Cord blood T cells retain early differentiation phenotype suitable for immunotherapy after TCR gene transfer to confer EBV specificity.

Adoptive T cell therapy can be effective for Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease and melanoma. Transducing high-affinity TCR genes into T lymphocytes is an emerging method to improve potency and specificity of tumor-specific T cells. However, both methods necessitate in vitro lymphocyte proliferation, generating highly differentiated effector cells that display reduced survival and antitumor efficacy postinfusion. TCR-transduction of naive lymphocytes isolated from peripheral blood is reported to provide superior in vivo survival and function. We utilized cord blood (CB) lymphocytes, which comprise mainly naive cells, for transducing EBV-specific TCR. Comparable TCR expression was achieved in adult and CB cells, but the latter expressed an earlier differentiation profile. Further antigen-driven stimulation skewed adult lymphocytes to a late differentiation phenotype associated with immune exhaustion. In contrast, CB T cells retained a less differentiated phenotype after antigen stimulation, remaining CD57-negative but were still capable of antigen-specific polyfunctional cytokine expression and cytotoxicity in response to EBV antigen. CB T cells also retained longer telomeres and in general possessed higher telomerase activity indicative of greater proliferative potential. CB lymphocytes therefore have qualities indicating prolonged survival and effector function favorable to immunotherapy, especially in settings where donor lymphocytes are unavailable such as in solid organ and CB transplantation.

ONIOM DFT/PM3 calculations on the interaction between dapivirine and HIV-1 reverse transcriptase, a theoretical study.

Theoretical investigations of the interaction between dapivirine and the HIV-1 RT binding site have been performed by the ONIOM2 (B3LYP/6-31G (d,p): PM3) and B3LYP/6-31G (d,p) methods. The results derived from this study indicate that this inhibitor dapivirine forms two hydrogen bonds with Lys101 and exhibits strong π-π stacking or H…π interaction with Tyr181 and Tyr188. These interactions play a vital role in stabilizing the NNIBP/dapivirine complex. Additionally, the predicted binding energy of the BBF optimized structure for this complex system is -18.20 kcal/mol.

Thalidomide induced remission of refractory diffuse large B-Cell Lymphoma post-allogeneic SCT.

Patients who relapse after High dose therapy and autologous stem cell transplant (ASCT) for Diffuse large B cell Lymphoma (DLBCL) have a poor prognosis with a median survival of only 3-6 month.1-2 This case demonstrates the ability of thalidomide at low doses to induce durable response in a patient with DLBCL who relapsed after full intensity allogeneic transplantation.

Can defects in transferrin receptor 2 and hereditary hemochromatosis genes account for iron overload in HbH disease?

Iron overload was found to be the major cause of disability in Chinese HbH disease patients although they were not on regular blood transfusion. The transferrin receptor 2 (TFR2) and hereditary hemochromatosis (HFE) genes were examined to see if inheritance of these gene defects may be a possible cause of iron overload in 45 HbH patients. A novel intronic (IVS6 (+6) T-->A) mutation of the TFR2 gene was identified in one patient, and six others were found to carry a known missense mutation (exon 5, I238M) that was also present in one normal control subject. One HbH patient and one normal control carried the H63D mutation of the HFE gene. Since only eight out of 45 iron-overloaded HbH patients carry a defect in the TFR2 or HFE gene in the heterozygote state and their iron loading status was comparable to the matched controls without such defects, it would appear that the accumulation of excess iron in HbH disease is more likely a result of increase dietary absorption secondary to ineffective erythropoiesis.

Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy.

