Understanding Concerns About Treatment-as-Prevention Among People with HIV who are not Using Antiretroviral Therapy.

The use of antiretroviral therapy to prevent HIV transmission is now advocated in many settings, yet little research has documented the views of people with HIV. Semi-structured interviews were conducted in Australia between 2012 and 2014 with 27 HIV-positive people not using treatment at the time of interview. Thematic analysis of views on treatment-as-prevention found that while many participants recognised potential prevention benefits, only a minority was in support of initiating treatment solely to achieve those benefits. A range of uncertain or critical views were expressed regarding who would benefit, risk reduction, and changing treatment norms. Participants resisted responsibility narratives that implied treatment should be used for the public good, in favour of making considered decisions about their preferred approach to managing HIV. Engaging communities in dialogue and debate regarding the risks and benefits of treatment will be critical if this new prevention strategy is to engender public trust.

Alloantigen persistence in induction and maintenance of transplantation tolerance.

Infusion of parental bone marrow cells into F1 hybrids conditioned by total lymphoid irradiation (TLI) results in chimeras with a high percentage of donor-type cells, and without clinical signs of graft-vs.-host reaction. In these chimeras, a state of tolerance has been shown to be associated with paucity of cytotoxic T lymphocyte percursors (pCTL) reactive with host-type alloantigens. To determine whether the presence of tolerizing alloantigens is essential for maintenance of unresponsiveness, lymphohematopoietic cells obtained from such tolerant chimeras were transferred into supralethally irradiated recipients of two different genotypes: in one case the adoptive recipients were syngeneic with host-type cells, and in the other they were syngeneic with donor-type cells of the original chimeras, thus providing the chimeric cells with a tolerogen-free environment. After "parking" for 4 d in syngeneic donor-type mice, the transferred cells displayed a marked increase in the frequency of pCTL directed against tolerizing alloantigens, whereas a low pCTL frequency directed against the same H-2 target cells was maintained in allogeneic tolerizing-type adoptive recipients. Multiple injections of adoptive donor-type mice with tolerizing-type cells of the original chimera reestablished a low level of cytotoxic precursors. Cytotoxic activity against unrelated alloantigens was independent of the presence of tolerogen-presenting cells in the adoptively transferred mice. Our experimental model suggests that persistence of cells bearing tolerizing alloantigens is an essential requirement for maintenance of previously established tolerance.

In vivo therapy of a murine B cell tumor (BCL1) using antibody-ricin A chain immunotoxins.

Prolonged remissions were induced in mice bearing advanced BCL1 tumors by the combined approach of nonspecific cytoreductive therapy and administration of a tumor-reactive immunotoxin. Thus, the vast majority of the tumor cells (approximately 95%) were first killed by nonspecific cytoreductive therapy using total lymphoid irradiation (TLI) and splenectomy. The residual tumor cells were then eliminated by intravenous administration of an anti-delta immunotoxin. In three of four experiments, all animals treated in the above fashion appeared tumor free 12-16 wk later. In one experiment, blood cells from the mice in remission were transferred to normal BALB/c recipients, and the latter animals have not developed detectable tumor for the 6 mo of observation. Because 1-10 adoptively transferred BCL1 cells will cause tumor in normal BALB/c mice by 12 wk, the inability to transfer tumor to recipients might indicate that the donor animals were tumor free. In the remainder of the animals treated with the tumor-reactive immunotoxin there was a substantial remission in all animals, but the disease eventually reappeared. In contrast, all mice treated with the control immunotoxin or antibody alone relapsed significantly earlier (3-4 wk after splenectomy).

Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: long-term survival of allogeneic bone marrow and skin grafts.

BALB/c mice were treated with fractionated high dose (3,400 rads) total lymphoid irradiation (TLI), and given semiallogeneic (BALB/c x C57BL/Ka) or allogeneic (C57BL/Ka) bone marrow and/or skin allografts. TLI alone prolonged the mean survival time (m.s.t.) of C57BL/Ka skin grafts to 49.1 days (control, 10.7 days). Shielding of the thymus during TLI produced only a slight increase in graft survival (m.s.t., 19 days). TLI combined with splenectomy was no more effective than TLI alone. Infusion of 10(7) semiallogeneic or allogeneic bone marrow cells after TLI produced stable chimeras in 7/8 and 8/15 recipients, respectively. Chimeras were specifically tolerant to donor tissues, since C57BL/Ka skin grafts were accepted for more than 250 days, but third-party (C3H/He) skin grafts were rejected rapidly. In addition, chimeric lymphocytes responded to C3H/He and C3H. Q but not to C57BL/Ka cells in the one-way mixed leukocyte reactions. BALB/c C57BL/Ka chimeras showed no clinical evidence of graft vs. host disease. These findings may have application of clinical organ transplantation, since (a) the recipient treatment (TLI) has already been shown to be safe in humans, (b) donors and recipients can be completely allogeneic, and (c) bone marrow and skin graft survival was permanent (greater than 250 days).

