Kv Doherty

Non-HLA immunogenetic polymorphisms and the risk of complications after allogeneic hemopoietic stem-cell transplantation.

Background. Existing data indicate that non-human leukocyte antigen (HLA) immunogenetic polymorphisms influence the risk of complications after allogeneic hemopoietic stem-cell transplantation. However, prior studies have been limited by small sample size and limited genotyping. Methods. We examined 22 polymorphisms in 11 immunoregulatory genes including cytokines, mediators of apoptosis, and host-defense molecules by polymerase chain reaction using sequence-specific primers in 160 related myeloablative transplants. Associations were confirmed in two independent cohorts. Results. An intronic polymorphism in the tumor necrosis factor gene (TNF 488A) was associated with the risk of acute graft-versus-host disease (GVHD) (odds ratio [OR] 16.9), grades II to IV acute GVHD (OR 3.3), chronic GVHD (OR 12.5), and early death posttransplant (OR 3.4). Recipient Fas −670G and donor interleukin (IL)-6 −174G were independent risk factors for acute GVHD. Recipient IL-10 ATA and Fas −670 genotype were independent risk factors for chronic GVHD. Recipient IL-1&bgr; +3953T was associated with hepatic acute GVHD, and Fas −670G was associated with major infection. Conclusions. These results highlight the potential importance of cytokine and apoptosis gene polymorphisms in stem-cell transplantation, and indicate that non-HLA genotyping may be useful to identify individuals at the highest risk of complications and new targets for therapeutic intervention.

Flow Cytometric Analysis of Cell Division by Dye Dilution

The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to tag proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division as well as to in vivo division of adoptively transferred cells and can resolve up to eight successive generations. CFSE is long lived, permitting analysis for several months after transfer, and has the same spectral characteristics as fluorescein, so monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes may be used to immunophenotype the dividing cells.

Mannose-binding lectin status is associated with risk of major infection following myeloablative sibling allogeneic hematopoietic stem cell transplantation

Mannose-binding lectin (MBL) is a mediator of innate immunity that influences the risk of infection in a range of clinical settings. We previously reported associations between MBL2 genotype and infection in a retrospective study of myeloablative allogeneic hematopoietic stem cell transplantation (allo-HCT). However, other studies have been inconclusive, and the role of MBL in reduced-intensity conditioning (RIC) transplantation is unknown. Here we report a prospective study examining MBL2 genotype, MBL levels, and risk of major infection following HLA-matched sibling myeloablative (n = 83) and RIC (n = 59) HCT. Baseline MBL levels were higher in recipients than donors (P < .001), and recipient MBL levels increased during the peritransplantation period (P = .001), most notably in MBL2 wild-type individuals receiving myeloablative total body irradiation (mTBI). MBL2 coding mutations were associated with major infection in recipients receiving mTBI. The cumulative incidence of major infection in recipient harboring an MBL2 mutation receiving mTBI was 70.6%, compared with 31.1% of those without mutations not receiving mTBI (P = .01). MBL status was not associated with infection in RIC transplants. These results confirm the association of MBL status with risk of infection in myeloablative, TBI-conditioned transplantation. Studies examining the role of MBL replacement therapy to prevent infection in this setting should be considered.

The impact of temporary deferral due to low hemoglobin: future return, time to return, and frequency of subsequent donation

BACKGROUND: This study investigated the effects of a 6‐month deferral due to low hemoglobin (Hb) on the subsequent donation patterns of Australian whole blood donors.

Imatinib inhibits the functional capacity of cultured human monocytes

Imatinib is a tyrosine kinase inhibitor that has been reported to specifically inhibit the growth of bcr‐abl expressing chronic myeloid leukaemia progenitors. This drug functions by blocking the ATP‐binding site of the kinase domain of bcr‐abl, and has also been found to inhibit the c‐abl, platelet‐derived growth factor receptor, ARG and stem cell factor receptor tyrosine kinases. Reports have recently emerged demonstrating that imatinib also inhibits the growth of non‐malignant haemopoietic cells. Here, we demonstrate that concentrations of imatinib within the therapeutic dose range inhibit the function of cultured monocytes (CM) from normal donors. A decrease in the response of CM to LPS was observed morphologically and functionally, with CM grown in the presence of imatinib showing decreased pseudopodia formation and inhibition of IL‐6 and TNF‐α production following LPS stimulation. Imatinib also reduced the ability of M‐CSF and GM‐CSF stimulated CM to phagocytose zymosan particles, with uptake of non‐opsonized zymosan by M‐CSF stimulated CM (M‐CM) being most affected. M‐CM that had been cultured in the presence of imatinib were also impaired in their ability to stimulate responder cells in a mixed lymphocyte reaction. These results demonstrate that human monocytes cultured in the presence of imatinib are functionally impaired, and suggest that imatinib displays inhibitory activity against other kinase(s) that play a role in monocyte/macrophage development.

Flow Cytometric Analysis of Cell Division by Dilution of CFSE and Related Dyes.

The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to track proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division, as well as to in vivo division of adoptively transferred cells and can resolve eight or more successive generations. CFSE is long lived, permitting analysis for several months after cell transfer, and has the same spectral characteristics as fluorescein, so monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes may be used to immunophenotype the dividing cells. In addition, information is given on a second‐generation dye, Cell Trace Violet (CTV), excited by 405‐nm blue laser light. CTV is chemically related to CFSE, but allows the 488‐nm line of the Argon laser to be used for other probes. Curr. Protoc. Cytom. 64:9.11.1‐9.11.12.

