Adoptive T cell immunotherapy for treatment of ganciclovir-resistant cytomegalovirus disease in a renal transplant recipient.

Cytomegalovirus (CMV) is a significant cause of morbidity, mortality and graft loss in solid organ transplantation (SOT). Treatment options for ganciclovir-resistant CMV are limited. We describe a case of ganciclovir-resistant CMV disease in a renal transplant recipient manifested by thrombotic microangiopathy-associated glomerulopathy. Adoptive T cell immunotherapy using CMV-specific T cells from a donor bank was used as salvage therapy. This report is a proof-of-concept of the clinical and logistical feasibility of this therapy in SOT recipients.

Novel loci associated with usual sleep duration: the CHARGE Consortium Genome-Wide Association Study.

Usual sleep duration is a heritable trait correlated with psychiatric morbidity, cardiometabolic disease and mortality, although little is known about the genetic variants influencing this trait. A genome-wide association study (GWAS) of usual sleep duration was conducted using 18 population-based cohorts totaling 47 180 individuals of European ancestry. Genome-wide significant association was identified at two loci. The strongest is located on chromosome 2, in an intergenic region 35- to 80-kb upstream from the thyroid-specific transcription factor PAX8 (lowest P=1.1 × 10(-9)). This finding was replicated in an African-American sample of 4771 individuals (lowest P=9.3 × 10(-4)). The strongest combined association was at rs1823125 (P=1.5 × 10(-10), minor allele frequency 0.26 in the discovery sample, 0.12 in the replication sample), with each copy of the minor allele associated with a sleep duration 3.1 min longer per night. The alleles associated with longer sleep duration were associated in previous GWAS with a more favorable metabolic profile and a lower risk of attention deficit hyperactivity disorder. Understanding the mechanisms underlying these associations may help elucidate biological mechanisms influencing sleep duration and its association with psychiatric, metabolic and cardiovascular disease.

Haploidentical peripheral blood stem cell infusion in combination with chemotherapy for acute myeloid leukaemia in elderly patients.

Elderly patients with acute myeloid leukaemia (AML) have a poor prognosis with standard chemotherapy. Two elderly AML patients treated with infusion of family-derived partially human leukocyte antigen (HLA)-matched peripheral blood stem cells following each cycle of chemotherapy entered morphological complete remission without graft versus host disease or major toxicity. Our results support this as a non-toxic approach for inducing a graft versus leukaemia effect in patients not suitable for allogeneic transplantation. Additional resources required for donor assessment and harvest may be reduced by using banked partially HLA-matched mononuclear cells from unrelated donors.

A single tube 10-color flow cytometry assay optimizes detection of minimal residual disease in chronic lymphocytic leukemia.

UNLABELLED: Levels of residual disease (RD) are an independent predictor of progression-free survival (PFS) and overall survival (OS) in patients treated for chronic lymphocytic leukemia (CLL). We modified the international standardized approach (ISA) to RD detection using flow cytometry by developing a single tube 10 color antibody assay. METHOD: A single tube incorporated the following monoclonal antibodies: CD81FITC, CD22PE, CD3ECD, CD5PercP5.5, CD20PECY7, CD79bAPC, CD38A700, CD43APC Alexa750, CD19eFluor 450, and CD45KO. A modified ISA gating strategy was developed that removed contaminating events. Sensitivity assays were performed using dilution with normal peripheral blood and bone marrow. Clinical samples were compared using the ISA and the single tube assay. RESULTS: Dilution studies showed that sensitivity of 0.001% was achievable when a minimum of 1.8 × 10(6) total events were acquired. One hundred twenty-nine samples were analyzed and showed RD levels from 0.003 to 22%. In 80 samples analyzed with both assays, there was an excellent correlation between the two methods (slope = 1.0, intercept = 0.07 and R2 = 0.992) and results from Bland-Altman analysis showed a bias of 0.04 ± 0.38 with 95% confidence interval of -0.71 to 0.79. Removal of contaminating events in the single tube assay led to a significant reduction in RD values (P = 0.0014). CONCLUSION: The single tube 10-color assay for the detection of RD in CLL provides equivalent results to the ISA but requires fewer cells, uses fewer reagents, and allows for simpler analysis. By directly removing contaminating events, it improves the accuracy of CLL RD detection and may reclassify the status of some patients following chemotherapy.

