Peripheral Blood CD34 Cells Research Articles

Very high efficacy of intermediate-dose cytarabine in combination with G-CSF as a second-line mobilization of hematopoietic stem cells

Autologous hematopoietic stem cell transplantation (autoHSCT) is used widely in the treatment of patients with lymphoid malignancies. Currently, 99 % of such procedures are performed using peripheral blood as a source of stem cells [1]. The generally accepted minimal level of CD34 cells required for rapid neutrophil and platelet recovery after autoHSCT is 2 9 10/kg. However, some data indicate that higher numbers are associated with less need for blood product transfusions and administration of antibiotics [2–4]. Mobilization regimens are based either on the use of granulocyte-colony stimulating factor (G-CSF) alone or G-CSF in combination with chemotherapy, most frequently cyclophosphamide 1.5–7 g/m or lymphoma-specific salvage regimens [5]. Unfortunately, a significant proportion of patients fail to mobilize sufficient number of CD34 cells, thus requiring additional attempts [6]. New mobilization strategies are being explored, including the use of plerixafor, CXCR4 inhibitor, in combination with G-CSF, with or without chemotherapy. This agent enabled sufficient CD34 cell harvest in 64.8–81.6 % of proven or predicted poor mobilizers [7, 8]. Unfortunately, such treatment is very expensive, which limits its worldwide application. Hence, the development of new strategies is still warranted. Although current studies focus on small molecules interfering with stem cell–stroma interactions, traditional chemotherapy-based salvage mobilization regimens have not been sufficiently explored. In our center, between November 2010 and September 2011, 14 patients who had failed chemotherapy-based mobilization were treated with salvage regimen including intermediate-dose cytarabine (ID-AraC) in combination with G-CSF. Most of the patients were referred for transplantation due to multiple myeloma (n = 8) or lymphoma (n = 5), while the remaining one had ovarian cancer. Patients had previously been treated with a median of 2 (range 1–5) lines of chemotherapy and most of them had received irradiation. Previous mobilization was usually based on cyclophosphamide plus G-CSF (Table 1). Six patients received AraC 400 mg/m (2 h infusions) every 12 h for three consecutive days (total dose, 2400 mg/m), while the following eight patients were treated with AraC for 2 days (total dose, 1600 mg/m). G-CSF 5–10 lg/kg was started on day 5 and continued daily until last leukapheresis. Leukaphereses were started once the peripheral blood CD34 cell count exceeded 15/lL and were performed using the Spectra-Optia Apheresis System (CaridianBCT Inc, Lakewood, CO, USA). The target number of CD34 cells collected was 5 9 10/kg for multiple myeloma (all planned for tandem autoHSCT) and 2 9 10/kg for the remaining patients. The maximum number of peripheral blood CD34 cells/lL after AraC ? G-CSF was 86 (17–312) and was significantly higher compared to the first-line mobilization: 8 (0–35) (Mann–Whitney U test, P = 0.002). No difference could be found according to the AraC dose: 80 (17–174) for 1.6 g/m versus 88 (24–312) for 2.4 g/m, P = 0.85. All patients collected required number of CD34 cells, which was achieved with a single apheresis in 10/14 (71.5 %) patients (Table 1). Although all patients experienced profound cytopenia and most required T. Kruzel M. Sadus-Wojciechowska J. Najda T. Czerw M. Glowala-Kosinska J. Holowiecki S. Giebel (&) Department of Bone Marrow Transplantation, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15 Street, 44-101 Gliwice, Poland e-mail: sgiebel@io.gliwice.pl

Abnormal Savda syndrome: Long-term consequences of emotional and physical stress on endocrine and immune activities in an animal model

To investigate the relationship between emotional status, cold-dry environment and long-term immune responses to the stressors, and the potential pathological mechanisms between causative factors of abnormal Savda syndrome (ASS) and the susceptibility to disease; thus to clarify the ASS, and secondly to identify the optimal ASS animal model for further studies on traditional Uighur therapeutical formulations. Sixty mice were randomly and equally divided into 4 groups: control and 3 stress groups. The cold-dry environment was applied by keeping the mice in a climatic chamber. The emotional stress was induced by the application of the repeated electric foot-shocks in the electric foot-shock apparatus. The mice of the combined stress group underwent the repeated electric foot-shock treatment before being housed in the climatic chamber. The experimental routine was repeated for 21 days. In order to look into endocrine and immune stress responses, ELISA was used to determine the serum levels of the hormones corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), Beta-endorphin (β-END) and corticosterone (CORT), of the cytokines interleukin 2 (IL-2), interleukin 6 (IL-6), interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α), and of the immunoglobulins immunoglobulin A (IgA), immunoglobulin M (IgM) and immunoglobulin G (IgG). Lymphocyte subsets were analyzed in duplicate in order to determine differences in the T cell ratio. In the cold-dry environment group, the serum levels of CRH, ACTH and CORT were significantly higher than those of the control group, whereas serum β-END was not found significantly different. In both the repeated electric foot-shock group as well as in the combined stress group the serum levels of CRH, ACTH, β-END and CORT were significantly higher. Compared to the control animals, the serum concentration of INF-γ was significantly lower in all three different stress groups. The serum level of IL-2 was decreased in the combined stress group whereas the serum TNF-α level was significantly higher. The serum IgG level was significantly higher in all three stress groups, whereas the IgA level was lower in both chronic electric foot-shock group and combined stress group. The IgM level was found significantly higher in the combined stress group only. The percentage of CD4(+) cells in peripheral blood was dramatically decreased in mice exposed to colddry environment, chronic electric foot-shock and combined stress, whereas the percentage of the CD8(+) subset was not significantly different. The CD4(+)/CD8(+) ratios were markedly lower in both cold-dry environment group and combined stress group. Combined stress can cause hyperactivity of the HPA axis, and an imbalance in the Th1/Th2 cell subset may contribute to illustrate the partial pathological mechanisms of ASS. This study identified this animal model of a combination of physical and emotional stress as an optimal model for further studies on ASS and relative therapies.

