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Biomedical subjects

A J Barrett

Publications and source records attributed to A J Barrett.

At least 37 records · Page 2Linked to original sources
PubMed2002

High-dose acyclovir and pre-emptive ganciclovir to prevent cytomegalovirus disease in myeloablative and non-myeloablative allogeneic stem cell transplantation.

We evaluated high-dose acyclovir and pre-emptive ganciclovir to prevent cytomegalovirus disease in myeloablative and non-myeloablative allogeneic stem cell transplantation. One hundred and seventy-four consecutive patients who were at risk for CMV infection (either recipient or donor seropositive) and received either intensive chemoradiotherapy and a T cell-depleted stem cell transplant followed by delayed add-back of donor T cells (TCDT: n = 98), or a non-myeloablative preparative regimen followed by an unmanipulated peripheral blood stem cell transplant (NMT: n = 76) from an HLA-identical sibling donor were studied. All received high-dose acyclovir (HDACV) from day - 7 for 3 months post-transplant in conjunction with weekly CMV pp65 antigenemia monitoring and pre-emptive treatment with intravenous immunoglobulin (not CMV-specific) and ganciclovir. The actuarial probabilities of developing pp65 antigenemia were 83 +/- 4% after TCDT and 41 +/- 6% after NMT (P < 0.00001) with reactivation occurring earlier in the TCDT group (the median 36 days vs 55 days). We observed no reactivation of CMV in seronegative recipients with a seropositive donor (n = 23). A total of 11 patients (5 in TCDT, 6 in NMT) developed CMV disease within 400 days after transplantation, and one death was clearly attributable to CMV interstitial pneumonitis (IP). This strategy was associated with effective control of CMV antigenemia in the majority of patients and near-complete eradication of fatal CMV IP.

Acyclovir↗
PubMed2002

Flow cytometric quantitation and characterization of the T-lymphocyte memory response to CMV in healthy donors.

BACKGROUND: Levels of circulating CMV Ag-specific lymphocytes determine CMV reactivation risk in immunocompromised individuals. METHODS: Frequencies of T cells producing cytokines after stimulation by CMV Ag were measured in hematopoietic stem-cell donors using flow cytometry. RESULTS: In seropositive individuals (n = 75) the mean number of CD8(+) (CD8(bright), CD8(dim)) and CD4(+) cells producing IFN-gamma was respectively 3.1% (12.6/microL) and 0.38% (3.2/microL), over 10-fold higher than in seronegative subjects (n = 22). CMV stimulation induced tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in both CD4(+) and CD8(+) cells (usually together), with a shift from memory- to effector-cell phenotype, while only a small proportion of CD4(+) cells produced IL-4. Although the normal range was wide, neither age, sex nor HLA type affected the frequency. DISCUSSION: These quantitative studies and the recognition of CD4(+) cells as potential effectors of CMV immunity are of relevance for immunotherapeutic approaches to prevent CMV disease after stem-cell transplantation.

Adoptive Transfer↗
PubMed2002

Optimized clinical-scale culture conditions for ex vivo selective depletion of host-reactive donor lymphocytes: a strategy for GvHD prophylaxis in allogeneic PBSC transplantation.

