Peripheral Blood CD34 Cells Research Articles

The effect of antiretroviral intensification with dolutegravir on residual virus replication in HIV-infected individuals: a randomised, placebo-controlled, double-blind trial

Whether ongoing virus replication occurs in HIV-infected individuals on antiretroviral therapy (ART) is unclear; therefore, whether residual virus replication is a barrier to achieving a cure for HIV is also unknown. We aimed to establish whether ART intensification with dolutegravir would reveal or affect residual virus replication in HIV-infected individuals on suppressive treatment. In this randomised, placebo-controlled, double-blind trial, we enrolled HIV-infected adults (aged 18 years and older) receiving combination ART (at least three agents) for at least 3 years from the Alfred Hospital and Melbourne Sexual Health Centre, Melbourne, VIC, Australia. Eligible participants had fewer than 50 copies per mL HIV-1 plasma RNA for more than 3 years and fewer than 20 copies per mL at screening and two CD4 counts higher than 350 cells per μL in the previous 24 months including screening. Participants were randomly assigned (1:1) to receive 50 mg oral dolutegravir or placebo once a day for 56 days in addition to background ART. Follow-up was done at days 1, 3, 7, 14, 28, 56, and 84. The primary outcome was the change from baseline in frequency of 2-long terminal repeat (2-LTR) circles in peripheral blood CD4 cells at day 7. This trial is registered with ClinicalTrials.gov, number NCT02500446. Between Sept 21, 2015, and Sept 19, 2016, 46 individuals were screened for inclusion. 40 were eligible for inclusion and were randomly assigned to the dolutegravir (n=21) or placebo group (n=19). All enrolled participants completed the study procedures and no individuals were lost to follow up. All participants were on suppressive ART with 12% receiving protease inhibitors and the others non-nucleoside reverse transcriptase inhibitors. Median 2-LTR circles fold-change from baseline to day 7 was -0·17 (IQR -0·90 to 0·90) in the dolutegravir group and -0·26 (-1·00 to 1·17) in the placebo group (p=0·17). The addition of dolutegravir to pre-existing ART regimens was safe and there were no treatment discontinuations or treatment-related serious adverse events. Our findings show that in HIV-infected individuals on modern suppressive ART regimens, residual replication is rare, if at all present, and was not recorded in blood after dolutegravir intensification. Because tissue biopsies were not done we cannot exclude the possibility of residual virus replication in tissue. Strategies other than ART alone are needed to eliminate HIV persistence on treatment. ViiV Healthcare.

Prognostic Models for 9-Month Mortality in Tuberculous Meningitis.

Tuberculous meningitis (TBM) is the most severe form of extrapulmonary tuberculosis. We developed and validated prognostic models for 9-month mortality in adults with TBM, with or without human immunodeficiency virus (HIV) infection. We included 1699 subjects from 4 randomized clinical trials and 1 prospective observational study conducted at 2 major referral hospitals in Southern Vietnam from 2001-2015. Modeling was based on multivariable Cox proportional hazards regression. The final prognostic models were validated internally and temporally and were displayed using nomograms and a Web-based app (https://thaole.shinyapps.io/tbmapp/). 951 HIV-uninfected and 748 HIV-infected subjects with TBM were included; 219 of 951 (23.0%) and 384 of 748 (51.3%) died during 9-month follow-up. Common predictors for increased mortality in both populations were higher Medical Research Council (MRC) disease severity grade and lower cerebrospinal fluid lymphocyte cell count. In HIV-uninfected subjects, older age, previous tuberculosis, not receiving adjunctive dexamethasone, and focal neurological signs were additional risk factors; in HIV-infected subjects, lower weight, lower peripheral blood CD4 cell count, and abnormal plasma sodium were additional risk factors. The areas under the receiver operating characteristic curves (AUCs) for the final prognostic models were 0.77 (HIV-uninfected population) and 0.78 (HIV-infected population), demonstrating better discrimination than the MRC grade (AUC, 0.66 and 0.70) or Glasgow Coma Scale score (AUC, 0.68 and 0.71) alone. The developed models showed good performance and could be used in clinical practice to assist physicians in identifying patients with TBM at high risk of death and with increased need of supportive care.

ID:3003 Increase in CD34 cells in peripheral blood following HLA randomized, ABO-matched cord blood transfusion in patients with advanced malignancy and anemia: Prognostic implications

