Peripheral CD34 Cell Count Research Articles

Peripheral blood stem cells mobilization in patients with relapsed or refractory lymphomas: A single-center experience.

High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (AHSCT) is an established treatment for chemosensitive patients with relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). We present the results of using different salvage chemotherapy plus granulocyte colony stimulating factor (G-CSF) for mobilization of peripheral blood stem cells in R/R lymphoma patients. For salvage chemotherapy, 93 patients received platinum-containing regimens, 4 patients received cytarabine-containing regimens, and 5 patients received other regimens. Patient distributions were HL (n = 35) and NHL (n = 67). In 87.2% of patients, first mobilization trial was successful (>2 × 10 6 CD34+ cells/kg). In 58.8% of patients, first apheresis season >5 × 10 6 CD34+ cells/kg collections was achieved. All 12.7% of patients were poorly mobilized at the first mobilization. There was no statistical difference between the previous chemotherapy numbers and failed mobilization ( P > 0.05). Five patients who were poorly mobilized and 4 patients who were successfully mobilized underwent a previous radiotherapy ( P < 0.05). Thirteen patients who were poorly mobilized in the first mobilization underwent a platinum-containing salvage regimen. At the time of the first mobilization, the average peripheral CD 34 counts in the successfully mobilized group were statistically higher than that in the poorly mobilized group ( P < 0.01). We demonstrated that peripheral CD 34 cell count in the peripheral blood on the first apheresis day was a significant factor for more stem cell mobilization, fewer apheresis sessions, less volume, and earlier neutrophil engraftment for patients with R/R lymphoma and eligible for AHSCT. The history of the previous radiotherapy was a significant factor for poor mobilization.

The Impact of Single Dose Plerixaforas an Adjunct to G-CSF Based Peripheral Blood Stem Cell Mobilisation on the Graft Composition and Outcome for T Replete Haploidentical PBSC Transplantation with Post Transplantation Cyclophosphamide: A Pilot Study

▪Introduction : We conducted a prospective study on T cell and NK cell subset composition of the graftand the transplant outcome with single dose of subcutaneous Plerixafor(Px) to GCSF based mobilisation in T-replete Haploidentical Peripheral blood Stem cell transplantation with post-transplantation cyclophosphamide (PTCy) in 25 donors (G+Px group). This was compared with 26 donors who received G-CSF alone for mobilisation (G group).Material and Methods : With the aim to collect 10 x 106/kg CD34 cells in a single apheresis, donors with peripheral CD 34 count less than 50 cells /µl in the peripheral blood on day 4 of mobilization were administered Injection Plerixafor at the dose of 0.12mcg/kg at 12 midnight in addition to routine GCSF. This was followed by PBSC harvest starting 11 hours later at 11 A.M on the next day. The rest were continued G-CSF only as per protocol.Results: CD 34 cell count[G+Px-136(range, 52-351)vs G-139 (range,72-240) cells/µl ] in the peripheral blood on day 5 as well as that in the graft [G+Px- 2.7(range 1.1-6.0) VsG-2.3(range 0.5-5.3) X106 /ml, p=0.05] comparable between the two groups, despite a significantly lower peripheral CD34 cell count on day 4 in the pleraxifor group (G+Px-33(range, 6-47) vs G-81(58-168) cells/µl, p=0.0001). The total nucleated cell count was higher in the G- group[ 3.4(range 1.7-5.0) vs 3.1(range 1.15-4.73) X108/ml, p=0.05 ]. Despite the lower trend of CD4+ T cells[ 2.3(range 0.3-6.8) Vs 2.7(range 0.53-6.8) X107/ml, p=0.09 ]in the G-CSF group, mobilisation of Tregs was similar(7.1% vs 6.9% in G+Px group,p=0.1). There were no differences noted in the absolute number of NK cells [G+Px-5.3(range 0.8-11.6) vs 5.7.(range 1.7-12.9) X106/ml, p=0.9 ], or the CD56+16+/16- subsets. The time to engraftment was similar in both groups. The incidences of acute and chronic GVHD, non-relapse mortality and relapse were also similar.Conclusion: Addition of single dose pleraxifor to G-CSF mobilisation improves CD34 recovery and does not significantly alter the T and NK cell composition of the graft, including Tregs. No adverse impact was noted on engraftment, GVHD, NRM and relapse. Our study demonstrates that pleraxifor can be safely used as an adjunct to G-CSF to boost CD34 mobilisation without impacting conventional or regulatory T cells and NK cells in the graft. The preliminary data suggests no adverse impact on transplant outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gut microbiota in HIV\u2013pneumonia patients is related to peripheral CD4 counts, lung microbiota, and in vitro macrophage dysfunction

