Search

GvHD still remains, despite the continuous improvement of transplantation platforms, a fearful complication of transplantation from allogeneic donors. Being able to separate GvHD from GvL represents the greatest challenge in the allogeneic transplant setting. This may be possible through continuous improvement of cell therapy techniques. In this review, current cell therapies are taken into consideration, which are based on the use of TCR alpha/beta depletion, CD45RA depletion, T regulatory cell enrichment, NK-cell-based immunotherapies, and suicide gene therapies in order to prevent GvHD and maximally amplify the GvL effect in the setting of haploidentical transplantation. [ABSTRACT FROM AUTHOR]

The management of patients with acute myeloid leukemia (AML) relapsed post allogeneic hematopoietic stem cell transplantation (HSCT) remains a clinical challenge. Intensive treatment approaches are limited by severe toxicities in the early post-transplantation period. Therefore, hypomethylating agents (HMAs) have become the standard therapeutic approach due to favorable tolerability. Moreover, HMAs serve as a backbone for additional anti-leukemic agents. Despite discordant results, the addition of donor lymphocytes infusions (DLI) generally granted improved outcomes with manageable GvHD incidence. The recent introduction of novel targeted drugs in AML gives the opportunity to add a third element to salvage regimens. Those patients harboring targetable mutations might benefit from IDH1/2 inhibitors Ivosidenib and Enasidenib as well as FLT3 inhibitors Sorafenib and Gilteritinib in combination with HMA and DLI. Conversely, patients lacking targetable mutations actually benefit from the addition of Venetoclax. A second HSCT remains a valid option, especially for fit patients and for those who achieve a complete disease response with salvage regimens. Overall, across studies, higher response rates and longer survival were observed in cases of pre-emptive intervention for molecular relapse. Future perspectives currently rely on the development of adoptive immunotherapeutic strategies mainly represented by CAR-T cells. [ABSTRACT FROM AUTHOR]

The treatment of acute myeloid leukemia (AML) with adverse genetics remains unsatisfactory, with very low response rates to standard chemotherapy and shorter durations of remission commonly observed in these patients. The complex biology of AML with adverse genetics is continuously evolving. Herein, we discuss recent advances in the field focusing on the contribution of molecular drivers of leukemia biogenesis and evolution and on the alterations of the immune system that can be exploited with immune-based therapeutic strategies. We focus on the biological rationales for combining targeted therapy and immunotherapy, which are currently being investigated in ongoing trials, and could hopefully ameliorate the poor outcomes of patients affected by AML with adverse genetics. [ABSTRACT FROM AUTHOR]

Today human leukocyte antigen-haploidentical transplantation is a feasible option for patients with high-risk acute leukemia who do not have matched donors. Whether it is T-cell replete or T-cell depleted, it is still, however, associated with issues of transplant-related mortality and posttrans-plant leukemia relapse. After reports that adoptive immunotherapy with T-regulatory cells controls the alloreactivity of conventional T lymphocytes in animal models, tomorrow's world of haploidentical transplantation will focus on new "designed" grafts. They will contain an appropriate ratio of conventional T lymphocytes and T-regulatory cells, natural killer cells, γ δ T cells, and other accessory cells. Preliminary results of ongoing clinical trials show the approach is feasible. It is associated with better immune reconstitution and a quite powerful graft-versus-leukemia effect with a low incidence of graft-versus-host disease and no need for posttransplant pharmacological prophylaxis. Future strategies will focus on enhancing the clinical benefit of T-regulatory cells by increasing their number and strengthening their function. [ABSTRACT FROM AUTHOR]

Abstract: We selected T regulatory cells (Tregs) from standard leukapheresis using double-negative selection (anti-CD8 and anti-CD19) followed by positive selection (anti-CD25) and 72 procedures were performed. A median of 263×106 cells (range 143–470×106) were recovered with a mean of CD4+/CD25+ cells of 94.5±2.4% (36.5±18.6% CD4+/CD25+hi). FoxP3+ cells were equal to 79.8%±22.2. CD127+ cells were 12.5%±8.2. The inhibition assay showed an inhibition rate of 67±22. Cells isolated by means of this approach can be used in allogeneic hematopoietic stem cell transplantation to reduce the incidence and severity of GvHD without bystander inhibition of general immunity. [Copyright &y& Elsevier]