Cord blood is a safe alternative of adult blood for transfusion given its content of fetal and adult haemoglobin, platelet and WBC counts, and a plasma filled with inflammatory and non-inflammatory cytokines and growth factors. It also has a hypoantigenic nature and altered metabolic profile and is rich in haematopoietic stem cells (CD34) . It has potential for treatment of anemia in diseases of different aetiologies including malignancy. 
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 STUDY DESIGN:
 In the present study, 46 sex and HLA-randomized patients suffering from advanced malignancy and anemia (Hb 8 Gm percent or less) were treated with placental cord blood transfusion. CD34 cells were collected from peripheral blood 72 hours after transfusion and variations were analyzed for prognostic implication. The study period was between 16 August 1999 and 16 May 2006.
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 RESULTS AND ANALYSIS:
 Among the forty-six cases included in this study , Case no 2 (breast sarcoma) received the lowest amount of card blood (6 units), while Case no 46 (breast cancer) received the largest amount (32 units). The youngest patient, suffering from non-Hodgkin's lymphoma (Case 3), was a 16-year-old boy who received eight units of cord blood to combat anemia. Other patients received amounts varying from 7-15 units: Case 14 received 15 units (metachronous lymph node metastatsis), Case 19 received 14 units (breast cancer), and Case 25 received seven units (lung cancer).
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 There was no transfusion-related visible immunological or nonimmunological reaction in any patient. Studies of CD34 levels after 72 hours of transfusion showed an initial rise followed by a fall in twenty-two cases, two cases registered very little effect on the CD34 level, i.e., no change from the baseline, and the rest of the cases demonstrated a sustained rise from the baseline, i.e, 56% to 99% of peripheral mononuclear cells and a sustained high CD34 level.
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 CONCLUSION:
 This preliminary study indicates that freshly collected cord blood transfusion may cause a transient transplant impact of transfused cord blood CD34 stem cells on the host without provoking clinical graft vs host disease perhaps due to a possible background structural or functional immune suppression in advanced malignancy.
 From the cord blood transfusion impact we could clinically divide the enrolled advanced cancer patients to a relatively good (Group A) and bad prognostic(Group B) variety of cancer. The survival rate of patients where we detected a persistant rise of CD34 in peripheral blood after 3 units or more of transfusion of HLA randomized ABO matched and screened cord blood was over 3 years and the cases where there are no rise of peripheral blood CD34 level in spite of repeated transfusion of 5- 32 units of cord blood died within 3 months or earlier (p value less than .01)

Immunologic and genomic characterization of high grade serous ovarian cancer (HGSOC) in patients (pts) treated with pembrolizumab (Pembro) in the phase II INSPIRE trial.

5581 Background: Checkpoint inhibitors have shown to be effective in different tumors and are under investigation in HGSOC. Methods: INSPIRE (NCT02644369) is a prospective multi-cohort study investigating tumor genomic and immune landscapes in pts treated with Pembro at 200 mg IV Q3W. Patients underwent tumor biopsy pre, on-treatment and at progression for DNA/RNA sequence, immune-profile, and PD-L1 expression by immunohistochemistry (IHC). Serial blood samples for immunophenotyping were collected. Correlative data are available for 6 pts: 3 with shrinkage in target lesion and 3 with progressive disease (PD). Results: At interim analysis as of January 2017, 18 pts with HGSOC have been enrolled and 16 have platinum-resistant disease, with median 3 prior lines of treatment (range 1-7). Of 14 evaluable pts, best response by RECIST 1.1 was stable disease (SD) in 5 (36%) and PD in 9 (64%). Mean Tumor Proportion Score of PD-L1 by IHC (Qualtek) was 6.4% (range 0-30%). Grade 3/4 adverse events possibly related to Pembro were observed in 4/18 (22%) pts; none was fatal and the most common were fatigue and hyponatremia. Preliminary correlative data showed no significant change in CD4, CD8 and myeloid-derived suppressor cells in peripheral blood after Pembro treatment. Mean PD-1 expression on CD4 and CD8 T cells on baseline tumor tissue (measured as product of PD-1+ cells and the per cell expression of PD-1 [% of mean fluorescence intensity]) was significantly higher in pts with tumor shrinkage compared to pts with PD (CD4: 2658 vs 678, p = .02; CD8: 1999 vs 451, p = .048). Genomic analysis of baseline tumor tissue was available for 3 pts with tumor shrinkage and 2 with PD. Mean mutation burden was higher for pts with tumor shrinkage (2.38 vs 1.0 mutations/Mb covered). The pt with the longest SD in our cohort (6 months) had the highest mutation burden (2.72), including somatic POLE (c.6331-6C > G) and germline BRCA2mutations. Conclusions: In HGSOC, pts with higher PD-1 level on tumor CD4 and CD8 T cells and higher mutation burden at baseline may have a better outcome following treatment with Pembro. POLE mutation is rare in HGSOC but may correlate with checkpoint inhibitor activity. Clinical trial information: NCT02644369.

Circulating immune cell phenotype can predict the outcome of lenalidomide plus low-dose dexamethasone treatment in patients with refractory/relapsed multiple myeloma.

Although the antimyeloma effect of lenalidomide is associated with activation of the immune system, the exact in vivo immunomodulatory mechanisms of lenalidomide combined with low-dose dexamethasone (Len-dex) in refractory/relapsed multiple myeloma (RRMM) patients remain unclear. In this study, we analyzed the association between immune cell populations and clinical outcomes in patients receiving Len-dex for the treatment of RRMM. Peripheral blood samples from 90 RRMM patients were taken on day 1 of cycles 1 (baseline), 2, 3, and 4 of Len-dex therapy. Peripheral blood CD3(+), CD4(+), and CD8(+) cell frequencies were significantly decreased by 3 cycles of therapy, whereas NK cell frequency was significantly increased after the 3rd cycle. For the myeloid-derived suppressor cell (MDSC) subset, the frequency of granulocytic MDSCs transiently increased after the 1st cycle, whereas there was an increase in monocytic MDSC (M-MDSC) frequency after the 1st and 3rd cycles. Among 81 evaluable patients, failure to achieve a response of VGPR or greater was associated with a decrease in CD8(+) cell frequency and increase in M-MDSC frequency after 3 cycles of Len-dex treatment. A high proportion of natural killer T (NKT)-like cells (CD3(+)/CD56(+)) prior to Len-dex treatment might predict a longer time to progression. In addition, patients with a smaller decrease in the frequency of both CD3(+) cells and CD8(+) cells by 3 cycles exhibited a longer time to the next treatment. These results demonstrated that early changes in immune cell subsets are useful immunologic indicators of the efficacy of Len-dex treatment in RRMM.

