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1.  West Nile virus nucleic acid persistence in whole blood months after clearance in plasma: implication for transfusion and transplantation safety 
Transfusion  2014;54(12):3232-3241.
Previous reports of WNV RNA persistence in blood compartments have raised concerns around the remaining risk of WNV transfusion-transmission. This study characterized the dynamics of WNV viremia in blood compartments in a longitudinal cohort of 54 WNV-infected blood donors.
Blood samples were collected throughout the year after WNV RNA+ blood donation (index) and characterized for anti-WNV IgM and IgG antibodies and for WNV RNA by real-time reverse-transcription polymerase chain reaction. WNV viral loads were compared in plasma and whole blood samples and correlated with blood groups and clinical outcomes.
WNV RNA persisted in the red blood cell (RBC) compartment up to three months post-index in 42% of the donors. Donors with the highest WNV RNA levels in plasma at index maintained the highest WNV RNA levels in whole blood over the three months post-index. Blood group A donors maintained higher post-index WNV viral load in whole blood than blood group O individuals (P=0.027). Despite a trend for WNV RNA to persist longer in whole blood from symptomatic subjects, no significant association was found between WNV RNA levels in whole blood and disease outcome.
This study confirmed that WNV RNA persists in the RBC fraction in whole blood and further suggested that the level of persistence in whole blood may be a reflection of initial viral burden in plasma. The association with blood groups suggests that WNV adherence to RBCs may be mediated by molecules overrepresented at the surface of blood group A RBCs.
PMCID: PMC4268370  PMID: 24965017
West Nile Virus; Viral persistence; Blood screening; Blood donors; Longitudinal cohort; Plasma; Whole blood; Red Blood Cells
2.  Antiretroviral-Free HIV-1 Remission and Viral Rebound Following Allogeneic Stem Cell Transplantation: A Report of Two Cases 
Annals of internal medicine  2014;161(5):319-327.
It is unknown if the reduction in HIV-1 reservoirs observed following allogeneic hematopoietic stem cell transplantation (HSCT) with susceptible donor cells is sufficient to achieve sustained HIV-1 remission.
To characterize HIV-1 reservoirs in blood and tissues, and to perform analytical antiretroviral treatment interruptions to determine the potential for allogeneic HSCT to lead to sustained antiretroviral-free HIV-1 remission.
Characterization of HIV-1 reservoirs and immunity before and after antiretroviral interruption.
Tertiary care center.
Two HIV-infected men with undetectable HIV-1 following allogeneic HSCT for hematologic malignancies.
Quantification of HIV-1 in various tissues after HSCT and the duration of antiretroviral-free HIV-1 remission after treatment interruption.
No HIV-1 was detected from peripheral blood or rectal mucosa prior to analytical treatment interruption. Plasma HIV-1 RNA and cell-associated HIV-1 DNA remained undetectable until 12 to 32 weeks after antiretroviral cessation. Both patients experienced rebound viremia with the development of acute retroviral syndrome within one to two weeks of the most recent negative viral load measurement. One patient developed new efavirenz resistance after re-initiation of antiretroviral therapy. Re-initiation of active therapy led to viral decay and resolution of symptoms in both patients.
The study was limited to 2 patients.
Allogeneic HSCT may lead to loss of detectable HIV-1 from blood and gut tissue and variable periods of antiretroviral-free HIV-1 remission, but viral rebound can occur despite a minimum 3-log10 reduction in reservoir size. Long-lived tissue reservoirs may have contributed to viral persistence. Defining the nature and half-life of such reservoirs is essential in order to achieve durable antiretroviral-free HIV-1 remission.
PMCID: PMC4236912  PMID: 25047577
4.  Antiretroviral Therapy Initiated Within 6 Months of HIV Infection Is Associated With Lower T-Cell Activation and Smaller HIV Reservoir Size 
The Journal of Infectious Diseases  2013;208(8):1202-1211.
Background. CD4+/CD8+ T-cell activation levels often remain elevated in chronic human immunodeficiency virus (HIV) infection despite initiation of antiretroviral therapy (ART). T-cell activation predicts early death and blunted CD4+ T-cell recovery during ART and may affect persistent HIV reservoir size. We investigated whether very early ART initiation is associated with lower on-therapy immune activation and HIV persistence.
Methods. From a cohort of patients with early HIV infection (<6 months duration since infection) we identified persons who started ART early (<6 months after infection) or later (≥2 years after infection) and maintained ≥2 years of virologic suppression; at-risk HIV-negative persons were controls. We measured CD4+/CD8+ T-cell activation (percent CD38+/HLA-DR+) and HIV reservoir size (based on HIV DNA and cell-associated RNA levels).