Background aims The impact of chronic lymphatic leukemia (CLL) tumor burden on the autologous immune system has already been demonstrated. This study attempted to elucidate the molecular mechanisms underlying T-cell immunologic deficiencies in CLL. Methods Freshly isolated CD3+ T cells from patients with a diagnosis of CLL and healthy donors were analyzed by gene expression profiling. Activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity against mutated and unmutated autologous B cells and DAUDI, K562 and P815 cell lines. To investigate T-cell mediated cytotoxicity in vivo, we co-transplanted OKT3-activated T lymphocytes and autologous B-cell CLL (B-CLL) cells into NOD/SCID mice. Results Gene expression profiles of peripheral blood T cells from B-CLL patients showed 25 down-regulated, and 31 up-regulated, genes that were mainly involved in cell differentiation, proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T-cell activation. After culture, the T-cell count remained unchanged, CD8 cells expanded more than CD4 and a cytotoxicity index >30% was present in 5/20 patients. Cytotoxicity against B autologous leukemic cells did not correlate with B-cell mutational status. Only activated T cells exerting cytotoxicity against autologous leukemic B cells prevented CLL in a human-mouse chimera. Conclusions This study indicates that patients with CLL are affected by a partial immunologic defect that might be somewhat susceptible to repair. This study identifies the molecular pathways underlying T-cell deficiencies in CLL and shows that cytotoxic T-cell functions against autologous B-CLL can be rebuilt at least in part in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Objective: Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Because multipotent mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation, this in vitro study investigated their role in Treg recruitment and function. Materials and Methods: Human MSCs and different T cell populations (CD3+, CD3+/CD45RA+, CD3+/CD45RO+, CD4+/CD25+, CD4+/CD25+/CD45RO+, CD4+/CD25+/CD45RA+) from healthy donors were cocultured for up to 15 days. Harvested lymphocytes were analyzed by flow cytometry and FoxP3 and CD127 expressions were measured by real-time polymerase chain reaction. Their regulatory activity was assessed. Results: We demonstrate MSC recruit Tregs from a fraction of CD3+ and from immunoselected CD3+/CD45RA+ and CD3+/CD45RO+ fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4+/CD25bright/FoxP3 subset and CD127 expression was downregulated. When purified Treg populations (CD4/CD25+, CD4/CD25+/CD45RA+, and CD4/CD25+/CD45RO+) are used in MSC cocultures, they maintain FoxP3 expression and CD127 expression is downregulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. Conclusions: In conclusion, our study demonstrates that MSCs recruit, regulate, and maintain T-regulatory phenotype and function over time. [Copyright &y& Elsevier]

Abstract: Although adoptive transfer of donor lymphocytes protects from infections and relapse after allogeneic hematopoietic stem cell transplantation in both mice and in men, it is associated with a high risk of graft versus host disease (GvHD) which rises with HLA mismatching and the number of T lymphocytes that are infused. Elimination/reduction of alloreactive donor T lymphocytes is an appealing approach and several strategies have been proposed. Here we describe generation of anti-3rd party T lymphocytes under conditions of IL-2 deprivation and their effects in a pre-clinical murine model. Our results clearly indicated that anti-3rd party T lymphocytes generated on a large scale by means of IL-2 deprivation maintain a broad T cell repertoire, do not proliferate in a mixed lymphocyte reaction and do not cause GvHD in NOD–SCID mice. These anti-3rd party lymphocytes contain a large adaptive T regulatory cell subset which might contribute to in vitro and in vivo immune modulation. [Copyright &y& Elsevier]

The herpes simplex virus thymidine kinase (HSV-tk) gene conferring ganciclovir (GCV)-specific sensitivity to transduced cells might control Graft-versus-Leukemia (GvL)/Graft-versus-Host Disease (GvHD). Human T lymphocytes were engineered with an LSN-tk retroviral vector encoding tk and neomycin resistance (NeoR) genes. A total of 80×106 tk+ lymphocytes were injected intraperitoneally in NOD-SCID mice. Engraftment was evaluated by human CD45+/CD3+ cytofluorimetric analysis and NeoR-based polymerase chain reaction (PCR) on peripheral blood, bone marrow, liver, thymus, and spleen on day +5. After 14 days, GCV (10 mg/kg daily) cytofluorimetric analysis and PCR were repeated (day +19). Immunohistological studies with anti-CD3 monoclonal antibody followed by alkaline phosphatase and monoclonal anti–alkaline phosphatase staining were performed on spleen and liver at the same time points. Human CD45+/CD3+ cells were engrafted in all tissues on day +5 according to cytofluorimetry, immunohistology, and PCR. Lymphocytes “homed” to the white pulp T-cell area and to the red pulp; liver localization is prevalently at the periportal area. After GCV (day +19), cytofluorimetry and immunohistology showed very few CD3+ cells. PCR identified the transgene in 22% tissue samples (positive only in thymus and spleen). GvHD did not occur in any animal. These data demonstrate elevated doses of human-transduced CD3+ cells engraft in NOD/SCID mice; after GCV, very few CD3+ cells can be detected and those that escape treatment can be found in the thymus and in the spleen on day +19. Lack of full response to GCV may account for cases of GvHD in patients receiving tk-transduced T lymphocytes. Cancer Gene Therapy (2002) 9, 756–761 doi:10.1038/sj.cgt.7700495 [ABSTRACT FROM AUTHOR]