BACKGROUND: Ex vivo selective depletion (SD) is a strategy to prevent GvHD, in which host-reactive donor lymphocytes are selectively eliminated from a PBSC allograft while conserving useful donor immune function. Prior to testing this strategy in patients, our goal was to develop a clinical-scale SD process, which involves co-culture of donor lymphocytes and irradiated recipient cells, followed by the addition of an immunotoxin (IT) directed against the alpha-chain of the IL-2 receptor (CD25), expressed on activated donor T cells. METHODS: Stimulator cells were generated from immunomagnetically selected and expanded recipient T lymphocytes. Donor PBMCs from G-CSF-mobilized peripheral blood were co-cultured for 72 h with irradiated stimulator cells. Alloreactive T cells were targeted for elimination by the addition of the anti-CD25 IT, RFT5-SMPT-dgA, and the IT enhancer, NH(4)Cl. RESULTS: Stimulator-cell selection/expansion yielded > 2 x 10(10) highly enriched CD3(+) cells (98.9 +/- 2.2%). After SD, cell recovery was 68.5 +/- 23.3% and viability was 84.6 +/- 6.4%. This permitted a potential T-cell dose >/= 1 x 10(8) CD3(+) cells kg(-1) to transplant recipients. Although SD donor lymphocytes retained little proliferative capacity against the original stimulator cells (2.6 +/- 0.6%), responses were conserved against third party cells (107.6 +/- 18.6%), the bacterial superantigen staphylococcus enterotoxin B (108.2 +/- 4.2%), and CMV Ag (72.1 +/- 3.8%). DISCUSSION: We have demonstrated that ex vivo SD is feasible in clinical-scale culture conditions. The ability of this strategy to prevent GvHD is the subject of an ongoing clinical trial, in which the SD lymphocyte product is transplanted in conjunction with a T cell-depleted PBSC allograft.

CD3 Complex↗
PubMed2001

Rabaptin-5 is a novel fusion partner to platelet-derived growth factor beta receptor in chronic myelomonocytic leukemia.

Chromosomal translocations involving the platelet-derived growth factor beta receptor (PDGFbetaR) gene have been reported in some patients with chronic myelomonocytic leukemia (CMML). The resultant fusion proteins have constitutive PDGFbetaR tyrosine kinase activity, but the partner genes previously reported (tel, Huntingtin interacting protein 1 [HIP-1], H4/D10S170) have poorly understood roles in the oncogenic activity of the fusion proteins. A novel PDGFbetaR fusion protein has been characterized in a patient with CMML and an acquired t(5;17)(q33;p13). Southern blot analysis on patient leukemia cells demonstrated involvement of the PDGFbetaR gene. Using 5' rapid amplification of complementary DNA ends-polymerase chain reaction (RACE-PCR) on patient RNA, rabaptin-5 was identified as a novel partner fused in-frame to the PDGFbetaR gene. The new fusion protein includes more than 85% of the native Rabaptin-5 fused to the transmembrane and intracellular tyrosine kinase domains of the PDGFbetaR. Transduction with a retroviral vector expressing rabaptin-5/PDGFbetaR transformed the hematopoietic cell line Ba/F3 to growth factor independence and caused a fatal myeloproliferative disease in mice. Rabaptin-5 is a well-studied protein shown to be an essential and rate-limiting component of early endosomal fusion through interaction with the Ras family GTPases Rab5 and Rab4. The fusion protein includes 3 of 4 coiled-coil domains (involved in homodimerization of native rabaptin-5), 2 caspase-3 cleavage sites, and a binding site for the tumor suppressor gene tuberin (tuberous sclerosis complex-2). Early endosomal transport is critical in regulation of various growth factor receptors, through ligand-induced clathrin-mediated endocytosis, and thus this new fusion protein links together 2 important pathways of growth regulation.

Adult↗
PubMed2001

Outbreak of human parainfluenza virus 3 infections in a hematopoietic stem cell transplant population.

Clinical manifestations and epidemiological features are described for a cluster of 12 cases of human parainfluenza virus 3 (HPIV3) infection that occurred among 64 allogeneic hematopoietic stem cell transplant (SCT) recipients in an 11-week period during spring 2000. Upper respiratory symptoms predominated. Pneumonia occurred in 3 patients and was a contributing factor in the death of 1 patient. Exposure histories and molecular analysis of HPIV3 isolates suggested that both community acquired and nosocomially transmitted infections occurred during this outbreak. A chain of transmission within the outpatient clinic appeared to have occurred in 4 outpatients and to have extended to 2 hospitalized patients. Molecular epidemiology was useful in discerning routes of transmission in this outbreak.

Disease Outbreaks↗
PubMed2001

Delayed donor red cell chimerism and pure red cell aplasia following major ABO-incompatible nonmyeloablative hematopoietic stem cell transplantation.

