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The effect of higher FOXP3 mRNA expression by recipient pre-transplant CD4+ T cells on leukaemia relapse was analysed in a series of 106 patients who received allogeneic haematopoietic stem cell transplantation after myeloablative conditioning with or without antithymocyte globulin (ATG) due to acute leukaemia in 1st or 2nd complete remission. FOXP3 mRNA was measured by qPCR in purified CD4+ T cells from blood obtained before conditioning. Higher FOXP3 mRNA expression was associated with an increased relapse risk when conditioning included ATG (n = 43, hazard ratio [HR] 11.0 [2.50-48.4], p = 0.00001). No effect was observed in patients not receiving ATG (HR 0.95 [0.53-1.81]).

BACKGROUND/AIMS Pre-transplant blood transfusions have previously shown a positive effect on organ allograft survival in humans and various animal species. The aims of this study were, first, to evaluate the effect of pre-transplant donor-specific blood transfusions on periodontal healing of fully developed allotransplanted teeth in monkeys; and second, to investigate the immune response against donor antigens and to determine a possible correlation between alloimmune reactions and histopathological signs of rejection. MATERIAL AND METHODS Twenty monkeys (Cercopithecus aethiops) were divided into ten pairs with similar sizes of incisors. One monkey in each pair gave three transfusions to the other monkey with 1-week intervals. One week after the last transfusion, each pair exchanged simultaneously a central maxillary incisor and a lateral mandibular incisor. The corresponding central maxillary and mandibular lateral incisors were autotransplanted to the contralateral sockets. All teeth were treated endodontically per-operatively. Histocompatibility was evaluated by mixed lymphocyte culture before the first transfusion, while alloantibodies and cell-mediated alloresponses were measured before transfusions and at 2 and 8 weeks after transplantation. All animals were sacrificed 8 weeks after tooth transplantation. Serial sections of the transplanted teeth were quantified histologically. RESULTS Mixed lymphocyte cultures showed positive reactions in 19 of 20 cases, indicating incompatibility. The majority of the monkeys developed antibodies towards the tooth donor. Cell-mediated cytotoxicity was negative in all monkeys. Histoquantification revealed a mean score of 70% normal periodontal ligament (PDL) in autotransplanted teeth, with 5% ankylosis. The allografts had a mean score of 17% normal PDL and 63% ankylosis, with no significant influence of transfusion. However, in the mandibular grafts, a tendency towards a positive transfusion effect was seen. CONCLUSIONS Donor-specific blood transfusion did not reduce ankylosis in tooth allografts. The healing of mandibular incisor tooth allografts was improved, but not that of maxillary incisors. Donor-specific antibodies showed no effect on the survival of allograft PDL.

OBJECTIVES: The curative effect of allogeneic haematopoietic stem cell transplantation (HSCT) for acute leukaemia is due in part to the donor T cell-mediated graft-versus-leukaemia immune reaction (GvL). Several studies have suggested that donor CD25+CD4+Foxp3+ regulator T cells (Tregs) may decrease graft-versus-host disease (GvHD) without abrogating GVL. This notion may need modification in acute lymphoblastic leukaemia (ALL).METHODS: Foxp3 mRNA level was measured by qPCR in pre-harvest donor blood CD4+ T cells. The study comprised 45 patients with ALL in 1st or 2nd CR who received myeloablative HSCT using T-replete bone marrow grafts.RESULTS: Relapse occurred in 17 patients median 363 days after HSCT. The relapse risk was estimated by Cox univariate and multivariate proportional hazard regression. The proportionality assumption was met by analysing the pre-harvest donor Foxp3 mRNA level as a time-dependent covariate. Early relapse was not modified by the Foxp3 mRNA level. However, a higher Foxp3 mRNA level was associated with a significantly increased relapse risk after day 363 after transplantation, compatible with inhibition of GvL. In contrast, a higher pre-harvest donor CD4+ T cell concentration was associated with reduced relapse risk.CONCLUSION: A higher pre-harvest donor Foxp3 mRNA level may be predictive of late ALL relapse after HSCT.

Introduction Hyperbaric oxygen, (HBO) is used to treat several conditions including late radiation tissue injury. Previous studies have suggested that HBO mobilizes bone marrow derived stem/progenitor cells (SPC) to the peripheral blood, however possible cumulative effects were highly variable. Methods We have investigated a possible HBO-induced mobilization of SPCs by determining CD34+CD45dim cell numbers, as well as SPCs in general. The latter were characterized by high aldehyde dehydrogenase (ALDH) activity by use of the Aldefluor® assay. We included ten patients admitted for HBO treatment of radiation tissue injury. Six patients completed the 29-30 HBO treatment exposures. We also investigated possible HBO-induced effects on platelet activation as measured by flow cytometry and functional analyses. Results We found a weak and insignificant tendency toward mobilization of CD34+CD45dim cells after a single HBO exposure versus before. Additionally, we found an additive effect of 15 HBO exposures on the increase in CD34+CD45dim cells relative to the pre-1st-HBO values. These changes were significantly more than zero but less than a doubling. We could not demonstrate a significant effect of HBO on the content of Aldefluor® positive SPCs in peripheral blood. There was no significant effect on platelet activation overall. However, in patients with increased expression of activation markers at baseline, we found a decrease after one exposure although this was not reflected in functional tests. Conclusion We found a minor statistically significant mobilizing effect of HBO treatment on the bone marrow derived stem/progenitor cell content in peripheral blood after 15 treatments (n = 10 patients), but no effect after 30 treatments (n = 6 patients). However, because of the low number of patients we cannot confidentially prove or disprove the null hypothesis. The possibility that HBO treatment reduces the number of activated platelets could not be demonstrated nor excluded.