Generation of an efficient graft-versus-leukemia (GVL) effect in patients with hematological malignancies who relapse after allogeneic bone marrow transplantation depends in part upon the number of infused T lymphocytes. Currently, a GVL reaction cannot be achieved without inducing concomitant graft-versus-host disease (GVHD); thus, one strategy is to try to modulate this GVL/GVHD ratio. We engineered human T lymphocytes with herpes simplex virus-thymidine kinase and neomycin resistance genes, with an LXSN-derived vector that confers a ganciclovir-specific sensitivity to the transduced T cells. We analyzed proliferation, interleukin-2 production, alloreactivity in a mixed lymphocyte culture, and clonogenicity during the different stages of retroviral infection and G418 selection. Our results confirm that a sufficient number of transduced T lymphocytes can be obtained after selection for clinical studies. Their proliferative activity, alloresponsiveness, and ability to produce and respond to interleukin-2 were retained. Compared with control populations, their clonogenicity, as assessed by limiting dilution assays, was reduced after retroviral infection and G418 selection by 1.6 and 2.9 logs, respectively, with both viral supernatant incubation and coculture procedures. This study shows that infection and selection with the thymidine kinase-neomycin resistance gene retroviral vector significantly reduces the number of functional T lymphocytes. This finding should be taken into account when establishing the dose of T lymphocytes necessary to trigger a modulated GVL/GVHD effect. [ABSTRACT FROM AUTHOR]

The article reports on a study determining whether NOTCH1 activating mutations are present in B-chronic lymphocytic leukemia (B-CLL) cells. As reported, Notch1 and Notch2 constitutive activation plays a critical role in survival and apoptosis resistance of B-CLL cells. It has been suggested in the study that for T-cell acute lymphoblastic leukemia (T-ALL), deletions of the COOH-terminal sequences in the PEST domain could enhance Notch-IC stability and signalling.

Simple Summary: Pancreatic cancer (PC) develops resistance to current therapeutic approaches with a consequent dismal prognosis. Immunotherapy is an effective approach in several tumors, but PC is resistant to immunotherapy. Tumor-derived extracellular vesicles (EVs) may modulate immune responses by either dampening or inducing antitumor immune responses. In this study, we explored the immunomodulatory potential of pancreatic-cancer-derived extracellular vesicles through proteomic and functional approaches. Notably, proteins involved in the "Immune System" were highly enriched in the protein cargo of PC-derived EVs, which also included immunostimulatory proteins. Interestingly, the treatment of healthy donor-derived peripheral blood mononuclear cells (PBMCs) with EVs from one of the PC cell lines analyzed induced early activation markers in CD8+ and CD4+ lymphocytes. This was consistent with the proteomic and ELISA analyses. Our study indicates that even if PC is an immune-cold tumor, in some cases, PC-EVs may activate early immune responses. This finding might be relevant for the development of effective immunotherapeutic strategies in PC. Pancreatic cancer (PC) has a poor prognosis and displays resistance to immunotherapy. A better understanding of tumor-derived extracellular vesicle (EV) effects on immune responses might contribute to improved immunotherapy. EVs derived from Capan-2 and BxPC-3 PC cells isolated by ultracentrifugation were characterized by atomic force microscopy, Western blot (WB), nanoparticle tracking analysis, and label-free proteomics. Fresh PBMCs from healthy donors were treated with PC- or control-derived heterologous EVs, followed by flow cytometry analysis of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated or untreated PBMCs was performed, and the IFN-γ concentration was measured by ELISA. Notably, most of the proteins identified in Capan-2 and BxPC-3 EVs by the proteomic analysis were connected in a single functional network (p = 1 × 10−16) and were involved in the "Immune System" (FDR: 1.10 × 10−24 and 3.69 × 10−19, respectively). Interestingly, the treatment of healthy donor-derived PBMCs with Capan-2 EVs but not with BxPC-3 EVs or heterologous control EVs induced early activation of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated PBMCs was consistent with their activation by Capan-2 EVs, indicating IFN-γ among the major upstream regulators, as confirmed by ELISA. The proteomic and functional analyses indicate that PC-EVs have pleiotropic effects, and some may activate early immune responses, which might be relevant for the development of highly needed immunotherapeutic strategies in this immune-cold tumor. [ABSTRACT FROM AUTHOR]

