non-Hodgkin Lymphoma.

Raman-enhanced spectroscopy (RESpect) probe, which enhances Raman spectroscopy technology through a portable fiber-optic device, characterizes tissues and cells by identifying molecular chemical composition showing distinct differences/similarities for potential tumor markers or diagnosis. In a feasibility study with the ultimate objective to translate the technology to the clinic, a panel of pediatric non-Hodgkin lymphoma tissues and non-malignant specimens had RS analyses compared between standard Raman spectroscopy microscope instrument and RESpect probe. Cryopreserved tissues were mounted on front-coated aluminum mirror slides and analyzed by standard Raman spectroscopy and RESpect probe. Principal Component Analysis revealed similarities between non-Hodgkin lymphoma subtypes but not follicular hyperplasia. Standard Raman spectroscopy and RESpect probe fingerprint comparisons demonstrated comparable primary peaks. Raman spectroscopic fingerprints and peaks of pediatric non-Hodgkin lymphoma subtypes and follicular hyperplasia provided novel avenues to pursue diagnostic approaches and identify potential new therapeutic targets. The information could inform new insights into molecular cellular pathogenesis. Translating Raman spectroscopy technology by using the RESpect probe as a potential point-of-care screening instrument has the potential to change the paradigm of screening for cancer as an initial step to determine when a definitive tissue biopsy would be necessary.

Raman-Enhanced Spectroscopy Distinguishes Anal Squamous Intraepithelial Lesions in Human Immunodeficiency Virus-Serodiscordant Couples.

HIV-positive individuals are at increased risk for precancerous anal squamous intraepithelial lesions (SILs). Anal cytology and digital rectal examination are performed as screening tools, but extensive training and appropriate instruments are required to follow up on an abnormal anal cytology. Thus, novel approaches to SIL evaluation could improve better health care follow-up by efficient and timely diagnosis to offer treatment options. Recently, Raman-enhanced spectroscopy (RESpect) has emerged as a potential new tool for early identification of SIL. RESpect is a noninvasive, label-free, laser-based technique that identifies molecular composition of tissues and cells. HIV-serodiscordant couples had anal biopsies obtained during high-resolution anoscopy. RESpect was performed on the specimens. Principal component analysis of the data identified differences between normal and abnormal tissue as well as HIV-positive and HIV-negative individuals of each couple even with similar pathologies. RESpect has the potential to change the paradigm of anal pathology diagnosis and could provide insight into different pathways leading to SIL in HIV-serodiscordant couples.

Elevated cerebrospinal fluid Galectin-9 is associated with central nervous system immune activation and poor cognitive performance in older HIV-infected individuals.

We previously reported that galectin-9 (Gal-9), a soluble lectin with immunomodulatory properties, is elevated in plasma during HIV infection and induces HIV transcription. The link between Gal-9 and compromised neuronal function is becoming increasingly evident; however, the association with neuroHIV remains unknown. We measured Gal-9 levels by ELISA in cerebrospinal fluid (CSF) and plasma of 70 HIV-infected (HIV+) adults stratified by age (older > 40 years and younger < 40 years) either ART suppressed or with detectable CSF HIV RNA, including a subgroup with cognitive assessments, and 18 HIV uninfected (HIV-) controls. Gal-9 tissue expression was compared in necropsy brain specimens from HIV- and HIV+ donors using gene datasets and immunohistochemistry. Among older HIV+ adults, CSF Gal-9 was elevated in the ART suppressed and CSF viremic groups compared to controls, whereas in the younger group, Gal-9 levels were elevated only in the CSF viremic group (p < 0.05). CSF Gal-9 positively correlated with age in all groups (p < 0.05). CSF Gal-9 tracked with CSF HIV RNA irrespective of age (ß = 0.33; p < 0.05). Higher CSF Gal-9 in the older viremic HIV+ group correlated with worse neuropsychological test performance scores independently of age and CSF HIV RNA (p < 0.05). Furthermore, CSF Gal-9 directly correlated with myeloid activation (CSF-soluble CD163 and neopterin) in both HIV+ older groups (p < 0.05). Among HIV+ necropsy specimens, Gal-9 expression was increased in select brain regions compared to controls (p < 0.05). Gal-9 may serve as a novel neuroimmuno-modulatory protein that is involved in driving cognitive deficits in those aging with HIV and may be valuable in tracking cognitive abnormalities.

In Vitro Blood-Brain Barrier Modeling adapted for Peripheral Blood Mononuclear Cell Transmigration from HIV-Positive Patients for Clinical Research on Therapeutic Drug Intervention.