BackgroundDimethyl sulfoxide (DMSO) is essential for the preservation of liquid nitrogen-frozen stem cells, but is associated with toxicity in the transplant recipient. Study Design and MethodsIn this prospective noninterventional study, we describe the use of DMSO in 64 European Blood and Marrow Transplant Group centers undertaking autologous transplantation on patients with myeloma and lymphoma and analyze side effects after return of DMSO-preserved stem cells. ResultsWhile the majority of centers continue to use 10% DMSO, a significant proportion either use lower concentrations, mostly 5 or 7.5%, or wash cells before infusion (some for selected patients only). In contrast, the median dose of DMSO given (20mL) was much less than the upper limit set by the same institutions (70mL). In an accompanying statistical analysis of side effects noted after return of DMSO-preserved stem cells, we show that patients in the highest quartile receiving DMSO (mL and mL/kg body weight) had significantly more side effects attributed to DMSO, although this effect was not observed if DMSO was calculated as mL/min. Dividing the myeloma and lymphoma patients each into two equal groups by age we were able to confirm this result in all but young myeloma patients in whom an inversion of the odds ratio was seen, possibly related to the higher dose of melphalan received by young myeloma patients. ConclusionWe suggest better standardization of preservation method with reduced DMSO concentration and attention to the dose of DMSO received by patients could help reduce the toxicity and morbidity of the transplant procedure.

In patients with stable coronary artery disease (CAD) and refractory angina, we performed direct intramyocardial injections of autologous mesenchymal stromal cells (MSC) and followed the safety and efficacy of the treatment for 12 months. A total of 31 patients with stable CAD, moderate to severe angina, normal left ventricular ejection fraction, and no further revascularization options were included. Bone marrow MSCs were isolated and culture expanded for 6–8 weeks and then stimulated with vascular endothelial growth factor (VEGF) for 1 week. The 12-month follow-up demonstrated that it was safe to culture expand MSCs and use the cells for clinical treatment. The patients' maximal metabolic equivalent (MET) during exercise increased from 4.23 MET at baseline to 4.72 MET at 12-month follow-up ( p < 0.001), Canadian Cardiovascular Society Class (CCS) was reduced from 3.0 to 0.8 ( p < 0.001), angina attacks per week from 13.8 to 3.2 ( p < 0.001), and nitroglycerin consumption from 10.7 to 3.4 per week ( p < 0.001). In addition, Seattle Angina Questionnaire (SAQ) evaluations demonstrated highly significant improvements in physical limitation, angina stability, angina frequency, and quality of life ( p < 0.001 for all). It is safe in the intermediate/long term to treat patients with stable CAD using autologous culture expanded MSCs. Previously reported, early and highly significant improvements in exercise capacity and clinical symptoms persist after 12 months. The results are encouraging, and a larger controlled study is warranted.

Objective Recently, it has been shown that a homozygous 32 base-pair deletion in the gene encoding CKR-5, a major coreceptor for HIV-1, leads to resistance to infection with HIV-1. We have investigated whether HIV-seropositive individuals who were heterozygous for the CKR-5 deletion had a different course of the disease. Design Thirty-five high-risk HIV-seronegative and 99 HIV-seropositive Danish homosexual men followed form 1985 to 1996 and 37 blood donors were analysed for their CKR-5 genotype by polymerase chain reaction. Results Two (6%) of the 35 HIV-seropositive subjects at high-risk of infection were homozygous and seven (20%) were heterozygous for the CKR-5 deletion. This was not significantly different from the distribution in normal donors. Twenty-two (22%) of the 99 HIV-seropositive subjects were heterozygous and none was homozygous. Two subgroups of patients who had an opposite course of the HIV disease were identified. Of nine long-term non-progressors, six (66%) were heterozygous for the deletion. This frequency is significantly higher than in nine rapid progressors of whom non was heterozygous. The frequency of heterozygotes in long-term non-progressors was also significantly higher than in the cohort as a whole. A Kaplan-Meier plot of the HIV-seropositive subjects, of whom 57 developed AIDS, showed a significantly better prognosis within the first 7 years of follow-up for those who were heterozygous for the deletion. Heterozygous individuals also had a significantly slower decrease in CD4 T-cell count per year. Conclusion Individuals who are heterozygous for the 32-base-pair deletion in the CKR-5 gene have a slower decrease in their CD4 T-cell count and a longer AIDS-free survival than individuals with the wild-type gene for up to 11 years of follow-up.

Aims . We evaluated the feasibility, safety and effi cacy of intra-myocardial injection of autologous mesenchymal stromal cells derived endothelial progenitor cell (MSC) in patients with stable coronary artery disease (CAD) and refractory angina in this fi rst in man trial. Methods and results. A total of 31 patients with stable CAD, moderate to severe angina and no further revascularization options, were included. Bone marrow MSC were isolated and culture expanded for 6 – 8 weeks. It was feasible and safe to establish in-hospital culture expansion of autologous MSC and perform intra-myocardial injection of MSC. After six months follow-up myocardial perfusion was unaltered, but the patients increased exercise capacity (p 0.001), reduction in CCS Class (p 0.001), angina attacks (p 0.001) and nitroglycerin consumption (p 0.001), and improved Seattle Angina Questionnaire (SAQ) evaluations (p 0.001). For all parameters there was a tendency towards improved outcome with increasing numbers of cells injected. In the MRI substudy: ejection fraction (p 0.001), systolic wall thickness (p 0.03) and wall thickening (p 0.03) all improved. Conclusions. The study demonstrated that it was safe to treat patients with stable CAD with autologous culture expanded MSC. Moreover, MSC treated patients had signifi cant improvement in left ventricular function and exercise capacity, in addition to an improvement in clinical symptoms and SAQ evaluations.