Delayed donor red cell engraftment and pure red cell aplasia (PRCA) are well-recognized complications of major ABO-incompatible hematopoietic stem cell transplantation (SCT) performed by means of myeloablative conditioning. To evaluate these events following reduced-intensity nonmyeloablative SCT (NST), consecutive series of patients with major ABO incompatibility undergoing either NST (fludarabine/cyclophosphamide conditioning) or myeloablative SCT (cyclophosphamide/high-dose total body irradiation) were compared. Donor red blood cell (RBC) chimerism (initial detection of donor RBCs in peripheral blood) was markedly delayed following NST versus myeloablative SCT (median, 114 versus 40 days; P <.0001) and strongly correlated with decreasing host antidonor isohemagglutinin levels. Antidonor isohemagglutinins declined to clinically insignificant levels more slowly following NST than myeloablative SCT (median, 83 versus 44 days; P =.03). Donor RBC chimerism was delayed more than 100 days in 9 of 14 (64%) and PRCA occurred in 4 of 14 (29%) patients following NST, while neither event occurred in 12 patients following myeloablative SCT. Conversion to full donor myeloid chimerism following NST occurred significantly sooner in cases with, compared with cases without, PRCA (30 versus 98 days; P =.008). Cyclosporine withdrawal appeared to induce graft-mediated immune effects against recipient isohemagglutinin-producing cells, resulting in decreased antidonor isohemagglutinin levels and resolution of PRCA following NST. These data indicate that significantly delayed donor erythropoiesis is (1) common following major ABO-incompatible NST and (2) associated with prolonged persistence of host antidonor isohemagglutinins. The clinical manifestations of these events are affected by the degree and duration of residual host hematopoiesis.

ABO Blood-Group System↗
PubMed2001

Transcription patterning of uncoupled proliferation and differentiation in myelodysplastic bone marrow with erythroid-focused arrays.

Because abnormal erythroid differentiation is the most common manifestation of the myelodysplastic syndromes (MDS), it was hypothesized that erythroid gene expression may be used to illustrate myelodysplastic transcription patterns. Ten normal bone marrow aspirates (NBM) were first analyzed using an erythroid-focused cDNA array to define steady-state transcription levels. Proliferation and differentiation gene subsets were identified by statistically significant differences between NBM and erythroleukemia gene expression. Next, cDNAs from 5 separate MDS aspirates were studied: refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation (RAEB-T), and RAEB-T/secondary MDS. A distinct pattern of significantly increased proliferation-associated and reduced differentiation-associated gene activity was established for MDS.

Aged↗
PubMed2001

Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome.

The myelodysplastic syndromes (MDSs) are characterized by bilineage or trilineage dysplasia. Although diagnostic criteria are well established for MDS, a significant number of patients have blood and bone marrow findings that make diagnosis and classification difficult. Flow cytometric immunophenotyping is an accurate and highly sensitive method for detection of quantitative and qualitative abnormalities in hematopoietic cells. Flow cytometry was used to study hematopoietic cell populations in the bone marrow of 45 patients with straightforward MDS. The results were compared with those obtained in a series of patients with aplastic anemia, healthy donors, and patients with a history of nonmyeloid neoplasia in complete remission. The immunophenotypic abnormalities associated with MDS were defined, and the diagnostic utility of flow cytometry was compared, with morphologic and cytogenetic evaluations in 20 difficult cases. Although morphology and cytogenetics were adequate for diagnosis in most cases, flow cytometry could detect immunophenotypic abnormalities in cases when combined morphology and cytogenetics were nondiagnostic. It is concluded that flow cytometric immunophenotyping may help establish the diagnosis of MDS, especially when morphology and cytogenetics are indeterminate. (Blood. 2001;98:979-987)

Adult↗
PubMed2001

Inhibition of distant caspase homologues by natural caspase inhibitors.