Pneumonia is common and frequently fatal in HIV-infected patients, due to rampant, systemic inflammation and failure to control microbial infection. While airway microbiota composition is related to local inflammatory response, gut microbiota has been shown to correlate with the degree of peripheral immune activation (IL6 and IP10 expression) in HIV-infected patients. We thus hypothesized that both airway and gut microbiota are perturbed in HIV-infected pneumonia patients, that the gut microbiota is related to peripheral CD4+ cell counts, and that its associated products differentially program immune cell populations necessary for controlling microbial infection in CD4-high and CD4-low patients. To assess these relationships, paired bronchoalveolar lavage and stool microbiota (bacterial and fungal) from a large cohort of Ugandan, HIV-infected patients with pneumonia were examined, and in vitro tests of the effect of gut microbiome products on macrophage effector phenotypes performed. While lower airway microbiota stratified into three compositionally distinct microbiota as previously described, these were not related to peripheral CD4 cell count. In contrast, variation in gut microbiota composition significantly related to CD4 cell count, lung microbiota composition, and patient mortality. Compared with patients with high CD4+ cell counts, those with low counts possessed more compositionally similar airway and gut microbiota, evidence of microbial translocation, and their associated gut microbiome products reduced macrophage activation and IL-10 expression and increased IL-1β expression in vitro. These findings suggest that the gut microbiome is related to CD4 status and plays a key role in modulating macrophage function, critical to microbial control in HIV-infected patients with pneumonia.

Significant correlation between peripheral blood CD34+ cell count in children prior to aphaeresis and CD34+ cell yield following aphaeresis: A single-center experience.

Numerous adults' studies demonstrated that preaphaeresis CD34+ cells significantly correlate with the number of CD34+ cells collected by the aphaeresis procedure. Equivalent studies in children are scarce. We studied retrospectively 92 aphaeresis procedures performed following chemotherapy (44) or in steady state (48) in 60 pediatric patients (40 males, 20 females), median age of 7.5years. Aphaeresis procedures were performed using a SPECTRA Optica (TERUMOBCT) continuous flow cell separator. CD34+ cell concentrations were assessed using flow cytometry. A highly significant correlation between peripheral CD34 cell count on the day of aphaeresis and CD34 cell yield per kg (R2 =.824, P<.0001) was demonstrated. A higher preaphaeresis CD34 cell count was demonstrated in patients with higher preaphaeresis white blood cell count, in patients with brain tumors, and in patients who received chemotherapy as part of their mobilization protocol. A threshold number of 20 peripheral CD34+ cell/μL was found to predict harvesting of 3×106 stem cells/kg, and 30 peripheral CD34+ cell/μL for harvesting of 5×106 stem cells/kg. This significant correlation between peripheral CD34 cell count and CD34 cell yield, and the threshold number of peripheral CD34 found to predict adequate harvesting can be useful in planning the optimal time for aphaeresis in children.

Phase 2 Study Of Bendamustine, Bortezomib (Velcade) and Prednisone (BVP) For Newly Diagnosed Multiple Myeloma (MM)

