Hypertension in people living with HIV on combined antiretroviral therapy in rural Tanzania.

Exposure to anti-retroviral therapy in HIV infection has been associated with hypertension, but whether and to what extent HIV-related factors and anti-retroviral treatment contribute to hypertension is not well defined; in addition, data are particularly scarce in Sub-Saharan Africa. Aim of the study was to investigate prevalence and awareness of hypertension in a cohort of people living with HIV (PLWHIV) on anti-retroviral therapy in rural Tanzania, and to identify possible predictors of hypertension. A cross-sectional study on hypertension in PLWHIV was conducted at Tosamaganga District Hospital, Iringa Region, Tanzania. Subjects on anti-retroviral therapy, age 26-80 years and with monthly attendance to the HIV clinic, were considered eligible. A total number of 242 patients were included in the analysis. Sixty-two subjects (26%) had hypertension, the majority (77%) of them not aware of the condition and/or not on treatment. Older age, higher BMI and lower baseline T-CD4 count were predictors of hypertension at multivariate analysis. The results of the study suggest that hypertension screening should become part of ordinary care of PLWHIV in Tanzania, particularly in subjects with more severe immunosuppression. Leveraging already existing HIV services could be an option to prevent the burden of non-AIDS complication and related deaths.

Community- and facility-based HIV testing interventions in northern Tanzania: Midterm results of Test & Treat Project.

Test & Treat Project offers universal HIV testing and access to antiretroviral treatment in Northern Tanzania. The current cross-sectional study provides midterm results on HIV testing and counseling activities through community outreaches and facility-based services. A total 255,329 HIV tests were performed: 198,451 (77.7%) during testing campaigns in the villages, 12,592 (4.9%) during special events outreach and 44,286 (17.4%) in the health facilities. Females represented 53.8% (23,809) among those tested in the health facilities, while males were the majority in the community (54.4%, 114,835). Over one third of tests (n = 104,605, 41%) were performed among first-time testers. The overall HIV positivity rate was 1.2%, ranging from 0.7% in the community to 3.8% in the health facilities and decreased over time. Using a multivariable analysis, a positive test result was associated with age ≥ 50 years (PR 1.22, 95% CI 1.11 to 1.34), with female gender (PR 1.61, 95% CI 1.50 to 1.73), being tested in health facilities (PR 5.00, 95% CI 4.65 to 5.36) and for the first time (PR 1.86, 95% CI 1.73 to 2.00). The estimated proportion of PLHIV who knew their status of the project area increased by 28.6% (from 35.7% to 64.3%) and 11.1% (from 57.7% to 68.8%) in the project areas of Shinyanga and Simiyu regions respectively. Reaching the first UNAIDS 90 target by the end of this project seems possible. Future strategies should focus on improving PITC coverage, implementing more targeted testing modalities, together with current universal community-based approach.

The antileukemic potential of natural killer cells.

The antileukemic potential of natural killer (NK) cells has over the years raised considerable interest and new immune-based treatment protocols characterized by the infusion of freshly isolated or ex vivo activated and expanded effectors have been designed. Several aspects still need to be addressed, including the optimal timing of NK infusion during the course of the disease, the best preparative regimen, the origin of NK cells and the possible need of ex vivo NK cell manipulation before the infusion. The aims of this review are to discuss the experimental and clinical data available on the role played by NK cells for leukemia patients and to revise the different good manufacturing practice protocols for ex vivo manipulation of these effector cells.

A good manufacturing practice method to ex vivo expand natural killer cells for clinical use.

BACKGROUND: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application. MATERIALS AND METHODS: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing. RESULTS: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing. DISCUSSION: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.

Immunophenotypic and functional characterization of ex vivo expanded natural killer cells for clinical use in acute lymphoblastic leukemia patients.