Results. In unadjusted analyses, early ART predicted lower on-therapy CD8+ T-cell activation (n = 34; mean, 22.1%) than achieved with later ART (n = 32; mean, 28.8%; P = .009), although levels in early ART remained elevated relative to HIV-negative controls (P = .02). Early ART also predicted lower CD4+ T-cell activation than with later ART (5.3% vs 7.5%; P = .06). Early ART predicted 4.8-fold lower DNA levels than achieved with later ART (P = .005), and lower cell-associated RNA levels (difference in signal-to-cutoff ratio (S/Co), 3.2; P = .035).
Conclusions. ART initiation <6 months after infection is associated with lower levels of T-cell activation and smaller HIV DNA and RNA reservoir size during long-term therapy.
PMCID: PMC3778965  PMID: 23852127
HIV antiretroviral therapy; early ART; T-cell activation; inflammation; HIVreservoir; HIV eradication; HIV cure
5.  Fetal production of growth factors and inflammatory mediators predicts pulmonary hypertension in congenital diaphragmatic hernia 
Pediatric research  2013;74(3):290-298.
Congenital diaphragmatic hernia (CDH) represents a spectrum of lung hypoplasia and consequent pulmonary hypertension is an important cause of postnatal morbidity and mortality. We studied biomarkers at the maternal-fetal interface to understand factors associated with the persistence of pulmonary hypertension.
Maternal and cord blood samples from fetuses with CDH and unaffected controls were analyzed using a human 39plex immunoassay kit. Cellular trafficking between the mother and the fetu was quantified using quantitative real-time PCR for non-shared alleles. Biomarker profiles were then correlated with CDH severity based on the degree of pulmonary hypertension.
Cord blood levels of epidermal growth factor, platelet-derived growth factor, and several inflammatory mediators increased significantly as the severity of CDH increased, while maternal levels growth factors and mediators decreased significantly with CDH severity. Maternal cells were increased in fetuses with severe CDH compared to controls, with elevated levels of the chemokine CXCL-10 in patients with the highest trafficking.
Patients with CDH demonstrate pro-inflammatory and chemotactic signals in fetal blood at the time of birth. Since some of these molecules have been implicated in the development of pulmonary hypertension, prenatal strategies targeting specific molecular pathways may be useful adjuncts to current fetal therapies.
PMCID: PMC4164304  PMID: 23770923
7.  Cell-Based Measures of Viral Persistence Are Associated With Immune Activation and Programmed Cell Death Protein 1 (PD-1)–Expressing CD4+ T cells 
Background. Studies aimed at defining the association between host immune responses and human immunodeficiency virus (HIV) persistence during therapy are necessary to develop new strategies for cure.
Methods. We performed a comprehensive assessment of ultrasensitive plasma HIV RNA levels, cell-associated HIV RNA levels, proviral HIV DNA levels, and T cell immunophenotyping in a cohort of 190 subjects in whom HIV levels were suppressed by highly active antiretroviral therapy.
Results. The median CD4+ T cell count was 523 cells/mm3, and the median duration of viral suppression was 31 months. Cell-associated RNA and proviral DNA levels (but not ultrasensitive plasma HIV RNA levels) were positively correlated with frequencies of CD4+ and CD8+ T cells expressing markers of T-cell activation/dysfunction (CD38, HLA-DR, CCR5, and/or programmed cell death protein 1 [PD-1]) (P < .05). Having a low CD4+ T-cell count despite receipt of virologically suppressive therapy was associated with high cell-associated RNA and proviral DNA levels (P < .01) and higher frequencies of CD4+ T cells expressing CD38, HLA-DR, CCR5, and/or PD-1 (P < .0001).
Conclusions. Cell-based measurements of viral persistence were consistently associated with markers of immune activation and the frequency of PD-1–expressing CD4+ T cells. Treated patients with a low CD4+ T-cell count had higher frequencies of PD-1–expressing CD4+ T cells and cell-based measures of viral persistence, suggesting that HIV infection in these individuals may be more difficult to cure and may require unique interventions.
PMCID: PMC3666131  PMID: 23089590
HIV; raltegravir intensification; 2-LTR circles; ongoing viral replication; D-dimer
8.  Analysis of maternal microchimerism in rhesus monkeys (Macaca mulatta) using real-time quantitative PCR amplification of MHC polymorphisms 
Chimerism  2014;5(1):6-15.