562. Fetal Hemoglobin Expression Can Be Increased by Targeted Disruption of the Gamma Hemoglobin Promoter in Human Peripheral Blood Stem Cells

Hemoglobinopathies such as sickle cell disease and β-thalassemia are both common and morbid inherited genetic disorders. Mutations in the β -globin (HBB) gene result in the expression of pathologic globin molecules or the reduction in expression of the wildtype allele. Despite the high prevalence of these disorders, current treatment options are limited. Patients with a hereditary persistence of fetal hemoglobin (HPFH) have disease modifying mutations that result in the compensatory expression of gamma or delta hemoglobin and these patients tend to have a much less severe phenotype. A previously reported unique 13bp deletion identified in the gamma hemoglobin promoter of two sickle cell patients with HPFH (HbF >30%) offers a novel target for gene editing using targeted endonucleases. This region contains known binding sites for multiple regulatory factors including the DRED repressor complex and the CAAT-box binding factor (CBF). TALENs designed to target this 13bp site have been generated and the mRNA successfully transfected into human peripheral blood CD34 cells. Genomic analysis of TALEN transfected cells reveals the introduction of INDELs by NHEJ in both the γ1 (HBG1) and γ2 (HBG2) genes (33% and 8% respectively). TALEN edited human peripheral blood stem cells were then differentiated into erythroid progeny that express increased levels of fetal hemoglobin detected by both flow cytometry (>50% increase over control) and HPLC (>5-fold increase over control). CRISPR guides (traditional and chemically modified RNA guides) targeting the same locus are currently being compared to TALENs. These data suggest that the introduction of INDELs in this locus is capable of de-repressing the gamma globin gene in human CD34 cells. In vivo mouse xenografts are underway to further assess this approach as a means for a functional cure of a wide array of hemoglobinopathies.

Cryptococcal Antigen Testing in Newly Diagnosed HIV-Positive Patients

Detection of cryptococcal antigen (CrAg) in serum or plasma is a sensitive predictor of cryptococcal meningitis (CM). Retrospective observations suggest that routine CrAg screening and preemptive antifungal therapy for those who are positive could prevent subsequent CM. To examine the effect of CrAg screening prospectively, 645 antiretroviral therapy (ART)-naive, HIV-infected patients with peripheral blood CD4 cell counts ≤100/µL in two South African clinics were screened for CrAg, first by serum latex agglutination (LA) …

Correlation of the lung microbiota with metabolic profiles in bronchoalveolar lavage fluid in HIV infection.

BackgroundWhile 16S ribosomal RNA (rRNA) sequencing has been used to characterize the lung’s bacterial microbiota in human immunodeficiency virus (HIV)-infected individuals, taxonomic studies provide limited information on bacterial function and impact on the host. Metabolic profiles can provide functional information on host-microbe interactions in the lungs. We investigated the relationship between the respiratory microbiota and metabolic profiles in the bronchoalveolar lavage fluid of HIV-infected and HIV-uninfected outpatients.ResultsTargeted sequencing of the 16S rRNA gene was used to analyze the bacterial community structure and liquid chromatography-high-resolution mass spectrometry was used to detect features in bronchoalveolar lavage fluid. Global integration of all metabolic features with microbial species was done using sparse partial least squares regression. Thirty-nine HIV-infected subjects and 20 HIV-uninfected controls without acute respiratory symptoms were enrolled. Twelve mass-to-charge ratio (m/z) features from C18 analysis were significantly different between HIV-infected individuals and controls (false discovery rate (FDR) = 0.2); another 79 features were identified by network analysis. Further metabolite analysis demonstrated that four features were significantly overrepresented in the bronchoalveolar lavage (BAL) fluid of HIV-infected individuals compared to HIV-uninfected, including cystine, two complex carbohydrates, and 3,5-dibromo-l-tyrosine. There were 231 m/z features significantly associated with peripheral blood CD4 cell counts identified using sparse partial least squares regression (sPLS) at a variable importance on projection (VIP) threshold of 2. Twenty-five percent of these 91 m/z features were associated with various microbial species. Bacteria from families Caulobacteraceae, Staphylococcaceae, Nocardioidaceae, and genus Streptococcus were associated with the greatest number of features. Glycerophospholipid and lineolate pathways correlated with these bacteria.ConclusionsIn bronchoalveolar lavage fluid, specific metabolic profiles correlated with bacterial organisms known to play a role in the pathogenesis of pneumonia in HIV-infected individuals. These findings suggest that microbial communities and their interactions with the host may have functional metabolic impact in the lung.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-016-0147-4) contains supplementary material, which is available to authorized users.

Extended Tumor Control after Dendritic Cell Vaccination with Low-Dose Cyclophosphamide as Adjuvant Treatment in Patients with Malignant Pleural Mesothelioma.