Caspases play an important role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, two of which, baculovirus p35 and members of the inhibitor of apoptosis (IAP) family, are thought to be caspase specific. However, caspases are members of the clan of cysteine proteases designated CD, which also includes animal and plant legumains, and the bacterial proteases clostripain, gingipain-R and gingipain-K. Since these proteases have been proposed to have a common mechanism and evolutionary origin, we hypothesized that the caspase inhibitors may also regulate these other proteases. We tested this hypothesis by examining the effect of the natural caspase inhibitors on other members of protease clan CD. The IAP family proteins were found to have only a slight inhibitory effect on gingipain-R. The cowpox viral cytokine-response modifier A (CrmA) serpin had no effect on any of the proteases tested but a single point mutation of CrmA (Asp-->Lys) resulted in strong inhibition of gingipain-K. More substantial, with respect to the hypothesis, was the strong inhibition of gingipain-K by wild-type p35. The site in p35, required for inhibition of gingipain-K, was mapped to Lys94, seven residues C-terminal to the caspase inhibitory site. Our data indicate that the virally encoded caspase inhibitors have adopted a mechanism that allows them to regulate disparate members of clan CD proteases.

Amino Acid Sequence↗
PubMed2001

Treatment of chronic granulomatous disease with nonmyeloablative conditioning and a T-cell-depleted hematopoietic allograft.

BACKGROUND: The treatment of chronic granulomatous disease with conventional allogeneic hematopoietic stem-cell transplantation carries a high risk of serious complications and death. We investigated the feasibility of stem-cell transplantation without ablation of the recipient's bone marrow. METHODS: Ten patients, five children and five adults, with chronic granulomatous disease underwent peripheral-blood stem-cell transplantation from an HLA-identical sibling. We used a nonmyeloablative conditioning regimen consisting of cyclophosphamide, fludarabine, and antithymocyte globulin. The allograft was depleted of T cells to reduce the risk of severe graft-versus-host disease. Donor lymphocytes were administered at intervals of 30 days or more after the transplantation to facilitate engraftment. RESULTS: After a median follow-up of 17 months (range, 8 to 26), the proportion of donor neutrophils in the circulation in 8 of the 10 patients was 33 to 100 percent, a level that can be expected to provide normal host defense; in 6 the proportion was 100 percent. In two patients, graft rejection occurred. Acute graft-versus-host disease (grade II, III, or IV) developed in three of the four adult patients with engraftment, one of whom subsequently had chronic graft-versus-host disease. None of the five children had grade II, III, or IV acute graft-versus-host disease. During the follow-up period, four serious infections occurred among the patients who had engraftment. Three of the 10 recipients died. Preexisting granulomatous lesions resolved in the patients in whom transplantation was successful. CONCLUSIONS: Nonmyeloablative conditioning followed by a T-cell-depleted hematopoietic stem-cell allograft is a feasible option for patients with chronic granulomatous disease, recurrent life-threatening infections, and an HLA-identical family donor.

Adolescent↗
PubMed2001

CD8+ T cells in large granular lymphocyte leukemia are not defective in activation- and replication-related apoptosis.

Persistent lymphocytosis in large granular lymphocyte leukemia (LGL) may result from defects in activation- or Fas crosslinking-induced cell death. Here we show that Fas crosslinking and CD3 activation causes apoptosis of in vitro activated CD8 T cells, but not of freshly isolated CD8 T cells. Death was partially blocked by a neutralizing antibody to FasL. Inhibition of metalloproteinase-mediated FasL solubilization significantly potentiated induction of cell death. Furthermore, CD3 plus CD28 stimulation resulted in telomeric erosion in LGL cells, and ultimately proliferation ceased. Together, these data indicate that activation- and proliferation-related cell death mechanisms are functional in LGL cells.

Adult↗
PubMed2001

Oligoclonal T cell expansion in myelodysplastic syndrome: evidence for an autoimmune process.

