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Damage to gut mucosa following conditioning regimens may favour bacterial infections that can trigger graft versus host disease (GvHD) in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Rifaximin, an oral and non-absorbable antibiotic, has been recently proposed as effective prophylaxis to reduce bacterial infections in the gut and consequently acute GvHD in this setting. The present study evaluated safety and outcomes of HSCT patients that were treated with rifaximin prophylaxis at Perugia University Hospital. Rifaximin prophylaxis was introduced as standard of care in HSCT patients in May 2018. We retrieved data from 118 consecutive transplants, and we compared the outcomes of rifaximin-treated patients with historical controls that did not receive antibiotic prophylaxis. While incidences of neutropenic fever, documented bacterial infections, and aGvHD were similar in the two groups, we found an increased frequency of invasive candidiasis and clinically relevant Candida spp. infections in rifaximin-treated patients (5 patients vs 1 patient, 25% [± 0.99%] vs 1% [± 0.01%], p < .0001). Three rifaximin-treated patients experienced life-threating candidemia (2 C. krusei, 1 C. orthopsilosis). Rifaximin was the only factor that increased the risk of Candida spp. infections. Rifaximin could have contributed to microbiome disruption which favoured an outbreak of life-threatening Candida infections. This important complication forced us to halt its use. Larger, prospective studies are needed to assess the impact of rifaximin prophylaxis on incidence of bacterial infections, aGvHD, and survival of HSCT patients., (© 2021. The Author(s).)

Allogeneic hematopoietic stem cell transplantation (HSCT) is the most effective treatment in eradicating high-risk acute myeloid leukemia (AML). Here, we present data from a novel HLA-haploidentical HSCT protocol that addressed the 2 remaining major unmet medical needs: leukemia relapse and chronic graft-versus-host disease (cGVHD). Fifty AML patients were enrolled in the study. The conditioning regimen included total body irradiation for patients up to age 50 years and total marrow/lymphoid irradiation for patients age 51 to 65 years. Irradiation was followed by thiotepa, fludarabine, and cyclophosphamide. Patients received an infusion of 2 × 106/kg donor regulatory T cells on day -4 followed by 1 × 106/kg donor conventional T cells on day -1 and a mean of 10.7 × 106 ± 3.4 × 106/kgpurified CD34+ hematopoietic progenitor cells on day 0. No pharmacological GVHD prophylaxis was administered posttransplantation. Patients achieved full donor-type engraftment. Fifteen patients developed grade ≥2 acute GVHD (aGVHD). Twelve of the 15 patients with aGVHD were alive and no longer receiving immunosuppressive therapy. Moderate/severe cGVHD occurred in only 1 patient. Nonrelapse mortality occurred in 10 patients. Only 2 patients relapsed. Consequently, at a median follow-up of 29 months, the probability of moderate/severe cGVHD/relapse-free survival was 75% (95% confidence interval, 71%-78%). A novel HLA-haploidentical HSCT strategy that combines an age-adapted myeloablative conditioning regimen with regulatory and conventional T-cell adoptive immunotherapy resulted in an unprecedented cGVHD/relapse-free survival rate in 50 AML patients with a median age of 53 years. This trial was registered with the Umbria Region Institutional Review Board Public Registry as identification code 02/14 and public registry #2384/14 and at www.clinicaltrials.gov as #NCT03977103., (© 2021 by The American Society of Hematology.)

Allogeneic hematopoietic cell transplantation from a human leukocyte antigen (HLA) haplotype mismatched donor (haploidentical transplantation) was not feasible for the treatment of hematologic malignancies until the early 1990s, due to the high risk of rejection and graft-versus-host disease (GVHD). The first successful protocol of haploidentical transplantation was based on a highly myeloablative and immunosuppressive conditioning regimen and the infusion of a "mega-dose" of T-cell-depleted hematopoietic stem cells. More than 90% of patients engrafted and <10% developed GVHD. The protocol did not include post-transplant immunosuppression, which favored the graft-versus-tumor effect mediated by alloreactive NK cells and residual alloreactive T cells. However, donor post-transplant immune reconstitution was slow with a high risk of infection-related mortality. More recently, T-cell-depleted haploidentical transplantation has become the platform for innovative cell therapies that aim to enhance T-cell immunity while preventing adverse reactions against host tissues. One strategy is adoptive immunotherapy with conventional T cells and regulatory T cells. Preclinical studies and clinical trials have proven that regulatory T cells control GVHD caused by co-infused conventional T cells while the graft-versus-tumor effect is retained. The use of regulatory T cells in the absence of any other form of immune suppression allowed for a conventional T cell-mediated full eradication of disease in the vast majority of high-risk acute leukemia patients.

