Allogeneic hematopoietic stem cell transplantation in the elderly.

The development of reduced intensity or non-myeloablative conditioning (NST) in preparation for allogeneic stem cell transplantation (SCT) revolutionized the field and led to reconsideration of the dogma of upper age limit that was set up by the transplant centers as an eligibility parameter. Analysis of the literature data showed that NST regimens are associated with decreased transplant related mortality, and graft-versus-host disease, in comparison with standard myeloablative conditioning, in patients above the age of 50-55 years, or in younger patients with significant comorbidities. However we have to mention, that our considerations are based on the retrospective analysis of the literature data, and that well controlled prospective randomized studies are needed in order to definitely assess the role of NST. Comorbidity indices might be proved as the most important parameters for the choice of the most proper regimen for each patient in need and should be included in future trials.

Peliosis hepatis following treatment with androgen-steroids in patients with bone marrow failure syndromes.

Androgens widely used in the treatment of bone marrow failure syndromes can in rare cases cause hepatic peliosis, a pathological entity characterized by multiple blood-filled cavities in the liver parenchyma. Bone marrow failure syndromes per se are associated with a low coagulation status, which is further magnified by bone marrow transplantation for aplastic anaemia due to deep thrombocytopenia. Both these conditions can cause bleeding; their combination is especially dangerous. We describe two cases of aplastic anaemia due to paroxysmal nocturnal hemoglobinuria and Fanconi syndrome, in which patients developed peliosis hepatis after prolonged treatment with androgens. One patient developed severe subcapsular bleeding, successfully treated with catheterization of the right hepatic artery and embolization of the bleeding site. The second patient bridged over deep post-transplant aplasia with high frequency platelet transfusions, and demonstrated an uncomplicated post-BMT course. We suggest avoiding or interrupting treatment with androgens in patients preparing for BMT.

The effect of high-dose thiotepa, alone or in combination with other chemotherapeutic agents, on a murine B-cell leukemia model simulating autologous stem cell transplantation.

The use of thiotepa (TH) is increasing, especially in stem cell transplantation, mainly due to its safety and blood-brain barrier penetration. We evaluated the use of TH in a murine model simulating autologous stem cell transplantation, with or without additional agents. Between 1 and 11 days following inoculation of BALB/c mice with 10(5)-10(8) B-cell leukemia (BCL1) cells (simulating pre-transplant leukemia loads), each group received an 'induction-like' irradiation and/or cytotoxic regimen. Animals were either followed without treatment, or an adoptive transfer (AT) was performed to untreated BALB/c mice. Administered alone without AT, high-dose TH did not change the time to appearance of leukemia. Nevertheless, in the AT experiments, TH as a single agent showed better antileukemic activity than busulfan (BU). Cyclophosphamide (CY)-containing regimens were the most effective, and the TH-CY combination was as effective as the commonly used BU-CY combination, and more effective than the BU-TH combination. Moreover, a synergistic effect was seen in the TH-CY combination (none of the animals developed leukemia, whereas 4/10 animals in the CY-TBI group developed leukemia (P=0.029)). In conclusion, although TH produced only a moderate effect against BCL1 leukemia when used alone, its combination with CY is promising and should be tested further in allogeneic murine models and clinical studies.

Interleukin-7 induced facilitation of immunological reconstitution of sublethally irradiated mice following treatment with alloreactive spleen cells in a murine model of B-cell leukemia/lymphoma (BCL1).

Interleukin-7 (IL-7) plays a key role in maturation and function of both T and B cells. We investigate the potential use of recombinant human IL-7 for facilitation of graft-versus-leukemia (GVL) effects mediated by T cells following transplantation in a murine model. Administration of IL-7 in vivo to allogeneic-transplanted mice improved disease-free survival: 67% of mice treated with IL-7 remained alive and disease free for more than 60 days, in comparison to 17% of the controls (P<0.05). Similar results were obtained when C57BL/6 spleen cells sensitized against irradiated B-cell leukemia (BCL(1)) cells in the presence of IL-7 were transplanted to F(1) mice, followed by IL-7 treatment in vivo. Of the BALB/c mice that received spleen cells from F(1) mice treated with IL-7 following transplantation of C57BL/6 spleen cells sensitized with irradiated BCL(1) in the presence of IL-7, only 29% developed leukemia, as compared to 79% in the control group (P<0.05). Mice treated with IL-7 showed increased splenic and thymic cellularity and improved T cell-dependent proliferative responses compared to the controls (P<0.05). IL-7 may provide a novel tool to enhance immune reconstitution following transplantation of mismatched stem cells and for enhancement of GVL effects mediated by alloreactive lymphocytes.