We demonstrated previously that autologous tumor lysate-pulsed dendritic cell-based immunotherapy in patients with malignant pleural mesothelioma is feasible, well-tolerated, and capable of inducing immunologic responses against tumor cells. In our murine model, we found that reduction of regulatory T cells with metronomic cyclophosphamide increased the efficacy of immunotherapy. To assess the decrease in number of peripheral blood regulatory T cells during combination therapy of low-dose cyclophosphamide and dendritic cell immunotherapy and determine the induction of immunologic responses with this treatment in patients with mesothelioma. Ten patients with malignant pleural mesothelioma received metronomic cyclophosphamide and dendritic cell-based immunotherapy. During the treatment, peripheral blood mononuclear cells were analyzed for regulatory T cells and immunologic responses. Administration of dendritic cells pulsed with autologous tumor lysate combined with cyclophosphamide in patients with mesothelioma was safe, the only side effect being moderate fever. Dendritic cell vaccination combined with cyclophosphamide resulted in radiographic disease control in 8 of the 10 patients. Overall survival was promising, with 7 out of 10 patients having a survival of greater than or equal to 24 months and two patients still alive after 50 and 66 months. Low-dose cyclophosphamide reduced the percentage of regulatory T cells of total CD4 cells in peripheral blood from 9.43 (range, 4.34-26.10) to 4.51 (range, 0.27-10.30) after 7 days of cyclophosphamide treatment (P = 0.02). Consolidation therapy with autologous tumor lysate-pulsed dendritic cell-based therapy and simultaneously reducing the tumor-induced immune suppression is well-tolerated and shows signs of clinical activity in patients with mesothelioma. Clinical trial registered with www.clinicaltrials.gov (NCT 01241682).

53. From GWAS To the Clinic: Genome-Editing the Human BCL11A Erythroid Enhancer for Fetal Globin Elevation in the Hemoglobinopathies

We describe here a fundamentally novel way to approach the development of a therapeutic: use of data from genome-wide association studies (GWAS) as a guide to create, in a targeted fashion, a disease-ameliorating genotype in the patient's own cells. We exemplify this by focusing on the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD): in both cases elevated levels of fetal hemoglobin (HbF) have been shown to lessen or eliminate disease symptoms. Thus, reversing HbF silencing in patients is an attractive strategy for the development of therapies. To this end, GWAS data pointed to loss-of-function variants in the erythroid-specific enhancer of the fetal globin repressor, BCL11A, as causative for HbF elevation. While such regulatory elements are challenging to affect in a clinical setting via a conventional small molecule approach, the development of genome editing with engineered nucleases allows, in principle, a targeted intervention at the DNA level to disable enhancer function. Here, we reduce this notion to practice.The BCL11A enhancer consists of three separate cis-regulatory elements spanning a total of~1,500 bp that together drive erythroid-specific expression of BCL11A and lead to a silencing of fetal hemoglobin post-birth. Guided in part by fine-scale in vivo protein-DNA interaction data, we performed a reverse-genetic analysis of the enhancer at its endogenous location in the physiologically relevant cell type, primary human erythroid cells, using zinc finger nucleases (ZFNs). We developed highly optimized ZFNs that yield 60-80% target locus editing in human mobilized peripheral blood CD34 cells. Remarkably, we find that the ZFN-driven ablation of a single 5 bp element, GATAA, resident in the enhancer reproducibly elevates fetal globin in erythroid progeny of these CD34 cells to levels indistinguishable from those resulting from an essentially complete ZFN-driven coding knockout of BCL11A itself. We demonstrate high-efficiency ZFN-driven marking at the enhancer in peripheral blood mobilized CD34 cells at clinical scale production in a GMP-compliant setting, observe the successful engraftment and differentiation of genome-edited cells in an immunodeficient mouse model, and use an unbiased deep-sequencing based assay to comprehensively characterize the nucleus-wide specificity profile of ZFN action.Together these data illustrate the feasibility of using findings from genome-wide association studies to pursue novel therapeutic approaches in monogenic disease. In particular, our work supports the further development of fetal globin elevation via the targeted genome editing of the BCL11A erythroid-specific enhancer in both peripheral blood- and bone marrow-derived CD34 cells as a potential treatment for the hemoglobinopathies.

A Suppressive Microenvironment in Acute Myeloid Leukemia Induces Global Alteration of T and NK Cell Profiles - Evidence for Immune-Editing Effect By Leukemia