Because activating killer cell immunoglobulinlike receptors (KIRs) are heterogeneously expressed in the population, we investigated the role of donor activating KIRs in haploidentical hematopoietic transplants for acute leukemia. Transplants were grouped according to presence vs absence of KIR-ligand mismatches in the graft-vs-host direction (ie, of donor-vs-recipient natural killer [NK]-cell alloreactivity). In the absence of donor-vs-recipient NK-cell alloreactivity, donor activating KIRs had no effects on outcomes. In the 69 transplant pairs with donor-vs-recipient NK-cell alloreactivity, transplantation from donors with KIR2DS1 and/or KIR3DS1 was associated with reduced risk of nonrelapse mortality, largely infection related (KIR2DS1 present vs absent: hazard ratio [HR], 0.25; P = .01; KIR3DS1 present vs absent: HR, 0.18; P = .006), and better event-free survival (KIR2DS1 present vs absent: HR, 0.31; P = .011; KIR3DS1 present vs absent: HR, 0.30; P = .008). Transplantation from donors with KIR2DS1 and/or KIR3DS1 was also associated with a 50% reduction in infection rate (P = .003). In vitro analyses showed that KIR2DS1 binding to its HLA-C2 ligand upregulated inflammatory cytokine production by alloreactive NK cells in response to infectious challenges. Because ∼40% of donors able to exert donor-vs-recipient NK-cell alloreactivity carry KIR2DS1 and/or KIR3DS1, searching for them may become a feasible, additional criterion in donor selection., (© 2015 by The American Society of Hematology.)

Posttransplant relapse is still the major cause of treatment failure in high-risk acute leukemia. Attempts to manipulate alloreactive T cells to spare normal cells while killing leukemic cells have been unsuccessful. In HLA-haploidentical transplantation, we reported that donor-derived T regulatory cells (Tregs), coinfused with conventional T cells (Tcons), protected recipients against graft-versus-host disease (GVHD). The present phase 2 study investigated whether Treg-Tcon adoptive immunotherapy prevents posttransplant leukemia relapse. Forty-three adults with high-risk acute leukemia (acute myeloid leukemia 33; acute lymphoblastic leukemia 10) were conditioned with a total body irradiation-based regimen. Grafts included CD34(+) cells (mean 9.7 × 10(6)/kg), Tregs (mean 2.5 × 10(6)/kg), and Tcons (mean 1.1 × 10(6)/kg). No posttransplant immunosuppression was given. Ninety-five percent of patients achieved full-donor type engraftment and 15% developed ≥grade 2 acute GVHD. The probability of disease-free survival was 0.56 at a median follow-up of 46 months. The very low cumulative incidence of relapse (0.05) was significantly better than in historical controls. These results demonstrate the immunosuppressive potential of Tregs can be used to suppress GVHD without loss of the benefits of graft-versus-leukemia (GVL) activity. Humanized murine models provided insights into the mechanisms underlying separation of GVL from GVHD, suggesting the GVL effect is due to largely unopposed Tcon alloantigen recognition in bone marrow., (© 2014 by The American Society of Hematology.)

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 posttransplant 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.

To detect factors associated with quality of life (QOL) of patients with myelodysplastic syndrome (MDS) and to compare the MDS patients' self-assessed QOL with that perceived by their physicians. In an observational, non-interventional, prospective, multicentre study, QOL was evaluated in 148 patients with newly diagnosed low- and intermediate-risk IPSS MDS. QOL measures (QOL-E v.2, LASA and EQ-5D) and patient-related candidate determinants of QOL were assessed for up to 18 months. Patients' QOL scores were compared with those obtained by appointed hematologists' assessment and with ECOG performance status (PS). Fatigue was not prevalent at diagnosis, though physical QOL and energy levels were low. Transfusion-dependent patients had worse QOL scores. In multivariate analysis, Hb levels and comorbidities were a major determinant of QOL. Physicians' perception of patients' well-being correlated with patients' QOL. Physicians underestimated the impact of disturbances on patients' QOL, mainly in the MDS-specific components. ECOG PS did not discriminate patients according to QOL status. In conclusion, the association of anemia with QOL is confirmed, while co-morbidities emerge as an independent predictor of QOL in MDS. Fatigue is not a major concern. ECOG PS is not a valuable surrogate of patient's QOL, thus highlighting that physician's judgment of patient's well-being must not substitute patient-reported outcomes. Appropriate questionnaires should be used to assess MDS patients' QOL in order to improve communication and therapeutic choice.