The use of plerixafor in hematopoietic progenitor cell collection in pediatric patients: a single center experience

Plerixafor was recently approved for use in combination with granulocyte-colony-stimulating factor (G-CSF) for hematopoietic progenitor cell (HPC) collection by apheresis in adults with multiple myeloma (MM) or non-Hodgkin lymphoma (NHL). However, its efficacy in pediatric patients is not well-studied; thus, we report on our institutional experience with this population. A retrospective observational analysis was performed using both stem cell-processing laboratory information as well as apheresis charts and medical records on all pediatric patients who received plerixafor as part of the mobilization regimen between December 2006 and December 2010. The primary outcome was collection yield. Secondary outcomes included the ability to undergo autologous hematopoietic stem cell transplantation (auto-HSCT) and engraftment status. Eighteen HPC collections by apheresis representing seven mobilization courses were performed on five pediatric patients with poor mobilization status (three males, two females; median age 14 years). Median pre-harvest peripheral blood CD34(+) cell (PB CD34(+)) count was 6.88/μL. A strong correlation between pre-harvest PB CD34(+) count and collection yield was observed. Median total collection yield was 2.26 × 10(6) CD34(+) cells/kg. Four patients achieved a minimum collection of 2 × 10(6) CD34(+) cells/kg. Three patients underwent auto-HSCT with a median neutrophil and platelet engraftment of 12 and 34 days, respectively. No major adverse events with plerixafor administration or apheresis collections were reported. Plerixafor in combination with G-CSF is a safe and potentially helpful mobilization agent in poor mobilizers. Further studies should be done to evaluate the true efficacy of plerixafor in the pediatric population.

Peripheral Lymphocyte Subsets as a Prognostic Indicator of Mortality and Morbidity in Healthy Dogs

To evaluate the relationship among immune status and increased morbidity and mortality, peripheral blood lymphocytes (CD3(+), CD4(+), CD8(+) and CD21(+) cells) from 32 healthy dogs over 8 years of age were analyzed. Twenty-five of the 32 dogs were followed-up for 3 years after the analysis; and 14 dogs were found to be diseased, and nine dogs died. There was no notable difference between the ages of the dogs that died compared with the ones that survived. The relative percentage of CD4(+) and the CD4(+):CD8(+) ratio decreased notably in dogs falling ill compared with healthy dogs. The relative percentage of CD3(+) lymphocytes showed a notable decrease in dogs that died within 3 years in comparison with dogs that survived. In a discriminant analysis of morbidity and mortality, most patients were correctly classified as diseased or not and surviving or dead, respectively. These results indicate that the immunophenotypes of peripheral blood lymphocytes in older dogs offer promise as parameters for evaluating mortality and morbidity.

Effectiveness and Potential Strategies to Improve Outcomes Following Chemomobilization with Etoposide + G-CSF in Patients Undergoing Autologous Stem Cell Transplantation for Lymphoma

Abstract 2083 INTRODUCTION:The addition of chemotherapy to G-CSF for stem cell mobilization prior to autologous stem cell transplantation (ASCT) provides the potential for increased cell yield and improved mobilization outcomes relative to G-CSF alone. We have investigated the use of mid-dose VP-16 plus G-CSF in pts with lymphoma and examined whether plerixafor might be incorporated into this chemomobilization backbone in a cost-effective way for a population with inferior outcomes. METHODS: Between June 2004 and September 2010, 159 pts with lymphoma underwent ASCT following the use of VP-16 (375mg/m2 on D#1 and D#2) and G-CSF (5mcg/kg twice daily from D#3 through the final day of collection) for mobilization. 26 pts also received a dose of Rituximab (375mg/m2) on D#1. Stem cell collection was initiated when the peripheral blood CD34 cell count was more than 7 per ul. Data on costs for fixed and variable expenditures associated with mobilization and collection were calculated on an individual patient basis. Costs also included unexpected complications such as inpatient hospitalizations, antibiotic use and blood product transfusions. “Poor mobilizers” were defined as pts failing to collect 5 × 106 cells in one or two days. Univariable and multivariate logistic regression were performed to identify predictive models for poor mobilization and to identify hypothetical breakpoint scenarios for the cost-effective utilization of plerixafor. For the breakpoint scenarios, a median of 3 doses of plerixafor was assumed based on the published phase III data with plerixafor plus G-CSF. RESULTS: Of 159 pts with lymphoma, 90 (57%) were identified as “good mobilizers,” 43% were “poor mobilizers”, and 150 (94%) collected at least 2 × 10 6th/kg CD34 cells in total (83% within 4 apheresis sessions), comparing favorably to published data with G-CSF alone or G-CSF + plerixafor. 51 (32%) required PRBC or platelet transfusion, 10 (6%) were admitted to the hospital during the mobilization period, and 8(5%) required a second mobilization or bone marrow harvest. There was no increased incidence of secondary malignancies. Average costs were $14923 ($6121-$24546) for good mobilizers and $27044 ($12206-$51846) for poor mobilizers (p<0.05). The first peripheral blood CD34 count (obtained between D9-D15, with 82% of first counts obtained on D12), accurately predicted “good” vs “poor” mobilizers (c statistic 0.941, CD34 cutpoint 27/uL). Using our data, we estimated that it would not be cost effective to give plerixafor to all patients, even if 100% of patients subsequently became “good” mobilizers (net loss $15,817/pt). Instead, by reserving plerixafor for only predicted “poor” mobilizers (probability<0.5) at the time of first CD34 count, we estimated that 64% (n=49) of predicted “poor” mobilizers would need to become “good” mobilizers in order to achieve cost neutrality. CONCLUSION: VP-16 and G-CSF is a safe and effective mobilization regimen for pts with lymphoma and compares favorably to published data with G-CSF alone or G-CSF + plerixafor. Mobilization outcomes after chemomobilization might be further improved in a cost-effective way by adding plerixafor in patients predicted by the first peripheral blood CD34 count to be poor mobilizers. This will be investigated prospectively. Disclosures:Shea:Otsuka Pharmaceuticals: Research Funding.