Allogeneic stem cell transplantation from matched related and unrelated donors in thalassemia major patients using a reduced toxicity fludarabine-based regimen.

The only radical cure for thalassemia major patients today is the replacement of the defective hematopoietic system by allogeneic stem cell transplantation (allo-SCT). The major obstacles for the application of allo-SCT even from matched family members have been the transplant-related morbidity and mortality and graft failure that is usually associated with the recurrence of the thalassemia hematopoiesis. The outcome of allo-SCT from HLA-identical family donors is largely dependent on the age of the recipient as well as on pretransplant parameters reflecting the degree of organ damage from iron overload. In this study we report our experience of allo-SCT from matched related and unrelated donors, using a reduced toxicity conditioning consisting of fludarabine, busulfan or more recently busulfex and antithymocyte globulin, in a cohort of 20 patients with thalassemia major. The regimen-related toxicity was minimal, while the incidence of acute grade II-IV and chronic GVHD was 25 and 25%, respectively. With a median follow-up period of 39 months (range: 5-112 months) the overall survival was 100%, while thalassemia-free survival was 80%. Although the results of our study look promising, larger cohorts of patients and prospective clinical trials are required to confirm the benefits of our approach as a possible better alternative to the existing protocols.

Treatment of post-hematopoietic stem cell transplantation hemorrhagic cystitis with intravesicular sodium hyaluronate.

Hemorrhagic cystitis (HC) is a well-known complication of HSCT. Its overall incidence has been reported to vary from 7-68%. The spectrum of clinical presentation varies from asymptomatic microhematuria to life-threatening bleeding. Sodium hyaluronate is a glycosaminoglycan present on the bladder mucosa, which serves as an important protective substance against uroepithelial damage. Preparations of this component have been shown to be effective in the treatment of interstitial cystitis. We report our experience in the treatment of post-transplant HC with intravesical instillation of sodium hyaluronate. Five out of the seven patients included in this study achieved complete response, while one patient had only partial response. Sodium hyaluronate administration was not associated with any local or systemic adverse effects. We consider that the results of our study are promising and the efficacy of sodium hyaluronate in the treatment of post-transplant HC should be tested in larger cohorts of patients.

An antibody to amphiregulin, an abundant growth factor in patients' fluids, inhibits ovarian tumors.

Growth factors of the epidermal growth factor (EGF)/neuregulin family are involved in tumor progression and, accordingly, antibodies that intercept a cognate receptor, epidermal growth factor receptor (EGFR)/ERBB1, or a co-receptor, HER2, have been approved for cancer therapy. Although they might improve safety and delay onset of chemoresistance, no anti-ligand antibodies have been clinically approved. To identify suitable ligands, we surveyed fluids from ovarian and lung cancer patients and found that amphiregulin (AREG) is the most abundant and generalized ligand secreted by advanced tumors. AREG is a low affinity EGFR ligand, which is upregulated following treatment with chemotherapeutic drugs. Because AREG depletion retarded growth of xenografted ovarian tumors in mice, we generated a neutralizing monoclonal anti-AREG antibody. The antibody inhibited growth of ovarian cancer xenografts and strongly enhanced chemotherapy efficacy. Taken together, these results raise the possibility that AREG and other low- or high-affinity binders of EGFR might serve as potential targets for cancer therapy.

Effect of Linomide on adhesion molecules, TNF-alpha, nitrogen oxide, and cell adhesion.

