Melissa Agsalda

Cognitive Performance Related to HIV-1-Infected Monocytes

The effect that HIV type 1 (HIV) has on neurocognition is a dynamic process whereby peripheral events are likely involved in setting the stage for clinical findings. In spite of antiretroviral therapy (ART), patients continue to be at risk for HIV-associated neurocognitive disorders (HAND), which might be related to persistence of inflammation. In a yearly assessment of HIV DNA levels in activated monocytes, increased HIV DNA copies were found in patients with persistent HAND. Furthermore, activated monocytes from patients with high HIV DNA copies secreted more inflammatory cytokines. Since these activated monocytes traffic to the CNS and enter the brain, they may contribute to an inflammatory environment in the CNS that leads to HAND.

HIV DNA Reservoir Increases Risk for Cognitive Disorders in cART-Naïve Patients

Objectives Cognitive impairment remains frequent in HIV, despite combination antiretroviral therapy (cART). Leading theories implicate peripheral monocyte HIV DNA reservoirs as a mechanism for spread of the virus to the brain. These reservoirs remain present despite cART. The objective of this study was to determine if the level of HIV DNA in CD14+ enriched monocytes predicted cognitive impairment and brain injury. Methods We enrolled 61 cART-naïve HIV-infected Thais in a prospective study and measured HIV DNA in CD14+ enriched monocyte samples in a blinded fashion. We determined HAND diagnoses by consensus panel and all participants underwent magnetic resonance spectroscopy (MRS) to measure markers of brain injury. Immune activation was measured via cytokines in cerebrospinal fluid (CSF). Results The mean (SD) age was 35 (6.9) years, CD4 T-lymphocyte count was 236 (139) and log10 plasma HIV RNA was 4.8 (0.73). Twenty-eight of 61 met HAND criteria. The log10 CD14+ HIV DNA was associated with HAND in unadjusted and adjusted models (p = 0.001). There was a 14.5 increased odds ratio for HAND per 1 log-value of HIV DNA (10-fold increase in copy number). Plasma CD14+ HIV DNA was associated with plasma and CSF neopterin (p = 0.023) and with MRS markers of neuronal injury (lower N-acetyl aspartate) and glial dysfunction (higher myoinositol) in multiple brain regions. Interpretation Reservoir burden of HIV DNA in monocyte-enriched (CD14+) peripheral blood cells increases risk for HAND in treatment-naïve HIV+ subjects and is directly associated with CSF immune activation and both brain injury and glial dysfunction by MRS.

Minimal disease assessment in the treatment of children and adolescents with intermediate-risk (Stage III/IV) B-cell non-Hodgkin lymphoma: a children’s oncology group report

Children/adolescents with mature B‐cell non‐Hodgkin lymphoma (B‐NHL) have an excellent prognosis but relapses still occur. While chromosomal aberrations and/or clonal immunoglobulin (Ig) gene rearrangements may indicate risk of failure, a more universal approach was developed to detect minimal disease (MD). Children/adolescents with intermediate‐risk B‐NHL were treated with French‐British‐American/Lymphome Malins de Burkitt 96 (FAB/LMB96) B4 modified chemotherapy and rituximab. Specimens from diagnosis, end of induction (EOI), and end of therapy (EOT) were assayed for MD. Initial specimens were screened for IGHV family usage with primer pools followed by individual primers to identify MD. Thirty‐two diagnostic/staging specimens screened positive with primer pools and unique IGHV family primers were identified. Two patients relapsed; first relapse (4 months from diagnosis) was MD‐positive at EOI, the second (36 months from diagnosis) was MD‐positive at EOT. At EOI, recurrent rates were similar between the MRD‐positive and MRD‐negative patients (P = 0·40). At EOT, only 13/32 patients had MRD data available with one relapse in the MRD‐positive group and no recurrences in the MRD‐negative group (P = 0·077). The study demonstrated molecular‐disseminated disease in which IgIGHV primer pools could be used to assess MD. This feasibility study supports future investigations to assess the validity and significance of MD screening in a larger cohort of patients with intermediate‐risk mature B‐NHL.