While chemotherapy is successful at inducing remission of acute myeloid leukemia (AML), the disease has a high probability of relapse. Immune-surveillance of AML and susceptibility of leukemia cells to both T cell and natural killer (NK) cell attack justifies the application of immune strategies to control residual AML persisting after remission induction. It is critical to understand the immunological landscape of AML at presentation and during the course of treatment to develop a rational approach for the utilization of immunotherapy in improving outcomes in AML. However, the abnormalities of immune function and possible immune deficits in AML have not been fully characterized.To explore cellular immunity in AML, we prospectively collected the samples from 32 patients (median age 63 years; range 24-80) with newly diagnosed AML or high-risk MDS under an IRB approved protocol. Mononuclear cells were isolated from blood and bone marrow at the onset of leukemia, 14-21 days (early phase) and 28-35 days (late phase) after the initiation of induction chemotherapy. Healthy donor blood from age and gender matched controls (n=31, median age 61 years) was concurrently analyzed. Extensive multi-color flow cytometry analysis was performed to characterize T cell subsets [regulatory T cells (Treg), natural killer (NK) cells, NKT cells and γδ T cells] with various functional markers representing T cell exhaustion (PD-1, LAG3, TIM-3, PDL-1), activating and inhibitory NK cell receptors (KIR, Lir-1, NKG2A, NKG2D) and the effector molecule (TRAIL).Both CD4 and CD8 T cell subsets showed phenotypic evidence of exhaustion with elevated expression of PD-1 (CD4: 30.1± 9.4% and CD8 32.8±11.6%: P<0.05), LAG3 (CD4: 28.3± 15.6% and CD8 25.3±16.3%: P<0.001), and TIM3 (CD4: 7.2± 5.0% and CD8 5.4±3.8%: P<0.001) at AML diagnosis compared to healthy controls. The markers of exhaustion were found in all memory subsets (stem cell memory, central memory, effector memory and effector cells) suggesting that T cells were affected during early maturation (p<0.05). Furthermore, a significantly higher frequency of exhausted T cells was found in bone marrow compared with peripheral blood in paired samples (p<0.01). These changes were accompanied with higher frequency of FoxP3+ CD4 cells in both peripheral blood and bone marrow mainly within the HeliosloFoxP3+Treg (p<0.01). Changes persisted in AML patients from the diagnosis to later phase after induction chemotherapy. NK cell subset analysis revealed dynamic NK cell reconstitution after induction chemotherapy: a significantly lower frequency of CD56dimNKG2A+KIR-CD57- NK cells at diagnosis suggesting NK cell maturation arrest, followed by a burst of expansion of CD56brightNK cells early post induction followed by an increase of the CD56dimNKG2A+KIR-CD57- NK subset. Reduced expression of the activating receptor, NKG2D was accompanied with higher expression of Lir-1, a NK cell inhibitory receptor, in all NK cell subsets throughout the course of treatment suggesting a dysfunctional NK cell population. TRAIL expression was significantly reduced in both NKT cells and CD8 cells. Finally, γδ T cells showed significantly lower NKG2D expression at diagnosis.AML is associated with a global abnormality of both innate and adaptive immunity with an increased exhaustion and regulatory phenotype changes across CD4, CD8, NK cell, NKT cells, and γδ T cells. Thus myeloid leukemia cells might create microenvironment that protects them against immune surveillance which persists into early remission through an immune-editing effect. Effective immunotherapy for AML should include strategies to overcome this unfavorable milieu. [Display omitted] DisclosuresSavona:Karyopharm Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Gilead Sciences, Inc.: Membership on an entity’s Board of Directors or advisory committees; Incyte: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees.

Factors affecting sperm fertilizing capacity in men infected with HIV.

Studies on the sperm-fertilizing capacity of HIV-seropositive men show conflicting results for reasons that are not yet clear. The aim of this study was to investigate the effects and relationships of some factors such as patient age, CD4(+) cells count, fathering offspring, concomitant sexually transmitted diseases (STD), and receipt of highly active anti-retroviral therapy (HAART) on sperm fertilizing capacity. Semen samples were collected from 33 HIV-seropositive men. Data on the above factors were acquired from a self-designed questionnaire. Computer-assisted sperm analysis, a hypo-osmotic swelling, and zona-free hamster oocyte penetration tests were performed according to criteria of the World Health Organization. CD4(+) cells in peripheral blood were examined using a flow cytometric (FCM) analyzer. Sperm vitality, sperm motility (grades a + b), total sperm motility, and sperm penetration rates were significantly higher in patients whose CD4(+) counts were ≧350/µl than in those whose CD4(+) counts were <350/µl (P < 0.05), and the parameters mentioned above were also significantly correlated with CD4(+) cell number (all P < 0.05). Significant differences in total sperm count and sperm tail swelling rate between patients co-infected with STD and without STD were observed (P < 0.05). Sperm penetration rate in patients receiving HAART was significantly higher than in those not receiving HAART (P < 0.05). Blood CD4(+) cell counts are an important indicator for evaluating sperm fertilizing capacity of HIV-seropositive men. After receiving HAART, the sperm penetration rate of HIV-seropositive men can be improved.

Role of leptin and ghrelin in induction of differentiation of IL-17-producing and T-regulatory cells.

We studied isolated and combined effects of leptin and ghrelin on the formation of IL-producing and T-regulatory cells. Leptin in concentrations comparable with its normal blood concentration during pregnancy (trimester II-III) promotes differentiation of peripheral blood CD4(+) cells to IL-17-producing cells and enhances IL-17A production, but suppresses the formation of T-regulatory cells in vitro. In contrast, ghrelin in a concentration typical of trimester I-II of pregnancy reduces the number of IL-17-producing cells, but stimulated the formation of T-regulatory cells. The effects of leptin and ghrelin in combination typical of trimester I-II of pregnancy stimulated the formation of T-regulatory cells, while in combination typical of trimester II-III did not shift the balance of T-regulatory cells and IL-17-producing cells, but stimulated the formation of IL-17A. We conclude that leptin and ghrelin play an important role in the maintenance of the balance of IL-17-producing and T-regulatory cells during pregnancy.