Fuzheng Yiliu Granule (扶正抑瘤颗粒) inhibits the growth of hepatocellular cancer by regulating immune function and inducing apoptosis in vivo and in vitro

To study the inhibitory effect of Fuzheng Yiliu Granule (FYG) on hepatocellular cancer (HCC) and investigate the mechanism mediating its bioactivity. H22 tumor-bearing ICR mice were treated with FYG [3.6 g/(kg·d)] for 5 days. Tumor volume and tumor weight, percentages of CD3(+), CD4(+), CD8(+), and natural killer (NK) cells in peripheral blood, tumor apoptosis and serum levels of interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) were evaluated. FYG-containing serum was prepared from SD rats treated for 7 days [high dose 3.6 g/(kg·d); middle dose 1.8 g/(kg·d); low dose 0.9 g/(kg·d)]. Cell cycle, cell viability, and apoptosis were evaluated after HepG2 cell line was cultured in FYG-containing serum for 48 h. The levels of IL-2 and TNF-α in FYG-containing serum were also determined. FYG produced a potent antitumor effect (P<0.01) and induced marked apoptosis of the tumor tissue (P<0.05). Mice treated with FYG had higher percentages of CD3(+) and CD4(+) (P<0.05), and more NK cells (P<0.01) in the peripheral blood than those in the animals treated with normal saline. Mice receiving FYG had the highest serum levels of IL-2 and TNF-α (P<0.01). High-dose FYG-containing serum significantly decreased HepG2 cell viability, inhibited cell proliferation (P<0.05), and induced apoptosis (P<0.01). In addition, the levels of IL-2 and TNF-α of high-dose-containing serum were higher than the blank serum (P<0.01). FYG could inhibit HCC growth by regulating immune function and inducing apoptosis of tumor cells in vivo and in vitro.

T-Regulatory Cells in Lymph Nodes

Because T-regulatory cells (Tregs) can harmfully impair HIV-specific responses or beneficially limit immune activation, we compared the number of Tregs in lymph nodes from 48 HIV+ patients and 106 HIV- subjects. By using a microscope counting grid, we found that the mean ± SD number of Tregs in lymph nodes was 3 times greater in HIV+ males than HIV+ females (23.5 ± 20.7 vs 7.8 ± 7.7; P = .0006) and almost twice as great in HIV+ males than HIV- males (23.5 ± 20.7 vs 13.5 ± 15.5; P = .04). There were fewer Tregs in HIV+ females than in HIV- females (mean ± SD, 7.8 ± 7.7 vs 13.4 ± 13.3; P = .04). HIV+ males compared with HIV+ females had higher viral loads (VLs) and lower peripheral blood (PB) CD4 cell counts (mean ± SD, 239,841 ± 307,494 vs 73,038 ± 146,763 copies/mL and 262 ± 207 vs 466 ± 278/mm(3); P = .02 and P = .01, respectively). Our data show that Tregs in lymph nodes from HIV+ patients are positively correlated with VL and negatively correlated with PB CD4 counts. These findings suggest that Tregs might impair an HIV-specific immune response, which could be modified by sex, or, alternatively, an increased VL causes increased Tregs.

CD4+CD25high Treg cells in peripheral blood during remission and exacerbation of allergic asthma in children

To determine the percentage of CD4+CD25(high) Treg cells in peripheral bloodCD4+ T cells of allergic asthmatic children during disease remission and exacerbation. Peripheral blood mononuclear cells (PBMC) and serum samples were collected from 6- to 11-year-old children with mild-to-moderate allergic asthma (n = 34)and from healthy controls (n = 15). CD4+CD25(high) T cells in PBMC were detected by flow cytometry. Total and specific IgE in serum were analysed by enzyme-amplified chemiluminescence, and IL-2 was measured by ELISA. There was no significant difference in CD4+CD25(high) T-cell proportions between asthmatic children in exacerbation and remission as compared with controls.CD4+CD25(high) T-cell percentages were not correlated with total and specific IgE. IL-2 was elevated in both disease remission and exacerbation but did not correlate significantly with CD4+CD25(high) T-cell percentages. CD4+CD25(high) T-cell proportion in the peripheral blood of total CD4+T cells is not reduced in children with allergic IgE-mediated asthma and does not differ between disease remission and exacerbation.