18 out of 40 healthy Danish type A haemophiliacs had antibodies against HTLV-III as measured by an enzyme linked immunosorbent assay (ELISA). The overall seropositivity was 45%. A significant positive correlation was found between seropositivity and total lifetime dose of factor VIII and the age of the patients. 63% and 79% of the patients predominantly treated with commercial American and European preparations, respectively, had antibodies, compared with 11% among patients predominantly treated with Danish cryoprecipitate. Patients exclusively treated with preparations from a single source in the year prior to investigation showed 69% seropositivity when treated with American and European preparations. None of the patients treated with Danish cryoprecipitates prepared from voluntary Danish donors had antibodies. No difference between seropositive and seronegative groups was found in total lymphocyte count, leu 2+ cells, leu 3 + cells and leu 2 +/leu 3 + ratio, but the seropositive group had significantly higher total IgG and lower skin test score. It is concluded that treatment with local European preparation carries less risk of HTLV-III infection compared with commercial preparations from either the USA or Europe. The results also suggested that T-cell subset alterations among haemophiliacs are not primarily due to HTLV-III infection.

Based on the literature and 2 patients studied, we suggest that at least 2 different clinical entities are included in the concept of T CLL: (i) a clinical variant characterized by a relatively benign course, splenomegaly without lymphadenopathy, low lymphocyte count and granulocytopenia; the proliferating lymphocyte is morphologically mature, of medium size and a cytoplasm with azurophilic granules staining positively for acid phosphatase and corresponding to parallel tubular arrays as demonstrated by electron microscopy. The cells form E-rosettes, have no surface-membrane-bound Ig, but Fc-receptors for IgG. With monoclonal antibodies, the phenotype is OKT3+, OKT4- and OKT8+, theoretically corresponding to the suppressor/cytotoxic T lymphocyte subset, but functionally the cells demonstrate killer cell (responsible for ADCC), but not natural or suppressor cell activity. (ii) another clinical variant with an aggressive course, massive hepato-splenomegaly, lymph node enlargement and very high lymphocyte counts; the lymphocytes are small without cytoplasmic granules; their immunological and functional characteristics have not been determined, but morphologically the cells correspond to the T helper/inducer lymphocyte subset. Thus, involvement of different T lymphocyte subsets may be the reason for the clinical variation in T CLL.

We analysed the outcome and hospitalization requirements of the first 100 patients (Hodgkin's disease (HD), N=13; multiple myeloma (MM), N=14; CLL, N=12; non-Hodgkin's lymphoma (NHL), N=17; myelodysplastic syndrome (MDS), N=18; AML, N=24 and CML, N=2) treated in Denmark with haematopoietic cell transplantation after non-myeloablative conditioning with TBI 2 Gy+/-fludarabine. The cumulative incidence of acute GVHD grade II-IV and extensive chronic GVHD was 67 and 49%. After a median follow-up of 534 days, the overall survival, PFS, relapse-related mortality and treatment-related mortality were 59, 50, 25 and 17%, respectively. Patients with CLL, NHL, AML and MDS with5% blasts at any time had a favourable outcome with a PFS of 61-71%. Patients with MM, HD and MDS and a history ofor =5% blasts had a less favourable outcome with a PFS of 19-38% (P=0.001). The cumulative incidence of discontinuation of immunosuppression was 37%. During the first and second year post transplant, patients experienced a mean of 41 and 13 outpatient clinic visits, and 53 and 16 days of hospitalization. Sixteen patients were admitted to the intensive care unit, of whom eight are still alive. In conclusion, transplantation outcomes were encouraging, but complications requiring admission and outpatient clinic visits occur frequently post transplant.

Five blood donors with occult persistent and one donor with an early window phase HBV infection were identified. They were negative in regular HBsAg screening and had low levels of HBV DNA that were probably not detectable by current mini-pool nucleic acid amplification testing. In four donors several mutations were found located in the HBs antigen loop. In three donors the mutations were predominantly outside the "a" determinant; one donor had a wild type HBsAg sequence. Fifty-five recipients of donations from the persistently infected donors could be tested for previous or ongoing HBV infection and of them 53% (29/55) were anti-HBc positive. Based on the prevalence of anti-HBc in Germany (7%), it was assumed that four of those recipients had already been positive before transfusion. In 22 cases, it was assumed that they acquired infection by the donations, but the infection remained asymptomatic and was resolved. In three cases transmission was proven by the time course of the acute infection and sequence identity The resulting infection was fatal and associated with immunological disorders at the time of transmission: in one case sepsis and in the othertwo cases immunosuppression. In a further asymptomatic case of proven transmission from the early window phase donation passively administered anti-HBs could not prevent spread of wildtype HBV but antiviral treatment lead to resolution. Surprisingly the typical acute hepatitis B was not observed in a single one of the 26 cases of assumed or proven transmission.

Cytokines are thought to play an important role in the pathophysiology of graft-versus-host disease (GVHD). To study the relationship between cytokines and GVHD, we obtained peripheral blood mononuclear cells (MNCs) from 21 patients with hematologic malignancies and their HLA-identical sibling donors before and sequentially after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning. The MNCs were cultured for 72 hours either alone or in mixed lymphocyte cultures with irradiated MNCs of recipient, donor, or HLA-mismatched third-party origin. The gene expression of interleukin (IL)–2, IL-4, IL-10, IL-18, tumor necrosis factor α, and transforming growth factor β in each culture was then measured by real-time quantitative reverse transcriptase-polymerase chain reaction. The composition of the responder MNCs differed between patients and donors and changed after HCT, with a possible influence on the results. Early after transplantation (day +14), the IL-10 messenger RNA (mRNA) level in response to recipient or donor antigens was higher in patients who did not develop clinically significant acute GVHD when compared with the level in patients who subsequently developed acute GVHD grades II to IV (P = .005 and P = .004, respectively). The IL-10 mRNA level on day +14 was highly correlated with the pretransplantation mRNA level of the recipient MNCs but not with the level of the donor MNCs; this suggests that the IL-10 mRNA detected on day +14 originated from responder cells of recipient origin. A higher IL-10 mRNA level was found in MNCs obtained before transplantation from recipients whose disease progressed or relapsed after the transplantation when compared with the level in patients whose disease did not progress or relapse (P = .03). In conclusion, a high IL-10 gene expression in the recipient MNCs may be related to a reduced incidence of acute GVHD grades II to IV and a reduced graft-versus-tumor effect after HCT with nonmyeloablative conditioning.