The management of acute lymphoblastic leukemia (ALL) patients has witnessed profound changes in recent years. Nonetheless, most patients tend to relapse, underlining the need for new therapeutic approaches. The anti-leukemic potential of natural killer (NK) cells has over the years raised considerable interest. In this study, we developed an efficient method for the expansion and activation of NK cells isolated from healthy donors and ALL patients for clinical use. NK cell products were derived from peripheral blood mononuclear cells of 35 healthy donors and 4 B-lineage ALL by immunomagnetic CD3 T cell depletion followed by CD56 cell enrichment. Isolated NK cells were expanded and stimulated in serum-free medium supplemented with irradiated autologous feeder cells and autologous plasma in the presence of clinical grade interleukin (IL)-2 and IL-15 for 14 days. Healthy donor NK cells expanded on average 34.9 ± 10.4 fold and were represented, after expansion, by a highly pure population of CD3(-)CD56(+) cells showing a significant upregulation of natural cytotoxicity receptors, activating receptors and maturation markers. These expanded effectors showed cytolytic activity against K562 cells and, most importantly, against primary adult B-lineage ALL blasts. NK cells could be efficiently isolated and expanded-on average 39.5 ± 20.3 fold-also from primary B-lineage ALL samples of patients in complete remission. The expanded NK cells from these patients showed a significantly increased expression of the NKG2D- and DNAM1-activating receptors and were cytotoxic against K562 cells. These data provide the basis for developing new immunotherapeutic strategies for the management of ALL patients.

Indoleamine 2,3-dioxygenase 1 (IDO1) activity in leukemia blasts correlates with poor outcome in childhood acute myeloid leukemia.

Microenvironmental factors contribute to the immune dysfunction characterizing acute myeloid leukemia (AML). Indoleamine 2,3-dioxygenase 1 (IDO1) is an interferon (IFN)-γ-inducible enzyme that degrades tryptophan into kynurenine, which, in turn, inhibits effector T cells and promotes regulatory T-cell (Treg) differentiation. It is presently unknown whether childhood AML cells express IDO1 and whether IDO1 activity correlates with patient outcome. We investigated IDO1 expression and function in 37 children with newly diagnosed AML other than acute promyelocytic leukemia. Blast cells were cultured with exogenous IFN-γ for 24 hours, followed by the measurement of kynurenine production and tryptophan consumption. No constitutive expression of IDO1 protein was detected in blast cells from the 37 AML samples herein tested. Conversely, 19 out of 37 (51%) AML samples up-regulated functional IDO1 protein in response to IFN-γ. The inability to express IDO1 by the remaining 18 AML samples was not apparently due to a defective IFN-γ signaling circuitry, as suggested by the measurement of signal transducer and activator of transcription 3 (STAT3) phosphorylation. Co-immunoprecipitation assays indicated the occurrence of physical interactions between STAT3 and IDO1 in AML blasts. In line with this finding, STAT3 inhibitors abrogated IDO1 function in AML blasts. Interestingly, levels of IFN-γ were significantly higher in the bone marrow fluid of IDO-expressing compared with IDO-nonexpressing AMLs. In mixed tumor lymphocyte cultures (MTLC), IDO-expressing AML blasts blunted the ability of allogeneic naïve T cells to produce IFN-γ and promoted Treg differentiation. From a clinical perspective, the 8-year event-free survival was significantly worse in IDO-expressing children (16.4%, SE 9.8) as compared with IDO-nonexpressing ones (48.0%, SE 12.1; p=0.035). These data indicate that IDO1 expression by leukemia blasts negatively affects the prognosis of childhood AML. Moreover, they speak in favor of the hypothesis that IDO can be targeted, in adjunct to current chemotherapy approaches, to improve the clinical outcome of children with AML.

Recognition of adult and pediatric acute lymphoblastic leukemia blasts by natural killer cells.