Peripheral Blood Stem Cell Collection In Patients Undergoing Induction Therapy with Lenalidomide Based Regimens: Failure Rates and Salvage Approaches

AbstractAbstract 2253 Background:Lenalidomide based combinations are among the most common initial therapies for myeloma. Previous studies have suggested that lenalidomide therapy can result in suboptimal stem cell collection in patients eligible to undergo autologous stem cell transplantation, especially older patients after prolonged exposure to the drug. Many salvage approaches are used when attempting repeat stem cell collection in this patient group. Patients and Methods:Two hundred twenty four patients who underwent stem cell collection following lenalidomide-dexamethasone induction from July 2004 and December 2009 were included in the current analysis. Data pertaining to the duration of lenalidomide therapy, stem cell mobilization regimen, and the collection yields were collected from the medical records. Results:The median age at mobilization was 60.6 years (range; 29, 76) and 136 (60%) were male. There were a total of 245 collection attempts from among 224 patients, 21 (9.8%) patients attempting to remobilize after failing to collect the desired numbers of stem cells at the first attempt. We first analyzed the results of the initial collection attempt. The median duration of lenalidomide therapy prior to stem cell collection was 4 months (range; 1, 26). The mobilization strategies were GCSF (Granulocyte Colony Stimulating Factor) alone in 151 (67%) patients, cyclophosphamide (CTX) followed by GCSF in 29 (13%) patients, and GCSF plus AMD3100 in 44 (20%) patients. Among those receiving AMD3100, it was added either due to peripheral blood CD34 cell count not reaching the threshold for initiation of harvest or for poor first day CD34 cells collection in 34 patients and given in a planned fashion in 10 patients. Overall 15 patients (7%) failed to reach the peripheral CD34 cell counts required to initiate apheresis, and among those starting apheresis 6 patients failed to collect at least 2 million CD34 cells/kg; a cumulative failure rate of 9%. Another 18 (8%) patients failed to collect at least 4 million CD34 cells /kg. The CD34 cells yield on day 1, the total yield, number of collections, the average daily yield and the percentage of the targeted cells collected for each mobilization strategy including failure rates are detailed in the table.Twenty-one patients reattempted stem cell mobilization; including 14 that failed a first attempt and 7 did who not achieve the intended goal even though they collected more than 2 million CD34 cells/kg. The mobilization regimens were GCSF alone, CTX + GCSF, GCSF + GM-CSF (Granulocyte Macrophage Colony Stimulating Factor) and GCSF + AMD in 5, 8, 3, and 4 patients respectively. All patients collected at least 2 million CD34 cells /kg and 14 patients (70%) collected more than 4 million CD34 cells /kg. The median CD34 cells collected with the second attempt was 5.4 million/kg (rang; 2, 19.5) bringing the median total collection for these 21 patients to 9.6 million/kg (2.6-19.6). Overall, of the 224 patients studied, all but the 6 patients who failed initially and did not attempt a second collection collected at least 2 million CD34 cells /kg and 197 (88%) collected at least 4 million CD34 cells/kg. Conclusion:While the overall failure rate of stem cell collection in patients receiving initial therapy with lenalidomide is 10%, a risk adapted approach of adding AMD3100 appear to decrease the risk of failure. However, majority of patients failing a stem cell harvest attempt can be salvaged with a second collection allowing these patients to proceed to a stem cell transplant if desired.AllG-CSFCTX +G-CSFAMD upfrontAMD rescuePN224151291034Day 1 CD34 collection Median(Range)2.0 (0–28.6)2.1 (0–10.1)4.2 (0–28.6)1.9 (0.7–15.3)1.3 (0.09–8.6)<0.001Total CD34 collection Median(Range)7.6 (0–28.6)7.5 (0–15.6)9.3 (0–28.6)8.5 (4.2–15.3)6.5 (0.2–13.5)0.02Number of collection days Median(Range)4 (0–12)4 (0–12)2 (1–11)2.5 (1–7)4 (1–9)<0.001Average daily collection Median(Range)1.6 (0–28.6)1.5 (0–10.1)4.4 (0–28.6)2.3 (1.6–15.3)1.2 (0.1–8.6)<0.001% of goal collected Median(Range)100 (0–715)97 (0–233)114 (0–715)113 (82–172)106 (2.5–141)0.001% patients failed (no apheresis or CD34 <2 million/kg)9111006NS% patients <4 million/kg172014015NS Disclosures:Gertz: Celgene: Honoraria; Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genzyme: Research Funding. Lacy: Celgene: Research Funding. Dispenzieri: Celgene: Honoraria, Research Funding; Binding Site: Honoraria. Micallef: Genzyme: Membership on an entity's Board of Directors or advisory committees. Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

CD4+ CD25+ but not CD4+ Foxp3+ T cells as a regulatory subset in primary biliary cirrhosis.