The immune function of retrovirus-mediated gene modified (GM) T cells is critical for a beneficial effect to follow their adoptive transfer into patients. Recent clinical data show that GM T cells expanded with PHA have reduced function in vivo. However, little functional analysis of PHA stimulation is available. Our results show that expansion of T cells with PHA impairs their ability to respond (proliferation, cytotoxicity and IFN gamma and perforin expression) to allogeneic stimulation or viral antigens in vitro. Conversely, CD3/CD28-based protocols can preserve this immune function. Retroviral transduction did not alter the functional profile induced by polyclonal stimulation. We investigated the mechanisms leading to this functional effect, and identified differential effects of PHA and CD3/CD28 on the distribution of CCR7/CD45RA T cell functional subsets, which may explain the functional differences observed. While CD3/CD28 stimulation parallels the lineage differentiation pattern induced by antigens in physiological conditions, PHA induces a skewed distribution of the CCR7/CD45RA functional T cell subsets, with near disappearance of the subpopulations that display the effector phenotype. Overall, this study demonstrates a functional disadvantage for transduction protocols based on PHA, uncovers mechanisms that may explain this functional effect, and provides us with information to design and select transduction protocols with an improved functional outcome.

HLA tetramers and anti-CMV immune responses: from epitope to immunotherapy.

BACKGROUND: Identification of HLA class I-restricted CMV epitopes, and the subsequent synthesis of HLA class I-peptide tetrameric complexes, have provided investigators with an important tool for visualising and quantifying the precise in vivo CTL response to CMV reactivation following stem cell transplantation. In conjunction with PCR-monitoring of the viral load, the magnitude and dynamic of the host's specific CD8(+) T cell response to viral replication can be studied. METHODS: CMV peptide epitopes can be identified be searching the CMV-pp65 antigen for HLA class I allele binding motifs, by testing their binding affinity and ability to generate CTLs, and by screening for CTL responses in as many individuals as possible to assess their general applicability for monitoring large number of patients. HLA tetramers are synthesized by refolding recombinant class I heavy chains and beta(2)m with CMV-pp65(495-503) peptide. After biotinylation and tetramerisation to PE-conjugated streptavidin, they are used to stain CD8(+) T cells taken from patients at different time points after SCT. RESULTS: The T-cell mediated immune response is mainly directed against epitopes derived from the CMV tegument protein pp65. CMV-specific CTL's confer protection against CMV reactivation above a threshold level of 10(7) to 2 x 10(7)/L. CMV reactivation is required to stimulate CTL responses. Transfer of CMV immunity from seropositive donors is associated with better outcome and steroids suppress the Ag-specific immune response. DISCUSSION: Initial studies with CMV-specific HLA class I tetramers have helped to define the nature of anti-CMV T cell response in SCT patients and to determine a threshold CTL level required for controlling CMV reactivation. Monitoring patients with HLA-tetramers should therefore allow clinicians to predict and assess the risk of reactivation and to balance the risks and benefits of early anti-viral treatment, thereby avoiding the hazards of anti-viral prophylaxis. HLA-tetramers can also be used to isolate antigen-specific cells for further in vitro expansion and transfer to patients for antiviral immunotherapy. The threshold level determined from patient monitoring can be used as a guide for estimating an effective target cell dose.

Helper T-lymphocyte precursor frequency predicts the occurrence of graft-versus-host disease and disease relapse after allogeneic bone marrow transplantation from HLA-identical siblings.

BACKGROUND AND OBJECTIVES: Donor helper T-lymphocytes may be involved in graft-versus-host disease (GVHD) and a graft-versus-leukemia effect after bone marrow transplantation (BMT). We assayed donor helper T-lymphocyte precursor frequencies (HTLP(f)) to see whether they could predict the severity of GVHD and disease relapse after transplantation, thereby facilitating donor selection, pre-transplant counselling and modification of GVHD prophylaxis after BMT. DESIGN AND METHODS: Thirty-six consecutive adult BMT recipients and their HLA-identical sibling donors were recruited. HTLP((f)) was measured as a function of interleukin-2 secretion by alloreactive donor T-cells using a limiting dilution assay. Patients were followed prospectively to assess the severity of GVHD and the status of the primary disease after BMT. RESULTS: Eight donors had HTLP((f)) less than or equal to 10(-6); no recipients of these grafts developed severe GVHD after transplantation. Twenty-eight donors had HTLP(f) greater than 10(-6) and 18 recipients of these grafts developed severe GVHD (> or = grade 2) (chi(2) test, p<0.01). Seven donors had HTLP(f) greater than 10(-5) and no recipient had disease relapse. Twenty-nine donors had HTLP(f) less than or equal to 10(-5), 11 recipients of these grafts developed disease relapse (chi(2) test, p=0.08). INTERPRETATION AND CONCLUSIONS: BMT recipients from HLA-identical sibling donors with low (<10(-6)) and high (>10(-5)) HTLP(f) may have a low risk of acute GVHD and disease relapse after transplantation.