There is accumulating evidence that the marrow-failure of myelodysplastic syndrome (MDS) is immune-mediated. We studied patients with MDS to look for oligoclonal or clonal expansion in T cells indicative of an autoimmune process. We used a PCR-based technique (spectratyping) to characterize the T cell repertoire in MDS (n=15; 9 RA, 4 RARS, 2 RAEB) and compared results with age-matched healthy donors (n=20) and transfusion-dependent (TD) patients with hemoglobinopathy (n=5). We found a significantly higher number of skewed Vbeta profiles in the MDS patients compared with controls. In peripheral blood T cells, 60/345 Vbeta profiles examined were skewed in MDS patients compared with 3/115 Vbeta profiles in TD controls (P<0.0001), and 58/460 Vbeta profiles in age-matched controls (P=0.05). A study of Jbeta region within the skewed Vbeta profiles revealed preferential usage of Jbeta 2.1 in MDS in contrast with a wide distribution over the entire Jbeta spectrum in controls, consistent with non-random T cell clonal expansion in MDS. These findings provide further evidence that T cell mediated immune processes are a feature of MDS.

Adult↗
PubMed2001

Dental health and viridans streptococcal bacteremia in allogeneic hematopoietic stem cell transplant recipients.

Viridans streptococci were the most common cause of bacteremia in 61 consecutive myeloablative allogeneic hematopoietic stem cell transplant (HSCT) recipients, occurring in 19 of 31 bacteremic patients (61%) during the period of post-transplant neutropenia. Seven of the 19 had more than one viridans streptococcus in the same blood culture. Twenty isolates from 15 patients were Streptococcus mitis. Most viridans streptococci were resistant to norfloxacin, used routinely for prophylaxis. Comparison of the 19 patients with viridans streptococcal bacteremia with a contemporaneous group of 23 allogeneic HSCT recipients with fever and neutropenia but no identified focus of infection found that patients with viridans streptococcal bacteremia were more likely to have severe intraoral pathology while neutropenic (26% vs 0%) and slightly shorter interval between the last dental procedure and the onset of neutropenia (11 vs 14 days). Poor underlying dental health and the use of norfloxacin thus appear to predispose to viridans streptococcal bacteremia.

Adult↗
PubMed2001

Large granular lymphocyte leukaemia is characterized by a clonal T-cell receptor rearrangement in both memory and effector CD8(+) lymphocyte populations.

Large granular lymphocyte (LGL) leukaemia is a disease with increased numbers of circulating granular lymphocytes and an increased percentage of clonally rearranged CD8(+)CD57(+) cells. To determine whether LGL cells are also found in other lymphocyte subsets, CD8(+) cells from 10 LGL patients were sorted into CD57(+) and CD57(-) fractions and analysed for clonality using a T-cell receptor gamma (TCR gamma) polymerase chain reaction (PCR). In nine patients, a clonal TCR rearrangement was identified in the CD8(+)CD57(+) cells, and in one patient, the TCR rearrangement was oligoclonal in the CD8(+)CD57(+) fraction. In eight out of nine of the clonally rearranged patients, the same band was also present in the CD8(+)CD57(-) fraction. To define the relationship between CD57(-) and CD57(+) LGL populations, CD8(+)CD57(-) and CD8(+)CD57(+) cells were sorted from five patients and cultured in the presence of anti-CD3 plus CD28 antibodies. The CD57(+) cells died of apoptosis before d 7, while the CD57(-) cells proliferated and differentiated into CD57(+) cells. Clonal analysis identified the same band in both cultured subpopulations and in the uncultured CD8(+) cells. Immunophenotypical analysis showed that CD8(+)CD57(-) cells expressed memory cell markers, while the CD8(+)CD57(+) cells exhibited effector characteristics. These results suggest that LGL disease originates in a CD57(-) memory T-cell compartment that continually generates CD57(+) (effector cell) progeny.

Adult↗
PubMed2001

Massive immune haemolysis after allogeneic peripheral blood stem cell transplantation with minor ABO incompatibility.