Acquisition of immune function during the development of the Langerhans cell network in neonatal mice

The immunological function of the Langerhans cell (LC) network in neonatal skin was examined by defining the development of cutaneous immunity relative to the structure, phenotype and function of the epidermal LC network in neonatal, juvenile and adult mice. Analysis of epidermal sheets showed the presence of major histocompatibility complex (MHC) II+, multilectin receptor DEC‐205– cells within the epidermis of 3‐day‐old mice; both cell density and DEC‐205 expression increased until day 14. When visualized with antibodies directed at MHC II, the network was poorly formed in 3‐ and 7‐day‐old mice, as there was a lower cell density and poor MHC II expression on dendritic processes, compared to mice at day14. Application of a fluorescent antigen to 3‐day‐old mice revealed that the LC were inefficient in transporting antigen to the draining lymph node. There was an improvement at day 7 and by day 14 comparable numbers of antigen carrying cells were detected in the lymph nodes of 6‐week‐old mice. The reduced antigen carriage in 3‐ and 7‐day‐old mice correlated with a poor contact sensitivity response. This was not simply due to failure to present antigen, but development of immunosuppression, as transfer of T cells from adult mice that were previously treated with antigen when they were 3 days old, to adult recipients resulted in antigen specific immunosuppression. Analysis of CD80 and CD86 expression showed that LC from day 3 skin expressed CD80, but not CD86 and application of antigen through this skin was inefficient in upregulating CD86. These findings indicate that when the neonatal LC network is poorly developed it is functionally immature and antigen applied through this ‘functionally immature network’ results in antigen specific immunosuppression.

Carcinogen‐modified dendritic cells induce immunosuppression by incomplete T‐cell activation resulting from impaired antigen uptake and reduced CD86 expression

Exposure of the skin to environmental stimuli, such as chemical or physical carcinogens, modifies the local skin environment, including depletion of epidermal Langerhans’ cells (LC). Any subsequent exposure of the LC‐depleted skin to antigen results in the generation of antigen‐specific tolerance. In this study we evaluated the antigen‐bearing cells in the draining lymph nodes by capitalizing on the fluorescent nature of the contact sensitizer, fluorescein isothiocyanate (FITC). When FITC was applied to the skin of normal mice, two distinct populations of antigen‐bearing cells were identified in the draining lymph nodes. They were classified as either FITChi or FITClo on the basis of their fluorescence intensity and thus the amount of antigen they internalized. Only FITClo cells were detected in the lymph nodes draining FITC‐treated murine skin that had been depleted of epidermal LC by prior treatment with the complete carcinogen 9,10‐dimethyl 1,2‐benzanthracene (DMBA). Functional analysis of these cells revealed that the FITChi cells, but not the FITClo cells, induced antigen‐specific T‐cell proliferation. Further analysis of the FITClo cells from the DMBA‐treated mice demonstrated that these cells had reduced levels of CD80 expression, had substantially reduced levels of CD86 expression and performed poorly as co‐stimulator cells in an anti‐CD3‐mediated proliferative assay. Nonetheless these cells still induced early signs of T‐cell activation and interleukin‐12 production. Consequently the FITClo cells migrating from the LC‐depleted skin, through a combination of reduced antigen presentation and reduced co‐stimulatory activity, induced a state of unresponsiveness or anergy in the responder T cells in a similar manner to that observed when antigen presentation occurs in the absence of co‐stimulation. We propose that these unresponsive, or anergic cells, account for the antigen‐specific tolerance observed in these experiments.

Validation of an automated immunoglobulin G–only cytomegalovirus (CMV) antibody screening assay and an assessment of the risk of transfusion transmitted CMV from seronegative blood

BACKGROUND: Cytomegalovirus (CMV) antibody donor screening assays have predominantly included both immunoglobulin G (IgG) and immunoglobulin M (IgM) detection. However, since in the majority of cases both CMV IgG and IgM are detected concomitantly during early seroconversion, CMV assays based only on IgG are now widely applied for donor screening.

Understanding non-return after a temporary deferral from giving blood: a qualitative study

BackgroundThe reasons why deferral from blood donation reduces the likelihood of future return remain unclear. This aim of this study was to investigate possible reasons why deferral has such a dramatic impact on donation patterns.MethodsQualitative methods were used to explore donors’ motivations to give blood, their experiences of temporary deferral, and their intentions to return once eligible. Semi-structured interviews were conducted with 23 donors in the two weeks following a temporary deferral due to a low haemoglobin concentration. The Framework approach was used to analyse data and identify themes associated with prompt return, ascertained from Blood Service records.ResultsWe found that, predominantly, individuals give blood because it represents an easy and convenient way to help others, and provides personal rewards, such as enhancing positive self-concepts and valuable knowledge about health. Deferral disrupts the habit of regular donation, and additionally, introduces an element of practical and emotional hassle to what is generally seen as an undemanding activity. Return after deferral was related to four aspects of a person and their context: an individual’s other obligations, especially parenting; whether donation arrangements were facilitated by a range of supports; the presence of a strong “blood donor” identity; and whether deferral left the donor feeling valued and appreciated.ConclusionsAspects of the deferral process need to be improved to ensure individuals feel valued, and continued attention should be given to the convenience of donation, especially for those with competing obligations.