Linomide (quinoline-3-carboxamide) is an immunomodulator with anti-inflammatory effects in rodents with autoimmune diseases. Its mode of action still remains to be elucidated. We hypothesized that an investigation of T cell interactions with the extracellular matrix (ECM), composed of glycoproteins such as fibronectin (FN) and laminin (LN), might provide better understanding of their in vivo mode of action in extravascular inflammatory sites. We examined the effect of Linomide on T cell adhesion to intact ECM, and separately to LN, and FN, and on the release and production of tumor necrosis factor (TNFalpha) and nitrogen oxide (NO) in relation to adhesive molecules in non-obese diabetic (NOD) female spleen cells, focusing on intracellular adhesion molecule-1 (ICAM-1) and CD44. NOD female mice that developed spontaneous autoimmune insulitis, which destroys pancreatic islets and subsequently leads to insulin-deficient diabetes mellitus, were studied. Linomide, given in the drinking water or added to tissue cultures in vitro, inhibited the beta1 integrin-mediated adhesion of T cells to ECM, FN and LN, as well as the production and release of TNFalpha and NO, which play a major role in the induction and propagation of T cell-mediated insulitis. In addition, exposure of T cells to Linomide resulted in increased expression of CD44 and ICAM-1 molecules on spleen cells of Linomide-treated mice; such an increase in adhesion molecule expression may lead to more effective arrest of T cell migration in vivo. The regulation of T-cell adhesion, adhesion receptor expression, and inhibition of TNFalpha and NO secretion by Linomide may explain its beneficial role and provide a new tool for suppressing self-reactive T cell-dependent autoimmune diseases.

Stimulation of immune reconstitution by interleukin-7 after syngeneic bone marrow transplantation in mice.

Successful outcome of autologous bone marrow transplantation (BMT) is severely handicapped by susceptibility to infection and by a high rate of relapse. While quantitative aspects of the immune system generally return to normal within the first 3-4 months after BMT, the recovery of qualitative immune functions is prolonged. Since interleukin-7 (IL-7) has growth-promoting and differentiating effects on pre-B cells and immature thymocytes, its role in the recovery of immune functions was investigated in BALB/c mice after syngeneic BMT (sBMT). After sBMT, mice treated with human recombinant IL-7 (rIL-7) showed an 11.9-fold increase in thymic cellularity associated with an enhanced response to a mitogenic stimulus compared with the controls. rIL-7 significantly increased RAG-1 expression and promoted V beta 8(D)J gene rearrangement of the T cell receptor in the thymus. Further, the cytokine boosted survival after challenge with influenza virus following sBMT. The finding that rIL-7 induces differentiation and proliferation of immature thymocytes and counteracts post-BMT immune deficiency makes it a promising medium for clinical application in BMT patients.

Involvement of interleukin-2 in immunologic reconstitution following bone marrow transplantation in mice.

Allogeneic or autologous bone marrow transplantation (BMT) is a curative form of treatment for patients with a variety of hematologic disorders. Impaired immune reconstitution following BMT may seriously impede successful outcome. In this study, the immune function of spleen and thymus was investigated in mice exposed to myeloablative total-body irradiation followed by syngeneic BMT. The T cell mitogen-induced proliferation of both splenic and thymic cells was delayed. Spleen cells started to respond only after 21 days, whereas thymic cells remained unresponsive. Kinetic analysis of surface markers revealed the early appearance of spleen cells with the CD3+CD4-CD8- phenotype, and the thymus, despite a low total number of cells, displayed fast recovery of CD3+CD4+CD8+. At the level of mRNA, a mild decrease in interleukin-2 (IL-2) induction following phytohemagglutinin activation of spleen cells correlated with a decrease in IL-2 secretion for only the first 2 weeks following transplantation. The early restoration of IL-2 implies other avenues for investigation of the immune dysfunction and its correction following syngeneic BMT.

Fludarabine in combination with cyclophosphamide decreases incidence of GVHD and maintains effective graft-versus-leukemia effect after allogeneic stem cell transplantation in murine lymphocytic leukemia.