A risk score for early cytomegalovirus reactivation after allogeneic stem cell transplantation identifies low-, intermediate-, and high-risk groups: reactivation risk is increased by graft-versus-host disease only in the intermediate-risk group.

BACKGROUND: This retrospective study was aimed at establishing a clinical score to stratify the risk of cytomegalovirus (CMV) reactivation in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) in order to direct strategies for post-transplant CMV monitoring and therapy. PATIENTS AND METHODS: In total, 335 adult patients undergoing HSCT were analyzed and divided into a training set (n = 235) and a validation set (n = 100). Logistic regression analysis on the training set identified recipient and donor CMV seropositivity, acute graft-versus-host disease (GVHD), and use of anti-thymocyte globulin or alemtuzumab as significant risk factors for CMV reactivation. Weighted scores were assigned to each factor. A weighted score (CMV scoring index [CSI]) was calculated for each patient using the scores of all risk factors except for GVHD. The index was collapsed into 3 risk groups - low risk (score of 0-2), intermediate risk (score of 3-5), and high risk (score of 6-7) - and reactivation rates were calculated. In the training set, CMV reactivation occurred in 5.8% in the low-risk group, 44.8% in the intermediate-risk group, and 67.7% in the high-risk group. RESULTS: In patients with an intermediate CSI only, significantly higher reactivation rates were seen in the presence of corticosteroid treatment for GVHD (57.8% vs. 24.5%, P < 0.01). These findings were similar in the validation set with reactivation rates of 0% in the low-risk, 46% in the intermediate-risk, and 68.4% in the high-risk groups. As seen in the training set, the presence of GVHD was associated with higher CMV reactivation rates only in the intermediate-risk group (64% vs. 28% in the absence of GVHD, P = 0.02). CONCLUSIONS: Identification of these 3 risk groups in association with the presence or absence of GVHD will help transplant units to make pre-transplant policy decisions about prophylactic, pre-emptive, or experimental CMV prevention strategies in groups of patients undergoing HSCT, as well as in those developing GVHD post transplant.

How we mobilize haemopoietic stem cells.

Mobilization and collection of haemopoietic stem and progenitor cells (HSPC) is the cornerstone of autologous and allogeneic stem cell transplantation for a wide variety of haematological and some non-haematological malignancies. Centres providing this service face the challenge of optimizing the likelihood of successful collection of transplantable doses of cells, while maximizing the efficiency of the apheresis unit and minimizing the risk of toxicity as well as mobilization failure. Recent developments in the understanding of the molecular mechanisms of mobilization have led to the emergence of novel strategies for HSPC mobilization, which may assist in meeting these imperatives. The task for clinicians is how to incorporate the use of these strategies into practice, in the light of emerging evidence for efficacy and safety of these agents. Herein, the literature is reviewed, and a proposed algorithm for HSPC mobilization is presented.

Adult B-cell acute lymphoblastic leukemia with two unrelated abnormal cytogenetic clones.

The presence of two different abnormal cell lines at diagnosis in hematologic malignancies is rare and raises the question of etiology and pathogenesis--two separate malignant lineages occurring together or a common stem cell malignancy? We present a 64-year-old woman who was evaluated for low platelet count and peripheral blasts. On the basis of the morphology, flow cytometry, and lack of myeloid-associated markers, a diagnosis of precursor B-cell acute lymphoblastic leukemia (B-ALL) was made. Cytogenetic analysis of the diagnostic bone marrow (BM) specimen revealed two unrelated abnormal clones--one had a dicentric (7;9)(p11;p11), resulting in the deletion of 7p and 9p, and the other had only trisomy 8. The dic(7;9) is a rare but recurrent abnormality in B-ALL, while trisomy 8 as a sole abnormality is most commonly associated with myeloid malignancies. After standard treatment for B-ALL, BM cytogenetic analysis showed disappearance of the dic(7;9) cell line but persistence of cells with trisomy 8. The presence of two unrelated clones suggestive of concomitant malignancies, possibly B-ALL with an underlying MDS, may have arisen by different mechanisms.

Excess mortality following hip fracture: the role of underlying health status.