Transplantation of allogeneic bone marrow without graft-versus-host disease using total lymphoid irradiation.

Bone marrow (BM) and skin allografts from C57BL/Ka (H-2b/b) mice were transplanted to BALB/c (H-2d/d) recipients treated with total lymphoid irradiation (TLI), whole-body irradiation (WBI), or fractionated thymic irradiation TLI prolonged skin allograft survival about five times as long as that in untreated controls, and allowed for permanent engraftment of BM cells in approximately equal to 90% of recipients. None of the BM recipients showed clinical signs of graft-versus-host disease (GVHD) (diarrhea, weight loss, hunched back, etc.). On the other hand, recipients given WBI and allogeneic BM cells developed severe clinical GVHD. The majority of the latter recipients died within 12 days after BM transplantation, and 95% died within 61 days. Although TLI protected BALB/c mice against GVHD induced by BM cells, all recipients given TLI and allogeneic spleen cells developed lethal GVHD. Thymic irradiation alone marginally prolonged skin allograft survival, and did not allow for allogeneic BM engraftment. These results suggest that TLI may be a useful regimen in clinical BM transplantation, since this form of radiotherapy is used extensively in humans and has few severe side effects.

Transplantation tolerance in adult rats using total lymphoid irradiation: permanent survival of skin, heart, and marrow allografts.

Lewis rats given total lymphoid irradiation (TLI) accepted bone marrow allografts from AgB-incompatible donors. The chimeras showed no clinical signs of graft-versus-host disease. Skin allografts from the marrow donor strain survived for more than 150 days on the chimeras. However, third-party skin grafts were rejected promptly. Although heart allografts survived more than 300 days in Lewis recipients given TLI and bone marrow allografts, detectable levels of chimerism were not required for permanent survival.

Correction of enzyme deficiency in mice by allogeneic bone marrow transplantation with total lymphoid irradiation.

Enzyme deficiency was corrected in mice after allogeneic bone marrow transplantation with occurrence of graft versus host disease. beta-Glucuronidase-deficient C3H/HeJ mice were treated with total lymphoid irradiation. Normal bone marrow cells (30 X 10(6)) from BALB/c to C3H/HeJ chimeras (>90 percent circulating donor-type cells) without graft versus host disease. beta-Glucuronidase activity increases to normal levels in all chimeras as measured in the liver and in the plasma. Activity was maintained throughout an observation period of 7 months.

Long-term survival of skin allografts in mice treated with fractionated total lymphoid irradiation.

Treatment of recipient Balb/c mice with fractionated, high-dose total lymphoid irradiation, a procedure commonly used in the therapy of human malignant lymphomas, resulted in fivefold prolongation of the survival of C57BL/Ka skin allografts despite major histocompatibility differences between the strains (H-2d and H-2b, respectively). Infusion of 10(7) (C57BL/Ka x Balb/c)F1 bone marrow cells after total lymphoid irradiation further prolonged C57BL/Ka skin graft survival to more than 120 days. Total lymphoid irradiation may eventually prove useful in clinical organ transplantation.

Mesenchymal progenitor cells in red and yellow bone marrow.

Marrow cavities in all bones of newborn mammals contain haematopoietic tissue and stromal microenvironment that support haematopoiesis (haematopoietic microenvironment), known as red bone marrow (BM). From the early postnatal period onwards, the haematopoietic microenvironment, mainly in tubular bones of the extremities, is replaced by mesenchymal cells that accumulate lipid drops, known as yellow BM, whereas haematopoietic tissue gradually disappears. We analysed the ability of mesenchymal cell progenitors in red and yellow BM to produce bone and haematopoietic microenvironment in vivo after transplantation into normal or haematopoietically deficient (irradiated and old) recipients. We found that (1) normal substitution of red with yellow BM results from a gradual loss of mesenchymal stem cells (MSCs) capable of developing bone and haematopoietic microenvironment; (2) the mesenchymal cell population in tubular bones still containing active haematopoietic tissue gradually becomes depleted of MSCs, starting from a young age; (3) haematopoietic microenvironment is incapable of self-maintenance and its renewal depends on the presence of precursor cells; (4) the mesenchymal cell population remaining in areas with yellow BM contains cells able to develop functionally active haematopoietic microenvironment in conditions of haematopoietic insufficiency. Our data also indicate the possible existence of bi-potential stromal precursor cells producing either bone in normal, or bone together with active haematopoietic microenvironment in irradiated or old recipients. This study opens a spectrum of opportunities for the extension of haematopoietic territories by substituting the fat contents of BM cavities with haematopoietic tissue, thereby improving haematopoiesis compromised by cytotoxic treatments, irradiation, ageing, etc.