Graft-versus-host disease (GVHD) is a severe disorder and despite therapeutic efforts to decrease its distressing clinical manifestations, treatment is still not optimal. Here we report the results of studies, in which the purine analogue, fludarabine phosphate, was used in an attempt to modify and decrease GVHD after stem cell transplantation, across major histocompatibility barriers for murine leukemia. B-cell leukemia (BCL-1) bearing (BALB/c x C57BL/6) F1 mice received two cycles of fludarabine (0.8 mg/kg) for 5 days every 2 weeks, followed by 400 mg/kg cyclophosphamide i.p. Animals were then transplanted with C57BL/6 precursor cells and the development of leukemia and extent of GVHD was monitored both clinically and histopathologically. In the fludarabine-treated group, only nine of 28 (32%) mice developed leukemia, compared to 25 of 33 (76%) of control animals (P=0.0006 ). Mice treated with fludarabine-containing regimens prior to transplantation also had much less GVHD both clinically and at autopsy, while graft-versus-leukemia appeared to be augmented in the same animals.

Allogeneic cell-mediated immunotherapy of leukemia with immune donor lymphocytes to upregulate antitumor effects and downregulate antihost responses.

Donor lymphocyte infusion mediates most effective graft- versus-leukemia (GVL) effects following induction of host-versus-graft tolerance by transplantation of donor stem cells. This study was designed to maximize GVL effects across both major (MHC) and minor (mHgs) histocompatibility barriers in recipients inoculated with murine B-cell leukemia (BCL1), using specifically immune donor lymphocytes. GVL effects were induced with donor spleen cells from mice immunized across MHC or mHgs barriers with BCL/1 cells or normal BALB/c spleen cells. Our data suggest that spleen cells from donor mice immunized against murine B-cell leukemia of BALB/c origin, or to a lesser extent against normal host alloantigens, induce better therapeutic GVL effects with less great-versus-host disease (GVHD) across both mHgs and MHC. The cytokine profile of effector cells inducing predominantly GVL effects with reduced GVHD across MHC and mHg barriers consisted preferentially of upregulated IFN-gamma, IL-2, IL-10 and IL-12 in donors, implying a Th-1 to Th-2 cytokine shift. We hypothesize that immunotherapy with immune donor lymphocytes sensitized in vivo or in vitro with allogeneic tumor cells or normal host cells together with allogeneic BMT may provide an effective approach for amplifying GVL effects, while reducing procedure-related morbidity and mortality due to uncontrolled GVHD.

Interleukin-7-enhanced cytotoxic T lymphocyte activity after viral infection in marrow transplanted mice.

Lethally irradiated BALB/c mice were reconstituted by syngeneic bone marrow transplantation (BMT), and injected with recombinant interleukin 7 (rIL-7), recombinant interleukin 2 (rIL-2), or saline 10 days post-transplantation. Intranasal infection with A/PR8/34 influenza virus 2 weeks after BMT was associated with the highest survival rate in the rIL-7 treated group. The protective mechanism elicited by rIL-7, as manifested by very low virus titers in the lung, involves T and B cell functions. High hemagglutinin inhibition antibody levels were observed on days 7 and 12 post-challenge in the rIL-7 mice. Moreover, the anti-influenza cytotoxic T lymphocyte activity was induced primarily by rIL-7, leaving the effect of rIL-2 on the same level as that of the control. Thus, rIL-7 promotes both T cell-mediated function and B cell production during the immunodeficient state after BMT. This cytokine may prove a potential immunotherapeutic modality in BMT recipients.

Activation of the recombination activating gene 1 (RAG-1) transcript in bone marrow of senescent C57BL/6 mice by recombinant interleukin-7.

The level of the recombination activating gene 1 (RAG-1) mRNA in bone marrow cells decreases to a minimal level by the age of 10 months. Recominbant interleukin-7 (rIL-7) is a potent proliferative stimulus for B cell progenitors and upregulates RAG-1 expression in lymphocyte precursors. To investigate the stimulatory effect of rIL-7 on the expression of RAG-1 in old mice, we compared the level of RAG-1 message in short-term bone marrow cultures of cells from mice aged 1 month and 18 months. We found similar levels of RAG-1 mRNA in bone marrow cells of young mice before and after 24 hours of incubation. No RAG-1 mRNA was detected in bone marrow cell cultures prepared from old mice after 24 hours of incubation. However, when rIL-7 was added to the culture medium, RAG-1 mRNA was detected after 24 hours of incubation and its level was similar to that measured in cells from young mice. The expression of RAG-1 was dose-dependent, with 20 ng of rIL-7 per 10(6) old nucleated cells yielding the maximal response. Our results indicate that despite the low or no RAG-1 expression in bone marrow cultures of old mice, the potential to activate RAG-1 in B-cell precursors is still present, and immunoglobulin heavy chain (V(H)D(H)J(H)) rearrangement may be enhanced by rIL7.