Although pregnancy-associated microchimerism is known to exist in humans, its clinical significance remains unclear. Fetal microchimerism has been documented in rhesus monkeys, but the trafficking and persistence of maternal cells in the monkey fetus and infant have not been fully explored. To investigate the frequency of maternal microchimerism in the rhesus monkey (Macaca mulatta), a real-time polymerase chain reaction (PCR) strategy was developed and validated to target polymorphic major histocompatibility complex (MHC) gene sequences. Informative PCR assays were identified for 19 of 25 dams and their respective offspring. Analyses were performed on tissues (thymus, liver, spleen, lymph nodes, and bone marrow) and peripheral blood mononuclear cells (PBMCs) collected prenatally and postnatally in a subset of animals. Seven of 19 monkeys had detectable maternal microchimerism in at least one compartment (range: 0.001–1.9% chimeric cells). In tissues, maternal microchimerism was found in 2 of 7 fetuses and 3 of 12 juveniles (1–1.5 years of age), and most of the animals that were positive had microchimeric cells in more than one tissue. Maternal microchimerism was detected in PBMCs from all (4 of 4) fetuses. These observations suggest that maternal microchimerism occurs in the rhesus monkey fetus and can be detected in tissues in a subset of offspring after birth.
PMCID: PMC3988117  PMID: 24451553
major histocompatibility complex; microchimerism; quantitative PCR; rhesus monkey; transplacental transfer
10.  Transfusion Associated Microchimerism: The Hybrid Within 
Transfusion medicine reviews  2012;27(1):10-20.
Microchimerism, the coexistence of genetically disparate populations of cells in a receptive host, is well described in both clinical and physiological settings, including transplantation and pregnancy. Microchimerism can also occur following allogeneic blood transfusion in traumatically injured patients, where donor cells have been observed decades after transfusion. To date, transfusion-associated microchimerism (TA-MC) appears confined to this clinical subset, most likely due to the immune perturbations that occur following severe trauma that allow foreign donor cells to survive. TA-MC appears to be unaffected by leukoreduction and has been documented following transfusion with an array of blood products. The only significant predictor of TA-MC to date is the age of red cells, with fresher units associated with higher risk. Thus far, no adverse clinical effect has been observed in limited studies of TA-MC. There are, however, hypothesized links to transfusion-associated graft vs. host disease (TA-GvHD) that may be unrecognized and consequently under-reported. Microchimerism in other settings has gained increasing attention due to a plausible link to autoimmune diseases, as well as its diagnostic and therapeutic potential vis-a-vis ante-natal testing and adoptive immunotherapy, respectively. Furthermore, microchimerism provides a tool to further our understanding of immune tolerance and regulation.
PMCID: PMC3518667  PMID: 23102759
Microchimerism; transfusion; chimerism; trauma; immunity; immune tolerance
Journal of Pediatric Surgery  2012;47(6):1089-1094.
Bi-directional trafficking of cells between the mother and the fetus is routine in pregnancy and a component of maternal-fetal tolerance. Changes in fetal-to-maternal cellular trafficking have been reported in prenatal complications, but maternal-to-fetal trafficking has never been studied in the context of fetal intervention. We hypothesized that patients undergoing open fetal surgery would have altered maternal-fetal cellular trafficking.
Cellular trafficking was analyzed in patients with myelomeningocele (MMC) who underwent open fetal surgical repair (n=5), MMC patients who had routine postnatal repair (n=6), and normal term patients (n=9). As a control for the fetal operation, trafficking was also analyzed in patients who were delivered by an ex utero intrapartum treatment (EXIT) procedure (n=6). Microchimerism in maternal and cord blood was determined using quantitative real-time PCR for non-shared alleles.
Maternal-to-fetal trafficking was significantly increased in patients who underwent open fetal surgery for MMC compared to normal controls, postnatal MMC repair, and EXIT patients. There were no differences in fetal-to-maternal cell trafficking between groups.
Patients undergoing open fetal surgery for MMC have elevated levels of maternal microchimerism. These results suggest altered trafficking and/or increased proliferation of maternal cells in fetal blood and may have important implications for preterm labor.
PMCID: PMC3377979  PMID: 22703775
Fetal surgery; myelomeningocele; spina bifida; maternal-fetal cellular trafficking; microchimerism; preterm labor; EXIT
12.  Challenges in Detecting HIV Persistence during Potentially Curative Interventions: A Study of the Berlin Patient 
PLoS Pathogens  2013;9(5):e1003347.