Chemotoxicity Recovery of Mitochondria in Non-Hodgkin Lymphoma Resulting in Minimal Residual Disease

The mechanisms responsible for resistant disease or recurrence of non‐Hodgkin lymphoma (NHL) in children cover a wide spectrum from drug resistance to genetic mutations. A unique mechanism suggesting the role of mitochondria as the key energy source is studied following a clinical observation where pediatric Burkitt lymphoma (BL) specimens from patients on therapy were found to have increased copies of mitochondria DNA (mtDNA) in specimens which were shown to be positive for minimal residual disease and/or persistent disease (MRD/PD). This study hypothesized that the mitochondria play an important role in a cells recovery from toxicity via a compensatory increase in mtDNA.

Mitochondrial DNA in residual leukemia cells in cerebrospinal fluid in children with acute lymphoblastic leukemia.

This feasibility study was designed to assess the ability to measure mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) cells that contributed to minimal disease/persistent or residual disease (MD/PRD) from children with acute lymphoblastic leukemia (ALL). Increase in mtDNA copies in cancer cells has been suggested to play a role in MD/PRD. CSF as well as blood specimens from 6 children were assayed for MD/PRD and mtDNA copy numbers by quantitative real-time polymerase chain reaction. Of 7 MD/PRD-positive specimens, 6 had increased mtDNA copy numbers; while 11 MD/PRD-negative specimens had no increase in mtDNA copy numbers, p < 0.003. This is the first proof-of-concept study to measure mtDNA copy numbers in MD/PRD-positive CSF specimens from children with ALL. Increase of mtDNA copy numbers in MD/PRD childhood ALL cells and its significance as a mechanism for recurrence requires further investigation. Keywords Minimal residual disease; Acute lymphoblastic leukemia; Central nervous system; Cerebrospinal fluid; Mitochondria

Characteristics of Activated Monocyte Phenotype Support R5-Tropic Human Immunodeficiency Virus.

BACKGROUND: Microbial translocation has been recognized as an important factor in monocyte activation and contributing to AIDS pathogenesis with elevated plasma lipopolysaccharide (LPS) levels, as a marker for microbial translocation, seen in advanced HIV disease. Therefore, the current study was undertaken to assess monocyte activation in vitro by LPS and to determine its impact on monocyte phenotype. METHODS: Monocytes from non-HIV-infected donors were analyzed for CD14, CD16, CD69, TNFα, and CCR5 by flow cytometry pre- and post-stimulation with LPS. In-vitro cultures were then set up to expose non-activated and activated monocytes to R5-, X4-, and dual (R5/X4)-tropic viruses; and the amount of HIV present on the cells was assayed. RESULTS: Non-HIV-infected monocytes, after LPS stimulation, were confirmed to have an activated phenotype with increase in CD16 and CD69 surface expressions (p<0.05). The activation phenotype was supported by increase in TNFα production, p<0.05. The activated monocytes had increased surface CCR5 (from 21% to 98%; p=0.05); and were found to have more R5-tropic virus than non-activated monocytes (p<0.05). CONCLUSIONS: Following activation by LPS, non-HIV-infected monocytes were found to have increase in surface CCR5. These activated monocytes, when exposed to R5-tropic virus, were found to have more virus compared to non-activated monocytes. The significance of the findings could lie in explaining how microbial translocation plays a role in HIV progression; and possibly promoting CCR5-directed strategies in treating HIV.

Screening for Residual Disease in Pediatric Burkitt Lymphoma Using Consensus Primer Pools

Assessing molecular persistent or minimal residual disease (PD/MRD) in childhood Burkitt lymphoma (BL) is challenging because access to original tumor is usually needed to design patient-specific primers (PSPs). Because BL is characterized by rearranged immunoglobulin heavy chain (IgVH) genes, IgVH primer pools from IgVH1–IgVH7 regions were tested to detect PD/MRD, thus eliminating the need for original tumor. The focus of the current study was to assess the feasibility of using IgVH primer pools to detect disease in clinical specimens. Fourteen children diagnosed with B-NHL had follow-up repository specimens available to assess PD/MRD. Of the 14 patients, 12 were PD/MRD negative after 2 months of therapy and remained in remission at the end of therapy; 2/14 patients were PD/MRD positive at 2-3 months and later relapsed. PSP-based assays from these 14 patients showed 100% concordance with the current assay. This feasibility study warrants further investigation to assess PD/MRD using IgVH primer pools, which could have clinical significance as a real-time assessment tool to monitor pediatric BL and possibly other B-cell non-Hodgkin lymphoma therapy.