In this study, we aimed to investigate the pathways of recognition of acute lymphoblastic leukemia blasts by natural killer cells and to verify whether differences in natural killer cell activating receptor ligand expression among groups defined by age of patients, or presence of cytogenetic/molecular aberrations correlate with the susceptibility to recognition and killing. We analyzed 103 newly diagnosed acute lymphoblastic leukemia patients: 46 adults and 57 children. Pediatric blasts showed a significantly higher expression of Nec-2 (P=0.03), ULBP-1 (P=0.01) and ULBP-3 (P=0.04) compared to adult cells. The differential expression of these ligands between adults and children was confined to B-lineage acute lymphoblastic leukemia with no known molecular alterations. Within molecularly defined subgroups of patients, a high surface expression of NKG2D and DNAM1 ligands was found on BCR-ABL(+) blasts, regardless of patient age. Accordingly, BCR-ABL(+) blasts proved to be significantly more susceptible to natural killer-dependent lysis than B-lineage blasts without molecular aberrations (P=0.03). Cytotoxic tests performed in the presence of neutralizing antibodies indicated a pathway of acute lymphoblastic leukemia cell recognition in the setting of the Nec-2/DNAM-1 interaction. These data provide a biological explanation of the different roles played by alloreactive natural killer cells in pediatric versus adult acute lymphoblastic leukemia and suggest that new natural killer-based strategies targeting specific subgroups of patients, particularly those BCR-ABL(+), are worth pursuing further.

Growth factor-associated graft-versus-host disease and mortality 10 years after allogeneic bone marrow transplantation.

This study analysed the effects of growth factor on outcome after haematopoietic stem-cell transplantation (HSCT) with >9 years follow-up. Of 1887 adult patients with acute leukaemia who received bone marrow from human leucocyte antigen (HLA)-identical siblings and were treated with myeloablative conditioning, 459 (24%) were treated with growth factor. Growth factor hastened engraftment of neutrophils (P < 0·0001), but reduced platelet counts (P = 0·0002). Graft-versus-host disease (GVHD)-free survival (no acute GVHD grade II-IV or chronic GVHD) at 10 years was 12 ± 2% (±SE) in the growth factor group, as opposed to 17 ± 2% in the controls [hazard ratio (HR) 0·81, P = 0·001]. Similar differences in GVHD-free survival were seen in patients with or without conditioning with total body irradiation (TBI). Non-relapse mortality (NRM) was higher in the growth factor group irrespective of whether or not TBI conditioning was included [HR = 1·48; 95% confidence interval (CI): 1·15-1·9; P = 0·002; HR = 1·59; 95% CI: 1·07-2·37; P = 0·02, respectively]. Both groups had similar probabilities of leukaemic relapse (HR = 0·96; 95% CI: 0·78-1·18; P = 0·71). Leukaemia-free survival (LFS) at 10 years was 35 ± 2% in those receiving growth factor prophylaxis, as opposed to 44 ± 1% in the controls (HR = 0·70; 95% CI: 0·60-0·82; P = 0·00001). Prophylaxis with growth factor increases the risk of GVHD, does not affect relapse, increases NRM and reduces LFS > 10 years after HSCT, regardless of conditioning with TBI.

The immune reconstitution after an allogeneic stem cell transplant correlates with the risk of graft-versus-host disease and cytomegalovirus infection.

Aim of the study was to correlate the clinical outcome of eighteen patients who have undergone an allogeneic stem cell transplant (SCT) with the concentration in the peripheral blood (PB) of lymphocyte subpopulations evaluated at 1 year from transplant. The occurrence of acute GVHD and CMV infection correlated with the concentration of Tregs in the PB; CMV infection also correlated with the content of NK cells. The obtained results document that the concentration of Tregs in the PB after an allogeneic SCT may protect from GVHD and from CMV infection; the potential anti-viral role of NK cells is confirmed.

B-cell concentration in the apheretic product predicts acute graft-versus-host disease and treatment-related mortality of allogeneic peripheral blood stem cell transplantation.

BACKGROUND: The influence of the graft composition on the clinical outcome after allogeneic peripheral blood stem cell (PBSC) transplantation is not well established. METHODS: The cellular composition of the apheretic products obtained from 63 human leukocyte antigen-identical siblings was prospectively correlated with the outcome of patients with hematological malignancies undergoing an allogeneic PBSC transplant after myeloablative conditioning. The concentration of nuclear, mononuclear, CD34+, T-cell subsets, B cells, and natural killer cells in the graft has been analyzed. RESULTS: In univariate analysis, acute graft-versus-host disease (GVHD) correlated with the disease (P=0.002), with the phase of disease at transplant (P=0.01), and with the number of CD20+ cells infused (P=0.05). In multivariate analysis, a dose of CD20+ cells in the graft higher than the median dose remained the only factor negatively affecting the incidence of acute GVHD (P=0.01; 95% confidence interval [CI]: 0.12-0.78). In univariate analysis, treatment-related mortality (TRM) correlated with the disease (P=0.04) and was negatively affected by a dose of infused B cells greater than the median value (28% versus 50%; P=0.02). In multivariate analysis, TRM was close to statistical correlation with the dose of CD20+ cells (P=0.06; 95% CI: 0.02-1.05). No other clinical parameter was influenced by the composition of the graft. CONCLUSIONS: Our results suggest that the concentration of B cells in the apheretic product may predict the incidence of acute GVHD and TRM in patients undergoing an allogeneic PBSC transplantation and open the way to the new preventive and therapeutic strategies for the management of GVHD.