Introduction: Haploidentical transplantation, with extensive T cell depletion to prevent GvHD, is associated with a high incidence of infection-related deaths. The key challenge is to improve immune recovery with allogeneic donor T cells without triggering GvHD. As T regulatory cells (Tregs) controlled GvHD in pre-clinical studies, the present study evaluated the impact of an infusion of donor CD4/CD25 + Tregs, followed by an inoculum of donor mature T cells (Tcons) and positively immunoselected CD34 + cells in the setting of haploidentical stem cell transplantation., Patients and Methods: Twenty-eight patients were enrolled in this study (22 AML; 5 ALL; 1 NHL). All received immunoselected Tregs (CliniMACS, Miltenyi Biotec) followed by positively immunoselected CD34 + cells together with Tcons 4 days later. No GvHD prophylaxis was administered., Results: 26/28 patients engrafted. No acute GvHD developed in 24/26 patients; 2 developed ≥ grade II acute GvHD. No patient has developed chronic GvHD. CD4 and CD8 counts rapidly increased after transplant. Episodes of CMV reactivation were significantly fewer than in controls., Conclusions: In the setting of haploidentical transplantation infusion of Tregs makes administration of a high dose of T cells feasible. This strategy provides a long-term protection from GvHD and robust immune reconstitution., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Hastening posttransplantation immune reconstitution is a key challenge in human leukocyte antigen (HLA)-haploidentical hematopoietic stem-cell transplantation (HSCT). In experimental models of mismatched HSCT, T-regulatory cells (Tregs) when co-infused with conventional T cells (Tcons) favored posttransplantation immune reconstitution and prevented lethal graft-versus-host disease (GVHD). In the present study, we evaluated the impact of early infusion of Tregs, followed by Tcons, on GVHD prevention and immunologic reconstitution in 28 patients with high-risk hematologic malignancies who underwent HLA-haploidentical HSCT. We show for the first time in humans that adoptive transfer of Tregs prevented GVHD in the absence of any posttransplantation immunosuppression, promoted lymphoid reconstitution, improved immunity to opportunistic pathogens, and did not weaken the graft-versus-leukemia effect. This study provides evidence that Tregs are a conserved mechanism in humans.

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.

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.

Background: We report a case of mucormycosis in a healthy 17-year-old accident victim with multiple abdominal injuries which was caused by infection with Absidia Corymbifera, a ubiquitous saphrophyte in the ground., Case Presentation: The patient was admitted to hospital with massive abdominal trauma. During an 8-hour emergency operation he received transfusions of compacted red blood cells, plasma, platelets and hemagel. He developed a crush syndrome with acute renal failure, resolved with extra-corporeal dialysis and had to undergo splenectomy because of spleen hematoma. As wound secretion and central venous catheter (CVC) blood cultures and drainage fluid were positive for Enterococcus Faecium, Providentia Rettgeri, Hafnia Alvei and Candida Albicans, tecoplanin, metronidazole, imipenem, and flucanozole were administered. Although the CVC was changed high fever persisted and discharge continued from the large abdominal wound. Repeated tampons in different sections and wound secretion smears were positive for A. corymbifera. Flucanozole was stopped and liposomal amphotericin (Ambisome; 5 mg/Kg i.v.) given for over 3 months. The patient improved; fever gradually disappeared. After 8 days, tampons and wound secretion smears were negative for A. corymbifera. No other fungal infections developed. Drainage fluid was later positive for tecoplanin-resistant E. faecium and Pseudomonas Aeroginosa responding only to meropenem and ciprofloxacin. Abdominal computerized tomography visualized fluid accumulation around the iliac-femoral bypass. Abcess was ruled out when scintigraphy showed no tracer uptake. The lesion was drained. Drainage fluid cultures were negative for bacteria and fungi. Fluid accumulation gradually disappeared with prolonged antibiotic and antifungal therapy. One year after the accident the patient is in good health, with normal quality of life., Conclusion: Successful outcome was due to early, specific antifungal therapy, at sufficiently high dosage which was prolonged for an adequate period of time. Early diagnosis of mucormycosis is essential for efficacious anti-fungal treatment and prevention of irreversible spread of mucormycosis to vital organs. It presupposes awareness that A. corymbifera infection can develop in healthy individuals who are stressed and traumatized through skin-ground contact in accidents.

Much progress has been made in the clinical, biological and technical aspects of the T-cell-depleted full-haplotype mismatched transplants for acute leukemia. Our experience demonstrates that infusing a megadose of extensively T-cell-depleted hematopoietic peripheral blood stem cells after an immuno-myeloablative conditioning regimen in acute leukemia patients ensures sustained engraftment with minimal graft-vs-host disease (GvHD) without the need of any post-transplant immunosuppressive treatment. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in more than 200 high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype mismatched donor, who is immediately available, a T-cell depleted mismatched transplant should be offered, not as a last resort, but as a viable option to high risk acute leukemia patients who do not have, or cannot find, a matched donor.