Increasing evidence indicates a role for regulatory T cells (Tregs) in the immune response and in autoimmune diseases, but the role of Tregs and cytokines in autoimmune hepatic diseases remains largely unclear and controversial, especially in patients with primary biliary cirrhosis (PBC). This study was undertaken to investigate Tregs and different cytokines in the liver and peripheral blood of PBC patients. We found that these patients demonstrated a reduction of CD4(+)CD25(+) T cells but elevated CD4(+)Foxp3(+) T cells in peripheral blood mononuclear cells (PBMCs) and CD4(+) T cells. The percentage of CD4(+)CD25(+) T cells in PBMCs was negatively correlated with elevated plasma interferon (IFN)-γ levels. A liver-specific analysis showed that the frequency of Foxp3(+) Tregs, transforming growth factor (TGF)-β1 and IFN-γ were increased in PBC patients. Our findings suggest that an imbalance between CD4(+)CD25(+) Tregs and cytotoxic cytokines plays a crucial role in the pathogenesis of PBC while the role of Foxp3 needs further investigation.

Effect of the Mutant Microphthalmia-Associated Transcription Factor Found in Tietz Syndrome on the In Vitro Development of Mast Cells

Mutations in microphthalmia-associated transcription factor (MITF) lead to Waardenburg syndrome type 2 (WS2), a dominantly inherited disorder involving hearing loss and pigment disturbances caused by a lack of melanocytes. On rare occasions, mutations in MITF lead to Tietz syndrome (TS), which is characterized by a severe WS2 phenotype. The MITF gene is the human homolog of the mouse microphthalmia (mi) gene in some families. Mi/mi mice show decreased numbers and an abnormal phenotype of mast cells (MC). In contrast, the number and phenotype of MC in WS2/TS patients who also have an alteration in their MITF gene are unclear. In this study, we identified a mutation in the MITF gene, delR217, which was equivalent to that found in mi/mi mice, in a case of TS. None of the MITF isoforms with the mutation were able to transactivate the tyrosinase gene promoter. In addition, mutant MITF-M showed dominant negative activity toward wild-type MITF-M, inhibiting its transactivation of the tyrosinase gene promoter. The patient's peripheral blood CD34 cells showed no differences with respect to total cell number or their expression levels of tryptase mRNA in a serum-deprived liquid culture system for 6 weeks when compared with normal control cells. These findings suggest that MITF does not play a critical role in MC development in humans.

Despite being highly diverse, immunovirological status strongly correlates with clinical symptoms during primary HIV-1 infection: a cross-sectional study based on 674 patients enrolled in the ANRS CO 06 PRIMO cohort

To analyse immunovirological status during primary HIV-1 infection (PHI) according to contemporary clinical status and time since infection. Plasma HIV-RNA and peripheral blood mononuclear cell (PBMC) HIV-DNA levels and CD4 cell counts were determined at enrolment in the ANRS PRIMO cohort. Time since infection was estimated based on both the number of antibodies on western blot at enrolment (0-1, 2-4 or > or =5 specific antibodies) and the estimated interval between infection and enrolment based on clinical and epidemiological features. Patients were classified according to the presence or absence of clinical symptoms at enrolment. Between 1996 and 2006, 674 patients were enrolled an estimated median of 47 days after infection. Median marker values were as follows: HIV-RNA 5.10 log(10) copies/mL (range <1.70-8.33); HIV-DNA 3.30 log(10) copies/10(6) PBMCs (<1.84-4.93); and 506 CD4 cells/mm(3) (40-1542). Median HIV-RNA and PBMC HIV-DNA levels were significantly higher in patients with 0 or 1 specific antibody (n = 71) than in patients with 2-4 (n = 228) or > or =5 antibodies (n = 375). Symptomatic patients had significantly higher HIV-RNA and PBMC HIV-DNA levels and lower CD4 cell counts. However, 10% of symptomatic patients recruited shortly after infection had favourable immunovirological status. Plasma HIV-RNA, PBMC HIV-DNA and CD4 cell count values were highly diverse and correlated strongly with clinical status during PHI. Early diagnosis was not always associated with severe PHI. Combining PBMC HIV-DNA with HIV-RNA, CD4 cell count and clinical symptoms would have allowed identification of 179 patients (26.5%) at high risk of rapid disease progression who did not meet current guidelines for early treatment initiation.

Mobilization of CD133+CD34− cells in healthy individuals following whole‐body acupuncture for spinal cord injuries

Acupuncture can alleviate symptoms of spinal cord injuries (SCI). The underlying mechanism, however, is unknown. We hypothesized that stem cells could be mobilized by acupuncture. Therefore, we enrolled 14 healthy study participants using acupuncture points for the treatment of SCI. The frequency of CD133 and CD34 cells in peripheral blood and the serum concentrations of matrix metalloproteinase (MMP)-9, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and interleukin-6 were determined before and after acupuncture (<1 hr, 24 hr, and 48 hr). CD133(+)34(-) cells were doubled 48 hr after acupuncture, with concomitant decreases in BDNF and MMP-9 levels. Interleukin-6 remained below detectable levels, eliminating a stress-induced cell release. Individuals acupunctured on control counterpoints showed no changes in CD133(+) cells. Our results indicate that acupuncture for SCI can mobilize human CD133(+)34(-) cells. (c) 2009 Wiley-Liss, Inc.

Memory CD8. + T Cells Specific for Minor Histocompatibility Antigen H60 Can Mediate the Graft-Versus-Leukemia Effect.