Completely mismatched allogeneic CD3/CD28 cross-linked Th1 memory cells elicit anti-leukemia effects in unconditioned hosts without GVHD toxicity.

Fully allogeneic CD3/CD28 cross-linked Th1 cells were found to elicit host-mediated anti-leukemia effects without GVHD toxicity. Mice inoculated with a lethal dose of BCL1 leukemia demonstrated significantly enhanced survival after allogeneic Th1 treatment. Cure rates of 12.5% with a single allogeneic cell infusion and 31.25% with multiple infusions were demonstrated. Cured mice were able to reject rechallenge with a lethal dose of tumor without further treatment. These results suggest that use of intentionally mis-matched, Th1 memory cells infused with cross-linked CD3/CD28 could represent a novel clinical approach to eliciting potent anti-tumor effects in patients without conditioning and without GVHD toxicity.

Time to engraftment following allogeneic stem cell transplantation is significantly longer in patients with myelodysplastic syndrome than with acute myeloid leukemia.

Patients with myelodysplastic syndrome (MDS) commonly present with pancytopenia, suggesting that the marrow stroma fails to support the growth of both malignant and normal stem cells. We therefore retrospectively analyzed the duration to engraftment of neutrophils (> or =0.5 x 10(9)/l and > or =1.0 x 10(9)/l) and platelets (> or =20 and > or =50 x 10(9)/l) in 37 MDS patients and 42 patients suffering from primary AML, following allogeneic SCT. A significantly shorter time to engraftment was documented in AML as compared to MDS patients in all four parameters. These results held true even when we subgrouped the patients according to gender, age (50 years being the cutoff age between young and elderly patients), patient-donor relationship, donor match and intensity of conditioning. To the best of our knowledge, this is the first time that such a comparison has been made. We suggest that the longer duration of post transplant pancytopenia that is frequently observed in MDS patients may also influence post transplant outcome.

Long-term follow-up of high-dose treatment with autologous haematopoietic progenitor cell support in 693 patients with follicular lymphoma: an EBMT registry study.

To evaluate the outcome of a large series of patients who received high-dose treatment (HDT) for follicular lymphoma (FL), 693 patients undergoing HDT (total-body irradiation (TBI)-containing regimen: 58%; autologous bone marrow (BM)/peripheral blood progenitor cells (PBPCs): 378/285 patients) were included in the study. A total of 375 patients (54%) developed recurrent lymphoma, 10-year progression-free survival (PFS) being 31%. On multivariate analysis, younger age (P=0.003) and HDT in first complete remission (CR1) (P<0.001) correlated with longer PFS. With a median follow-up of 10.3 years, 330 patients died. Ten-year overall survival (OS) from HDT was 52%. Shorter OS was associated on multivariate analysis with older age (P<0.001), chemoresistant disease (P<0.001), BM+PBPC as source of stem cells (P=0.007) and TBI-containing regimens (P=0.004). Thirty-nine patients developed secondary myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML), in 34 cases having received TBI as the conditioning regimen. The 5-year non-relapse mortality (NRM) was 9%. On multivariate analysis, older age (P<0.001), refractory disease (P<0.001) and TBI (P=0.04) were associated with a higher NRM. This long follow-up study shows a plateau in the PFS curve, suggesting that a selected group of patients might be cured with HDT. On the downside, TBI-containing regimens are associated with a negative impact on survival.

The anti-tumor effect of allogeneic bone marrow/stem cell transplant without graft vs. host disease toxicity and without a matched donor requirement?