The number of infused CD34+ cells does not influence the incidence of GVHD or the outcome of allogeneic PBSC transplantation, using reduced-intensity conditioning and antithymocyte globulin.

The influence of graft composition on the outcome of reduced-intensity (RIC) allogeneic PBSC transplantation (allo-PBSC) remains controversial. In this study, we analyzed the impact of CD34+ cell dose on the incidence of GVHD, and on the outcome after allo-PBSC, in 103 patients with hematological malignancies, using a uniform RIC regimen. The following variables were included in statistical analysis: (1) number of C34+ cells, (2) high-risk vs low-risk disease status, (3) matched related vs matched unrelated donor, (4) female donor to male recipient vs any other combination, (5) age of recipient (above vs below the median). Univariate and multivariate analysis did not reveal any association between CD34+ cell dose and acute grade-2 to grade-4, cGVHD, non-relapse mortality (NRM), relapse rate (RR) and OS. High-risk disease status was the only variable independently associated with increased NRM (P=0.001), increased RR (P=0.012) and decreased OS (P<0.001). The same results were obtained when analysis was restricted to a subgroup of 55 patients with myeloid neoplasms. The influence of graft composition on the outcome of RIC allo-PBSC should be further investigated via well-controlled randomized prospective studies.

Alefacept treatment for refractory chronic extensive GVHD.

Alefacept (Amevive) is an immunosuppressive dimeric fusion protein that is used for psoriasis control. We recently showed its effect in acute steroid-resistant/dependent GVHD. In this study, we describe the effect of alefacept treatment on chronic extensive GVHD (cGVHD). Twelve patients were included in this study; of these 8 (9 of 13 episodes) showed response. The median time to initial response was 2.25 weeks and the response was marked (n=3), moderate (n=2) or minimal (n=4). In two responding patients, the response was only temporary. Complications that appeared during treatment included infection, pericarditis and squamous cell carcinoma of the lip. All these events may be related to other drugs given simultaneously. With a 30-month median follow-up, 6 of 12 patients are alive, with all but one with stable or improved cGVHD. Six patients died because of GVHD progression, whereas none of the patients experienced relapse of the disease for which the transplantation was done. As reported earlier in psoriatic patients treated with alefacept, we found a consistent increase in the percentage of naive T cells as a consequence of treatment. In conclusion, alefacept is effective for the treatment of cGVHD, and dose and time intervals of treatment should be explored further.

Reviewing the potential utility of interleukin-7 as a promoter of thymopoiesis and immune recovery.

The identification of interleukin-7 (IL-7) as a critical cytokine in early B- and T-cell development, combined with the discovery that it acts on mature T cells, opens new avenues for investigating the thymopoietic machinery and manipulation of the immune system. Initially, IL-7 was thought to be a growth factor in the context of the B-cell lineage in that it stimulates proliferation of early B-cell progenitors. However, it appears that this cytokine has a much broader field of activity within the network of signal transduction. Indeed, evidence exists to support the pivotal involvement of IL-7 in the gene rearrangement of the T-cell receptor repertoire that ultimately leads to thymocyte commitment. The finding that IL-7 is an inducer of both cytotoxic T-cell- and lymphocyte-activated killer cells is one of the significant recent developments in the field of tumor immunology. Lately, it has been demonstrated that administration of IL-7 to mice after myeloablative treatment accelerates immune recovery via a unique pathway. This review of the literature dealing with IL-7 in the realm of immune function shows, inter alia, the value of the cytokine in immunosuppressed animals. The collection of findings noted in this paper may be considered the forerunner for clinical application of IL-7 in a variety of conditions of hematolymphopoietic failure.