BackgroundBortezomib-based combinations, including alkylating agents (VMP or CyBorD) or immunomodulatory drugs (VTD or RVD) have been established as regimens widely used in newly diagnosed MM patients. Bendamustine is a bifunctional alkylating agent effective in relapsed and/or refractory MM patients, and approved in Europe in combination with prednisone for elderly newly diagnosed MM. Since bendamustine may be more efficient than other alkylators, an attractive possibility would be to explore it in combination with bortezomib and prednisone (BVP) in newly diagnosed MM patients both transplant and non transplant candidates. Patients and Methods60 newly diagnosed MM patients were included in the trial. The first cycle consisted on bendamustine at 90 mg/m2 given IV on days 1 and 4, in combination with bortezomib at 1,3 mg/m2 given IV on days 1, 4, 8, 11, 22, 25, 29 and 32 and prednisone at 60 mg/m2 given PO on days 1 to 4. In the following cycles, bendamustine was given on days 1 and 8, and bortezomib on days 1, 8, 15 and 22 (weekly schedule), and prednisone as it was previously described. Patients younger than 65 years proceeded to peripheral blood stem cell collection (PBSC) using growth factors alone after 4 cycles; HDT-ASCT was performed after 6 cycles. Patients older than 65 years received up to nine 28-day cycles. ResultsBetween May 2011 and July 2012 enrollment was completed (60 pts). Median age was 61 years (range 38-82; 18 pts ≥65), 67% had ISS stage II/III, and 67% had unfavorable cytogenetics: t(4;14), t(14;16), del 17p or 1q gains by FISH.After a median of 6 cycles (2-9), 75% of patients achieved at least PR, including 16% of sCR, 9% CR and 28% of VGPR. Although the differences were not statistically significant, there was a trend to higher CR rate in the group of patients <65 years (31%) compared with elderly patients (11%). No differences were observed in overall response rates and CR rates in patients with standard and high risk cytogenetic abnormalities.Forty patients proceeded to stem cell collection after a median of 4 cycles of BVP. Upon using G-CSF alone, 14 pts (35%) failed to collect a minimun of 2 x 106 CD34+ cells/Kg. An ammendment was done and plerixafor was recomended for poor mobilizers (peripheral CD34 cell count inferior to 10/μL on day 4); all patients but 2 achieved, with G-CSF plus plerixafor, the minimum of CD34+ cells required to proceed to ASCT. These 2 patients successfully collected CD34+ cells using chemotherapy plus G-CSF and plerixafor. Of the 31 patients who received HDT-ASCT, sCR and CR rate before transplant was 18% and 13%, respectively, upgrading up to 39% of sCR and 13% CR after transplant. 7 pts (22%) achieved immunophenotypic CR.After a median f/u of 12 months (5-25), 8 pts have progressed, resulting in a 15-m TTP of 85%. Concerning OS, 89% of patients remained alive at 15 months. None of patients achieving sCR and CR have progressed and all of them are alive at 15 months. Regarding cytogenetic abnormalities, although there were not significant differences, one patient progressed in standard risk group and five in the high risk subgroup resulting in a 15 m-TTP of 93% vs 85%. No significant differences have been observed in terms of 15 m-OS between standard and high risk cytogenetic subgroup (100% vs 92%, respectively).As far as toxicity is concerned, hematologic toxicities included: G3/4 anemia (11%), neutropenia (23%), and thrombocytopenia (14%). The most common G3/4 non-hematologic toxicities were: asthenia (10%), infections (9%), and peripheral neuropathy (4%). ConclusionsIn patients candidates to HDT-ASCT, response rates obtained before and after transplant are comparable to other three drug bortezomib-based combinations, such as VTD or CyBorD. However, growth factors alone for stem cell collection after four BVP cycles as induction resulted in a 35% of poor mobilizers who were rescued with plerixafor. In the elderly population, although the number of patients included was small, BVP seems not superior to VMP in response rates. Disclosures:Mateos:Janssen, Mundipharma: Honoraria. Off Label Use: bendamustine plus bortezomib and prednisone is not an approved combination for first line of therapy. Oriol:Celgene: Consultancy. Ocio:Onyx: Consultancy, Research Funding; Novartis: Consultancy; Array Biopharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding. Alegre:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Bladé:Janssen, Mundipharma: Honoraria. San Miguel:Janssen, Mundipharma: Membership on an entity's Board of Directors or advisory committees.

High Correlation of Pre-Apheresis Peripheral Blood CD34+ Cell Counts with Final Apheresis Product CD34+ Cell Count by Using a More Accurate Determination of Patient Blood Volume