HIV DNA in CD14+ reservoirs is associated with regional brain atrophy in patients naive to combination antiretroviral therapy.

Objective:To examine associations between regional brain volumes and HIV DNA in peripheral CD14+ cells (monocytes) among HIV-infected individuals naive to combination antiretroviral therapy (cART). Design:A prospective study of HIV-infected Thai individuals who met Thai national criteria for cART initiation. Enrolment was stratified by HIV DNA in a blinded fashion. Methods:CD14+ cells were isolated from peripheral mononuclear cells to high purity (median 91.4% monocytes by flow cytometry), and HIV DNA was quantified by multiplex real-time PCR. Baseline regional brain volumes obtained by T1-weighted 1.5-Tesla MRI were compared between HIV DNA groups using analysis of covariance (ANCOVA). Results:We studied 60 individuals with mean (SD) age of 34.7 (7.0) years, CD4+ T-lymphocyte count of 232 (137) cells/&mgr;l and log10 plasma HIV RNA of 4.8 (0.73). Median (interquartile range, IQR) HIV DNA copy number per 106 CD14+ cells was 54 (102). Using our previously determined optimal cut-point of 45 copies/106 cells for this cohort, a threshold value above which CD14+ HIV DNA identified HIV-associated neurocognitive disorders (HANDs), we found that CD14+ HIV DNA ≥ 45 copies/106 cells was associated with reduced volumes of the nucleus accumbens (P = 0.021), brainstem (P = 0.033) and total gray matter (P = 0.045) independently of age, CD4+ cell count and intracranial volume. Conclusion:HIV DNA burden in CD14+ monocytes is directly linked to brain volumetric loss. Our findings implicate peripheral viral reservoirs in HIV-associated brain atrophy and support their involvement in the neuropathogenesis of HAND, underscoring the need for therapies that target these cells.

Correction: HIV DNA Reservoir Increases Risk for Cognitive Disorders in cART-Naïve Patients

There were errors in multiple affiliations for multiple authors. Please see the following correct list of authors and affiliations: Victor G. Valcour 1,2*,Jintanat Ananworanich, 3,4,5,6, Melissa Agsalda 6, Napapon Sailasuta 7, Thep Chalermchai 3, Alexandra Schuetz 8, Cecilia Shikuma 6, Chin-Yuan Liang 6, Supunee Jirajariyavej 9, Pasiri Sithinamsuwan 10, Somporn Tipsuk 3, David B. Clifford 11, Robert Paul 12, James L. K. Fletcher 3, Mary A. Marovich 13, Bonnie M. Slike 14, Victor DeGruttola 15, Bruce Shiramizu 6 1. Memory and Aging Center, Department of Neurology, University of California, San Francisco, CA USA 2. Division of Geriatric Medicine, Department of Medicine, University of California, San Francisco CA, USA 3. SEARCH, Thai Red Cross AIDS Research Centre, Bangkok, Thailand 4. HIV-NAT, Thai Red Cross AIDS Research Center, Bangkok, Thailand 5. Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 6. Hawaii Center for AIDS, University of Hawaii, Honolulu HI USA 7. Huntington Medical Research Institutes, Pasadena CA USA 8. Department of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand 9. Taksin Hospital, Bangkok, Thailand 10. Division of Neurology, Department of Medicine, Phramongkutklao Hospital, Bangkok, Thailand 11. Department of Neurology, Washington University, St. Louis MO USA 12. Department of Psychology, University of Missouri, St. Louis MO, USA 13. Division of AIDS, National Institute of Allergy and Infection Diseases of the National Institutes of Health, Bethesda MD, USA 14. US Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring MD, USA 15. Department of Biostatistics, Harvard School of Public Health, Boston MA, USA