The anti-tumor immune response that occurs in allogeneic bone marrow/stem cell transplant (BMT) settings is capable of eradicating tumors that are resistant to chemotherapy/radiation treatment. This anti-tumor immune response, known as the graft vs. tumor (GVT) effect, is the most effective immunotherapy treatment ever discovered. Unfortunately, the clinical application of GVT is severely limited due to the intimate association of GVT with the extremely toxic and often lethal side-effect known as graft vs. host disease (GVHD). It is a major research focus in the field of BMT to develop methods to separate the beneficial GVT effect from the detrimental GVHD toxicity. However, due to the intimate association of these effects, attempts to limit GVHD also have a tendency to limit the GVT effect. We propose a new concept for harnessing the power of the GVT effect without the toxicity of GVHD. Rather than trying to separate GVT from GVHD, we propose that these naturally coupled effects can 'mirrored' onto the host immune system and maintain their intimate association. The 'mirror' of GVHD is a host rejection of a graft (HVG). As rejection of an allograft would not be toxic, an HVG effect coupled to a host vs. tumor (HVT) effect, the 'mirror' of the GVT effect, would provide the anti-tumor effect of BMT without GVHD toxicity. In the 'mirror' setting, the HVT effect must occur against syngeneic tumors, while in the BMT setting the GVT effect occurs in the allogeneic setting. Previous attempts to elicit syngeneic anti-tumor immunity using therapeutic tumor vaccines have had disappointing results in the clinic due to the influence of tumor immunoavoidance mechanisms. We propose that the 'danger' signals that are released as a result of GVHD in the allogeneic BMT setting serve as an adjuvant to the GVT effect disabling tumor immunoavoidance. The chemotherapy/radiation conditioning prior to transplant is a required initiating event to the coupled GVT/GVHD effects. The conditioning releases 'danger' signals that mediate this adjuvant effect. To imitate this immunological event in immunocompetent, non-conditioned patients we propose that infusion of freshly activated, polyclonal CD4+ memory Th1 cells which express CD40L on the cell surface will stimulate a HVT/HVG 'mirror' effect, providing a non-toxic means to elicit the effective immune-mediated anti-tumor effect of BMT without the GVHD toxicity and without the requirement for a matched donor.

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.

Prevention and reversal of adoptively transferred, chronic relapsing experimental autoimmune encephalomyelitis with a single high dose cytoreductive treatment followed by syngeneic bone marrow transplantation.

A chronic relapsing form of experimental autoimmune encephalomyelitis (CR-EAE) was induced in SJL/J mice by adoptive transfer of lymph node cells (LNC) sensitized to guinea pig myelin basic protein (GMBP). We examined the efficacy of high dose immunosuppressive regimens (cyclophosphamide [CY] 300 mg/kg or total body irradiation [TBI] 900 cGy) followed by syngeneic bone marrow transplantation (SBMT) in prevention and treatment of already established CR-EAE. Treatment with TBI and SBMT on day 5 after the induction of CR-EAE, just before the onset of clinical signs, completely inhibited the appearance of the paralytic signs. The same treatment, applied 4 d after the clinical onset of the disease, led to a significant regression of the paralytic signs and to a total inhibition of spontaneous relapses during a follow-up period of 2 mo. Challenge of mice with GMBP+CFA 78 d after the passive induction of CR-EAE induced a relapse of the disease 7 d later in almost all of the untreated mice; in contrast, the same challenge given to TBI+SBMT-treated mice caused a delayed relapse (30 d later) in only a minority (3/7) of the challenged mice. In vitro lymphocytic proliferative responses to GMBP and purified protein derivative were significantly lower in TBI/SBMT-treated mice before and after the GMBP challenge, although these mice were fully immunocompetent, as evidenced by their normal lymphocytic proliferation to concanavalin A (ConA) and the FACS analysis of their lymphocytic subpopulations. A similar beneficial therapeutic effect was observed in mice treated with CY followed by SBMT, after the onset of CR-EAE. Our results could support possible clinical applications of similar therapeutic strategies, involving acute immunosuppression followed by stem cell transplantation and retolerization of the reconstituting immune cells in life-threatening neurological and multisystemic autoimmune diseases.

New strategies for bone marrow transplantation.

Adoptive immunotherapy of hematologic malignancies and metastatic solid tumors by donor lymphocyte infusion following induction of host-versus-graft transplantation tolerance against can best be achieved following nonmyeloablative stem-cell transplantation (NST). Induction of mixed chimerism may represent the best approach for induction of transplantation tolerance to donor alloantigens. Thus NST may become the optimal approach for the treatment of nonmalignant diseases, where replacement of host with donor hematopoietic cells is indicated: for correction of genetic or stem cell deficiency diseases; as a platform for immunotherapy of autoimmune or infectious diseases; or for induction of tolerance to organ allografts.

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.