Background A reliable in vitro test that estimates the level of ongoing alloreactivity would be valuable in allogeneic hematopoietic cell transplantation (HCT) as a help to guide clinical interventions such as donor lymphocyte infusions and changes in the immunosuppression. In the present study, the use of limiting dilution analysis of interleukin-2 (IL-2) producing helper T lymphocyte frequencies (HTL assay) as a way to quantify alloreactivity following HCT was investigated. Methods Serial HTL assays were performed following allogeneic HCT with myeloablative or nonmyeloablative conditioning in 26 patients with hematologic malignancies. Results Deviations from single-hit kinetics were frequently observed in the HTL assays and a nonlinear model was therefore used for analysis. The results of this analysis suggested the presence of an inhibitory cell population. Inhibition was observed in the majority of patients and was not restricted to a specific transplant regimen. Inhibition occurred more often with high frequencies of IL-2 producing cells, indicating a physiological role of the putative inhibitory cell population in the regulation of an immune response. Higher frequencies of IL-2 producing cells were observed in patients with acute graft-versus-host disease grades II-IV than in patients with grades 0-I (P = 0.046), indicating that the degree of ongoing alloreactivity is indeed quantified by the HTL assay. Conclusions We find that the HTL assay may yield interesting insight into regulation of immune responses following allogeneic HCT, but because of the complexity of the results obtained, its use as a routine procedure to guide immunosuppression cannot be recommended.

Thirty patients with haematological malignancies received peripheral blood stem cells from human leucocyte antigen (HLA)-identical sibling donors after non-myeloablative conditioning with fludarabine and total body irradiation. Twenty-seven patients received the transplant as an outpatient procedure. All patients engrafted. The probability of acute graft-versus-host disease (GVHD) grades II-IV and extensive chronic GVHD was 57% and 80%, respectively. Patients alive on day +365 experienced a median of 44 d (range 4-151) of hospitalization during the first year. In the entire cohort, GVHD accounted for 22%, infections for 18%, thrombotic thrombocytopenic purpura (TTP) for 16% and engraftment syndrome for 14% of the time in hospital. The 1-year risk of TTP was 26%. Acute GVHD was a risk factor for the development of TTP (P = 0.008). With a median follow-up of 602 d, the 2-year estimates for overall survival, progression-free survival, non-relapse mortality and relapse related mortality were 68%, 43%, 22% and 13%, respectively. This transplantation regimen is feasible and induces long-term remissions in heavily pretreated patients. The procedure can be performed in the outpatient setting, but complications could result in a substantial number of admissions during the first year.

The present study compared the contents of pluripotent and lineage-committed hematopoietic progenitor cells (HPCs) in various types of allografts. Bone marrow (BM) allografts and single leukapheresis products (LPs) collected from G-CSF-mobilized donors contained similar amounts of pluripotent HPCs (CD34(+)CD38(-)) and total CD34(+) cells. However, the content of late-myeloid HPCs (CD34(+)CD33(+)CD15(+)) were significantly higher in BM grafts compared to LPs (P>0.02), whereas the contents of early-myeloid HPCs (CD34(+)CD33(+)CD15-) were 2.5-fold higher in LPs (P

Udgivelsesdato: 2003-Jan-15 The relationship among CCR5 genotype, cytomegalovirus infection, and disease progression and death was studied among 159 human immunodeficiency virus (HIV)-infected patients with hemophilia. One patient (0.6%) had the CCR5Delta32/CCR5Delta32 genotype (which occurs in approximately 2% of the Scandinavian population) and a rapid disease course. His HIV V3 region contained genotypic features attributable to X4 virus and resembled functionally verified X4 virus and virus from patients treated with a CD4 cell-stimulating drug, tucaresol. Age-related differences in disease progression rate and survival time were seen for CCR5/CCR5 patients. Surprisingly, no protective effect of the CCR5/CCR5Delta32 genotype on disease progression or survival was seen for children but was evident for adults. Age group-related immunologic differences might explain this variation, and transmission route and/or viral phenotype variation within donor virus may be related to the limited protection of the CCR5Delta32/CCR5Delta32 genotype. Sequence comparisons indicate that X4 virus can be selected in vivo due to either absence of CCR5 receptors or relative increase of CXCR4 receptors.

To quantify HIV-RNA in plasma, in lymphoid tissue and proviral DNA in peripheral blood mononuclear cells and to relate these to immunological markers among patients with plasma viral load counts of/= 200 HIV-RNA copies/mL.A prospective study of one hundred and three patients was undertaken with an inclusion criteria of plasma viral load of/= 200 copies/mL. The patients had advanced HIV infection; 25% had developed AIDS. Patients were seen every 6 months for a period of 2 years.The median plasma viral load was20 copies/mL with no increase during follow-up. Thirty-one per cent had plasma viral load of/= 20 copies/mL at all visits, 44% had/= 1 measurement with 21-200 and 25% had/= 1 sample with plasma HIV-RNA200 copies/mL. Lymphoid tissue viral load was low at enrolment and declined further during follow-up. Baseline HIV-DNA and immunoglobulin (IgA) differed significantly between the plasma viral load rebound groups (P0.05).In this cohort, selected solely on the basis of having a plasma viral load of/= 200 copies/mL, we found stable or declining viral loads in the measured compartments during 2 years of follow-up. Baseline HIV-DNA and IgA levels were higher among patients with less complete virological suppression relative to patients with persistently undetectable plasma HIV-RNA. Hence, a high cellular level of HIV-DNA and high plasma IgA may predict subsequent development of low-grade viraemia.