There is intense interest in developing curative interventions for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV-infected adult who has exhibited evidence of cure after allogeneic hematopoietic stem cell transplant from a homozygous CCR5Δ32 donor. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV DNA or RNA was detected in peripheral blood mononuclear cells (PBMC), spinal fluid, lymph node, or terminal ileum, and no replication-competent virus could be cultured from PBMCs. However, HIV RNA was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in ART-suppressed patients. It was not possible to obtain sequence data from plasma or gut, while an X4 sequence from PBMC did not match the pre-transplant sequence. HIV antibody levels were readily detectable but declined over time; T cell responses were largely absent. The occasional, low-level PCR signals raise the possibility that some HIV nucleic acid might persist, although they could also be false positives. Since HIV levels in well-treated individuals are near the limits of detection of current assays, more sensitive assays need to be developed and validated. The absence of recrudescent HIV replication and waning HIV-specific immune responses five years after withdrawal of treatment provide proof of a clinical cure.
Author Summary
There is intense interest in developing a cure for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV+ adult who has exhibited evidence of cure after a stem cell transplant. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV was detected in blood cells, spinal fluid, lymph node, or small intestine, and no infectious virus was recovered from blood. However, HIV was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in antiretroviral treated patients. The occasional, low-level HIV signals might be due to persistent HIV or might reflect false positives. The sensitivity of the current generation of assays to detect HIV RNA, HIV DNA, and infectious virus are close to the limits of detection. Improvements in these tests will be needed for future curative studies. The lack of rebounding virus after five years without therapy, the failure to isolate infectious virus, and the waning HIV-specific immune responses all indicate that the Berlin Patient has been effectively cured.
PMCID: PMC3649997  PMID: 23671416
13.  Absence of Transfusion-Associated Microchimerism in Pediatric and Adult Recipients of Leukoreduced and Gamma Irradiated Blood Components 
Transfusion  2011;52(5):936-945.
Transfusion-associated microchimerism (TA-MC), the persistence of significant levels of donor leukocytes in blood recipients for prolonged periods, has been demonstrated following non-leukoreduced and leukoreduced transfusion to patients with severe traumatic injury. Development of TA-MC has not been rigorously studied in settings that do not involve massive trauma where the blood is leukoreduced and irradiated.
A cohort of 409 prospectively followed medical and surgical adult and pediatric female recipients of leukoreduced and mostly irradiated allogeneic red blood cell and platelet transfusions were evaluated to determine development of TA-MC. Four and eight-week post-transfusion samples were analyzed using quantitative real-time polymerase chain reaction (RT-PCR) for Y-chromosome sequences in leukocyte DNA, the marker for microchimeric cells in female blood recipients. Repeat testing was performed on Y-chromosome positive samples to confirm microchimerism (MC), and subsequent post-transfusion samples were tested to investigate persistence of MC.
On initial testing, forty of 207 (19%) adult and forty-four of 202 (22%) pediatric female blood recipients demonstrated low level MC. On repeat testing of these and additional specimens, twelve (3%) recipients demonstrated low level transient MC, but none had persistent TA-MC similar to that seen in transfused trauma patients.
Persistence of MC was not demonstrated in adult and pediatric recipients of leukoreduced and mostly irradiated blood components. The risk of TA-MC appears to be dependent on the clinical setting and is rare other than in patients sustaining severe traumatic injury.
PMCID: PMC3257351  PMID: 21981710
microchimerism; transfusion; irradiation; pediatric; blood recipients
14.  Distribution of Parvovirus B19 DNA in Blood Compartments and Persistence of Virus in Blood Donors 
Transfusion  2011;51(9):1896-1908.
Because the receptor for Parvovirus B19 (B19V) is on erythrocytes, we investigated B19V distribution in blood by in-vitro spiking experiments and evaluated viral compartmentalization and persistence in natural infection.
Two whole blood protocols (ultracentrifugation and a rapid RBC lysis/removal protocol) were evaluated using quantitative real-time PCR. Whole blood (WB) was spiked with known concentrations of B19V and recovery in various blood fractions was determined. The rapid RBC lysis/removal protocol was then used to compare B19V concentrations in 104 paired whole blood and plasma samples collected longitudinally from 43 B19V infected donors with frozen specimens in the REDS Allogeneic Donor and Recipient Repository (RADAR).