Simple Summary: Chronic lymphocytic leukemia (CLL) is an incurable hematological disorder, representing an unmet need within the field of cancer therapy. In this study, we highlighted the cytotoxicity of silver nanoparticles (AgNPs) against CLL cells and demonstrated the synergistic activity of AgNPs with drugs currently used in CLL, such as Venetoclax and Ibrutinib, which can be exploited for potential combined therapies. Furthermore, the conjugation of AgNPs with the anti-CD20 antibody Rituximab (AgNPs@Rituximab) increased the specificity and cytotoxicity of AgNPs toward CLL cells in vitro. AgNPs@Rituximab also extended the survival of CLL xenograft models compared to each unconjugated single agent. These data provided evidence that AgNPs@Rituximab could also overcome the non-specific distribution of AgNPs in vivo, thus increasing the selective elimination of CLL cells. The findings that emerged from this study could provide the rationale for further investigations aimed at defining a potential clinical application of nanotechnologies in the context of CLL therapy. Background: Chronic lymphocytic leukemia (CLL) is an incurable disorder associated with alterations in several pathways essential for survival and proliferation. Despite the advances made in CLL therapy with the new target agents, in some cases, relapses and resistance could occur, making the discovery of new alternatives to manage CLL refractoriness necessary. To provide new therapeutic strategies for CLL, we investigated the anti-leukemic activity of silver nanoparticles (AgNPs), whose impact on CLL cells has been poorly explored. Methods: We studied the action mechanisms of AgNPs in vitro through flow cytometry and molecular analyses. To improve the bioavailability of AgNPs, we generated AgNPs coated with the anti-CD20 antibody Rituximab (AgNPs@Rituximab) and carried out imaging-based approaches and in vivo experiments to evaluate specificity, drug uptake, and efficacy. Results: AgNPs reduced the viability of primary CLL cells and the HG-3 cell line by inducing an intrinsic apoptotic pathway characterized by Bax/Bcl-2 imbalance, caspase activation, and PARP degradation. Early apoptotic events triggered by AgNPs included enhanced Ca2+ influx and ROS overproduction. AgNPs synergistically potentiated the cytotoxicity of Venetoclax, Ibrutinib, and Bepridil. In vitro, the AgNPs@Rituximab conjugates were rapidly internalized within CLL cells and strongly prolonged the survival of CLL xenograft models compared to each unconjugated single agent. Conclusions: AgNPs showed strong anti-leukemic activity in CLL, with the potential for clinical translation in combination with agents used in CLL. The increased specificity of AgNPs@Rituximab toward CLL cells could be relevant for overcoming in vivo AgNPs' non-specific distribution and increasing their efficacy. [ABSTRACT FROM AUTHOR]

Background: Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is a valid treatment for hematological oncological or metabolic diseases. Despite its therapeutic efficacy, it is an aggressive treatment that impacts negatively on quality of life (QoL) and may result in Post-Traumatic Stress Disorder (PTSD) symptoms. The aim of this study is to explore rates and risk factors for PTSD symptoms, and fatigue in post-HSCT patients with hematological malignancies. Methods: A total of 123 patients after HSCT were evaluated for PTSD symptoms, QoL and fatigue. PTSD symptoms were assessed with the Impact of Event Scale- Revised (IES-R), QoL was measured with Functional Assessment of Cancer Therapy-Bone Marrow Transplant (FACT-BMT) and fatigue symptoms were assessed with Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F). Results: A total of 58.54% of the sample developed PTSD symptoms after transplant. Patients with PTSD symptoms reported significantly lower QoL total scores and significantly higher fatigue than those without PTSD symptoms (p < 0.001). The SEM analysis showed that worse QoL and fatigue affected PTSD symptomatology along different pathways. Fatigue was found as a major influencing factor of PTSD symptoms directly (β = 0.31 **), while QoL only through the mediation of fatigue at a lesser extent. (β = 0.33 *). Conclusions: Our findings indicate that QoL is a concurrent causative factor to the development of PTSD symptomatology through the mediating role of fatigue. Innovative interventions before transplantation to prevent PTSD symptoms should be investigated to improve survival and QoL in patients. [ABSTRACT FROM AUTHOR]