CD4 Cell Levels during Treatment for Tuberculosis (TB) in Ethiopian Adults and Clinical Markers Associated with CD4 Lymphocytopenia

BackgroundThe clinical correlations and significance of subnormal CD4 levels in HIV-negative patients with TB are unclear. We have determined CD4 cell levels longitudinally during anti-tuberculosis treatment (ATT) in patients, with and without HIV co-infection, and their associations with clinical variables.MethodAdults diagnosed with TB (maximum duration of ATT for 2 weeks, and with no history of antiretroviral therapy (ART) in HIV-positive subjects) were included consecutively in eight out-patient clinics in Ethiopia. Healthy individuals were recruited for comparison at one of the study health centers. Data on patient characteristics and physical findings were collected by trained nurses following a structured questionnaire at inclusion and on follow-up visits at 2 and 6 months. In parallel, peripheral blood CD4 cell levels were determined. The evolution of CD4 cell levels during ATT was assessed, and the association between clinical characteristics and low CD4 cell levels at baseline was investigated using regression analysis.ResultsIn total, 1116 TB patients were included (307 HIV-infected). Among 809 HIV-negative patients, 200 (25%) had subnormal CD4 cell counts (<500 cells/mm3), with <350 cells/mm3 in 82 (10%) individuals. CD4 cell levels increased significantly during the course of ATT in both HIV+ and HIV- TB-patients, but did not reach the levels in healthy subjects (median 896 cells/mm3). Sputum smear status, signs of wasting (low mid upper arm circumference (MUAC)), and bedridden state were significantly associated with low CD4 cell counts.ConclusionA high proportion of Ethiopian TB patients have subnormal CD4 cell counts before starting treatment. Low CD4 cell levels are associated with smear positive disease and signs of wasting. The continuous increase of CD4 cell counts during the course of ATT suggest a reversible impact of active TB on CD4 cell homeostasis, which may be considered in interpretation of CD4 cell counts in HIV/TB co-infected subjects.

Bendamustine (Treanda®)-Based Regimens Are Effective In Mobilizing Peripheral Blood Hematopoietic Stem Cells For Autologous Transplantation

▪ BackgroundHigh dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard of care for patients with advanced or treatment refractory multiple myeloma (MM) and non-Hodgkin lymphoma (NHL). Stem cell proliferation and mobilization can be enhanced though the addition of myelosuppressive chemotherapy prior to GCSF administration. Chemotherapeutic agents without cross resistance to prior therapies may support peripheral blood stem cell (PBSC) collection and improve patient outcomes by exacting a more potent direct anti-tumor effect prior to ASCT. Bendamustine (Treanda®) is a synthetic chemotherapeutic agent that shares structural similarities to both purine analog and alkylating agents without significant cross resistance to other compounds in either drug class. Bendamustine appears to have low stem cell toxicity in vitro, is well tolerated, and has activity in MM and NHL, but the potential for the purine moiety to adversely impact stem cell reserve is unknown. We hypothesized that bendamustine's activity in patients with disease resistant to first line therapies makes it a logical candidate for chemotherapy based PBSC mobilization and tested its impact on stem cell yield. MethodsPatients were eligible if they had relapsed or refractory MM, B-cell NHL or T-cell NHL and were candidates for ASCT. Other criteria included: age >18 years, ANC >1,500/mm3, platelets >100,000/mm3, adequate renal and hepatic function, <3 prior myelotoxic regimens, <6 cycles of lenalidomide, no failed mobilization attempt, and no prior pelvic/spinal irradiation. Patients received 1 cycle of BED therapy [bendamustine (120 mg/m2 IV d 1, 2 - provided along with financial support for this study by Teva Pharmaceuticals), etoposide (200 mg/m2 IV d 1- 3), dexamethasone (40 mg PO d 1- 4), delivered as an outpatient, followed by filgrastim (initially 10 mcg/kg/d sc; starting on d 5 through end of collection)]. Apheresis was initiated when peripheral blood CD34 cell counts were >5/µL. The primary endpoint was successful mobilization, defined as collection of >2.0 x 106CD34 cells/kg. AEs were graded using the CTCAE v4.0. ResultsThirty-seven patients (32 MM, 4B-cell NHL, 1 NK/T-cell NHL) were treated. The median age was 60 years (range 43-70). The median number of prior therapies was 1 (range 1-3) for MM and 2 (range 1-3) for NHL patients. All patients (37/37) were successfully mobilized. The median number of CD34+ cells collected was 19.43 x 106/kg (range 4.35 to 55.51 x 106). All MM patients collected >10 x 106 CD34+cells/kg. The median time from the start of BED mobilization therapy to the first day of CD34 stem cell collection was 12 days (range 9 to 20 days). The median number of apheresis days was 1 (range 1 to 4). A predictable pattern of leucocyte nadir and recovery was demonstrated (95% of patients started apheresis between days 9-13). Two patients (5%) were given plerixafor and for 2 patients (5%) GCSF was increased to 16 mcg/kg twice daily. Among the 37 patients mobilized and collected, 31 have thus far undergone ASCT and 100% (31/31) achieved an unsupported neutrophil count >500/µL at a median of 15 days (range 7-19) after PBSC infusion and a platelet count >20K/µL at a median of 11 days (range 8-14). Ten SAEs were observed in 8 patients and 1 patient died due to disease progression prior to ASCT. SAEs include: neutropenic fever (1, grade [GR] 3), bone pain (2, GR 3), renal insufficiency (1, GR 1), atrial fibrillation (1, GR 2), hypotension (1, GR 3), stroke (1, GR 2), and one patient accounted for 3 SAEs including GR 3 tumor lysis syndrome and sepsis and GR 5 disease progression. Among twenty-nine evaluable patients to date, responses include: CR= 4 PR=2, SD=19 and PD=4. The ORR to this single cycle of therapy was 21%. ConclusionsPBSC mobilization with BED is safe and effective. BED is not an acute stem cell toxin. Large numbers of stem cells were rapidly mobilized and resulted in short durations of apheresis. No patient with MM collected <10 x 106 CD34+ cells/kg (sufficient for 2 ASCTs). Twenty-one percent of patients demonstrated a measurable response to a single cycle of BED therapy and an additional 65% of patients had stable disease. In patients who were transplanted, the time to neutrophil and platelet engraftment was comparable to other chemotherapy based mobilization regimens. The BED regimen was well tolerated and these findings suggest that the role of BED in PBSC mobilization should be further explored. Disclosures:No relevant conflicts of interest to declare.