BACKGROUND: Dimethyl sulfoxide (DMSO) is essential for the preservation of liquid nitrogen-frozen stem cells, but is associated with toxicity in the transplant recipient. STUDY DESIGN AND METHODS: In this prospective noninterventional study, we describe the use of DMSO in 64 European Blood and Marrow Transplant Group centers undertaking autologous transplantation on patients with myeloma and lymphoma and analyze side effects after return of DMSO-preserved stem cells. RESULTS: While the majority of centers continue to use 10% DMSO, a significant proportion either use lower concentrations, mostly 5 or 7.5%, or wash cells before infusion (some for selected patients only). In contrast, the median dose of DMSO given (20 mL) was much less than the upper limit set by the same institutions (70 mL). In an accompanying statistical analysis of side effects noted after return of DMSO-preserved stem cells, we show that patients in the highest quartile receiving DMSO (mL and mL/kg body weight) had significantly more side effects attributed to DMSO, although this effect was not observed if DMSO was calculated as mL/min. Dividing the myeloma and lymphoma patients each into two equal groups by age we were able to confirm this result in all but young myeloma patients in whom an inversion of the odds ratio was seen, possibly related to the higher dose of melphalan received by young myeloma patients. CONCLUSION: We suggest better standardization of preservation method with reduced DMSO concentration and attention to the dose of DMSO received by patients could help reduce the toxicity and morbidity of the transplant procedure.

Cytokine-mobilized peripheral blood progenitor cells (PBPCs) have been used successfully for hematopoietic reconstitution following allogeneic transplantation. The ease of harvest, the faster engraftment and the high yield of CD34+ cells have made this source of hematopoietic progenitor cells (HPCs) an attractive alternative to bone marrow (BM). In the present study we compared the engraftment potential of conventional BM allografts and single leukapheresis products (LPs) collected from healthy donors following the administration of granulocyte colony-stimulating factor (G-CSF). For this, lineage-committed and primitive HPCs were assessed by flow cytometry and by colony- and cobblestone area-forming cell (CFC, CAFC) assays. Mean numbers of CD34+ cells in LPs (n = 11) were similar to that of BM grafts (n = 12) (278+/-57 vs 227+/-34 x 10(6) CD34+ cells). The frequencies of CFCs, week 5 CAFCs and week 8 CAFCs were 1.6-, 8.4- and 10.3-fold higher in the CD34+ compartment of mobilized blood than that of marrow, resulting in significantly higher yields of clonogenic HPCs in LPs when compared to BM grafts. We conclude that G-CSF preferentially mobilizes clonogenic progenitors capable of short- and, in particular, longterm reconstitution, and that the engraftment potential of single LPs is superior to that of BM allografts. Hence, the use of PBPCs may be favorable for protocols that include graft manipulations with expected cell loss (eg T cell depletion, CD34+ selection). PBPCs may also be advantageous for gene therapy trials due to their high numbers of potential target cells (eg CAFCs).

In allogeneic hematopoietic stem cell (HSC) transplantation, collection of a sufficient number of HSCs at a fixed time point is crucial. For HSC mobilization into the peripheral blood, the standard regimen, that is, granulocyte-colony-stimulating factor (G-CSF), may be inadequate. Use of plerixafor as adjuvant to G-CSF is so far off-label in healthy donors.We present six cases in which the "just-in-time" addition of plerixafor ensured proper CD34+ collection from healthy donors with insufficient G-CSF mobilization. In four of these cases a high number of CD34+ cells was needed due to subsequent CD34+ selection or haploidentical transplantation.From all six donors a sufficient number of CD34+ cells was obtained by using plerixafor as an adjuvant to G-CSF. This treatment regimen resulted in only mild side effects for the donor.We have presented six cases with different causes leading to insufficient G-CSF mobilization in allogeneic donors and in which the administration of plerixafor just-in-time ensured a proper graft for transplantation.

Cytotoxicity mediated by natural killer (NK) and lymphokine-activated killer (LAK) cells may be of significance in host defense against viral infections. This study included 347 patients infected with human immunodeficiency syndrome virus (HIV) type 1 and 110 controls. The NK cell activity, either unstimulated or stimulated with interferon-alpha (IFN-alpha) or interleukin-2 (IL-2), and the LAK cell activity were suppressed in patients, but the NK/LAK cell activity did not differ between patients with AIDS and patients without AIDS. However, the IFN-alpha-stimulated NK cell activity and LAK cell activity were reduced in patients with symptoms of HIV disease (CDCIV) when compared with asymptomatic patients (CDCII+III). When the data were analyzed by multiple linear regression, the percentage of CD4+ cells had a positive effect on these two parameters in patients without AIDS, whereas the percentage of CD4+ cells had no significant effect on unstimulated and IL-2-stimulated NK cell activity in these patients. In controls and AIDS patients, the percentage of CD4+ cells had no effect on NK/LAK cell activity in multiple linear models. The total number of CD16+ cells was low in patients compared to controls, whereas the percentages of CD16+, CD56+, and CD16+CD56+ were either normal or elevated. Therefore, the decrease in NK cell subpopulations did not contribute to the observed depression in NK/LAK cell activity in vitro. It is concluded that natural immunity is suppressed in HIV-seropositive patients primarily because of a qualitative defect of the NK/LAK cells. This qualitative defect includes a reduced responsiveness to IFN-alpha, which is progressive until the onset of symptoms, and possibly related to the loss of CD4+ cells.