Abstract 1332Poster Board I-354In allogeneic hematopoietic stem cell transplantation (alloSCT), donor T cells mediate graft-vs-leukemia (GVL) but also cause graft-vs.-host disease (GVHD). Our previous studies indicate that memory T cells (TM) induce less GVHD than do naïve T cells (TN). Therefore the selective infusion of memory T cells could improve immune reconstitution with less GVHD. However, human memory T cells have a more restricted TCR repertoire than do TN and therefore may be less effective in mediating GVL. One approach for addressing this concern would be to vaccinate donors against a single miHA expressed by host leukemic cells. Then the selective transfer of memory T cells could improve both GVL and immune reconstitution with less GVHD. Questions remain about how best to apply this strategy. For example, differences in target antigen distribution could affect the re-expansion of transferred memory cells and determine whether vaccination augments GVHD. Also, different types of memory cells could behave differently. To begin to optimize this approach in mouse models we chose as our target antigen the H-2Kb-restricted minor histocompatibility antigen (miHA) H60. We cloned the H60 peptide sequence (LTFNYRNL) into a genetic construct encoding the heavy chain of a monoclonal antibody against DEC205. Donor C3H.SW (H-2b, H60-) mice were vaccinated with a single injection of anti-DEC205-H60 plus an agonist antibody against CD40 (FGK45). By two months post vaccination, H60-reactive memory cells were a stable population that comprised approximately 4-8% of splenic and peripheral blood CD8 cells. Approximately 50% of H60-tetramer+ cells had central memory phenotype, which could be ideal as central memory T cells mount strong anti-tumor responses. Similar results were obtained by using a prime/boost approach with H60-pulsed DCs. To test their function, CD44+ memory CD8 cells from H60-vaccinated mice were sort-purified and transferred into recipient B6 mice congenic for H60 (B6.H60; expression hematopoietically restricted ). By day 7 post transplant, H60-specific CD8+ TM from vaccinated mice comprised 70-90% of total splenic and blood CD8 cells, as compared to 1-5% in recipients of TM from unvaccinated mice. In contrast to anti-H60 responses by naïve CD8 cells, expansion of H60-tetramer+ cells from H60-vaccinated mice did not require CD4 help. To test whether target antigen distribution affects the re-expansion of H60-reactive cells from H60-vaccinated mice, expansion was compared in B6.H60 and B6.H60<right arrow>B6.actH60 (ubiquitous expression of H60 driven by an actin promoter) bone marrow chimeras. Approximately 60% of splenic and lymph node CD8 cells in both recipient groups were H60-tetramer+, though there was a trend towards increased overall numbers of tetramer+ cells in B6.H60 recipients. We are currently testing the GVL potency of TM from H60-vaccinated against mouse models of chronic phase (CP-CML) and blast crisis chronic myelogenous leukemia (BC-CML). In an ongoing experiment, very low numbers of CD8+ TM from H60-vaccinated mice mediate potent GVL against CP-CML relative to CD8+ TM from unvaccinated donors. Small numbers of CD8+ TM cells from vaccinated mice also mediated GVL against BC-CML, which is typically GVL resistant. Future studies will better define the potency of TM from H60-vaccinated mice and test these cells against CP-CML and BC-CML that do or do not express H60 but are otherwise identical. GVHD effect will also be tested. DisclosuresNo relevant conflicts of interest to declare.

The Efficacy and Safety of VP-16 and G-CSF as a Mobilization Regimen Prior to Autologous Stem Cell Transplantation (ASCT) for Patients (Pts) with Multiple Myeloma (MM) and Lymphoma.