Genetic and clinical features of hemoglobin H disease in Chinese patients.

BACKGROUND: Normally, one pair of each of the two alpha-globin genes, alpha1 and alpha2, resides on each copy of chromosome 16. In hemoglobin H disease, three of these four alpha-globin genes are affected by a deletion, a mutation, or both. We studied the alpha1-globin gene abnormalities and the clinical and hematologic features of Chinese patients with hemoglobin H disease in Hong Kong. METHODS: We assessed the clinical features, hematologic values, serum ferritin levels, and liver function of 114 patients with hemoglobin H disease. We also performed echocardiography and magnetic resonance imaging of the liver and examined the two pairs of alpha-globin genes. RESULTS: Hemoglobin H disease in 87 of the 114 patients (76 percent) was due to the deletion of three of the four alpha-globin genes (--/-alpha), a combination termed the deletional type of hemoglobin H. The remaining 27 patients (24 percent) had the nondeletional type of hemoglobin H disease, in which two alpha-globin genes are deleted and a third is mutated (--/alphaalphaT). All 87 patients with the deletional type of hemoglobin H were double heterozygotes in whom there was a deletion of both alpha-globin genes from one chromosome, plus a deletion of the alpha1 or alpha2 gene from the other chromosome (--/alpha- or --/-alpha). A variety of mutated alpha-globin genes was found in the patients with nondeletional type of hemoglobin H disease. Patients with the nondeletional type of the H disease had more symptoms at a younger age, more severe hemolytic anemia, and larger spleens and were more likely to require transfusions than patients with deletional hemoglobin H disease. The severity of iron overload was not related to the genotype. CONCLUSIONS: Chinese patients in Hong Kong with the nondeletional type of hemoglobin H disease have more severe disease than those with the deletional type of the disease. Iron overload is a major cause of disability in both forms of the disease.

Regulation of DNA binding by Rel/NF-kappaB transcription factors: structural views.

Rel/NF-kappaB transcription factors form homo- and heterodimers with different DNA binding site specificities and DNA binding affinities. Several intracellular pathways evoked by a wide range of biological factors and environmental conditions can lead to the activation of Rel/NF-kappaB dimers by signaling degradation of the inhibitory IkappaB protein. In the nucleus Rel/NF-kappaB dimers modulate the expression of a variety of genes including those encoding cytokines, growth factors, acute phase response proteins, immunoreceptors, other transcription factors, cell adhesion molecules, viral proteins and regulators of apoptosis. The primary focus of this review is on structural and functional aspects of Rel/NF-kappaB:DNA complexes and their formation. The salient features of the Rel/NF-kappaB dimer:DNA structure are described, as are modes of transcriptional regulation by phosphorylation, altered DNA binding properties, varying protein conformations, and interactions with IkappaB proteins.

Outcome of Chinese patients with chronic myeloid leukaemia (CML) underwent allogeneic bone-marrow transplantation (BMT).