Abstract 2996The final apheresis product CD34+ cell count per Kg of recipient weight is used to determine if additional apheresis procedures are necessary to collect the targeted amount of hematopoietic stem cells for autologous peripheral blood stem cell transplantation. Flow cytometric analysis of CD34+ cells can take several hours. A more timely technique to predict apheresis product CD34+ cell counts during apheresis may help determine if further administration of cytokines is necessary or if apheresis catheters can be removed, and overall improve the efficiency of patient (pt) care. We performed a retrospective review of all pts undergoing autologous peripheral blood stem cell mobilization with granulocyte-colony stimulating factor (G-CSF) alone, G-CSF and plerixafor, or with chemotherapy followed by G-CSF, from July 2010 through May 2011 who underwent peripheral blood stem cell collection with apheresis on the COBE Spectra cell separator. Linear regression models were used to formulate the calculation of pt blood volume based on the pre-apheresis CD34+ cell count per micro liter of blood, the final apheresis product CD34+ cell counts, and the amount of blood processed during the apheresis procedure. This calculated blood volume is expressed by the formula BV = 82.5(patients weight in Kg) + 793. We then prospectively evaluated the next consecutive pts who underwent stem cell mobilization and apheresis in June and July 2011. Twenty-seven apheresis collections were done on 26 pts. Fourteen pts were female, and 12 pts were male. Seventeen pts were diagnosed with myeloma, 6 pts with NHL, and 3 pts with other diseases. Twenty pts were mobilized with G-CSF (10 ug/kg daily) with apheresis to begin on day 5. Fifteen of the 20 pts required plerixafor on day 4 because of low peripheral blood CD34+ cell counts (< 10/ul). Six pts were mobilized with chemotherapy followed by G-CSF 10 ug/kg daily until peripheral CD34 cell counts recovered greater than 10/ul and then apheresis was started. Each pt had their blood volume calculated according to the formula above and the peripheral blood CD 34+ cell count was measured on the first and second day of apheresis. The peripheral blood CD 34+ cell count/ul was multiplied by 1000 and this product was multiplied by the calculated blood volume and then divided by the pts weight [(PBCD34+ cell count × 1000) × BV]/Kg to determine the predicted apheresis product CD34+ cell count, which was then compared to the actual apheresis product final CD34+ cell count. On the first day of collection the mean for the predicted product CD34+ cell count was 4.98 × 106 +/− 3.1 × 106, and the actual apheresis product CD34+ cell count was 4.61 × 106 +/− 2.90 × 106 (Pearson correlation r value of 0.913 and a p value <0.001)(see figure). Nineteen collections were evaluable on the second day of collection with the mean for the predicted product CD34 + cell count of 2.08 × 106 +/− 1.64 × 106, and the mean for the actual apheresis product CD34+ cell count of 2.29 × 106 +/− 0.768 × 106 (Pearson correlation r value of 0.620 with a p value of 0.005). There was no significant difference in the correlation between patients mobilized with G-CSF alone, G-CSF and plerixafor or after chemotherapy and G-CSF. In conclusion, a more accurate determination of patient blood volume allowed for a high degree of correlation on the first day of peripheral blood stem cell collection on the COBE Spectra machine between the predicted product CD34+ cell count and the actual apheresis product CD34+ cell count. An accurate prediction of the final apheresis product CD34+ cell count may allow for less cytokine administration, quicker removal of apheresis catheters, and more efficient disposition of patients undergoing peripheral blood stem cell collection. [Display omitted] Disclosures:Shaughnessy:Otsuka: Honoraria, Speakers Bureau; Millenium: Honoraria, Speakers Bureau; Genzyme: Consultancy, Honoraria, Research Funding, Speakers Bureau.

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.

Targeting Trojan Horse leukocytes for HIV prevention

…. is sure design'd, by fraud or force: trust not their presents, nor admit the horse. Virgil, Aeneid Human immunodeficiency virus type 1 (HIV-1), the lymphotropic virus that causes AIDS, has infected more than 60 million people worldwide since its clinical appearance in 1981. Despite intensive prevention efforts, the HIV/AIDS epidemic continues to spread, particularly in developing countries in sub-Saharan Africa, southeast Asia and the Caribbean, as well as the developed world [1]. Although HIV can be transmitted very efficiently parenterally, the advent of routine blood screening prior to transfusion and harm reduction programs for injection drug users, have made this mode of transmission much less common than mucosal transmission. Most new HIV infections are attributable to mucosal transmission: through genital and rectal mucosae in the case of sexual transmission and through oral or gastrointestinal mucosae in the case of mother-to-child transmission [2]. Much has been learned about HIV pathogenesis and infection mechanisms at the molecular level, but the scientific community has yet to develop an effective vaccine or microbicide for HIV prevention. Many unanswered questions remain concerning HIV-1 sexual transmission. In 1983, barely 2 years into the AIDS epidemic, we hypothesized that the agent that was subsequently identified as HIV-1 may be sexually transmitted by infected ‘Trojan Horse’ leukocytes in semen [3]. This hypothesis was based on our knowledge at the time that human semen contains substantial numbers of T lymphocytes and macrophages, which could host a T-cell tropic virus, and the following assumptions: intracellular virus would be better protected than free virus from adverse effects of antiviral factors in the genital environment such as antiviral antibodies likely to be present in genital secretions of the virus-infected transmitter, as well as antimicrobial peptides that play an important role in genital innate immune defense; and virus-infected allogeneic cells could also escape early detection by major histocompatibility complex (MHC)-restricted cytotoxic T cells in a new host. Over the intervening 25+ years, others have also championed this cause [4,5], and convincing evidence has emerged from clinical research as well as in-vitro and animal studies that infected leukocytes indeed play a role in HIV transmission. Yet, most recent research on sexual HIV transmission has focused on cell-free HIV in genital secretions because of the wide availability of HIV RNA quantification assays. Furthermore, the majority of HIV vaccines and microbicides have been designed to block transmission of cell-free virus and have been tested in animal and in-vitro models that use cell-free virus as the only infectious inoculum. As the molecular events underlying cell-associated HIV transmission differ from those underlying cell-free virus transmission, many of the current vaccine and microbicide candidates might not be expected to protect against cell-associated HIV transmission. The failure of several recent vaccine and microbicide clinical trials may be due in part to this oversight. It should be possible to design strategies that block cell-associated HIV transmission as well as cell-free HIV transmission. In this article, we present an overview of research that has been conducted on cell-associated HIV mucosal transmission and recommendations for future research. We focus on sexual HIV transmission, but this review also has relevance for mother-to-child HIV transmission, which may occur through mucosal transmission of cell-associated HIV from maternal genital or mammary gland secretions [6–8]. We review published reports that describe and enumerate HIV-infected cells in genital secretions, and compelling evidence from clinical, animal and in-vitro studies demonstrating that such cells can transmit HIV across genital tract epithelial surfaces; potential molecular mechanisms underlying cell-associated HIV transmission that could be specifically targeted by future HIV prevention strategies; and in-vitro and animal cell-associated HIV transmission models currently used for studies on cell-associated HIV transmission mechanisms and for testing vaccine and microbicide candidates. Using this information as a foundation, we discuss the evidence and probability that various current microbicide and vaccine approaches prevent cell-associated HIV transmission, and suggest additional microbicide and vaccine concepts and experiments that will move this field forward.