Abstract 4225 INTRODUCTIONAlthough the number of ASCTs performed for myeloma and lymphoma continues to increase, there remains little consensus about the best means for mobilizing stem cells prior to transplantation and whether cytokine alone or cytokine plus chemotherapy is preferred. Cytokine alone, usually G-CSF, provides easy scheduling while the combination approaches generally provide higher cell yields and additional cytoreduction. Because of its value as a cytoreductive agent, and its lack of stem cell damaging properties or other toxicities such as hemorrhagic cystitis or significant liver toxicity, we investigated the use of mid-dose VP-16 plus G-CSF as a mobilizing regimen. Here, we report our experience with this regimen prior to ASCT for pts with various diseases. METHODSBetween May 2004 and June 2009, 296 pts with the following diseases underwent autologous stem cell transplantation following the use of VP-16 (375mg/m2 on D#1 and D#2) and G-CSF (5mcg/kg twice daily from D#3 through the final day of collection) for mobilization: Multiple Myeloma (152), Non-Hodgkin Lymphoma (98), Hodgkin Disease (38), Acute Promyeloyctic Leukemia (4), Acute Myelogenous Leukemia (1), Chronic Lymphocytic Leukemia (1), Plasma Cell Leukemia (1), and testicular cancer (1). In 14 pts, one dose of Rituximab (375mg/m2) was also given on D#1. 125 pts were female, 171 were male, and median age at the time of transplant was 55 years (range 19-79). Stem cell collection was initiated when the peripheral blood CD34 cell count was more than 7 per microliter. The target total CD34 cell collection was 5 × 106 cells. RESULTSAmong all pts, 286 (97%) had stem cells successfully collected after one mobilization. An additional 7 (2%) pts required more than one mobilization or bone marrow harvest, with one pt experiencing progressive disease after failed mobilization. 117 (40%) pts required either treatment for infection (inpatient hospitalization and/or intravenous antibiotics; 14% of all pts) or at least one supportive transfusion with PRBCs or platelets (33% of all pts). 1 pt is known to have developed secondary myelodysplastic syndrome, and there are no known cases of secondary AML. The median number of days collecting stem cells was 1 for MM pts and 2 for lymphoma pts, and the median total number of CD34 cells collected was 12 × 106 for MM pts and 6 × 106 for lymphoma pts. 94% of MM pts and 43% of lymphoma pts collected in a single day. The median time to neutrophil engraftment was 11 days for both MM and lymphoma pts, and the median time to a sustained platelet count > 20,000 without transfusions was 16 days for MM pts and 15 days for lymphoma pts. “Poor mobilizers” were defined as pts who failed to collect 5 × 106 cells in one or two days, and included 19% of all pts. 1% of MM pts and 40% of lymphoma pts were “poor mobilizers,” a statistically significant difference (p<0.001). 6 lymphoma pts failed to collect at least 2 × 106 cells, though all MM pts achieved this minimum target. The pre-mobilization white blood count (p=0.01) and platelet count (p=0.006) were associated with poor mobilization in lymphoma pts by univariable logistic regression analysis. Pts requiring more than 14 days to engraft neutrophils or 27 days to engraft platelets (> 1 S.D. above the mean) were considered “poor engrafters,” and included 16 pts (6%). We failed to detect statistically significant associations between “poor engrafters” (n=6 for lymphoma, n=10 for MM) and the covariates of “poor mobilizers,” age, disease type, amount and type of prior chemotherapy, radiation therapy, marrow cellularity or disease involvement, or the pre-mobilization complete blood count. CONCLUSIONVP-16 and G-CSF is an effective and safe mobilization regimen for pts undergoing ASCT with a low incidence of requiring hospitalization and/or IV antibiotics for fever, or of association with subsequent myelodysplasia or secondary AML. A diagnosis of lymphoma (HD or NHL) as opposed to MM, and low platelet or WBC count pre-mobilization predicted for poor CD34 cell yield. Further research is needed to help predict “poor mobilizers” and “poor engrafters” prior to transplant, as are the use of novel agents, such as the CXCR4 antagonist plerixafor, to improve outcomes in these populations. Disclosures:Shea:Genzyme, Millenium, Otsuka: Research Funding; Board of Directors, CALGB; CIBMTR Scientific Advisors, President-elect: Membership on an entity's Board of Directors or advisory committees; Genzyme, Genentech, Novartis: Consultancy.

Ex vivoexpansion and lentiviral transduction ofMacaca nemestrinaCD4+T cells

Macaca nemestrina is a nonhuman primate used as a model in preclinical studies of hematopoietic stem cell transplantation and adoptive transfer of T cells. Adoptive T cell transfer studies typically require ex vivo expansion of substantial numbers of T cells prior to their reinfusion into the subject. Pigtailed macaque peripheral blood CD4(+) cells were expanded using CD3 and CD28 antibody-coated beads. These cells were transformed using Herpesvirus saimiri and were also transduced with HIV-1 based lentiviral vectors. We report an efficient method for the ex vivo expansion of CD4(+) T cells from Macaca nemestrina peripheral blood. With this protocol, primary CD4(+) T cells can be expanded between 300- to 6000-fold during 24-day period and can be efficiently transduced with lentiviral vectors. Furthermore, these T cells can be transformed by Herpesvirus saimiri and maintained in culture for several months. The transformed T cell lines can be productively infected with the simian immunodeficiency virus (SIV) strain SIV(mac239). We have established methods for the expansion and transformation of primary M. nemestrina CD4(+) T cells and demonstrated the utility of these methods for several applications.

Evidence for Translocation of Microbial Products in Patients with Idiopathic CD4 Lymphocytopenia

Translocation of microbial products has been described in chronic human immunodeficiency virus (HIV) infection and correlates with activation of the immune system. We investigated the potential translocation of microbial products in idiopathic CD4 lymphocytopenia (ICL), a rare disorder characterized by low CD4 T cell counts in the absence of HIV infection. Plasma lipopolysaccharide (LPS) levels and T cell activation were measured in a cross-sectional cohort study of patients with ICL and HIV infection and healthy control subjects. Increases in CD4 T cell proliferation but not CD8 T cell proliferation were observed in patients with ICL. LPS levels were significantly elevated both in patients with ICL and in patients with HIV infection, and they were strongly correlated with the proportion of proliferating CD4 T cells in the cohort of patients with ICL (r = 0.88; P= .003). The proportions of T helper (Th) 17 and Th1 CD4 cells in peripheral blood were similar between patients with ICL, patients with HIV infection, and control subjects. These findings suggest a potential association of translocation of microbial products with perturbed CD4 T cell homeostasis in individuals with CD4 lymphopenic states other than HIV infection.