Simple Summary: In this review we aim to address the potential of extracellular vesicles stemming from chimeric antigen receptor T (CAR-T) lymphocytes as therapeutic agents in tumors. We underlined how CAR-T-lymphocytes, representing one of the new frontiers of immunotherapy for the fight against refractory neoplastic diseases, demonstrated their potential effectiveness in cancer. However, the presence of physical barriers that prevent the entry of CAR-T and other immune effector cells, the hostile microenvironment that hampers persistence and activity of immune cells, as well as tumor heterogeneity resulted in their variable or low efficacy against solid tumors. The application of CAR-T-derived extracellular vesicles as therapeutic agents may improve the homing of CAR-T effector functions through their facilitated diffusion within solid tumors and at the same time might circumvent some of the adverse effects that are induced by the cellular counterpart. Extracellular vesicles (EVs) are a heterogenous population of plasma membrane-surrounded particles that are released in the extracellular milieu by almost all types of living cells. EVs are key players in intercellular crosstalk, both locally and systemically, given that they deliver their cargoes (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells, crossing biological barriers. Those mechanisms further trigger a wide range of biological responses. Interestingly, EV phenotypes and cargoes and, therefore, their functions, stem from their specific parental cells. For these reasons, EVs have been proposed as promising candidates for EV-based, cell-free therapies. One of the new frontiers of cell-based immunotherapy for the fight against refractory neoplastic diseases is represented by genetically engineered chimeric antigen receptor T (CAR-T) lymphocytes, which in recent years have demonstrated their effectiveness by reaching commercialization and clinical application for some neoplastic diseases. CAR-T-derived EVs represent a recent promising development of CAR-T immunotherapy approaches. This crosscutting innovative strategy is designed to exploit the advantages of genetically engineered cell-based immunotherapy together with those of cell-free EVs, which in principle might be safer and more efficient in crossing biological and tumor-associated barriers. In this review, we underlined the potential of CAR-T-derived EVs as therapeutic agents in tumors. [ABSTRACT FROM AUTHOR]

We developed a good manufacturing practices–compatible expansion protocol to improve number and purity of regulatory T cells (Tregs) available for clinical trials. Six clinical-grade separation procedures were performed, followed by expansion with high-dose interleukin (IL)-2, anti-CD3/anti-CD28 TCR stimulation, and rapamycin for 19 days achieving a median of 8.5-fold (range, 6.25 to 13.7) expansion. FOXP3 expression was stably maintained over the culture period, while the percentage of CD127 was significantly reduced. The in vitro suppression assay showed a strong Mixed Lymphocytes Reaction inhibition. In vitro amplification did not induce any karyotypic modification. To evaluate the graft-versus-host disease (GVHD)/graft-versus-leukemia (GVL) bifunctional axis, expanded Tregs and conventional T cells (Tcons) were tested in NOD/SCID/IL2Rgnull mice injected with primary acute myeloid leukemia (AML) cells, AML cell line, acute lymphoid leukemia Philadelphia cell line, or Burkitt-like lymphoma cell line. All mice that received leukemia cells together with expanded Tregs and Tcons were rescued from leukemia and survived without GVHD, showing that Treg expansion procedure did not compromise GVHD control and the strong Tcon-mediated GVL activity. This report might represent the basis for treating high-risk leukemia and/or relapsed/refractory leukemia patients with high-dose Treg/Tcons. [ABSTRACT FROM AUTHOR]

The iron chelator deferasirox (DFX) prevents complications related to transfusional iron overload in several haematological disorders characterized by marrow failure. It is also able to induce haematological responses in a percentage of treated patients, particularly in those affected by myelodysplastic syndromes. The underlying mechanisms responsible for this feature, however, are still poorly understood. In this study, we investigated the effect of DFX-treatment in human haematopoietic/progenitor stem cells, focussing on its impact on the redox balance, which proved to control the interplay between stemness maintenance, self-renewal and differentiation priming. Here we show, for the first time, that DFX treatment induces a significant diphenyleneiodonium-sensitive reactive oxygen species (ROS) production that leads to the activation of POU5F1 (OCT4), SOX2 and SOX17 gene expression, relevant in reprogramming processes, and the reduction of the haematopoietic regulatory proteins CTNNB1 (β-Catenin) and BMI1. These DFX-mediated events were accompanied by decreased CD34 expression, increased mitochondrial mass and up-regulation of the erythropoietic marker CD71 (TFRC) and were compound-specific, dissimilar to deferoxamine. Our findings would suggest a novel mechanism by which DFX, probably independently on its iron-chelating property but through ROS signalling activation, may influence key factors involved in self-renewal/differentiation of haematopoietic stem cells. [ABSTRACT FROM AUTHOR]