Expansion of natural killer cells with lytic activity against autologous blasts from adult and pediatric acute lymphoid leukemia patients in complete hematologic remission.

BACKGROUND AND OBJECTIVES: Natural killer (NK) cells constitute an important area of research for hematologic malignancies. The anti-leukemic activity of NK cells against acute myeloid leukemia (AML) blasts has been described, but very few data are available for acute lymphoid leukemia (ALL). The present study was designed to investigate whether: (i) NK effectors could be expanded from adult and pediatric ALL patients in complete remission; (ii) the signal transduction machinery of these cells was preserved; (iii) NK cells showed cytotoxic activity against autologous blasts; (iv) interleukin (IL)-2, IL-12 and IL-15 were able to increase lytic activity in our in vitro model; (v) any differences in cytotoxic activity could be found between expanded effectors from adult and pediatric patients. DESIGN AND METHODS: We co-cultured patients' peripheral blood mononuclear cells (PBMC) with the feeder cell line RPMI 8866 and analyzed the NK cells' expansion capacity by cell count and cytofluorimetric analyses. Signal transduction of expanded effector cells was evaluated by Western blot. 51Cr release assays, before and after stimulation with activating cytokines, were performed to analyze the cytotoxic potential of effector cells against tumor cell lines and autologous blast cells. Data were analyzed with t-tests for paired data. RESULTS: We obtained an average 40-fold increase in NK cells. Signal transduction through the CD16 receptor was preserved. Patients' expanded cells showed cytotoxic activity against target cell lines comparable to that of normal donors. More significantly, these cells also exerted a lytic effect against autologous blasts. In addition, incubating these effectors for 24 hours with IL-2 + IL-15 significantly increased this cytotoxic function. No differences in expansion and cytotoxic activity were found between pediatric and adult patients. INTERPRETATION AND CONCLUSIONS: These findings document for the first time the possibility of expanding ex vivo cytotoxic effectors with autologous killing capacity from ALL patients in remission, and suggest a new potential immunotherapeutic strategy for the management of early disease recurrence or of residual disease.

FLT3 inhibition in t(4;11)+ adult acute lymphoid leukaemia.

The present study was designed to investigate, in t(4;11)+ adult lymphoid leukaemia (ALL) blast cells, the pathogenetic role of the FLT3 protein, its level of mRNA and protein expression, the degree of constitutive phosphorylation, the possible presence of mutations of the sequence, the capacity of signal transduction and the potential therapeutic role of specific inhibitors. We evaluated nine adult ALL patients carrying this translocation. The increased FLT3 mRNA levels, determined by oligonucleotide microarray analysis, was in agreement with the increased protein expression evaluated by Western blot. The protein was constitutively phosphorylated in all cases analysed. Polymerase chain reaction detected no internal tandem duplication or point mutations. The signal transduction apparatus, after stimulation with the specific ligand, was preserved. We then investigated the effect of specific FLT3 inhibition on signal transduction and survival. The PKC412 inhibitor specifically inhibited ligand-induced phosphorylation; the same inhibitor reduced the survival of leukaemic cells when compared with untreated cells. These data indicate that the FLT3 protein might play a role in this subgroup of ALL with a particularly poor prognosis. Specific inhibition of the kinase receptor must be hypothesised as an innovative therapeutic tool for t(4;11)+ ALL patients.

Defective expression of the T-cell receptor-CD3 zeta chain in T-cell acute lymphoblastic leukaemia.