Medical injections have recently been suggested to cause a large proportion of African HIV infections. If this hypothesis is true, high prevalences of other infections transmitted by injections should be expected. In a cohort of 145 HIV-negative and 124 HIV-positive individuals from a rural area of Zimbabwe with a high HIV prevalence we only found one with antibodies to hepatitis C virus. This does not support injections playing a major role in HIV transmission in sub-Saharan Africa.

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Graft rejection after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning is a rare but serious clinical problem. Graft rejection and salvage therapy in eight patients in a retrospective analysis of 124 consecutive patients is reported. The patients were conditioned with low-dose fludarabine and total body irradiation (TBI). The association of pretransplantation risk factors with rejection and the effect of chimerism and graft-versus-host disease on rejection were analyzed. Overall survival (OS) and progression free survival (PFS) were compared between patients with and without rejection. Retransplantation was performed with increased TBI conditioning for all patients, and with increased mycophenolate mofetil doses for recipients with HLA-identical sibling donors. No known pretransplantation risk factors were confirmed in this study. Rejection episodes were unevenly distributed over time. The storage temperature of the apheresis products was identified as a risk factor for rejection. Storage of the apheresis products at 5 degrees C diminished the risk of rejection. Low donor T cell chimerism at Day +14 significantly increased the risk of rejection. Seven patients were retransplanted. All but one engrafted successfully, but with decreased OS and PFS. Two patients received pentostatin infusion prior to donor lymphocyte infusions in unsuccessful attempts at reversing rejection. Storage temperature and donor chimerism had a significant effect on rejection. Following rejection, patients are at greater risk of dying from infections and progression/relapse of their malignancy. Retransplantation is feasible and well tolerated after HCT with nonmyeloablative conditioning and should be performed without delay in patients with imminent and manifest graft rejection.

Seven consecutive patients who presented with a severe acute mononucleosis-like illness associated with HIV seroconversion were evaluated by T-cell subset enumerations and measurements of lymphocyte transformation responses to mitogens and antigen during both their primary illness and a 1-year follow-up period. We observed a characteristic pattern of response to primary HIV infection; initial lymphopenia was followed by CD8 lymphocytosis and inversion of the CD4:CD8 ratio. During follow-up, the CD8 count gradually returned to normal, whereas the CD4:CD8 ratio remained inverted because of a relatively low number of CD4 lymphocytes. Primary infection was followed by prolonged and severe cellular hyporesponsiveness to both mitogens and antigen. At the last follow-up, responses to pokeweed mitogen were still severely impaired, with a median 19% (range 7-50%) of that observed in healthy controls. We conclude that severe primary HIV infection may be followed by sustained lymphocyte hyporesponsiveness, a sustained low percentage of CD4 lymphocytes and sustained inversion of the CD4:CD8 ratio.

We present a reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD-women. This test is needed for a future antenatal RH prophylaxis. A new real time PCR based assay targeting RHD exon 7 and a published assay for RHD exon 10 were used to determine the fetal RHD status in DNA extracted from plasma from 56 pregnant women in 15th-36th week of gestation. Prediction of fetal RhD type was compared with the phenotype determined after birth, and showed 100% concordance. This setup will be of value in antenatal RH prophylaxis and in the management of immunised women.

Objectives The objective of this study was to establish a reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD negative women. This test is needed for future prenatal Rh prophylaxis. Methods A novel real-time PCR-based assay targeting RHD exon 7 combined with a published assay for RHD exon 10 were used to determine the fetal RHD status in DNA extracted from plasma, sampled from 56 pregnant RhD negative women in 15th–36th week of gestation. Thirty-eight samples were from ongoing pregnancies of Danish women and 21 samples from 18 pregnant women were stored anonymized samples from the International Blood Group Reference Laboratory, Bristol, United Kingdom. Prediction of fetal RhD type was compared with the serological result obtained after birth. Results The prediction of the fetal RhD type was in 100% concordance with the serological RhD type from the 16th week of gestation. One sample from the 15th week of gestation was inconclusive. The number of copies of fetal RHD DNA was found to increase with gestational age. Low levels of DNA were found to follow the Poisson distribution (p = 1.0000). Conclusion Our set-up was very reliable for determination of fetal RhD genotype, and thus will be of value in prenatal Rh prophylaxis and in the management of immunized women. Copyright © 2005 John Wiley & Sons, Ltd.