OBJECTIVE: HIV-associated cognitive impairment (HACI) continues to persist for HIV-seropositive individuals who are on antiretroviral therapy (ART). HACI develops in part when HIV-infected monocytes (MOs) transmigrate through the blood-brain barrier (BBB) and secrete pro-inflammatory cytokines and chemokines, which leads to neuronal damage. In vitro BBB models are important tools that can elucidate mechanisms of MO transmigration. Previously described in vitro BBB models relied on pathology specimens, resulting in potentially variable and inconsistent results. This project reports on a reliable and consistent alternative in vitro BBB model that has the potential to be used in clinical research intervention studies analyzing the effects of ART on the BBB and on MO transmigration. METHODS: A bilayer BBB model was established with commercially available astrocytes and endothelial cells on a 3µm PET membrane insert to allow the contact of astrocytic foot processes with endothelial cells. Inserts were cultured in growth medium for 7 days before exposure to HIV- or HIV+ peripheral blood mononuclear cells (PBMCs). PBMCs were allowed to transmigrate across the BBB for 24 hours. RESULTS: Confluency and integrity measurements by trans-endothelial electrical resistance (TEER) (136.7 ± 18.3Ω/cm2) and permeability (5.64 ± 2.20%) verified the integrity of the in vitro BBB model. Transmigrated MOs and non-MOs were collected and counted (6.0x104 MOs; 1.1x105 non-MOs). Markers indicative of glial fibrillary acidic protein (GFAP), von Willebrand factor (vWF), and p-glycoprotein (Pgp) were revealed in immunofluorescence staining (IF), indicating BBB phenotype and functionality. CONCLUSION: Potential applications for this model include assessing the HIV DNA copy numbers of transmigrated cells (pre- and post-targeted ART) and understanding the role of oxidative stress related to HIV DNA and HACI.

Human Papillomavirus-16 DNA Quantitation Differentiates High-Grade Anal Neoplasia.

Background: Due to their higher rates of anal dysplasia/cancer, human immunodeficiency virus (HIV)-positive individuals are recommended to undergo anal dysplasia screening, which consists of anal cytology (AC) and high resolution anoscopy (HRA) with anal biopsy (AB) after abnormal AC result. However, AC variability limits its usefulness. Our objective was to evaluate human papillomavirus (HPV)-16 DNA quantitation as part of the screening algorithm. Methods: HPV-16 was detected in AC specimens from 75 HIV-positive participants using quantitative real-time polymerase chain reaction. AB results were available from 18/44 patients who had abnormal AC. Statistical tests included Mann-Whitney U, Kruskal-Wallis, receiver operating characteristic (ROC) analysis and Kappa coefficient tests. Results: HPV-16 copy numbers differed significantly across AC (p = 0.001) and AB grades (p = 0.009). HPV-16 ≥ 65 copies/cell predicted high-grade AB (p = 0.04). Using this cut-off in comparison to AB, it had better specificity (1.00) than AC (0.75) and specificity (0.77) than qualitative HPV-16 detection (0.38). Also, the Kappa coefficient of the cut-off (κ = 0.649) was higher than AC (κ = 0.557) and qualitative HPV-16 detection (κ = 0.258) to AB. Conclusion: Higher HPV-16 copy numbers corresponded to higher AC and AB grades, suggesting the importance of HPV burden on disease stage. Furthermore, HPV-16 ≥ 65 copies/cell distinguished high-grade disease and demonstrated better sensitivity, specificity, and agreement with AB than AC or qualitative HPV-16 detection. These results support the potential use of HPV quantitation in conjunction with AC in anal dysplasia screening.

Brief Report: CD14+ Enriched Peripheral Cells Secrete Cytokines Unique to HIV-Associated Neurocognitive Disorders.

Monocytes play a vital role in HIV-associated neurocognitive disorder (HAND), postulated to transport HIV into the brain and secrete pro-inflammatory cytokines. We analyzed cytokines released by cultured peripheral blood mononuclear cells enriched with the CD14 marker isolated from HIV-infected individuals with HAND and normal cognition (NC) in combination antiretroviral therapy naive and after 1 year on treatment. Interleukin-8 and monocyte chemoattractant protein-1 levels were higher in HAND compared with NC at baseline (P = 0.002 and P < 0.0001). These cytokines remained higher in HAND patients 1 year after combination antiretroviral therapy and were significant when NC patients who were initially HAND were excluded (P = 0.012 and P = 0.002). Both correlated with baseline CD14 peripheral blood mononuclear cell HIV DNA levels supporting the role of HIV DNA reservoir size and monocyte cytokines in HAND persistence.