This study analysed the T-cell receptor (TCR)-CD3 zeta complex and the signal transduction apparatus of T-acute lymphoblastic leukaemia (T-ALL) blasts, and investigated the function of the ubiquitin-proteasome system. In all nine T-ALL samples studied, the leukaemic cells showed a marked reduction in the expression of the zeta chain, while a variety of tyrosine kinases (p56lck, ZAP70 and SYK) were normally present. There was no expression of the FcepsilonRIgamma chain. To confirm that this aberration was specific to immature T-ALL blasts, we investigated two patients with lymphoproliferative disorders of granular lymphocytes (LDGL), characterized by the expansion of mature T lymphocytes and found normal zeta chain expression. The reduction of the zeta chain protein was not reversible after 72 h stimulation with the anti-CD3 monoclonal antibody and interleukin 2, either alone or in combination. Northern blot analysis indicated that the reduced protein expression did not correspond to a defect at the mRNA level, nor were mutations in the coding region of the zeta chain found. We, therefore, hypothesized that the observed reduction of protein expression in T-ALL blasts could be secondary to an increased degradation at the proteasome level. Following selective inhibition of the proteasome, a marked increase of the zeta chain expression was observed. Moreover, an increase in the surface expression of CD3 was also documented. Taken together, these results indicate that the expression of the zeta subunit of the TCR-CD3 complex is consistently reduced in T-ALL blasts and that degradation of the protein is mediated by the proteasome system.

Human acute stem cell leukemia with multilineage differentiation potential via cascade activation of growth factor receptors.

The morphologic, immunophenotypic, genotypic, genomic, and functional features of an undifferentiated acute leukemia with stem cell features are reported. At light and electron microscopy, the leukemic population was represented by primitive progenitor cells with no evidence of differentiation. The blasts were CD34(+), AC133(+), CD71(-), HLA-DR(-), CD38(-/dim+), CD90(+), CD117(dim+), flt3(+); did not express B, T, or myeloid-associated antigens; and showed a germline configuration of the immunoglobulin and T-cell receptor. Genomic profiling documented the expression of early stem cell and myeloid-associated genes. Receptors for early-acting hemopoietic growth factors (HGFs) were detected, while receptors for unilineage HGF were not expressed. Incubation with the flt3 or Kit ligand induced the expression of unilineage HGF receptors, allowing these cells to respond to their respective ligands. Growth without differentiation was sustained only in the presence of early-acting HGF, namely flt3 ligand, while early and unilineage HGF gave rise to all types of hemopoietic colonies.

Expansion of cytotoxic effectors with lytic activity against autologous blasts from acute myeloid leukaemia patients in complete haematological remission.

New therapeutic approaches are needed to improve the cure rates in acute myeloid leukaemia (AML). The present study was designed to investigate whether: (1) cytotoxic lymphocytes could be expanded from AML patients in complete remission; (2) their signal transduction machinery was preserved; (3) these cells were capable of producing cytokines involved in the cytolytic process; and (4) these cells showed cytotoxic activity against allogeneic and autologous blasts. By co-culturing blood mononuclear cells with feeder cells, we obtained an average 5.3-fold increase in the total cell number and a 35-fold increase in natural killer (NK) cells. Expression of the zeta chain and of tyrosine kinases of the Src and Syk-ZAP families involved in the triggering of NK functions was analysed on expanded cells. The results demonstrated a signal transduction apparatus preserved and quantitatively similar to that of normal donors. After phorbol myristate acetate and ionomicin stimulation, the ability of expanded cells to produce interferon gamma and tumour necrosis factor alpha was documented. Patients' expanded cells showed a cytotoxic activity against target lines and allogeneic blasts which was similar to that of normal donors. Purification experiments indicated that the NK cell fraction was responsible for most of the lytic effect. More significantly, these cells also exerted a lytic effect against autologous blasts that could be further enhanced following incubation with low-dose interleukin 2. These findings document the possibility of expanding cytotoxic effectors with preserved signal transduction machinery and autologous killing capacity from AML patients in remission, and suggest a new potential immunotherapeutic strategy for the management of early disease recurrence or of residual disease.