Depletion of CD4+ T Cells in Semen During HIV Infection and Their Restoration Following Antiretroviral Therapy

Information concerning the effects of HIV-1 infection, disease progression, and antiretroviral therapy (ART) on male genital white blood cell (WBC) profiles could provide important insight into genital immune defense in HIV-infected men and seminal HIV transmission mechanisms. To compare concentrations of WBC populations in semen from HIV-1-seronegative (HIV) and HIV-1-seropositive (HIV) men and determine whether HIV disease stage and ART are associated with alterations in seminal WBC profiles. Subjects were 102 HIV men, 98 ART-naive (ART) HIV men, and 22 HIV men on dual nucleoside ART, before and 6 months after addition of indinavir. Seminal WBCs, macrophages (MØ), and T-lymphocyte subpopulations were enumerated by immunohistology technique. Seminal CD4 and CD8 T-cell populations were severely depleted in most ART HIV men regardless of peripheral blood CD4 cell count. Seminal MØ counts were reduced by 50%. HIV men on dual nucleoside ART had significantly higher seminal MØ, CD4, and CD8 T-cell counts than ART HIV men; addition of indinavir led to a dramatic (>25-fold, P < 0.001) increase in seminal CD4 T-cell counts which paralleled an increase in blood CD4 cell counts. Two outlier ART HIV men with notably elevated seminal WBC profiles (>20 x 10 WBCs/mL) and infectious cell-associated HIV in semen are described. HIV infection severely depletes CD4 T cells in the male genital tract as it does at other mucosal sites. This provides evidence that ART HIV men have depressed T cell-dependent genital immune defense functions and are vulnerable to other genital infections that could promote HIV transmission. Seminal CD4 T-cell counts rebounded after treatment with a viral-suppressing ART regimen, indicating that ART may reverse HIV-associated genital immunosuppression. The relative abundance of seminal MØ in HIV men suggests that these cells may be predominant HIV host cells in the male genital tract and vectors of HIV transmission. A subgroup of HIV men with exceptionally elevated seminal MØ and CD4 T-cell counts and HIV titers may be highly infectious and contribute disproportionately to HIV transmission.

Indicaciones de la punción lumbar en pacientes con sífilis precoz activa coinfectados por el VIH. Casuística en un hospital terciario de La Coruña (España) 2003-2006

IntroductionIn the last years, the incidence of syphilis has incremented in Spain and coinfection with HIV occurs in a high percentage. In HIV-infected patients with syphilis, neurological complications, treatment failure and relapse appear to be slightly raised. Therefore, careful follow-up must be carried out because of the risk of developing neurosyphilis. According to the guidelines, lumbar puncture (LP) is indicated in HIV-infected patients with late latent syphilis or syphilis of unknown duration, but it is discussed in HIV-infected patients with early active syphilis. Recent research has been developed in order to determine clinical and analytical findings for identification of patients with high neurosyphilis risk. We review different opinions about this topic and report our experience. MethodsWe have performed LP in all HIV-infected patients with early active syphilis during 2003-2006. ResultsOf the eight studied patients, none met criteria for neurosyphilis. Three of eight (38 %) had a peripheral blood CD4 cell count CD4 +≤350 cells/μL. Seven of eight (87’5%) had RPR≥1:32. ConclusionIn these patients, performance of LP could be over indicated because of lack of well-established criterion. Our results are in agreement over to recent studies which restrict indication of LP to specific groups.

In vitro production of interferon-gamma by peripheral blood from patients with Graves' disease, Hashimoto's thyroiditis and rheumatoid arthritis

The production of interferon-gamma (IFN-gamma) by peripheral blood mononuclear cells (PBMC), CD4 cells, or CD8 cells in response to interleukin-2 (IL-2) stimulation has been studied; the samples were obtained from 12 healthy control subjects, 19 patients with Graves' disease (10 hyperthyroid and nine euthyroid), 13 patients with Hashimoto's thyroiditis (four hypothyroid and nine euthyroid), and 15 patients with rheumatoid arthritis (11 active and four inactive). A dose of IL-2 (25 U/ml) was utilized to induce IFN-gamma by PBMC from all four groups. The incremental increase in IFN-gamma values (with IL-2 stimulation minus without stimulation) was significantly less in PBMC from patients with Graves' disease, Hashimoto's thyroiditis, and rheumatoid arthritis than that in PBMC from control subjects. The values from PBMC in patients with Graves' disease in a euthyroid state were below normal but greater than those from patients with Graves' disease in a hyperthyroid state. The incremental increase in IFN-gamma values from Graves' disease PBMC correlated with the serum TSH values (r = 0.622, P less than 0.01), but not with thyroid autoantibodies (anti-thyroid microsomal antibodies, anti-thyroid microsomal antibodies, nor TSH-binding inhibitory immunoglobulin activities). The incremental increase in IFN-gamma from PBMC from both control subjects and Graves' disease was correlated with that from CD4 cells (r = 0.711, P less than 0.01), but not with that from CD8 cells. The production of IFN-gamma in response to IL-2 from PBMC in Graves' disease correlated inversely with thyroid function, appearing to reflect the very effect of hyperthyroidism in this process. The precise explanation of these phenomena remains unclear. The decreased response of IFN-gamma to IL-2 stimulation by PBMC from patients with Graves' disease, Hashimoto's thyroiditis, and rheumatoid arthritis seems to be a non-specific phenomenon occurring in both organ specific autoimmune disease and systemic autoimmune disease. It may be due to a down-regulation in autoimmune disease of CD4 cells in response to IL-2, a decreased level of IL-2 cellular receptors or a decreased receptor affinity, associated increased soluble IL-2 receptors, or a defect of the intra-CD4 cellular IL-2 signal to produce or release IFN-gamma in the conditions studied.