The Ikaros gene encodes a Krüppel-like zinc-finger transcription factor involved in hematopoiesis regulation. Ikaros has been established as one of the most clinically relevant tumor suppressors in several hematological malignancies. In fact, expression of dominant negative Ikaros isoforms is associated with adult B-cell acute lymphoblastic leukemia, myelodysplastic syndrome, acute myeloid leukemia and adult and juvenile chronic myeloid leukemia. Here, we report the isolation of a novel, non-canonical Ikaros splice variant, called Ikaros 11 (Ik11). Ik11 is structurally related to known dominant negative Ikaros isoforms, due to the lack of a functional DNA-binding domain. Interestingly, Ik11 is the first Ikaros splice variant missing the transcriptional activation domain. Indeed, we demonstrated that Ik11 works as a dominant negative protein, being able to dimerize with Ikaros DNA-binding isoforms and inhibit their functions, at least in part by retaining them in the cytoplasm. Notably, we demonstrated that Ik11 is the first dominant negative Ikaros isoform to be aberrantly expressed in B-cell lymphoproliferative disorders, such as chronic lymphocytic leukemia. Aberrant expression of Ik11 interferes with both proliferation and apoptotic pathways, providing a mechanism for Ik11 involvement in tumor pathogenesis. Thus, Ik11 could represent a novel marker for B-cell lymphoproliferative disorders. [ABSTRACT FROM AUTHOR]

Objective: Discovery of genetic variations in the genes encoding for Toll-like receptors (TLRs) has highlighted a potential link between genomic variation of the host and susceptibility to infections. Materials and Methods: We investigated the association between polymorphisms in the TLR2, TLR4, and TLR9 genes in recipients of allogeneic hematopoietic stem cell transplant and susceptibility to infections caused by cytomegalovirus and filamentous fungi. Results: A significant association was observed between the presence of the T-1237C polymorphism (TLR9) and susceptibility to viral pneumonia (p =0.04; odds ratio [OR]: 1.73). For fungi, a significant association was observed between the presence of the cosegregating Asp299Gly/Thr399Ile polymorphisms (TLR4) and fungal colonization (p =0.003; OR: 10.6). However, susceptibility to fungal infections, predominantly fungal pneumonia, was instead significantly decreased in the presence of the same polymorphisms (p =0.03; OR: 0.23). Conclusion: Thus, fungal colonization may not predict susceptibility to infection in the presence of these single nucleotide polymorphisms. The finding that defective viral but not fungal sensing may predict susceptibility to infection highlights the divergent function of TLRs in the pathogenesis of opportunistic infections. [Copyright &y& Elsevier]

Patients who underwent autologous stem cell transplantation (ASCT) are prone to decreased bone mineral density (BMD). We measured BMD in 180 patients who underwent ASCT for hematologic malignancies. Patients were evaluated with a median of 6.2 years after ASCT. Twenty patients who received only chemotherapy were evaluated as controls. The loss of bone mass was greater during the first year after ASCT, since majority of patients recover BMD and normalize bone turnover markers during the following years. After ASCT, over half of the patients show osteopenia or osteoporosis independent of the sex. According to the results of other groups, our results emphasize the potential usefulness of antiresorptive agents to prevent or treat post-ASCT osteopenia or osteoporosis, and the importance of the measurement of BMD as an integral component to the follow-up of ASCT. [ABSTRACT FROM AUTHOR]

Abstract: T-cell homeostasis is regulated by several molecules; among these, interleukin (IL)-7 plays an essential role in the survival and homeostatic proliferation of peripheral naive T cells. In a previous study, we investigated whether human mesenchymal stromal cells (MSCs) could be engineered with the IL-7 gene to produce functional level of this cytokine. In the present study, we analyzed the impact of different quantities of IL-7 produced by MSCs on the survival and proliferation of a negative immunoselected naive (CD3+/CD45RA+) T-cell population. Co-cultivation of peripheral naive T cells with MSCs producing low (16 pg/mL) or high (1000 pg/mL) IL-7 levels or in the presence of exogenous IL-7 (0.01 ng/mL and 100 ng/mL) maintained the CD3+/CD45RA+ naive T-cell phenotype. Chemokine receptor CCR7+ expression was also maintained among this T-cell population. Naive T-cell molecular characteristics were maintained as assessed by the Vβ spectratyping complexity score, which showed the maintenance of a broad T-cell repertoire. No Th1 or Th2 differentiation was observed, as assessed by interferon-γ or IL-4 accumulation. In contrast, only MSCs producing high amounts of IL-7 caused increased activation (CD25 31.2% ± 12% vs 10% ± 3.5%; P < .05), proliferation (CD71 17.8±7% vs 9.3%±3, P < .05), apoptosis (assessed by annexin V: 18.6% ± 5% vs 14.9% ± 2.6%; P > .05), and the phase S cell cycle (15% vs 6.9%, P > .05). Exogenous IL-7 exhibited no significant effect. In conclusion, we demonstrated that IL-7 produced by MSCs has a dose-independent effect on naive T-cell survival while exerting a dose-dependent effect on activation/proliferation. Due to the continuous production of IL-7 by engineered cells, our system is more efficacious than exogenous IL-7. [Copyright &y& Elsevier]