UNLABELLED: Clinical studies have shown that patients with chronic myeloid leukaemia (CML) treated with allogeneic bone-marrow transplantation (BMT) experience not only prolonged disease-free survival but also complete cure in some. Therefore, we followed a cohort of 81 Chinese patients who received allogeneic BMT. PATIENTS AND METHODS: The donors were either relatives (65 siblings, 1 parent) or unrelated volunteers (15). BMT was performed at a median interval of 11.6 months from diagnosis of CML, and the stages of disease before BMT were: first chronic phase (60 patients), accelerated or second chronic phase in (10 patients), and blastic crisis (11 patients). Three conditioning regimens were employed: Bu-Cy, Cy-TBI, or Bu-Cy-TBI. Standard cyclosporin and short methotrexate protocol were used for acute graft-versus-host disease (GvHD) prophylaxis. RESULTS: There were five graft failures with three after related BMT. Patients after related or unrelated BMT had a comparable rate of neutrophil recovery (median = 22 days), but significant delay in platelet recovery occurred after unrelated BMT (median = 34 vs. 20 days, P < 0.05). The latter also had higher incidence of acute GvHD (73% vs. 41%, P < 0.05), although the incidence of chronic GvHD was not different between groups. At a median follow-up of 43.5 months, patients after related BMT had a significantly better rate of disease-free survival (68% vs. 37.3%, P < 0.05) and overall survival (81% vs. 38.9%, P < 0.05) at 4 years. Subgroup analysis of patients after related BMT showed the outcome was better when they were transplanted at first chronic phase. Multivariate analysis showed that advanced disease (RR = 2.01, 95% CI = 1.48-2.73) significantly worsened the outcome of BMT, whereas the presence of chronic GvHD had a protective effect against relapse and survival (RR = 0.09, 95% CI = 0.02-0.38). CONCLUSION: Allogeneic BMT is a curative form of treatment for patients with CML. Treatment outcome is best for those who undergo transplants from HLA-matched siblings during the first chronic phase.

Construction, expression, purification and functional analysis of recombinant NFkappaB p50/p65 heterodimer.

NFkappaB plays an important role in mediating the gene expression of numerous cellular processes such as growth, development, the inflammatory response and virus proliferation. The p50/p65 heterodimer is the most abundant form of the NFkappaB dimers and plays a more elaborate role in gene regulation. Biochemical research on p50/p65 NFkappaB has not benefited however from the availability of easily purified recombinant protein. We report two methods for the large scale expression and purification of recombinant NFkappaB p50/p65 heterodimer. The first utilizes a bacterial double expression vector which contains two ribosomal binding sites to facilitate the coexpression of the polypeptides in the p50/p65 NFkappaB heterodimer. The second method uses a mixed protein refolding strategy. Both methods yield crystallizable protein. Electrophoretic mobility shift assays confirm that the DNA binding affinity is independent of the method used to purify the protein. These methods will facilitate the numerous studies on various NFkappaB/Rel family members.

A reverse dot-blot method for rapid detection of non-deletion alpha thalassaemia.

A reverse dot blot method based on membrane-bound allele-specific oligonucleotides as hybridization targets for amplified alpha-gene fragments has been developed for the rapid detection of four non-deletion alpha thalassaemia defects found in the Chinese. Since these non-deletion defects account for 22 8% of haemoglobin H disease, a sensitive, specific and rapid screening method should be of value.

Qualitative and quantitative magnetic resonance imaging in haemoglobin H disease: screening for iron overload.

