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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 tecoplaninresistant 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 antifungal 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 skinground contact in accidents. [ABSTRACT FROM AUTHOR]

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]

A letter to the editor is presented which reports on the prevalence of absidia corymbifera necrotizing cellulitis in immunocompromised patients treated with voriconazole.

A letter to the editor is presented which discusses a case study of a 49-year-old with lymphoma-induced bone marrow aplasia.

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]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 × 106/kg), Tregs (mean 2.5 × 106/kg), and Tcons (mean 1.1 × 106/kg). No posttransplant immunosuppression was given. Ninety-five percent of patients achieved full-donor type engraftment and 15% developed sgrade 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. [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: 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]

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]

Immunotoxins were prepared with three ribosome-inactivating proteins (RIP), momordin, pokeweed antiviral protein from seeds (PAP-S) and saporin-S6, linked to the anti-CD22 monoclonal antibody OM124. These immunotoxins inhibited protein synthesis by CD22-expressing cell lines Daudi, EHM, BJAB, Raji and BM21 with IC50 (concentration causing 50% inhibition) ranging from < 5 × 10-15 to 7.6 × 10-11 M as RIP, and IC90 (concentration causing 90% inhibition) ranging from 5 × 10-14 to 5 × 10-8 M, with no effect on a CD22-negative HL60 cell line at the highest concentration tested (5 × 10-8 M). Apoptosis was induced in sensitive cells. The formation of bone marrow colonies was inhibited by no more than 40% by the immunotoxins at concentrations up to 10-9 M. Treatment with the immunotoxins, alone or in combination, significantly extended the survival time of mice bearing transplanted Daudi cells. A treatment with cyclophosphamide and OM124/saporin immunotoxin was particularly effective in SCID mice transplanted with a low number of cells (3 × 10-6), when 60% of the animals remained tumour-free. [ABSTRACT FROM AUTHOR]

Allogeneic hematopoietic stem cell transplantation (HSCT) is the most powerful therapy for patients with high risk of relapse. In spite of that, no matter the donor source or conditioning regimen used, leukemia relapse is still the leading cause of HSCT failure. In HLA-haploidentical HSCT, we recently applied a clinical protocol consisting of total body irradiation (TBI)-based conditioning regimen and a peripheral blood CD34+ cell graft combined with the adoptive transfer of naturally occurring regulatory T cells (Tregs) and conventional T cells (Tcons). No post-transplant pharmacologic GvHD prophylaxis was given. Such protocol was associated with low GvHD and relapse rate (Martelli et al., Blood 2014). To further reduce leukemia relapse in Treg/Tcon-based haploidentical HSCT (Treg/Tcon haplo-HSCT) we used high dose hyper-fractionated TBI (HF-TBI) in the conditioning regimen. We also extended Treg/Tcon haplo-HSCT to patients that are unfit (because of previous comorbidities) and/or too old to withstand high intensity regimens. In these patients the extra-hematologic toxicity of irradiation was reduced with the use of targeted total marrow and lymph node irradiation (TMLI). 40 patients with high risk acute leukemia (36 AML, 4 ALL) received Treg/Tcon haplo-HSCT. All but 3 patients were transplanted in complete remission. 12 younger patients (median age: 28, range: 20-43) received HF-TBI, while 28 older or unfit patients (59, 40-70) received TMLI in the conditioning regimen. HF-TBI (14.4 Gy) was administered in 12 fractions, 3 times a day for 4 days. TMLI was administered by means of Helical Tomotherapy HI-ART (9 fractions, 2 times a day for 4.5 days). Irradiation was followed by chemotherapy with Thiotepa, Fludarabine, and Cyclophosphamide. 2 × 106/kg freshly isolated CD4+CD25+FOXP3+ Tregs were transferred 4 days before the infusion of 1 × 106/kg Tcons and a mega-dose of CD34+ hematopoietic stem cells. No post-transplant pharmacologic GvHD prophylaxis was given. 38/40 patients engrafted. 12 (31%) developed aGvHD grade ³2 (10 are alive and off-therapy). 6 (16%) died because of transplant related complications (2 because of aGvHD, 2 infections, 1 veno-occlusive disease, 1 intracranial hemorrhage). Strikingly, despite the high risk diseases, no patient relapsed after a median follow up of 13 months (range 1-36, Fig. A). Further, only 1 patient developed cGvHD. Thus, cGvHD/Leukemia-free survival was 82% (Fig. B). Treg adoptive transfer allows for the safe infusion of an otherwise lethal dose of donor alloreactive Tcons in the absence of any other form of immune suppression. Our results demonstrate that the potent graft versus leukemia effect of Treg/Tcon adoptive transfer was boosted by high dose marrow irradiation. Thus, this study proves that the right combination of haploidentical Treg/Tcon immunotherapy plus a powerful conditioning regimen can fully eradicate leukemia. [ABSTRACT FROM AUTHOR]

Background: In this study we tried to achieve successful transplantation in patients with acute leukemia with the use of hematopoietic stem cells from donors who shared only one HLA haplotype with the recipient (a “full-haplotype mismatch”). To prevent graft failure, large doses of T-cell–depleted hematopoietic stem cells were transplanted after a conditioning regimen of enhanced myeloablation and immunosuppression was administered to the recipient. Methods: Forty-three patients with high-risk acute leukemia who were scheduled for transplantation received total-body irradiation, thiotepa, fludarabine, and antithymocyte globulin. The graft consisted of peripheral-blood progenitor cells that had been mobilized in the donor with recombinant granulocyte colony-stimulating factor and also, in 28 cases, bone marrow. Bone marrow from the donor was depleted of T lymphocytes by processing with soybean agglutinin and E-rosetting. T-cell depletion of peripheral-blood mononuclear cells was achieved by E-rosetting followed by positive selection of CD34+ cells. No post-transplantation prophylaxis against graft-versus-host disease (GVHD) was administered. Results: In all the patients, full donor-type engraftment was achieved. In none of the patients who could be evaluated did acute or chronic GVHD develop. Regimen-related toxicity was minimal. Eleven of the 23 patients with acute lymphoblastic leukemia had a relapse, as did 2 of the 20 patients with acute myeloid leukemia. Transplantation-related mortality was 40 percent. After a median follow-up of 18 months (range, 8 to 30), 12 of the 43 patients were alive and free of disease. All surviving patients had a good quality of life. Conclusions: The main limitations of transplantation of bone marrow from donors who are matched with the recipient for only one HLA haplotype — GVHD and graft failure — can be overcome. Since most patients have a relative with one haplotype mismatch, advances in this method will increase the availability of hematopoietic-cell transplantation as curative therapy for acute leukemia. (N Engl J Med 1998;339:1186-93.) [ABSTRACT FROM AUTHOR]