Abstract: Tumor necrosis factor α (TNFα) is constitutively produced by B-chronic lymphocytic leukemia (B-CLL) cells and may act as an autocrine factor for their growth and survival. However, very few data are available on the possible cytotoxic effect of TNFα produced by B-CLL cells. This study investigated whether B-CLL cells exert in vitro cytotoxicity by TNFα and if so, whether this cytotoxicity can be modulated by cytokines. In 8 of 12 patients (66.6%), B-CLL cells in vitro constitutively produced TNFα and exerted a TNFα-mediated cytotoxicity, evaluated in an 18-h 51Cr release assay, against the TNFα-sensitive Jurkat, U937 and K562 cell lines but not against the TNFα-resistant Raji cell line. Involvement of TNFα in B-CLL cell cytotoxicity is demonstrated by the fact that anti-TNFα antibodies strongly inhibited it and supernatants of cytotoxic cultures contained TNFα and mediated a completely TNFα-dependent cytotoxicity. When the cytotoxic B-CLL cells were stimulated with interleukin (IL)-2 plus IL-12, there was increased TNFα mRNA expression, TNFα production and TNFα-mediated cytotoxicity. All eight patients with cytotoxic leukemic cells had progressive disease and six of these also expressed high levels of ZAP-70 protein. In the other four patients (33.3%), B-CLL cells did not produce TNFα in vitro and were not cytotoxic, either spontaneously or after IL-2 plus IL-12 stimulation. Of these four patients, three had stable disease and one had progressive disease. The patient with progressive disease and one of the three with stable disease expressed low levels of ZAP-70 protein. We conclude that a group of B-CLL patients with progressive disease have leukemic B cells able to exert in vitro a TNFα-mediated cytotoxicity, which is modulated by cytokines. [Copyright &y& Elsevier]

Haploidentical stem cell transplantation has became a clinical reality in the last 10 years as it provides the chance of transplant for about 50% of patients with hematological malignancies who do not have a matched related or unrelated donor. Proper graft preparation for this type of transplant is crucial and this paper analyses our work over the past decade in the search for the optimal graft processing procedure moving from E-rosetting and soybean agglutination, through a combination of negative or positive selection of hematopoietic stem cells to the current method of one-step positive selection. In preparing a graft for haploidentical transplant, three essential requisites must be met. It must contain (1) a megadose (>10 × 106 × kg recipient b.w.) of hematopoietic stem cells to overcome the HLA histocompatibility barrier; (2) very few T-lymphocytes (CD3+ cells <3 × 104/kg recipient b.w.) to prevent severe acute and chronic graft-versus-host disease (GvHD); (3) very few B-lymphocytes to prevent Epstein–Barr virus-related lymphoproliferative disorders.With current graft processing technologies based on positive selection of hematopoietic stem cells, these requirements can be met. A 70–80% hematopoietic stem cell recovery ensures the target megadose is achieved in over 70% of cases with a T-cell depletion of more than 4 logs and a B-cell depletion of over 3 logs.Progress in graft processing has ensured primary, sustained engraftment rates of over 90% and has significantly reduced the incidence of severe acute GvHD and EBV-related lymphoproliferative disorders.Modern time-saving automated graft processing devices ensure reproducibility, reliability, and biological safety, which make widespread application of the haploidentical transplant currently feasible. [Copyright &y& Elsevier]