Compartmentalized Human Immunodeficiency Virus Type 1 Originates from Long-Lived Cells in Some Subjects with HIV-1–Associated Dementia

Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) shortly after systemic infection and can result in the subsequent development of HIV-1–associated dementia (HAD) in a subset of infected individuals. Genetically compartmentalized virus in the CNS is associated with HAD, suggesting autonomous viral replication as a factor in the disease process. We examined the source of compartmentalized HIV-1 in the CNS of subjects with HIV-1–associated neurological disease and in asymptomatic subjects who were initiating antiretroviral therapy. The heteroduplex tracking assay (HTA), targeting the variable regions of env, was used to determine which HIV-1 genetic variants in the cerebrospinal fluid (CSF) were compartmentalized and which variants were shared with the blood plasma. We then measured the viral decay kinetics of individual variants after the initiation of antiretroviral therapy. Compartmentalized HIV-1 variants in the CSF of asymptomatic subjects decayed rapidly after the initiation of antiretroviral therapy, with a mean half-life of 1.57 days. Rapid viral decay was also measured for CSF-compartmentalized variants in four HAD subjects (t1/2 mean = 2.27 days). However, slow viral decay was measured for CSF-compartmentalized variants from an additional four subjects with neurological disease (t1/2 range = 9.85 days to no initial decay). The slow decay detected for CSF-compartmentalized variants was not associated with poor CNS drug penetration, drug resistant virus in the CSF, or the presence of X4 virus genotypes. We found that the slow decay measured for CSF-compartmentalized variants in subjects with neurological disease was correlated with low peripheral CD4 cell count and reduced CSF pleocytosis. We propose a model in which infiltrating macrophages replace CD4+ T cells as the primary source of productive viral replication in the CNS to maintain high viral loads in the CSF in a substantial subset of subjects with HAD.

Primary macrophages from HIV-infected adults show dysregulated cytokine responses to Salmonella, but normal internalization and killing

Adults with advanced HIV are susceptible to invasive and recrudescent infections with nontyphoidal salmonellae. To examine whether persistence and recurrence of salmonella infection results from HIV-related defects in macrophage internalization and intracellular killing or from ineffective type 1 cytokine responses. Such defects could be a direct consequence of macrophage HIV infection or secondary to reduced enhancement of macrophage effector functions by interferon-gamma (IFNgamma) as CD4 cell count falls. Ex-vivo scientific case-control study. Primary ex-vivo human alveolar macrophages (huAM) from HIV-negative and HIV-positive subjects were challenged with Salmonella typhimurium under unprimed and IFNgamma-primed conditions to study internalization and intracellular killing of bacteria and cytokine responses of huAM. Priming of huAM with IFNgamma reduced bacterial internalization but enhanced microbicidal activity against intracellular salmonellae. HuAM from HIV-positive subjects showed unimpaired internalization and intracellular killing of salmonellae, with and without IFNgamma priming. Opsonic and mannose receptor (CD206)-mediated entry was not required for optimal internalization. HuAM from HIV-positive subjects, however, exhibited increased secretion of tumour necrosis factor alpha (TNFalpha), interleukin (IL)-10 and IL-12 in response to S. typhimurium challenge, regardless of IFNgamma priming. This cytokine dysregulation showed a trend to a curvilinear relationship with peripheral CD4 cell count, with marked decline at values < 250 cell/mul. Dysregulation of proinflammatory cytokine release, including IL-12, by macrophages during salmonella infection may underlie the susceptibility to severe salmonellosis in patients with AIDS. This defect was not reversed by IFNgamma and may represent a proinflammatory effect of HIV infection upon the macrophage or the alveolar milieu.