Neurotrophins, originally identified as neuronal survival and differentiation factors, exert their actions through tyrosine kinase receptors such as TrKA, in the case of the nerve growth factor. Neurotrophins also interact with p75, a common receptor devoid of kinase activity and connected to apoptosis. Here we show that nerve growth factor, TrKA and p75 are expressed in cell lines of human cancers of various non-neuronal lineages, including a panel of muscular sarcomas, and we show that all cell lines investigated actively release nerve growth factor into the medium. Treatment by AG879 (a tyrosine kinase inhibitor that inhibits TrKA phosphorylation, but not TrKB and TrKC) or by neutralizing antibodies anti-nerve growth factor and anti-TrKA dramatically decreases their proliferation with a variable increase in apoptosis. Similarly, p75 transfection induced a significant increase in apoptosis. Furthermore, for the first time we have determined by high-performance liquid chromatography the pharmacokinetic profile of a novel preparation of AG879 and we have established an optimal plasmatic concentration for in-vivo administration. Treatment with AG879 in immunodepressed mice grafted with leiomyosarcoma or promyelocytic leukemia cells resulted in dramatic reductions in tumor sizes. In conclusion, our data have a novel preclinical potential for revealing a possible therapeutical utility in targeting in-vivo nerve growth factor/TrKA by AG879 or neutralizing antibody anti-TrKA in cancer proliferation and in muscle sarcomas, in particular.

In this study we determined whether human stromal cells could be engineered with a retroviral vector carrying the interleukin 7 (IL-7) gene and investigated the effects on T cells in vitro and in vivo in a murine model. Transduced mesenchymal cells strongly express CD90 (98.15%), CD105 (87.6%), and STRO-1 (86.7%). IL-7 production was 16.37 (+/-2 SD) pg/ml, which remained stable for 60 days. In vitro-immunoselected naive T cells maintained the CD45RA+ CD45RO- naive phenotype (4.2 times more than controls) after 7 days of culture with IL-7-engineered stromal cells. The apoptosis rate (4.7%) of the naive T cells cultured with transduced stromal cells overlapped with that of freshly isolated cells. Immunohistological analysis detected stromal cells in bone marrow, spleen, and thymus. Cotransplantation of IL-7-engineered stromal cells with CD34+ cells improved engraftment in terms of CD45+ cells and significantly increased the CD3+ cell count in peripheral blood, bone marrow, and spleen. These data demonstrate the following: (1) human stromal cells can be transduced, generating a normal layer; (2) transduced stromal cells in vitro maintain the naive T cell phenotype; and (3) IL-7-transduced stromal cells in vivo home to lymphoid organs and produce sufficient IL-7 in loco, supporting T cell development in a cotransplantation model. Because of their efficient cytokine production and homing, IL-7-engineered stromal cells might be an ideal vehicle to hasten immunological reconstitution in T cell-depleted hosts.

Purpose: Establishment of hematopoietic stem-cell (HSC) transplantation from mismatched relatives is feasible for patients with acute leukemia. As our original method of graft processing was unsuitable for large-scale clinical studies, we use automated devices for CD34+ cell purification., Patients and Methods: Sixty-seven patients with acute myeloid leukemia (AML; 19 complete remission [CR] 1, 14 CR 2, nine CR > 2, 25 in relapse) and 37 with acute lymphoid leukemia (ALL; 14 CR 1, eight CR 2, two CR > 2, 13 in relapse) were conditioned with total-body irradiation, thiotepa, fludarabine, and antithymocyte globulin. Peripheral-blood progenitor cells were mobilized with recombinant human granulocyte colony-stimulating factor and depleted of T-cells using CD34+ cell immunoselection. No post-transplantation graft-versus-host disease (GvHD) prophylaxis was administered., Results: Primary engraftment was achieved in 94 of 101 assessable patients. Six of the seven patients who rejected the primary graft, engrafted after a second transplantation. Overall, 100 of 101 patients engrafted. Acute GvHD developed in eight of 100 patients, and chronic GvHD, in five of 70 assessable patients. Thirty-eight patients died of nonleukemic causes. Relapse occurred in nine of 66 patients receiving transplantation in remission and in 17 of 38 receiving transplantation in relapse. Median follow-up of the 40 patients who survived event-free was 22 months (range, 1 to 65 months). Event-free survival (+/- standard deviation) rate was 48% +/- 8% and 46% +/- 10%, respectively, for the 42 AML and 24 ALL patients receiving transplantation in remission., Conclusion: Our transplantation procedure provides reliable, reproducible CD34+ cell purification, high engraftment rates, and prevention of GvHD. The mismatched-related transplant emerges as a viable, alternative source of stem cells for acute leukemia patients without matched donors and/or those who urgently need transplantation.

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 x 10(6) 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.