In B19V spiking experiments, ~one-third of viral DNA was recovered in plasma and two-thirds was loosely bound to erythrocytes. In the IgM positive stage of infection in blood donors when plasma B19V DNA concentrations were > 100 IU/mL, median DNA concentrations were ~30-fold higher in WB than in plasma. In contrast, when IgM was absent and when the B19V DNA concentration was lower, the median whole blood to plasma ratio was ~1. Analysis of longitudinal samples demonstrated persistent detection of B19V in WB but declining ratios of WB/plasma B19V with declining plasma VL levels and loss of IgM-reactivity.
The WB/plasma B19V DNA ratio varies by stage of infection. Further study is required to determine if this is related to the presence of circulating DNA-positive erythrocytes derived from B19V infected erythroblasts, B19V-specific IgM mediated binding of virus to cells, or other factors.
PMCID: PMC3591477  PMID: 21303368
15.  Development and Application of a High-Throughput Micro-Neutralization Assay - Lack of XMRV/MLV Detection in Blood Donors 
Transfusion  2012;52(2):332-342.
Xenotropic murine leukemia virus (MLV)-related virus (XMRV) and other related MLVs have been described with chronic fatigue syndrome (CFS) and certain types of prostate cancer. In addition, prevalence rates as high as 7% have been reported in blood donors, raising the risk of transfusion-related transmission. Several laboratories have utilized micro-neutralization assays as a surrogate marker for detection of anti-MLV serological responses – with up to 25% of prostate cancer patients reported to harbor neutralizing antibody responses.
We developed a high-throughput micro-neutralization assay for research studies on blood donors using retroviral vectors pseudotyped with XMRV-specific envelopes. Infection with these pseudotypes was neutralized by sera from both macaques and mice challenged with XMRV, but not pre-immune serum. 354 plasma samples from blood donors in the Reno/Tahoe area were screened for neutralization.
6.5% of donor samples gave moderate neutralization of XMRV, but not control pseudotypes. However, further testing by Western blot revealed no evidence of antibodies against MLVs in any of these samples. Furthermore, no evidence of infectious virus or viral nucleic acid was observed.
A micro-neutralization assay was developed for detection of XMRV, and can be applied in a high-throughput format for large scale studies. Although a proportion of blood donors demonstrated the ability to block XMRV envelope-mediated infection, we found no evidence that this inhibition was mediated by specific antibodies elicited by exposure to XMRV/MLV. It is likely that this moderate neutralization is mediated through another, non-specific mechanism.
PMCID: PMC3299481  PMID: 22239212
High-throughput micro-neutralization assay; XMRV; MLV; Pseudoviruses; Donor screening
16.  The impact of HFE mutations on haemoglobin and iron status in individuals experiencing repeated iron loss through blood donation 
British journal of haematology  2011;156(3):388-401.
Frequent blood donors become iron deficient. HFE mutations are present in over 30% of donors. A 24-month study of 888 first time/reactivated donors and 1537 frequent donors measured haemoglobin and iron status to assess how HFE mutations impact the development of iron deficiency erythropoiesis. Donors with two HFE mutations had increased baseline haemoglobin and iron stores as did those with one mutation, albeit to a lesser extent. Over multiple donations haemoglobin and iron status of donors with HFE mutations paralleled those lacking mutations. The prevalence of HFE mutations was not increased in higher intensity donors. Thus, in general, HFE mutations do not temper donation-induced changes in haemoglobin and iron status. However, in Black donors there was an increase of H63D carriers at baseline, from 3.7% in first time/reactivated donors to 15.8% in frequent donors, suggesting that the relative effects of HFE mutations on iron absorption may vary between racial/ethnic groups. In secondary analyses, venous haemoglobin decreased more slowly in donors with ferritin ≥ 12 μg/l; and haemoglobin recovery time was shorter in donors with reticulocyte haemoglobin (CHr) ≥ 32.6 pg, indicating that these biochemical measures are better indicators of a donor’s response to phlebotomy than their HFE mutation status.
PMCID: PMC3508695  PMID: 22118647
HFE; blood donor; iron deficiency; haemoglobin; ferritin
17.  The role of transplacental microtransfusions of maternal lymphocytes in HIV transmission to newborns 
AIDS (London, England)  2008;22(17):2251-2256.
Perinatal HIV transmission could occur via microtransfused maternal blood during delivery. If so, detecting maternal cells in umbilical cord blood should correlate with infection risk.
To develop sensitive assays for maternal DNA in infant's blood stored as dried blood spots (DBS) and examine the correlation between microtransfusion and perinatal HIV infection risk.