Peripheral Hematopoietic Stem Cell Mobilization and Collection Efficacy Is Not an Independent Prognostic Factor for Autologous Stem Cell Transplantation.

Background: High dose chemotherapy with autologous hematopoietic stem cell rescue has become one of the standard treatment options for many hematopoietic malignancies. Mobilization by granulocyte colony-stimulating factor (GCSF) with or without chemo priming and followed by peripheral blood stem cell (PBSC) collection is the preferred method to obtain progenitor cells. Successful mobilization and collection of stem cells is dependent upon a number of clinical factors including previous chemotherapy history and disease stage. One question concerns whether efficacy of mobilization and collection, as measured by the number of days or number of collections to reach a defined CD34 cell target dose, is an independent prognostic factor for autologous stem cell transplantation outcome.Method: A total of 358 patients transplanted from January 2003 to December 2004 (201 males and 157 females, ages from 2.7 to 77.3 years old with median of 53 years of age) underwent autologous PBSC collection after mobilization with GCSF at 10 mcg/kg to 16mcg/kg with or without chemo priming. PBSC collections are typically initiated on day 5 post GCSF injection, or day 10 after chemo priming and GCSF injection without information about their absolute peripheral CD34 cell count. This retrospective study included patients with the following diagnoses: 45 (13%) acute Myelogenous leukemia (AML), 124 (35%) non-Hodgkin's lymphoma (NHL) 33 (9%) Hodgkin's disease (HD), 123 (34%) multiple myeloma (MM), and 33 (9%) solid tumors. All patients underwent 4 hours of daily PBSC collection with an average processed volume of 12 to 15 liters at average flow rate of 40ml to 60ml/minute (by Cobe Spectra, Gambro BCT, Denver, CO) until a minimum CD34 count of 2×106/kg of body weight with a target of CD34 cell count of 5×106/kg is reached. The mean number of collections to reach this minimum CD34 cell dose was 2.7 (median 1, range 1 to 14). Collected cells were cryopreserved and stored at −140°C in the vapor phase of a liquid nitrogen freezer prior to their transplantation. Patients received a median CD34 cell dose of 6.4×106/kg with range 2.0 to 67.6×106/kg for their transplants. Correlations were performed between PBSC mobilization/collection efficacy and transplantation outcomes.Results: Both greater CD34 cell dose used in the transplant (r= −0.24, p<0.0001) and shorter number of days PBSC to reach the minimum 2×106/kg (r=0.10, p=0.06) fostered quicker engraftment. Regardless, most patients engrafted within a reasonable time, with outliers not explained by cell dose or days to collect 2×106 CD34 cells. There was only one graft failure. CD34 cell dose and number of days PBSC collection to reach the minimum 2×106/kg of CD34 cell dose did not independently affect the overall survival or disease-free-survival for this study population as a whole and per each diagnostic group. Patient age at transplantation did not confound the result.

Amount of lymphatic tissue fibrosis in HIV infection predicts magnitude of HAART-associated change in peripheral CD4 cell count

The structure of lymphatic tissues is an important component of lymphatic tissue T-cell homeostasis. Collagen deposition in lymphatic tissues (common in HIV infection) disrupts the niche and limits the size of the resident CD4 cell population. In this report we show that a single measurement of lymphatic tissue collagen predicts the magnitude of recovery of the peripheral CD4 cell pool with HAART (P < 0.001). This suggests that collagen-targeted therapies might be of benefit.

Altered expression of CD43-hexasaccharide isoform on peripheral T lymphocytes from HIV-infected individuals.

To examine if peripheral T lymphocytes from HIV-infected individuals show abnormalities in the surface expression of CD43, the major sialoglycoprotein of leukocytes. A series of 86 HIV-positive individuals was studied. The subjects, grouped by their peripheral CD4 cell count, were in different stages of the disease as defined by the Centers for Disease Control and Prevention (CDC). Peripheral leukocytes and isolated lymphocytes were examined by double and triple immunofluorescence flow cytometric and Western blot analyses with monoclonal antibodies, which discriminate between CD43 isoforms. We found elevated percentages of the surface expression of CD43-hexasaccharide isoform on T lymphocytes from 82 out of 86 individuals tested. Increasing percentages are progressively found in CDC groups 1, 2 and 3 patients. The expression of the molecule is remarkably biased towards the CD8 cell subpopulation. The percentage of cells bearing human leukocyte antigen-DR locus molecules (HLA-DR) is also augmented. Two subsets expressing T305 have been identified: a minor subset that co-expresses HLA-DR and T305; and a second population formed by the majority of T305-positive cells, which lack surface HLA-DR. Finally, we found CD43 bands with altered electrophoretic mobility in lysates from peripheral lymphocytes from all HIV-positive individuals tested. The augmented expression of CD43-hexasaccharides and the observed cellular distribution suggest an important regulatory role for this molecule in HIV-specific responses.