Pharmacokinetic and pharmacodynamic analysis of (E)-2-ene valproyl derivatives of glycine and valproyl derivatives of nipecotic acid.

GABA is a major inhibitory neurotransmitter in mammals, whose uptake in glial cells is inhibited by nipecotic acid. In addition to GABA, glycine is an important inhibitory neurotransmitter. Valproic acid (VPA) is one of the four established antiepileptics and (E)-2-ene valproic acid ((E)-2-ene VPA) is its major active metabolite. The described structure-pharmacokinetic-pharmacodynamic relationship (SPPR) study explored the possibility of utilizing valproyl derivatives of glycine and nipecotic acid as new antiepileptics. The pharmacokinetics and pharmacodynamics (anticonvulsant activity and neurotoxicity) of the following conjugation products were investigated: (E)-2-ene valproyl glycinamide (between (E)-2-ene VPA and glycinamide) and valproyl nipecotic acid and valproyl nipecotamide (between VPA and nipecotic acid). Out of the investigated compounds only (E)-2-ene valproyl glycinamide showed a good anticonvulsant profile in both mice and rats due to its better pharmacokinetic and pharmacodynamic profile. (E)-2-ene valproyl glycinamide was more potent than VPA and showed an activity and a safety margin similar to those of its analogous compound valproyl glycinamide. The investigated valproyl derivatives did not operate as chemical drug delivery systems (CDDSs) of glycine or nipecotic acid, but, rather, acted as drugs on their own. (E)-2-ene valproyl glycinamide was partially excreted unchanged in the urine (fe = 7.4%), while its urinary metabolite was (E)-2-ene valproyl glycine. Unlike the new antiepileptic tiagabine, in which nipecotic acid is attached to 4, 4-di-(3-methylthien-2-yl)-3-butenyl and yields an active compound, the conjugation between nipecotic acid or its amide and VPA yielded inactive entities. In contrast to nipecotic acid, the conjugation between VPA or (E)-2-ene VPA and glycinamide gave two active compounds with similar pharmacokinetic and pharmacodynamic profiles.

Pharmacokinetic analysis and antiepileptic activity of tetra-methylcyclopropane analogues of valpromide.

PURPOSE: The described structure pharmacokinetic pharmacodynamic relationships (SPPR) study explored the utilization of tetramethylcyclopropane analogues of valpromide (VPD), or tetramethylcyclopropane carboxamide derivatives of valproic acid (VPA) as new antiepileptics. METHODS: The study was carried out by investigating the pharmacokinetics in dogs and pharmacodynamics (anticonvulsant activity and neurotoxicity) of the following three cyclopropane analogues of VPD: 2,2,3,3-tetramethylcyclopropane carboxamide (TMCD), N-methyl TMCD (M-TMCD) and N-[(2,2,3,3-tetramethylcyclopropyl)carbonyl]-glycinamide (TMC-GLD). RESULTS: The three investigated compounds showed a good anticonvulsant profile in mice and rats due to the fact that they were metabolically stable VPD analogues which were not biotransformed to their nonactive acid, 2,2,3,3-tetramethylcyclopropane carboxylic acid (TMCA). M-TMCD was metabolized to TMCD and TMC-GLD underwent partial biotransformation to its glycine analogue N-[(2,2,3,3-tetramethylcyclopropyl)carbonyl]-glycine (TMC-GLN). Unlike TMC-GLN, the above mentioned amides had low clearance and a relatively long half life. CONCLUSIONS: In contrast to VPD which is biotransformed to VPA, the aforementioned cyclopropane derivatives were found to be stable to amide-acid biotransformation. TMCD and M-TMCD show that cyclic analogues of VPD, like its aliphatic isomers, must have either two substitutions at the beta position to the carbonyl, such as in the case of TMCD, or a substitution in the alpha and in the beta positions like in the VPD isomer, valnoctamide (VCD). This paper discusses the antiepileptic potential of tetramethylcyclopropane analogues of VPD which are in animal models more potent than VPA and may be non-teratogenic and non-hepatotoxic.