Blood-in-blood serial dilutions were prepared as DBS. Extracted DNA was amplified for unique minor-population sequences using 24 allele-specific polymerase-chain-reaction (AS-PCR) assays. Using newborns born to HIV+ mothers, paired mother-infant samples were similarly examined to identify unique maternal sequences targeted by AS-PCR of DNA extracted from cord blood DBS. Cord-blood PCR-negative infants were categorized as uninfected or perinatally infected by HIV PCR on samples collected 4–8 weeks after birth.
Sequences from added cells were detected at ≤1:1000 dilutions in 19 of 20 aliquots, and ≤1:10,000 dilutions in 7 of 20 aliquots; the median limit of detection (probit analysis) was 1 added genomic sequence in 9500 background sequences of amplifiable DNA. Maternal sequences were detected in cord-blood DBS of 50% of infected infants (N=18) and 44% of uninfected infants (N=43). Infection did not correlate with more frequent detection of maternal sequences.
This semi-quantitative assay reliably detected maternal DNA sequences in DBS at levels of less than 1:1000 cells. Maternal sequences were frequently detected but did not correlate infection risk with detection or level of maternal DNA in umbilical cord blood. Therefore we could not demonstrate that microtransfusions at parturition were responsible for perinatal HIV transmission.
PMCID: PMC3509793  PMID: 18981764
18.  The role of transplacental micro-transfusions of maternal lymphocytes in in utero HIV transmission 
The mechanisms of HIV transmission from mothers to infants are poorly understood. A possible mechanism of in utero transmission is transplacental transfer of HIV-infected maternal leukocytes into the fetal circulation during pregnancy.
To determine if the frequency of in utero HIV infection correlates with presence or levels of maternal cells (MC) in placenta-derived cord blood.
DNA was extracted from dried cord blood spots (DBS) from newborns born to HIV+ mothers and corresponding maternal DBS specimens. Paired mother-infant samples were probed to identify unique maternal sequences targeted by 24 allele-specific real-time PCR assays. Infant DBS-derived DNA was then probed in replicate analyses for non-inherited maternal allelic-sequences. Rates of detection and levels of maternal cells in DBS samples of HIV(+) and HIV(−) newborns were compared.
Of 114 mother-infant pairs with informative alleles, 38 newborns were HIV(+) and 76 HIV(−), based on detection of HIV DNA/RNA at birth. MC were detected in 23 of 38 HIV(+) newborns (60.5%) and in 47 of 76 HIV(−) newborns (61.8%). The mean and median concentrations of nucleated maternal cells in DBS for the HIV(+)/MC(+) newborns (N=23) were 0.33% and 0.27%, respectively, compared with 0.09% and 0.10% for the HIV(−)/MC(+) newborns (N=47) (Two-Sample T-test for means: p=0.78).
There was no significant difference in rates of detection or concentrations of MC in DBS between HIV(+) and HIV(−) newborns. Therefore, we could not demonstrate a correlation between MC in DBS, assumed to reflect levels of in utero maternal-fetal cell trafficking, and the risk of in utero HIV transmission.
PMCID: PMC3509795  PMID: 20683195
HIV; maternal-fetal transmission; polymerase chain reaction; PCR; microchimerism; placenta
19.  Lack of Evidence for mtDNA as a Biomarker of Innate Immune Activation in HIV Infection 
PLoS ONE  2012;7(11):e50486.
Many human immunodeficiency virus (HIV) infected individuals suffer from persistent immune activation. Chronic inflammation and immune dysregulation have been associated with an increased risk of age-related diseases even among patients on highly active antiretroviral therapy. The factors leading to immune activation are complex, but have been hypothesized to include persistent viral replication with cellular death as well as microbial translocation across the gastrointestinal tract. Both processes may trigger innate immune responses since many native molecules released from dying cells are similar in structure to pathogen associated molecular patterns. These damage associated molecular patterns include mitochondrial DNA and formylated peptides. We hypothesized that circulating mitochondrial nucleic acid could serve as a biomarker for HIV-associated cell death and drive innate immune activation in infected individuals. We developed a quantitative polymerase chain reaction assay for plasma mitochondrial DNA and validated it on normal blood donors. We then measured mitochondrial DNA levels in acute and chronic HIV infection. While the assay proved to be accurate with a robust dynamic range, we did not find a significant association between HIV disease status and circulating mitochondrial DNA. We did, however, observe a negative correlation between age and plasma mitochondrial DNA levels in individuals with well-controlled HIV.
PMCID: PMC3510194  PMID: 23209754
20.  Absence of reproducibly detectable low-level HIV viremia in highly exposed seronegative men and women. 
AIDS (London, England)  2011;25(5):619-623.