Immune haemolysis as a result of minor ABO incompatibility is an underappreciated complication of haematopoietic transplantation. The increased lymphoid content of peripheral blood stem cell (PBSC) transplants may increase the incidence and severity of this event. We observed massive immune haemolysis in 3 out of 10 consecutive patients undergoing HLA-identical, related-donor PBSC transplants with minor ABO incompatibility. Non-ablative conditioning had been given in 9 of these 10 cases, including two with haemolysis. Cyclosporin alone was used as prophylaxis against graft-vs.-host disease (GVHD). Catastrophic haemolysis of 78% of the circulating red cell mass led to anoxic death in the first case seen, but severe consequences were avoided by early, vigorous donor-compatible red cell transfusions in the subsequent two cases. Haemolysis began 7-11 d after PBSC infusion and all patients with haemolysis had a positive direct antiglobulin test (DAT), with eluate reactivity against the relevant recipient antigen. However, neither the intensity of the DAT, the donor isohaemagglutinin titre, nor other factors could reliably be used to predict the occurrence of haemolysis. Our data indicate that haemolysis may be frequent and severe after transplantation of minor ABO-incompatible PBSCs when utilizing cyclosporin alone to prevent GVHD. Meticulous clinical monitoring and early, vigorous donor-compatible red cell transfusions should be practiced in all instances.

ABO Blood-Group System↗
PubMed2001

In vitro T-cell receptor V beta repertoire analysis may identify which T-cell V beta families mediate graft-versus-leukaemia and graft-versus-host responses after human leucocyte antigen-matched sibling stem cell transplantation.

We studied oligoclonal T-cell expansions of 24 T-cell receptor (TCR) V beta families in normal donor lymphocytes stimulated with patient's cells and in recipient blood after transplant, using a polymerase chain reaction-based assay (spectratyping). T cells from donor blood were incubated with separated myeloid leukaemia cells or T cells from the HLA-identical sibling recipient. In five of the six patients tested, the T-cell V beta skewing pattern observed in vitro was seen in vivo after transplant. After transplant, the myeloid-specific V beta skewing coincided with the disappearance of residual disease in three patients and in one patient skewing was lost at the time of leukaemic relapse. In functional tests, T cells generated against leukaemic cells in vitro produced interferon gamma in response to the leukaemia. Removal of the leukaemia-expanded skewed V beta families significantly decreased cytotoxic killing of the leukaemia. However, while there was a general concordance in the V beta family exhibiting clonal expansion in vitro and in vivo, the exact clonotype expanded in vitro and in vivo differed. These findings suggest that alloresponses involve multiple T-cell clones within a restricted TCR V beta repertoire that undergo different selection pressures in vitro and in vivo.

Acute Disease↗
PubMed2001

Transplant dose of CD34(+) and CD3(+) cells predicts outcome in patients with haematological malignancies undergoing T cell-depleted peripheral blood stem cell transplants with delayed donor lymphocyte add-back.

We sought to optimize and standardize stem cell and lymphocyte doses of T cell-depleted peripheral blood stem cell transplants (T-PBSCT), using delayed add-back of donor T cells (DLI) to prevent relapse and enhance donor immune recovery. Fifty-one patients with haematological malignancies received a T-PBSCT from an HLA-identical sibling, followed by DLI of 1 x 10(7) and 5 x 10(7) CD3(+) cells/kg on d +45 and +100 respectively. Twenty-four patients were designated as standard risk and twenty-seven patients with more advanced leukaemia were designated as high risk. Median recipient age was 38 years (range 10-56). Median (range) of CD34(+) and CD3(+) cell transplant doses were 4.6 (2.3-10.9) x 10(6)/kg and 0.83 (0.38-2) x 10(5)/kg respectively. The cumulative probability of acute GVHD was 39%. No patient died from GVHD or its consequences. The probability of developing chronic GVHD was 54% (18% extensive). The probability of relapse was 12% for the standard-risk patients and 66% for high-risk patients. In multivariate analysis, the risk factors for lower disease-free survival and overall survival were high-risk disease, CD34(+) dose < 4.6 x 10(6)/kg and CD3(+) dose < 0.83 x 10(5)/kg. Predictive factors for chronic GVHD were a T-cell dose at transplant > 0.83 x 10(5) CD3(+) cells/kg. These results further define the impact of CD34 and CD3 cell dose on transplant outcome and show that careful dosing of stem cells and lymphocytes may permit the control and optimization of transplant outcome.

Adolescent↗