UNLABELLED: We evaluated the long-term excess mortality associated with hip fracture, using prospectively collected data on pre-fracture health and function from a nationally representative sample of U.S. elders. Although mortality was elevated for the first six months following hip fracture, we found no evidence of long-term excess mortality. INTRODUCTION: The long-term excess mortality associated with hip fracture remains controversial. METHODS: To assess the association between hip fracture and mortality, we used prospectively collected data on pre-fracture health and function from a representative sample of U.S. elders in the Medicare Current Beneficiary Survey (MCBS) to perform survival analyses with time-varying covariates. RESULTS: Among 25,178 MCBS participants followed for a median duration of 3.8 years, 730 sustained a hip fracture during follow-up. Both early (within 6 months) and subsequent mortality showed significant elevations in models adjusted only for age, sex and race. With additional adjustment for pre-fracture health status, functional impairments, comorbid conditions and socioeconomic status, however, increased mortality was limited to the first six months after fracture (hazard ratio [HR]: 6.28, 95% CI: 4.82, 8.19). No increased mortality was evident during subsequent follow-up (HR: 1.04, 95% CI: 0.88, 1.23). Hip-fracture-attributable population mortality ranged from 0.5% at age 65 among men to 6% at age 85 among women. CONCLUSIONS: Hip fracture was associated with substantially increased mortality, but much of the short-term risk and all of the long-term risk was explained by the greater frailty of those experiencing hip fracture.

Failure to achieve a threshold dose of CD34+ CD110+ progenitor cells in the graft predicts delayed platelet engraftment after autologous stem cell transplantation.

In this study, we retrospectively analysed the utility of CD110 expression on CD34(+) cells as a predictor of delayed platelet transfusion independence in 39 patients who underwent autologous peripheral blood stem cell transplantation. Absolute CD34(+) cells and CD34(+) subsets expressing CD110 were enumerated using flow cytometry. Of the 39 patients, 7 required 21 days or more to achieve platelet transfusion independence. Six of the seven patients received a dose of CD34(+)CD110(+) cells below 6.0 x 10(4)/kg while 30 of 32 patients who achieved platelet transfusion independence in <21 days received a dose of CD34(+)CD110(+) cells >6.0 x 10(4)/kg (P<0.001). Patients with delayed platelet engraftment received a median dose of 5.2 x 10(4) CD34(+)CD110(+) cells/kg compared with a median dose of 16.4 x 10(4) cells/kg for those engrafting within 21 days (P=0.003). Further analysis showed that >6.0 x 10(4) CD34(+)CD110(+) cells/kg was highly sensitive (93.8%) and highly specific (85.7%) for achieving platelet transfusion independence within 21 days. Delay in platelet transfusion independence translated into an increased requirement for platelet transfusion (median 6 vs 2 transfusions, P<0.0001). The dose of CD34(+)/CD110(+) cells/kg infused at time of transplantation appears to be an important factor identifying patients at risk of delayed platelet engraftment.

Rapid expansion of tumor-reactive cells from HLA-matched siblings for adoptive immunotherapy of melanoma.

BACKGROUND: Administration of expanded tumor-infiltrating lymphocytes in association with lymphodepleting chemotherapy is effective in some patients with advanced malignant melanoma. However, obtaining lymphocytes and subsequent expansion is labor intensive, making it impractical for broad clinical application. Allogeneic transplantation may have anti-melanoma efficacy because of a graft vs. tumor effect. The disappointing tumor control observed post-transplant suggests that adoptive immunotherapy using melanoma-reactive cells will be essential for sustained responses. METHODS: Melanoma cell lines were grown from two patients with advanced disease. High-level CD80 and CD86 expression was obtained in the tumor lines using a retroviral vector for gene transfer. Transduced melanoma and controls were cultured with mononuclear cells from HLA-identical sibling donors. RESULTS: Expression of CD80 and CD86, particularly the former, promoted marked expansion of lymphocytes from HLA-matched sibling donors. Proliferation of up to 300-fold after 4 weeks of culture was observed. Lymphocytes from cultures stimulated with CD80 demonstrated cytotoxicity against recipient-untransfected melanoma (45-75% specific lysis at an E:T ratio of 50:1). Although expanded lymphocytes were predominantly CD4(+), cytotoxicity was greatest in the numerically smaller CD8(+) subpopulation. Both CD4(+) and CD8(+) cells secreted IFN-gamma (but not IL-4) on exposure to untransduced stimulator melanoma cells. DISCUSSION: Our strategy generates a large number of melanoma-reactive lymphocytes from HLA-identical siblings using a 4-week culture strategy. Lymphocytes expanded in this way offer an alternative to tumor-infiltrating lymphocytes for allogeneic cellular immunotherapy after stem cell transplantation in young patients.