The greatest transfusion-transmitted disease risk facing a transfusion recipient is that of bacterial sepsis. The prevalence of bacterial contamination in platelets and red blood cells is approximately 1 in 3000 units transfused. The available data indicate that transfusion-associated sepsis develops after 1 in 25 000 platelet transfusions and 1 in 250 000 red blood cell transfusions. One of the most widely used strategies for decreasing bacterial sepsis risk is bacterial detection. A roundtable meeting of experts was convened during the XXVIII Annual Congress of the International Society of Blood Transfusion (Edinburgh, UK, July 2004) to provide a forum for experts to share their experiences in the routine bacterial detection of platelet products. This article summarizes the presentations, discussions, and recommendations of the panel. The data presented indicate that some of the current bacterial screening technology is useful for blocking the issuance of platelet units that contain relatively high levels of contaminating bacteria. Platelet units are usually released based on a test-negative status, which often become test-positive only upon longer storage. These data thus suggest that bacterial screening may not prevent all transfusion-transmitted bacterial infections. Two transfusion-transmitted case reports further highlighted the limitation of the routine bacterial screening of platelet products. It was felt that newer technologies, such as pathogen inactivation, may represent a more reliable process, with a higher level of safety. The panel thus recommended that the Transfusion Medicine community may need to change its thinking (paradigm) about bacterial detection, toward the possibility of the pathogen inactivation of blood products, to deal with the bacterial contamination issue. It was suggested, where permitted by regulatory agencies, that blood centers should consider adopting first-generation pathogen inactivation systems as a more effective approach to reducing the risk of transfusion-associated sepsis than some of the approaches currently available. AN EXPERT PANEL roundtable meeting on bacterial detection convened was during the XXVIII Annual Congress of the International Society of Blood Transfusion in Edinburgh, UK, on July 13, 2004, under the chairmanship of Dr MA Blajchman. Appendix A provides a list of participants who attended. This roundtable meeting provided a forum for the panelists to discuss the current transfusion risks due to the bacterial contamination of platelet concentrates, the impact of the implementation of routine bacterial screening, the testing methods available, the necessary actions to be taken when a positive test is obtained, and the benefits and limitations of routine bacterial testing. The panelists presented the protocols they have in place in their institutions for bacterial detection and their experiences since first application of bacterial detection. Clinical cases were presented to highlight the problems associated with transfusion-associated sepsis. The panel recognized that the bacterial contamination of blood products is the most common microbiologic risk of transfusion. The prevalence of contamination of cellular blood products is reported to be approximately 1 in 3000 blood product units. The prevalence of significant clinical events is reported to be approximately 1 in 25 000 for platelet transfusions and 1 in 250 000 for red blood cell (RBC) transfusions.1 Underreporting and underrecognition are acknowledged as being considerable problems. The panel discussed such subjects as screening technology, when to sample, the sample volume, the clinical significance of anaerobic organisms, whether RBCs should be screened, the clinical significance of low levels of bacterial contamination, and which universally effective measures might be taken to reduce this readily preventable transfusion risk. The ultimate purpose of this roundtable meeting was to provide guidance on issues related to the bacterial detection in blood products. This report summarizes the presentations, discussions, and recommendations of the panel.

Chimerism analysis of hematopoietic cells has emerged as an essential tool in nonmyeloablative hematopoietic stem cell transplantation. We have investigated the development of donor chimerism in granulocytes and CD4+ and CD8+ T cells in blood and bone marrow of 24 patients with hematologic malignancies who received HLA-identical sibling peripheral blood stem cell grafts after conditioning with fludarabine and 2 Gy of total body irradiation. The T-cell chimerism of blood and bone marrow was tightly correlated. Complete donor chimerism was reached earlier in the granulocytes than in the T cells. Mixed T-cell chimerism was common at the time of onset of acute graft-versus-host disease (aGVHD), and both CD4+ and CD8+ donor T-cell chimerism increased with the occurrence of aGVHD grades II to IV (P = .0002 and P = .019, respectively). The rate of disappearance of recipient CD8+ T cells was faster in patients with aGVHD grades II to IV than in patients without clinically significant aGVHD (P = .016). This observation indicates a role of graft-versus-lymphohematopoietic tissue reactions in creating complete donor T-cell chimerism. A donor CD8+ T-cell count above the median on day +14 increased the risk of subsequent development of aGVHD grades II to IV (P = .003).

Thirty patients with haematological malignancies received peripheral blood stem cells from human leucocyte antigen (HLA)-identical sibling donors after non-myeloablative conditioning with fludarabine and total body irradiation. Twenty-seven patients received the transplant as an outpatient procedure. All patients engrafted. The probability of acute graft-versus-host disease (GVHD) grades II-IV and extensive chronic GVHD was 57% and 80%, respectively. Patients alive on day +365 experienced a median of 44 d (range 4-151) of hospitalization during the first year. In the entire cohort, GVHD accounted for 22%, infections for 18%, thrombotic thrombocytopenic purpura (TTP) for 16% and engraftment syndrome for 14% of the time in hospital. The 1-year risk of TTP was 26%. Acute GVHD was a risk factor for the development of TTP (P = 0.008). With a median follow-up of 602 d, the 2-year estimates for overall survival, progression-free survival, non-relapse mortality and relapse related mortality were 68%, 43%, 22% and 13%, respectively. This transplantation regimen is feasible and induces long-term remissions in heavily pretreated patients. The procedure can be performed in the outpatient setting, but complications could result in a substantial number of admissions during the first year.

Blood infectivity in transmissible spongiform encephalopathies (TSE) is reviewed with special emphasis on transmission by blood transfusion in human beings. It is concluded that transmission by transfusion seems biologically plausible as regards variant Creutzfeld-Jakob Disease (vCJD), albeit present knowledge suggests that it is extremely uncommon. Precautionary measures against the putative risk of vCJD transmission by blood transfusion are discussed.