OBJECTIVES: To evaluate the clinical utility of magnetic resonance imaging (MRI) in screening for iron overload in non-transfusion dependent Haemoglobin (Hb) H disease. PATIENTS AND METHODS: Thirty-six non-transfusion dependent HbH patients were evaluated with axial spin echo T1 and gradient echo T2 MRI of the abdomen and heart. The ratios of signal intensities (SIR) of the liver, spleen, pancreas and heart to paraspinous muscles were calculated. SIR <1 was taken as indicative of iron overload. Qualitative grading (0-4 scale) of iron overload was also performed. The relationship between T1 and T2 SIR and serum ferritin, and that between qualitative grading and serum ferritin were examined using standard statistical methods. Comparisons were also made between qualitative grading and quantitative T1 and T2 SIR data in diagnosing iron overload. Six patients underwent liver biopsies. RESULTS: T2 SIR was more sensitive in detecting iron overload than T1 SIR. Thirty-three livers, 13 spleens, six pancreas and one heart were diagnosed as having iron overload with T2 SIR, including three patients with normal serum ferritin. A positive diagnosis by T2 SIR was more closely related to that of qualitative grading than T1 SIR. Serum ferritin was negatively correlated with hepatic SIR (T1 and T2), and with T2 SIR of the spleen and pancreas, even after adjustment for age. Liver haemosiderosis was confirmed in all six patients who underwent liver biopsies. Liver iron concentration of only one and a half times the normal was found in one patient with positive MR findings. CONCLUSION: MR is a non-invasive, effective method for early detection of iron overload particularly in the liver and spleen. Qualitative grading and quantitative T2 SIR data are equivalent in diagnosing iron overload. Routine screening of non-transfusion dependent HbH patients will identify high risk patients in whom early therapeutic intervention may prevent further complications and morbidity.

Unique risk factors for bacteraemia in allogeneic bone marrow transplant recipients before and after engraftment.

A study of the risk factors associated with bacteraemia in 191 allogeneic bone marrow transplant (BMT) recipients (1991-1996) was performed. In contrast to risk factors commonly cited for cancer chemotherapy, mucositis, degree of conditioning toxicity of the gut and lungs, duration of neutropenia, and severity of neutropenia and monocytopenia were not associated with bacteraemia in the pre-engraftment period, during which the only significant risk factor was late stage underlying disease (P < 0.05). After engraftment, Hickman catheter infection, and severe acute and chronic graft-versus-host disease (GVHD) were found to be independently associated with bacteraemia by multivariate analysis (P < 0.001, <0.05 and <0.05, respectively). This might be explained by intense antimicrobial prophylaxis, early empirical treatment, and non-routine use of haemopoietic growth factors. No significant difference in mortality was detected between bacteraemic and non-bacteraemic patients in both periods. Allogeneic BMT recipients are therefore a group of patients distinct from other cancer patients receiving chemotherapy at risk of developing bacteraemia. The study findings prompt consideration of a management protocol incorporating early and routine use of haemopoietic growth factors before engraftment in high-risk patients with late stage underlying malignancies, routine antimicrobial prophylaxis for acute GVHD with intense immunosuppression, and intravenous immunoglobulin therapy for chronic GVHD. Further cost-benefit analyses are warranted.

Recurrent isolated extramedullary relapses as granulocytic sarcomas following allogeneic bone marrow transplantation for acute myeloid leukemia.

Isolated extramedullary relapses as granulocytic sarcomas (GS) following allogeneic bone marrow transplantation (BMT) for acute myeloid leukemia (AML) are rare events. We describe three such patients who presented with a unique pattern of GS relapse post-BMT. The clinical features included repeated relapses in multiple sites, absence of marrow involvement, and prolonged survival. Fluorescence in situ hybridization (FISH) demonstrated persistence of donor hematopoiesis despite disseminated GS. The findings indicated that a graft-versus-leukemia (GVL) effect might operate more strongly in the marrow than in peripheral sites. Finally, our observations suggest that isolated GS relapses after BMT might be compatible with long survivals, and that in patients with marrow hematopoiesis of donor origin, augmentation of the GVL effect might be of use.

Crystal structure of p50/p65 heterodimer of transcription factor NF-kappaB bound to DNA.