INTRODUCTION:: High-dose chemotherapy conditioning regimens for autologous stem cell transplantation generally give similar results in multiple myeloma. We compared two regimens: melphalan versus melphalan plus busulphan. METHODS:: In all, 30 untreated patients with stage III low-risk multiple myeloma were studied. After induction with three VAD courses and mobilization with cyclophosphamide 7?g/m2 and recombinant human granulocyte-colony stimulating factor (rHuG-CSF) (10?µg/kg b.w./die), they received melphalan 200?mg/m2 (arm A) or busulphan 16?mg/kg plus melphalan 100?mg/m2 (arm B) for conditioning for transplantation. All patients received maintenance therapy with Interferon 3 MU × 3/week. RESULTS:: Time to engraftment after transplantation was similar in both groups. All patients received rHuG-CSF after reinfusion of peripheral stem cells. No differences emerged in transplant-related infective and noninfective complications. There were no transplant-related deaths. A better response was observed in the melphalan plus busulphan regimen (85 versus 75%, P<0.05). The 5-year overall survival with this regimen was 56 versus 49% with melphalan, and the median survival was 126 months versus 108 months for melphalan (P=0.7). The median progression-free survival was 121 months for melphalan plus busulphan versus 97 months for melphalan (P=0.05). CONCLUSION:: These two conditioning regimens showed similar overall response rate and overall survival, though progression-free survival was better with busulphan plus melphalan.The Hematology Journal (2004) 5, 118-122. doi:10.1038/sj.thj.6200369 [ABSTRACT FROM AUTHOR]

The EGFP-tk retroviral vector, encoding enhanced green fluorescent protein (EGFP) and the herpes simplex virus thymidine kinase (HSV-tk) packaged in a Phoenix amphotropic cell line, was used to transduce healthy donor T lymphocytes. Infection yielded a mean of 41·8 ± 9·3% SD (range 31·1–48·4%) EGFP-positive cells and a mean of 92 ± 2% SD (range 90–94%) after cell sorting. EGFP expression remained stable for 30 d after infection. The entire gene transfer procedure had no significant effect on lymphocyte subsets and slightly reduced clonogenicity. Ganciclovir (gcv) treatment (1 µg/ml × 10 d) killed all EGFP-positive cells in the transduced and transduced/sorted populations, but had no effect on untransduced controls. Our results show that primary T lymphocytes can be transduced using an EGFP-tk vector that yields a homogeneous infected population without affecting lymphocyte subsets, function and clonogenicity. [ABSTRACT FROM AUTHOR]

Key features of chronic lymphocytic leukemia (CLL) are defects in the immune system and the ability of leukemic cells to evade immune defenses and induce immunosuppression, resulting in increased susceptibility to infections and disease progression. Several immune effectors are impaired in CLL, including T and natural killer (NK) cells. The role of T cells in defense against CLL and in CLL progression and immunotherapy has been extensively studied. Less is known about the role of NK cells in this leukemia, and data on NK cell alterations in CLL are contrasting. Besides studies showing that NK cells have intrinsic defects in CLL, there is a large body of evidence indicating that NK cell dysfunctions in CLL mainly depend on the escape mechanisms employed by leukemic cells. In keeping, it has been shown that NK cell functions, including antibody-dependent cellular cytotoxicity (ADCC), can be retained and/or restored after adequate stimulation. Therefore, due to their preserved ADCC function and the reversibility of CLL-related dysfunctions, NK cells are an attractive source for novel immunotherapeutic strategies in this disease, including chimeric antigen receptor (CAR) therapy. Recently, satisfying clinical responses have been obtained in CLL patients using cord blood-derived CAR-NK cells, opening new possibilities for further exploring NK cells in the immunotherapy of CLL. However, notwithstanding the promising results of this clinical trial, more evidence is needed to fully understand whether and in which CLL cases NK cell-based immunotherapy may represent a valid, alternative/additional therapeutic option for this leukemia. In this review, we provide an overview of the current knowledge about phenotypic and functional alterations of NK cells in CLL and the mechanisms by which CLL cells circumvent NK cell-mediated immunosurveillance. Additionally, we discuss the potential relevance of using NK cells in CLL immunotherapy. [ABSTRACT FROM AUTHOR]

The article presents a case study of a novel NOTCH1 mutation of the proline, glutamic acid, serine and threonine (PEST) domain in a 65-year-old male with chronic lymphocytic leukemia (CLL). He received three cycles of hemoimmunotherapy with rituximab and fludarabine due to disease progression. He died after one month due to worsening of his conditions associated with persistent thrombocytopenia, anemia and lymphocytosis.

The article offers information on the advancement in the process of diagnosis, prognosis, and treatment of B-cell chronic lymphocytic leukaemia (B-CLL). It mentions that high expression of zeta-chain-associated protein kinase 70 (ZAP-70) and unmutated immunoglobulin heavy variable (IGHV) genes are considered poor factors for prognosis. It informs that NOTCH1 and PEST mutations are considered as marker of poor prognosis in a minority of B-CLL patients.