Mobilized peripheral blood stem and progenitor cells (PBPCs) are increasingly used to restore hematopoiesis after myeloablative treatment. To obtain a sufficient number of CD34(+) cells, many studies have focused on the improvement of the collection technique during the leukapheresis procedure (LP), and so-called large-volume leukapheresis (LVL) procedures have been developed. Such procedures can be performed by extending the duration of the LP and/or by increasing the inlet flow rate. However, no previous studies have compared the efficiency of these procedures. In the present study, we compared the kinetics of PBPCs recruitment (including CD34(+) cell subsets), the PBPCs yield, and the collection efficiency as well as the overall feasibility of the procedures during a single LVL performed by standard (group I) (median 85 ml/min; range 50-97 ml/min) and high inlet flow rates (group II) (median 130 ml/min; range 110-150 ml/min). Seven patients with hematological malignancies were enrolled and allocated to each group. The patients' blood volumes (BV) were processed four times. The apheresis product (AP) was collected in four separate bags, which were changed every time one BV had been processed. The CD34(+) cell number and CD34(+) cell subsets were assessed in the four collection bags and in peripheral blood (PB) before every time one BV had been processed and after the leukapheresis. The CD34(+) cell yield exceeded the pre-apheresis CD34(+) cell number per ml BV in 6 out of 7 patients in group I and in 3 out of 7 patients in group II. In group II, the recruitment of CD34(+) cells from the bone marrow (BM) to PB starts in the second collection period--as early as 30-60 min after initiating the procedure. No exhaustion in the recruitment was observed in the two groups for at least 5 h during the leukapheresis, and all CD34(+) cell subsets were recruited at a steady rate. However, the collection efficiency in group II was only half of that in group I. In addition, we experienced many technical problems during the leukapheresis in group II. Thus, in 4 out of 7 patients in this group, it was not possible to perform the maximal inlet flow rate because of catheter problems. In conclusion, due to the technical problems associated with the high inlet flow rate procedure and the fact that the relative number of CD34(+) cells harvested and recruited during the leukapheresis was higher in group I than II and, also reflected an approximately two-fold higher collection efficiency, we recommend that LVL be performed by standard inlet flow rate.

Currently, bone marrow (BM), cord blood (CB), and G-CSF-mobilized peripheral blood progenitor cells (PBPCs) are the most commonly used sources for allogeneic stem cell transplantation (SCT). The aim of this study was to assess the yields and distribution of lymphocyte subsets, lymphocyte progenitors and hematopoietic stem cells (HSC) in each type of allograft by three-color flow cytometry. The yields of CD34(+)CD38(-) HSCs did not differ significantly between BM grafts (2.80 +/- 0.74 x 10(6)) and leukapheresis products (LPs) (1.82 +/- 0.64 x 10(6)), and were lowest in CB grafts (0.21 +/- 0.05 x 10(6)). For most lymphocyte subsets yields were lowest in CB grafts and significantly higher in LPs than in BM grafts. BM grafts, however, contained the highest yields of CD34(+)CD19(+)CD20(-) B cell progenitors and CD19(+)CD20(-) B cells. The relative frequencies of the naive CD45RA(+)CD45RO(-) phenotype among CD4(+) and CD8(high) T cells were highest in CB grafts (P < or = 0.001), and higher in LPs than in BM grafts (P < or = 0.02). The latter finding was in accordance with a preferential G-CSF mobilization of naive T cells relative to the total lymphocyte population (P < or = 0.014). CD3(+)CD8(low) and CD3(+)CD8(low)CD4(-) subsets, which facilitate engraftment in murine transplantation models, demonstrated a tendency towards lower frequencies among T cells in CB grafts and LPs compared to BM grafts. This observation coincided with a significantly reduced mobilization of subsets potentially enriched for facilitating cells as compared to the total lymphocyte population (P < or = 0.036). The CD34(+) compartment of CB grafts contained a significantly higher percentage (12.1%) of CD34(+)CD7(+)CD3(-) T cell progenitors than those of BM grafts (5.1%) and LPs (3.6%). In addition, CB lymphocytes contained the highest fraction of CD3(-)CD16/56(+) NK cells (P < or = 0.013) and almost no CD3(+)CD16/56(+) NKT cells (P < 0.001) compared to adult cell sources. In summary, LPs, CB allografts and BM allografts differ widely with respect to the cellular composition of their lymphocyte compartments, which is partially affected by a varying mobilization efficiency of G-CSF for distinct lymphocyte subsets.

In the case reported here, transfusion of HIV RNA–positive, HIV-seronegative blood did not lead to infection of the recipient, probably because potent postexposure antiretroviral therapy was initia...

Two monoclonal antibodies, anti-CD45RA and anti-CD29, reciprocally divide the CD4+ and CD8+ lymphocytes into CD4+ CD45RA+, CD4+ CD29+, CD8+ CD45RA+ and CD8+ CD29+ subsets. The CD4+ CD45RA+, CD4+ CD29+ and CD8+ CD45RA+ possess suppressor-inducer, helper-inducer and suppressor-effector functions respectively. Since the role of these subsets has not been established after allogenic bone marrow transplantation we studied lymphocyte subpopulations in 12 patients 45-227 days after the procedure. The fraction of CD4+ lymphocytes was significantly (P = 0.0005) decreased to 20 +/- 9% versus 43 +/- 3% in controls. Within the CD4+ compartment, we found an increase in the fraction of CD4+ cells that co-expressed CD29 (CD29+/CD4+) to 92 +/- 10% versus 48 +/- 15% (P = 0.008) in controls and a concomitant decrease in CD45RA+/CD4+ to 16 +/- 12% versus 56 +/- 25% (P = 0.008). Patients were also noted to have an increase in the percentage of CD8+ lymphocytes to 41 +/- 5% compared to 23 +/- 4% in controls (P = 0.0004). Examination of the CD8+ subsets revealed a significant increase in the CD29+/CD8+ fraction to 97 +/- 3% versus 64 +/- 2% in controls (P = 0.008) and a decrease in the CD45RA+/CD8+ fraction to 36 +/- 11% versus 70 +/- 21% (P = 0.008). The number of cells co-expressing CD57 were also determined within the CD4+ and CD8+ subsets. In patients CD57+/CD4+ were increased to 29 +/- 7% versus 1 +/- 1% in controls (P = 0.04), and CD57+/CD8+ to 49 +/- 12% versus 23 +/- 9% (P = 0.02). Since CD29+ and CD57+ cells have a poor capability for IL-2 production and proliferation this shift in subset distribution may account for some of the defects in cellular immunity seen within the first year after allogeneic bone marrow transplantation.