Highly active antiretroviral therapy decreases the incidence of visceral leishmaniasis in HIV-infected individuals.

In Mediterranean countries visceral leishmaniasis (VL) is a zoonosis caused by the protozoon Leishmania infantum. In the early 1990s, the incidence of VL increased in southern European countries, probably because of the increased number of cases that occurred in patients with HIV infection [1]. Recently, highly active antiretroviral therapy (HAART), using a combination of protease inhibitors and reverse transcriptase inhibitors [2], has been demonstrated not only to have a powerful activity on HIV, as shown by the reduction in plasma HIV RNA and the increase in peripheral CD4 cells, but also to cause a drop in the incidence of opportunistic infections and deaths [3,4]. However, no data are presently available on the effect, if any, of HAART on the incidence of VL in HIV-infected individuals. Therefore, the aim of this study was to assess the incidence of VL, before and after the introduction of HAART, as standard therapy in HIV-infected patients in a large cohort of Italian HIV-infected subjects. In particular, we performed a 9 year (1991–1999) retrospective cohort study considering all cases of VL admitted to the Department of Infectious Diseases of a large university hospital in Rome, Italy. Over the study period, we observed 36 cases of leishmaniosis, 20 cases (55%) in HIV-infected patients and 16 (35%) in immunocompetent individuals. For a better evaluation of the differences in VL incidence in HIV-infected patients attributed to HAART, two periods of time were compared: January 1991–December 1995 (period A), and January 1997–December 1999 (period B). HAART became the standard of care in the majority of our patients after September 1996, when protease inhibitors became available in Italy, and for this reason we have not considered, in the statistical analysis, cases of VL occurring in 1996. In particular, we observed 18 episodes of VL in period A and 13 in period B. HIV infection was present in 78% (14/18) of the total VL cases observed in period A and in 15% (2/13) in period B (P < 0.01; odds ratio 19.25; 95% confidence interval 2.37–220.18). Comparing the incidence of VL in HIV-infected patients in periods A and B, a statistically significant reduction in the incidence of VL, from 0.7 episodes per 100 person/years to 0.13 episodes per 100 person/years, was observed (P < 0.01; odds ratio 5.25; 95% confidence interval 1.20–47.65). The majority of HIV-infected patients with VL were men (72%) and in the C3 category of HIV infection (75%) without any significant statistical differences between period A and period B. The mean number of peripheral CD4 cells was 54/mm3 (range 1–207/mm3). The mean level of plasma HIV RNA, available in patients observed in period B only, was 105 000 copies/ml. The analysis of risk factors for VL, including the geographical area of origin of the patients, did not differ significantly between the two study periods. In conclusion, our study confirms that HIV is a relevant predisposing condition for VL, in Italy as well as in other Mediterranean countries [5,6], and indicates, as a novel observation, a significant decrease in the incidence of VL in HIV-infected patients after the introduction of HAART. The reduction in the incidence of VL is probably the consequence of the well-known immune restoration induced by HAART [2], as witnessed by the increase in median peripheral CD4 cell counts and the decrease in HIV viraemia (data not shown). Such a reduced incidence of VL in HIV-infected patients could have a substantial impact on future morbidity and health costs in Mediterranean countries as well as in other geographical areas where VL is endemic. Mario Tumbarello Evelina Tacconelli Silvia Bertagnolio Roberto Cauda

Quantification of HIV-1-specific T-cell responses at the mucosal cervicovaginal surface.

To characterize HIV-1 specific cellular immune responses at mucosal surfaces using a rapid, sensitive enzyme-linked immuno-spot (ELISPOT) technique. Cervicovaginal mononuclear cells obtained from cytobrush and cervicovaginal lavage were assessed for production of interferon-gamma (IFN-gamma) in response to stimulation by HIV-1 antigens. HIV-1 specific responses were compared in a cross-sectional study of two HIV-1-positive patient groups: women not currently on antiretroviral therapy with peripheral CD4 cell counts > 250 x 10(6)/l (n = 12); and women on highly active antiretroviral therapy (HAART) (n = 9). Mononuclear cells from peripheral blood or cervicovaginal specimens were assessed in an ELISPOT assay for responses to HIV-1 antigens expressed by recombinant vaccinia viruses. This assay detects primarily CD8 T cells and shows good correlation with MHC class I tetramer staining of cytotoxic T lymphocytes. HIV-1 specific IFN-gamma spot-forming cells were detected in cervicovaginal samples of one out of nine women (11%) on HAART and five out of 12 women (42%) not currently on HAART. In peripheral blood mononuclear cells, HIV-1 specific IFN-gamma spot-forming cells were significantly more numerous in women not currently on HAART than in women on HAART (P = 0.009). In most cases, antigens recognized by mucosal T cells were also recognized by PBMC; however, there were exceptions. HIV-1-specific antigen-reactive T cells may be detected in routine, noninvasive gynecological specimens. The results suggest that cervicovaginal HIV-1-specific T cells may be less numerous in individuals on HAART than in those not on HAART, as shown previously for HIV-1-specific cytotoxic T lymphocytes in the peripheral blood.