Interleukin‐17 Immunity in Pediatric Crohn Disease and Ulcerative Colitis

The present understanding of inflammatory bowel disease pathogenesis mainly relies on studies of adult patients. Therefore, we studied the balance between T-effector and regulatory cells in pediatric inflammatory bowel disease. Quantitative polymerase chain reaction and immunohistochemistry served to quantify the expression of immunological markers in mucosal biopsies and flow cytometry analysis was used in peripheral blood mononuclear cells. Colonic interleukin (IL)-17+, IL-22, and IL-6 mRNA upregulation and increase in the number of colonic IL-17 cells were demonstrated in both Crohn disease (CD) and ulcerative colitis (UC). Likewise, colonic forkhead box P3 (FOXP3+) mRNA expression and the number of colonic FOXP3 cells were increased both in CD and in UC and were accompanied in CD also with increased numbers of FOXP3+CD25 High CD4 cells in peripheral blood. Ileal relation of IL-17/CD4 cells was increased only in CD. We showed activation of colonic IL-17/IL-22 axis and upregulation of FOXP3 to occur both in pediatric CD and in UC, indicating shared immunological characteristics. Upregulation of IL-17 was restricted to colon in UC, but existed in the ileum and in the colon in active CD.

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

Exhausted T Helper Cells in Lung Transplantation Portend Worse Outcome

Purpose In chronic viral infections, CD4 exhaustion correlates with poor containment of virus. We predicted that high levels of CD4 exhaustion would be beneficial in lung transplantation. Methods and Materials A retrospective review was performed on 96 lung transplant recipients from Feb 2006 to April 2010 with at least 1 bronchoscopy or peripheral blood (PB) in the first post transplant year. CD4 exhaustion was measured using the sample closest to the 1-year post transplant date. CD4 cells in bronchoalveolar lavage fluid (BALF) and peripheral blood were assessed by flow cytometry and considered exhausted if PD1hi, CD127lo, and GranzymeB negative. Samples were analyzed for the association between exhausted CD4 cells and survival and developing chronic rejection. We performed univariate Kaplan-Meier analyses between CD4 exhaustion and chronic rejection or death. Chronic rejection was defined as BOS 1 or higher by ISHLT criteria. Results A modest rejection free benefit was associated with lower CD4 exhaustion. There was a statistically significant increased freedom from rejection in the patients with a lower degree of CD4 exhaustion in the BALF (p=.04 by Log Rank). Conclusions We hypothesized that high levels of CD4 exhaustion would be fortuitous as this would serve as a biomarker for anergy toward the lung allograft. Our findings indicate that CD4 exhaustion is in fact a negative predictor of lung function. We predict that high levels of CD4 exhaustion are associated with substantial defects in protective immunity thereby leading to impaired pathogen containment. Our findings are consistent with prior reports showing an association with low humoral immunity and chronic rejection. 1-, 3-, and 5-year Rate of Survival and Chronic Rejection Free in Different Levels of Exhausted CD4 in BALF and PB Survival Rate (%) Chronic Rejection Free (%) Exhausted CD4 Frequency 1-year 3-year 5-year 1-year 3-year 5-year Low in BALF 90.6 77.4 65.8 98.1 81.0 67.5 High in BALF 100 84.7 67.8 93.9 63.6 50.0 Low in PB 91.8 77.0 64.6 95.1 73.5 56.6 High in PB 100 86.2 56.6 100 72.4 67.2

Interleukin‐17A‐producing T lymphocytes in chronic active Epstein–Barr virus infection

T helper (Th) 17 cells are reportedly effector T cells that produce interleukin (IL)-17A and play a significant role in the development of autoimmune diseases and immune responses for antimicrobial host defense. Production of IL-17A in chronic active Epstein-Barr virus infection (CAEBV) was studied to investigate its contribution to pathogenesis of this disease. Significantly more IL-17A-producing cells were detected in the peripheral blood of CAEBV patients than in that of healthy controls, although a significant difference in serum IL-17A production was not confirmed. Of the IL-17A-producing cells, 91.8% were cluster of differentiation (CD)4-positive Th17 cells. Moreover, there were significantly more IL-17A-producing cells among CD4(+) cells in peripheral blood of CAEBV patients than in that of controls (1.97 ± 0.69% vs. 1.09 ± 0.53%, P = 0.0073). These data suggest that IL-17A-producing cells may influence the pathophysiology of CAEBV.