Transient HIV infections have been invoked to account for the cellular immune responses detected in highly virus-exposed individuals who have remained HIV seronegative. We tested for very low levels of HIV RNA in 524 seronegative plasma samples from 311 highly exposed women and men from 3 longitudinal HIV cohorts.
2073 transcription mediated amplification (TMA) HIV RNA tests were performed for an average of 3.95 TMA assays per plasma sample. Quadruplicate TMA assays, analyzing a total of 2 ml of plasma, provided an estimated sensitivity of 3.5 HIV RNA copies/ml.
Four samples from subjects who did not sero-convert within the following six months were positive for HIV RNA. For one sample, human polymorphism DNA analysis indicated a sample mix up. Borderline HIV RNA detection signals were detected for the other three positive samples and further replicate TMA testing yielded no positive results. Nested PCR assays (n=254) for HIV proviral DNA on PBMC from these 3 subjects were negative.
Transient viremia was not reproducibly detected in highly HIV exposed seronegative men and women. If transient infections do occur, plasma HIV RNA levels may remain below the detection limits of the sensitive assay used here, be of very short duration, or viral replication may be restricted to mucosal surfaces or their draining lymphoid tissues.
PMCID: PMC3458706  PMID: 21297421
21.  A Randomized, Controlled Trial of Raltegravir Intensification in Antiretroviral-treated, HIV-infected Patients with a Suboptimal CD4+ T Cell Response 
The Journal of Infectious Diseases  2011;203(7):960-968.
Background. Some human immunodeficiency virus (HIV)–infected individuals are not able to achieve a normal CD4+ T cell count despite prolonged, treatment-mediated viral suppression. We conducted an intensification study to assess whether residual viral replication contributes to replenishment of the latent reservoir and whether mucosal HIV-specific T cell responses limit the reservoir size.
Methods. Thirty treated subjects with CD4+ T cell counts of <350 cells/mm3 despite viral suppression for ≥1 year were randomized to add raltegravir (400 mg twice daily) or matching placebo for 24 weeks. The primary end points were the proportion of subjects with undetectable plasma viremia (determined using an ultrasensitive assay with a lower limit of detection of <.3 copy/mL) and a change in the percentage of CD38+HLA-DR+CD8+ T cells in peripheral blood mononuclear cells (PBMCs).
Results. The proportion of subjects with undetectable plasma viremia did not differ between the 2 groups (P = .42). Raltegravir intensification did not have a significant effect on immune activation or HIV-specific responses in PBMCs or gut-associated lymphoid tissue.
Conclusions. Low-level viremia is not likely to be a significant cause of suboptimal CD4+ T cell gains during HIV treatment.
Clinical Trials Registration. NCT00631449.
PMCID: PMC3068029  PMID: 21402547
22.  HIV-Specific CD4+ T Cells May Contribute to Viral Persistence in HIV Controllers 
Among HIV controllers, higher activated and HIV-specific CD4+ T cell frequencies were strongly associated with a greater burden of pro-viral DNA, suggesting that the very immune response helping control viral replication may be contributing to viral persistence.
Background. Human immunodeficiency virus (HIV)–-infected individuals maintaining plasma HIV RNA levels <75 copies/mL in the absence of therapy (“HIV controllers”) often maintain high HIV-specific T cell responses, which likely contribute to the control of viral replication. Despite robust immune responses, these individuals never eradicate HIV infection. We hypothesized that HIV-specific CD4+ T cells might serve as target cells for HIV, contributing to viral persistence in this setting.
Methods. We measured frequencies of activated (CD38+ HLA-DR+) and HIV Gag-specific CD4+ and CD8+ T cells and plasma- and cell-associated levels of HIV RNA and DNA in a cohort of 38 HIV controllers.
Results. Although there was no evidence of a relationship between the extent of low-level viremia and the frequency of either activated or HIV-specific CD4+ T cells, controllers with higher HIV-specific CD4+ T cell frequencies had higher cell-associated HIV DNA levels (ρ = 0.53; P = .019). Higher activated CD4+ T cell frequencies were also associated with higher levels of cell-associated DNA (P = .027) and RNA (P = .0096). However, there was no evidence of a relationship between cell-associated HIV RNA or DNA levels and HIV-specific CD8+ T cell frequencies.
Conclusions. These data support a model in which strong HIV-specific CD4+ T cell responses in HIV controllers, while contributing to a potent adaptive immune response, may also contribute to viral persistence, preventing the natural eradication of HIV infection.