Generation of tumour-specific cytotoxic T-cell clones from histocompatibility leucocyte antigen-identical siblings of patients with melanoma.

Lymphodepletion and infusion of autologous expanded tumour-infiltrating lymphocytes is effective therapy for patients with malignant melanoma. Antitumour responses are likely to be mediated by HLA class I- and II-restricted immune responses directed at tumour antigens. We assessed whether the peripheral blood of normal HLA-matched siblings of patients with melanoma could be used to generate lymphocytes with antimelanoma activity for adoptive immunotherapy after allogeneic blood or marrow transplantation. Melanoma cell lines were derived from two donors and were used to stimulate the mononuclear cells of three HLA-identical siblings. CD4(+) clones dominated cultures. Of these, approximately half were directly cytotoxic towards recipient melanoma cells and secreted interferon-gamma in response to tumour stimulation. More than half of the noncytotoxic clones also secreted interferon-gamma after melanoma stimulation. No CD4(+) clones responded to stimulation with recipient haemopoietic cells. The majority of CD8(+) clones directly lysed recipient melanoma, but did not persist in long-term culture in vitro. No crossreactivity with recipient haemopoietic cells was observed. The antigenic target of one CD4(+) clone was determined to be an HLA-DR11-restricted MAGE-3 epitope. Antigenic targets of the remaining clones were not elucidated, but appeared to be restricted through a non-HLA-DR class II molecule. We conclude that the blood of allogeneic HLA-matched sibling donors contains melanoma-reactive lymphocyte precursors directed at tumour-associated antigens. Adoptive immunotherapy with unselected or ex vivo-stimulated donor lymphocytes after allogeneic stem cell transplantation has a rational basis for the treatment of malignant melanoma.

Unrelated umbilical cord blood transplantation for adults with haematological malignancies: results from a single Australian centre.

BACKGROUND: A number of haematological malignancies can be cured by allogeneic stem cell transplantation but only approximately 30% of Australians have a suitable histocompatible related donor. Matched donors can be found on international registries of unrelated volunteers for a proportion of the remaining patients. For those patients in need of an allogeneic transplant, but for whom a suitable matched related or unrelated adult donor cannot be found, the use of banked unrelated umbilical cord blood has emerged as a potential option. However, there is uncertainty about the applicability of this technique for the majority of adult patients as a result of limitations in the number of cells in banked cord blood units and the degree of mismatching for histocompatibility antigens. AIMS: The aim of this study was to define the feasibility of allogeneic stem cell transplantation using single unrelated cord blood units in a cohort of adults with poor prognosis leukaemia or lymphoma. METHODS: Nine patients with haematological malignancies (five with acute myeloid leukaemia, one with acute lymphoblastic leukaemia, one with Hodgkin lymphoma and two with non-Hodgkin lymphomas) received transplants of cryopreserved cord blood after conditioning therapy with high-dose cyclophosphamide, total body irradiation and antithymocyte globulin. Cord units contained a median 2.6 x 10(7) nucleated cells/kg recipient bodyweight and were matched for four (seven cases) or five (two cases) major histocompatibility complex class 1 and 2 antigens. Patients were given post-transplant immunosuppression with cycosporin and methylprednisolone. RESULTS: Neutrophil recovery to 0.5 x 10(9)/L was seen by median day 30 after transplant in all seven patients who survived more than 1 month post-transplant. Platelet recovery to 50 x 10(9)/L occurred by median day 81 in five evaluable patients. Acute graft versus host disease (GVHD) grades II-IV was seen in four of seven evaluable patients and limited chronic GVHD was seen in four of five. Infection was the most common complication. Four patients died before day 100 of infection (methicillin-resistant Staphylococcus aureus septicaemia, respiratory syncitial virus pneumonia), GVHD and multi-organ failure, and intracranial bleeding. Five patients survived 7-69 months post-transplant, without evidence of relapse of the underlying malignancy. CONCLUSION: Unrelated cord blood transplantation is feasible in adults with high-risk malignancy, with infection relating to immunocompromise being the major limitation.