Bendamustine (Treanda®), Etoposide and Dexamethasone (BED) Followed by GCSF Effectively Mobilizes Autologous Peripheral Blood Hematopoietic Stem Cells

AbstractAbstract ▪4126▪This icon denotes a clinically relevant abstract Background:High dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard of care for patients with advanced or treatment refractory multiple myeloma (MM) and non-Hodgkin lymphoma (NHL). Stem cell proliferation and mobilization can be enhanced though the addition of myelosuppressive chemotherapy to GCSF administration. Chemotherapeutic agents without cross resistance to prior therapies may support peripheral blood stem cell (PBSC) collection and improve patient outcomes by exacting a more potent direct anti-tumor effect prior to ASCT. Bendamustine (Treanda®) is a synthetic chemotherapeutic agent that shares structural similarities to both purine analog and alkylating agents without significant cross resistance to other compounds in either drug class. Bendamustine appears to have low stem cell toxicity in vitro, is well tolerated, and has activity in MM and NHL. We hypothesized that bendamustine’s activity in patients with disease resistant to first line therapies makes it a logical candidate for chemotherapy based PBSC mobilization. Methods:Patients were eligible if they had relapsed or refractory MM, B-cell NHL or T-cell NHL and were candidates for ASCT. Other criteria included: age >18 years, ANC >1,500/mm3, platelets >100,000/mm3, adequate renal and hepatic function, <3 prior myelotoxic regimens, <6 cycles of lenalidomide, no prior failed mobilization attempt, and no prior pelvic/spinal irradiation. Patients received 1 cycle of BED therapy [bendamustine (120 mg/m2 IV d 1, 2 - provided along with financial support for this study by Teva Pharmaceuticals), etoposide (200 mg/m2 IV d 1– 3), dexamethasone (40 mg PO d 1– 4), delivered as an outpatient, followed by filgrastim (10 mcg/kg/day; starting on d 5 through end of collection)]. Apheresis was initiated when peripheral blood CD34 cell counts were >5/μL. The primary endpoint was successful mobilization, defined as collection of >2.0 × 106CD34 cells/kg. Adverse events (AEs) were graded using the CTCAE v4.0. Results:Twenty patients (16 MM, 3 B-cell NHL, 1 NK/T-cell NHL) were treated. The median age was 59 years (range 43–70), and the median number of prior therapies was 1 (range 1–3) for MM and 2 (range 2–3) for NHL patients. All patients (20/20) were successfully mobilized. The median number of CD34+ cells collected was 19.11 × 106/kg (Mean 22.49; range 4.35 to 55.51 × 106). All MM patients collected >10 × 106 CD34+cells/kg. The median time from BED mobilization therapy to the first day of CD34 stem cell collection was 12 days (mean 12.05; range 10 to 20 days). The median number of days of apheresis was 1 (mean 1.45; range 1 to 4). A predictable pattern of leucocyte nadir and recovery was demonstrated (88% of patients started apheresis between days 10–12). One patient (5%) was given plerixafor and for 2 patients (10%) the dose of GCSF was increased to 16 mcg/kg twice daily. Among the 20 patients mobilized and collected, 12 have thus far undergone ASCT and 100% (12/12) have achieved an unsupported neutrophil count >500/μL at an average of 14.3 days after PBSC infusion and a platelet count >20K/μL at an average of 10 days. Serious AEs (SAEs) were observed in 5 patients and 1 patient died due to disease progression. No unexpected grade 3 or greater treatment related SAEs were seen. Disease response assessments are ongoing. The original protocol design involved 3 agents (bendamustine, dexamethasone and GCSF [BDG]). After the first 3 patients enrolled, the mobilization regimen was modified to include etoposide because BDG did not yield a predictable pattern of leucocyte nadir and recovery, thus complicating timing for apheresis (median time to collection 22 days). The first 3 patients were censored from the analysis, however all 3 patients were successfully mobilized and collected. Conclusions:The initial experience with PBSC mobilization after BED in this phase II study suggests the regimen is safe and effective, while the use of BDG does not yield predictable CD34 kinetics. Time to neutrophil and platelet engraftment after ASCT appears unimpaired when compared with other chemotherapy based mobilization regimens. Large numbers of stem cells were rapidly mobilized and resulted in short durations of apheresis. No patient with MM collected <10 × 106 CD34+ cells/kg (sufficient for 2 ASCTs). The regimen was very well tolerated and these findings suggest that the role of bendamustine in PBSC mobilization should be further explored. Disclosures:Green:Teva Pharmaceuticals: Research Funding. Holmberg:Millenium: Research Funding; Otsuka: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Sanofi: Research Funding. Budde:Teva Pharmaceuticals: Research Funding. Gopal:Teva Pharmaceuticals: Research Funding.