HIV DNA Is Frequently Present within Pathologic Tissues Evaluated at Autopsy from Combined Antiretroviral Therapy-Treated Patients with Undetectable Viral Loads.

UNLABELLED: 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. IMPORTANCE: 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.

Loss of CCR2 expressing non-classical monocytes are associated with cognitive impairment in antiretroviral therapy-naïve HIV-infected Thais.

HIV DNA in monocytes has been linked to HIV-associated neurocognitive disorders (HAND), however, characterization of monocyte subsets associated with HAND remains unclear. We completed a prospective study of antiretroviral therapy-naïve, HIV-infected Thais, with varying degrees of cognitive impairment, compared to HIV-uninfected controls. Monocyte subsets' CCR2, CCR5 and CD163 expression were profiled and inflammatory markers in plasma and cerebrospinal fluid (CSF), measured. Lower numbers of CCR2(+)non-classical monocytes were associated with worse neuropsychological test performance (r=0.43, p=0.024). CCR2(+)non-classical monocyte count inversely correlated with CSF neopterin (r=-0.43, p=0.035) and plasma TNF-α levels (r=-0.40, p=0.041). These data benchmark CCR2(+)non-classical monocytes as an independent index of cognitive impairment.

Impact of persistent minimal residual disease post-consolidation therapy in children and adolescents with advanced Burkitt leukaemia: a Children's Oncology Group Pilot Study Report.

Patient-specific primers from 10 children/adolescents with Burkitt leukaemia (BL) ± central nervous system disease who were treated with French-British-American/Lymphome Malins de Burkitt 96 C1 plus rituximab were developed from diagnostic blood/bone marrow. Minimal residual disease (MRD) was assessed by real-time polymerase chain reaction at the end of induction (EOI) and consolidation (EOC). Seventy per cent (7/10) and 71% (5/7) were MRD-positive at EOI and EOC, respectively, with no disease recurrences. MRD after induction and consolidation did not predict relapse and subsequent therapy appeared to eliminate MRD. Thus, assessing MRD at a later time point is warranted in future trials to determine its clinical significance.

Different levels of HIV DNA copy numbers in cerebrospinal fluid cellular subsets.

Inequities in the incidence of HIV infection and AIDS with continued persistence of HIV-associated neurocognitive disorders (HAND) exist in populations in Hawaii (HI) and Puerto Rico (PR). We previously reported that peripheral monocyte HIV DNA levels are high in patients in Hawaii with HAND and we now hypothesize that similar findings would be observed in the cerebrospinal fluid (CSF) cellular subsets. Cerebrospinal fluid cells were obtained from patients from PR and HI undergoing neurocognitive testing and sorted into monocytes (CD14+) and lymphocytes (CD14-) and HIV DNA was measured. From six PR subjects (three HAND, three normal cognition, NC) and six HI subjects (three HAND, three NC), HIV DNA burden in CD14+ cells was higher in HAND than NC patients; NC patients had higher HIV DNA burden in CD14-cells versus HAND. Differences in HIV DNA burden in particular CSF cellular subsets suggest that HIV DNA burden may play a role in HAND neuropathogenesis.

Presence of high-risk human papillomavirus genotype and human immunodeficiency virus DNA in anal high-grade and low-grade squamous intraepithelial lesions.

Human immunodeficiency virus type 1 (HIV)-infected individuals are at risk for anal cancer, which is caused by human papillomavirus (HPV). The relationship between HIV and HPV that leads to anal cancer remains unclear. Recent data, however, suggest that the continued persistence of HIV DNA in patients treated with combined antiretroviral therapy leads to progression of HIV disease and other HIV-associated complications. Therefore, we investigated the relationship among anal low- and high-grade squamous intraepithelial lesions (LGSIL/HGSIL), high-risk HPV genotypes, and high HIV DNA copy numbers. Anal cytology specimens were assayed for HPV genotype and HIV DNA copy number. High-risk HPV genotypes (odds ratio OR: 3.73; 95% confidence interval CI: 1.08-12.91; p=0.04) and high HIV DNA copy numbers (OR(per 100 HIV DNA copies): 1.13; 95% CI: 1.01-1.27, p=0.04) were both associated with LGSIL/HGSIL. When considering both high-risk HPV genotypes and HIV DNA copy numbers in predicting LGSIL/HGSIL, HIV DNA copy number was significant (OR(per 100 HIV DNA copies): 1.09; 95% CI: 0.96-1.23, p=0.04) but not high-risk HPV genotypes (OR: 2.30, p=0.28), which did not change when adjusted for nadir CD4 cell count and HIV RNA levels. The findings warrant further investigation of HIV DNA and its relationship with HPV in LGSIL/HGSIL pathogenesis.