Cerebrospinal fluid viral load, intrathecal immunoactivation, and cerebrospinal fluid monocytic cell count in HIV-1 infection.

To assess the association between cerebrospinal fluid (CSF) viral load, intrathecal immunoactivation, and immunosuppression in HIV-1-infected individuals with no antiretroviral treatment experience a cross-sectional study of stored frozen CSF and plasma samples were conducted. The study population included a total of 120 antiretroviral-naive HIV-1-infected patients, 110 neuroasymptomatic patients, and 10 with neurologic complications. HIV-1 RNA was quantified in cell-free CSF and plasma using polymerase chain reaction (PCR; Roche Amplicor HIV-1 Monitor version 1.5, Roche Diagnostic Systems, Hoffmann-La Roche, Inc., Base, Switzerland). Immunoactivation was measured by CSF-serum IgG index, CSF neopterin concentrations, and CSF monocytic cell count. The CSF HIV-1 RNA load did not differ significantly between patients with or without neurologic complications. In patients without neurologic symptoms, the CSF monocytic cell counts were correlated to the CSF viral load (r(s) = 0.40, p < .001), whereas IgG index and CSF neopterin concentrations were correlated to the viral load only in the subgroup of patients with CD4 counts > or =200 x 10(6) cells/L. In this subgroup of patients, the peripheral CD4 cell count was, as expected, inversely correlated to the CSF viral load (r. = -0.36, p < .01), whereas in patients with CD4 counts <200 x 10(6) cells/L, an unexpected, significant positive correlation (r(s) = 0.43, p < .01 ) was found. In HIV-1-infected patients with neurologic complications, no significant correlations were found between immune activation, CSF viral load, and immunosuppression.

The prevalence of skin disease in HIV infection and its relationship to the degree of immunosuppression.

A cross-sectional study of human immunodeficiency virus (HIV) positive patients who attended the HIV clinic in Brighton over a 4-month period was carried out to describe the prevalence and severity of skin manifestations in HIV-positive patients and to elucidate their association with the peripheral CD4 cell count and with the HIV disease stage. The subjects were consecutively examined by an experienced dermatologist. Skin manifestations were classified into infections, dermatoses, pruritus and neoplasm. A severity index was derived by scoring each condition as either absent, mild, moderate or severe. One hundred and fifty-one patients were enrolled with a mean age of 38.3 years. One hundred and thirty-nine were homo/bisexual men; 58 were asymptomatic and 35 had acquired immune deficiency syndrome (AIDS); 37 had CD4 counts below 200. Skin conditions were present in 138 of the 151 subjects (91.4%). The total number of events was 331. The most frequent problem was infection followed by dermatoses, pruritus and malignancy. The most frequent condition was seborrhoeic eczema followed by tinea and xerosis. We have demonstrated a statistically significant association between CD4 count, disease stage and skin manifestations in HIV-positive individuals.

Factors associated with increased levels of human immunodeficiency virus type 1 DNA in semen.

Human immunodeficiency virus type 1 (HIV-1)-infected cells have been isolated from semen and may be a major source of transmissible virus. Quantitative polymerase chain reaction (PCR) assay was used to determine HIV proviral DNA load in cellular fractions of semen from 74 antiviral therapy-naive HIV-1-seropositive men and 53 paired blood samples. HIV-1 DNA was detected in 65% of semen (range: <10-5000 copies/mL) and 100% of blood samples (range: 20-2500 copies/mL). HIV-1 DNA copy numbers in semen correlated significantly with those in blood, but for most cases, the concentration of blood HIV-1 DNA was higher (mean blood-to-semen ratio = 2.9). Factors associated with elevated HIV-1 provirus levels in semen included reduced peripheral CD4 cell count and asymptomatic genital tract inflammation (>10(6) white blood cells/mL of semen). These data provide evidence that genital tract inflammation and reduced peripheral CD4 cell count may be associated with enhanced sexual transmission of HIV-1 because of increased numbers of HIV-1-infected cells in semen.