Cardiac autonomic function in older adults with sleep-disordered breathing.

We searched for possible associations between various measures of severity of sleep-disordered breathing (SDB) and indices of cardiac autonomic function in older subjects (>60 years). Twenty four overnight unattended homebased based polysomnograms obtained from the Sleep Heart Health Study were analyzed using spectral analysis. For each subject, six autonomic indices reflecting heart rate variability were quantitatively determined during wakefulness, REM sleep and non-REM sleep. Each individual autonomic marker was regressed against each of 4 measures of SDB, including the respiratory disturbance index (RDI), respiratory oscillation index, cumulative oxygen desaturation, and arousal index. In general, we found no correlation between any of these measures of SDB severity and each of the autonomic indices. However, mean heart rate was found to decrease as RDI increased. As well, the ratio of low-frequency to high-frequency power (LHR) decreased with increasing RDI. Contrary to previous reports, our preliminary findings suggest that sympathetic activity decreases with increasing severity of SDB. This paradoxical association between SDB and cardiac autonomic function may be the result of natural compensatory mechanisms at work, allowing some subjects with SDB to be protected from systemic hypertension or other cardiovascular diseases. Supported in part by NIH Grants HL076375, EB001978, HL63463 and HL53941.

APOE epsilon4 is associated with obstructive sleep apnea/hypopnea: the Sleep Heart Health Study.

BACKGROUND: Obstructive sleep apnea/hypopnea (OSAH) has a strong heritable component, although its genetic basis remains largely unknown. One epidemiologic study found a significant association between the APOE epsilon4 allele and OSAH in middle-aged adults, a finding that was not replicated in a cohort of elderly adults. The objective of this study was to further examine the association of the APOE epsilon4 allele with OSAH in a community-dwelling cohort, exploring age dependency of the association. METHODS: A genetic association study was performed, nested within a prospective cohort study of the cardiovascular consequences of OSAH. Unattended, in-home nocturnal polysomnography was used to measure apnea-hypopnea index (AHI) in 1,775 participants age 40 to 100 years. OSAH was defined as an AHI > or = 15. The relation of APOE genotype to prevalent OSAH was analyzed using generalized estimating equations to account for non-independent observations of individuals from the same sibship. RESULTS: At least one APOE epsilon4 allele was present in 25% of subjects, with 1.3% epsilon4/epsilon4 homozygotes. The prevalence of OSAH was 19%. After adjustment for age, sex, and BMI, the presence of any APOE epsilon4 allele was associated with increased odds of OSAH (OR 1.41, 95% CI 1.06 to 1.87, p = 0.02). The effect was approximately twice as great in subjects <75 (OR 1.61, CI 1.02 to 2.54) as in those > or =75 years old (OR 1.32, CI 0.91 to 1.90). Exploratory analyses revealed that the strongest effect of APOE epsilon4 was in subjects age <65 (OR 3.08, CI 1.43 to 6.64), and was stronger in those with hypertension or cardiovascular disease than in those without. CONCLUSION: The APOE epsilon4 allele is associated with increased risk of OSAH, particularly in individuals under age 65. The mechanisms underlying this association are uncertain. Age-dependency of the APOE-OSAH association may explain previous conflicting results.

Effects of inhibitors of the chemokine receptor CXCR4 on acute lymphoblastic leukemia cells in vitro.

Stromal cell-derived factor-1 (SDF-1) is a key regulator of the behavior of normal and leukemic precursor-B (pre-B) cells. It is possible that inhibiting SDF-1-driven processes in pre-B acute lymphoblastic leukemia (ALL) may have therapeutic implications. In this study, we examined the ability of SDF-1 inhibitors to modulate pre-B ALL cell responses to SDF-1, including chemotaxis, migration into bone marrow stroma, and stroma-supported survival and proliferation on human bone marrow stromal layers. The polyphemusin II-derived inhibitors, T140, TC140012, and T134, and the bicyclam AMD3100, effectively inhibited binding of the anti-CXCR4 monoclonal antibody 12G5 on the pre-B ALL cell line NALM6, with IC(50) values of 0.9, 0.9, 0.9, and 1.9 nM, respectively. Similar results were obtained with ALL samples. T140 (0.1 micro M) and AMD3100 (1 micro M) completely blocked SDF-1-induced chemotaxis and attenuated the migration of pre-B ALL cells into bone marrow stromal layers. AMD3100 and TC140012 at a concentration of 50 micro M significantly inhibited stroma-dependent proliferation of six and four of the eight cases tested, respectively, without reducing the cell viability. In addition, AMD3100 and TC140012 enhanced the cytotoxic and antiproliferative effects of the cytotoxic agents vincristine and dexamethasone. The ability of SDF-1 inhibitors to modulate these biologically important functions of leukemic cells warrants further investigation.