PMCID: PMC3060894  PMID: 21245154
23.  Male microchimerism in peripheral blood leukocytes from women with multiple sclerosis 
Chimerism  2011;2(1):6-10.
Fetal microchimerism (F-MC), the persistence of fetal cells in the mother, is frequently encountered following pregnancy. The high prevalence of F-MC in autoimmune disease prompts consideration of the role for immune tolerance and regulation. This study examines the association between F-MC and multiple sclerosis (MS), an autoimmune disorder, of undetermined etiology.
21 out of 51 MS-positive subjects (41%) were classified as positive for F-MC; 4 of 22 (18%) of MS-negative sibling controls, were also positive for MC (p = 0.066). Unanticipated F-MC in controls lead to re-evaluation using 30 female singleton cord blood units (CBUs) as a biological control. Four CBUs were low-level positive.
Study Design and Methods:
Seventy-three female subjects were assigned to three groups according to disease status and pregnancy history: (1) MS positive (+) women with a history of one male pregnancy before symptom onset (n = 27); (2) MS negative (−) female siblings of MS+ women with a history of one male pregnancy (n = 22); and (3) MS+ women that reported never having been pregnant (n = 24). Ten micrograms of genomic DNA obtained from peripheral blood leukocytes of each subject were analyzed for F-MC using allele-specific real-time PCR targeting the SR-Y sequence on the Y-chromosome. MC classification was dichotomous (positive vs. negative) based on PCR results.
The association between F-MC and MS warrants further study to define this relationship. F-MC in women self-reporting as nulligravid, supports previous findings that a significant proportion of pregnancies go undetected. This lead to re-validation of a Y-chromosome based assay for F-MC detection.
PMCID: PMC3084949  PMID: 21547029
multiple sclerosis; microchimerism; fetal cells; autoimmune disease; twinning; pregnancy
24.  No Evidence for XMRV Nucleic Acids, Infectious Virus or Anti-XMRV Antibodies in Canadian Patients with Chronic Fatigue Syndrome 
PLoS ONE  2011;6(11):e27870.
The gammaretroviruses xenotropic murine leukemia virus (MLV)-related virus (XMRV) and MLV have been reported to be more prevalent in plasma and peripheral blood mononuclear cells of chronic fatigue syndrome (CFS) patients than in healthy controls. Here, we report the complex analysis of whole blood and plasma samples from 58 CFS patients and 57 controls from Canada for the presence of XMRV/MLV nucleic acids, infectious virus, and XMRV/MLV-specific antibodies. Multiple techniques were employed, including nested and qRT-PCR, cell culture, and immunoblotting. We found no evidence of XMRV or MLV in humans and conclude that CFS is not associated with these gammaretroviruses.
PMCID: PMC3219700  PMID: 22114717
25.  Evidence of Persistent Low-level Viremia in Long-term HAART-suppressed, HIV-Infected Individuals 
AIDS (London, England)  2010;24(16):2535-2539.
Highly active antiretroviral therapy (HAART) can effectively reduce plasma HIV RNA levels to below the level of detection in most HIV-infected patients. The degree to which residual low-level viremia persists during HAART remains unclear.
We identified 180 subjects (median duration of HIV infection 12 years) who had ≥2 consecutive plasma HIV-1 RNA levels below the level of detection (<50-75 copies/mL) while taking antiretroviral drugs; 36/180 had been virologically suppressed for >5 years. Longitudinal plasma samples that were taken from these subjects during periods of viral load suppression were selected and analyzed. The isothermal Transcription Mediated Amplification (TMA) (limit of detection <3.5 copies RNA/mL) assay was used to measure persistent viremia. A “detuned” EIA assay was used to obtain quantitative HIV antibody levels.
A total of 1606 TMA assays were performed on 438 specimens in 180 HAART-suppressed subjects (median 3 replicates per specimen). In the first year of viral suppression, plasma RNA levels declined significantly (p=0.001), but after month 12 there was no evidence for a continued decline (p=0.383). In the first year of viral suppression, HIV antibody levels also declined (p=0.054), but after month 12 there was no evidence for a continued decline (p=0.988).
Viremia continued to decline during the first 12 months after viremia became undetectable using conventional methods, and then remained stable. HIV antibody levels also decreased in the first year of viral suppression and then remained stable. Viremia and the HIV-associated host response appear to achieve a steady-state “set-point” during long-term combination therapy.
PMCID: PMC2954261  PMID: 20651585
Residual viremia; HAART-suppressed; HIV antibody levels

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