The NF-kappaB p50/p65 heterodimer is the classical member of the Rel family of transcription factors which regulate diverse cellular functions such as immune response, cell growth, and development. Other mammalian Rel family members, including the proteins p52, proto-oncoprotein c-Rel, and RelB, all have amino-terminal Rel-homology regions (RHRs). The RHR is responsible for the dimerization, DNA binding and cytosolic localization of these proteins by virtue of complex formation with inhibitor kappaB proteins. Signal-induced removal of kappaB inhibitors allows translocation of dimers to the cell nucleus and transcriptional regulation of kappaB DNA-containing genes. NF-kappaB specifically recognizes kappaB DNA elements with a consensus sequence of 5'-GGGRNYYYCC-3' (R is an unspecified purine; Y is an unspecified pyrimidine; and N is any nucleotide). Here we report the crystal structure at 2.9 A resolution of the p50/p65 heterodimer bound to the kappaB DNA of the intronic enhancer of the immunoglobulin light-chain gene. Our structure reveals a 5-base-pair 5' subsite for p50, and a 4-base-pair 3' subsite for p65. This structure indicates why the p50/p65 heterodimer interface is stronger than that of either homodimer. A comparison of this structure with those of other Rel dimers reveals that both subunits adopt variable conformations in a DNA-sequence-dependent manner. Our results explain the different behaviour of the p50/p65 heterodimer with heterologous promoters.

Human herpesvirus-6 and human herpesvirus-7 infections in bone marrow transplant recipients.

Human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6), and human herpesvirus-7 (HHV-7) DNA in peripheral blood leukocytes (PBL) of 61 bone marrow transplant recipients was monitored weekly during the first 12 weeks post-transplantation by a nested polymerase chain reaction (PCR). Thirty-seven (61%), 17 (28%), and 32 (53%) of patients had one or more PBL specimens positive for HCMV, HHV-6 or HHV-7 DNA, respectively. HHV-7 DNA in PBL during the early post-transplant period was associated with a longer time to neutrophil engraftment (mean 28.8 days vs 19.8 days; P = 0.01). In two patients who failed to engraft, HHV-6 DNA and HHV-7 DNA was detected in plasma and PBL, respectively, early in their post-transplant period. Patients with HCMV disease were more likely to have concurrent HHV-7 DNA in PBL prior to onset of disease than were patients with asymptomatic HCMV infection, suggesting that HHV-7 may be a cofactor in the progression from HCMV infection to HCMV disease. In the 17 patients (179 specimens) in whom viral DNA in plasma was studied (in addition to PBL), a positive result was found only in 3. In each, viral DNA in plasma appeared to correlate with clinically significant disease. HHV-7 DNA in plasma was associated with encephalitis in an allograft recipient.

Late onset haemolysis and red cell autoimmunisation after allogeneic bone marrow transplant.

We reviewed the medical records of 293 patients who underwent allogeneic bone marrow transplants at the Hammersmith Hospital between 1989 and 1994. There was clinical evidence of an autoimmune reaction against red cells in nine patients. Seven of these patients had significant haemolysis; the other two had red cell autoagglutination. Haemolysis was resistant to treatment in three cases. Six of the nine patients had monoclonal Ig bands identified within 1 year of transplant. The autoimmune reaction could be classified broadly into two types: an early onset type (n = 4) beginning 2 to 8 months post-transplant associated with a cold antibody, and a late onset type (n = 5) beginning 6 to 18 months post-transplant associated with warm antibodies. The predominant antibody in the two categories described may reflect the kinetics of immune reconstitution post-transplant, since serum IgM levels typically return to normal 2 to 6 months post-transplant, while IgG levels may not reach normal levels until 12-18 months post-transplant. We speculate that unbalanced reconstitution of B and T cell lymphopoiesis post-transplant may favour emergence of oligoclonal proliferation and that some of the resulting antibodies may have activity against red cells.

Treatment of adenovirus-associated haemorrhagic cystitis with ganciclovir.

We report a 47-year-old bone marrow transplant recipient with haemorrhagic cystitis caused by adenovirus successfully treated with ganciclovir. This is the first report on the use of ganciclovir for the successful treatment of adenoviral infection. Shell vial culture may be more sensitive than conventional culture in the detection of adenovirus in such patients.