The burden of prevention: downstream consequences of Pap smear testing in the elderly.

CONTEXT: Although cervical cancer is an unusual cause of death among women 65 and older, most elderly women in the US report continuing to undergo periodic Pap smear screening. OBJECTIVE: To describe the incidence of Pap smears and downstream testing among elderly women. SETTING: Claims-based analysis of female Medicare enrollees age 65 and older. METHODS: Using three years of Medicare Part B 5% Files (1995-1997), we differentiated between women undergoing screening Pap smears and those undergoing Pap smears for surveillance of previous abnormalities or Pap smear follow-up. We determined the proportion of elderly women undergoing Pap smear testing and rates of downstream testing and procedures after an initial Pap smear. RESULTS: Four million female Medicare beneficiaries over 65 years underwent Pap smear testing between 1995 and 1997, representing 25% of the eligible population. After adjusting for underbilling for Pap smears under Medicare, 43% of women over 65 are estimated to have undergone Pap smear testing during the 3-year period. The large majority (90%) of Pap smears were for screening, while 10% were done for surveillance or follow-up. For every 1000 women with a screening Pap smear, 39 had at least one downstream intervention within eight months of the initial Pap smear, including seven women who underwent colposcopy and two women who had other surgical procedures. Rates of downstream interventions were considerably higher for women undergoing Pap smear follow-up (302 per 1000 with at least one downstream intervention), and surveillance of previous abnormalities (209 per 1000 with a downstream intervention). CONCLUSION: Cervical cancer screening is widespread among elderly American women, and follow-up testing is not uncommon, particularly among the ten percent of women who appear to be in a cycle of repeated testing. This substantial volume of testing occurs despite the rarity of cervical cancer deaths and unknown benefits of screening in this age group.

The chemokine receptor CXCR4 enhances integrin-mediated in vitro adhesion and facilitates engraftment of leukemic precursor-B cells in the bone marrow.

OBJECTIVE: It has been demonstrated that acute lymphoblastic leukemia (ALL) blasts migrate into layers of bone marrow fibroblasts (BMF) in vitro using the beta1 integrins VLA-4 and VLA-5, and that the chemokine SDF-1 and its receptor CXCR4 influences ALL migration. We investigated whether this effect was due to SDF-1-mediated induction of adhesion through beta1 integrins. METHODS: Adhesion of pre-B ALL cells or the cell line NALM6 to extracellular matrix proteins was examined using short-term in vitro binding assays. The effects of exposure of cells to SDF-1, antibodies to CXCR4, and the G protein inhibitor pertussis toxin (PTX) were assessed. The consequences of down regulation of CXCR4 on the in vivo behavior of pre-B ALL cells after injection into sublethally irradiated NOD/SCID mice was studied. RESULTS: Treatment with SDF-1 of NALM6 cells or cells from cases of precursor-B ALL resulted in a doubling of adhesion to fibronectin, laminin, and VCAM-1, but had no effect on binding to collagens I or IV. Antibodies to CXCR4 and PTX inhibited SDF-1-induced adhesion on these substrates. NALM6 cells with CXCR4 expression downregulated by SDF-1 exposure demonstrated a reduced capacity to engraft into the bone marrow of NOD/SCID mice, with only 22 +/- 11% of marrow cells being of human origin in mice receiving SDF-1-treated cells compared to 48 +/- 5% in mice receiving untreated cells (p < 0.001). The homing of SDF-1-treated cells to the bone marrow after 24 hours was also reduced by 72 +/- 16% compared to control cells. CONCLUSIONS: These data show that SDF-1 and CXCR4 are involved in regulation of beta1 integrin function, and are important for the localization of pre-B cells to the bone marrow in vivo.