Immunoablation and autologous hematopoietic stem cell transplantation in the treatment of new-onset type 1 diabetes mellitus: long-term observations.

The activity of the autoimmune mechanism underlying type 1 diabetes mellitus (T1DM) can be suppressed when immunoablation and autologous hematopoietic stem cell transplantation (AHSCT) are applied early in the course of the disease. We report here a single centre experience with this treatment modality. Twenty-four patients underwent a AHSCT preceded by immunoablative conditioning with high-dose cyclophosphamide and anti-thymocyte globulin. During the 52-month median time of follow-up 20 out of 23 patients (87%) remained for at least 9.5 months without the use of exogenous insulin. The median time of T1DM remission for these patients was 31 months (range of 9.5-80 months). Among the patients available for follow-up (n=20), four remain insulin free (for 80, 61, 42 and 34 months). The average glycated hemoglobin (HbA1c) concentrations were 10.9% at diagnosis, 5.9% at 1 year, 6.4% at 2 years, 6.8% at 3 years and 7.1% at 4 years after AHSCT. No severe complications of diabetes were seen, however one of the patients died of pseudomonas sepsis in the course of neutropenia after AHSCT. AHSCT leads to a remission of T1DM with good glycemic control in the vast majority of patients, with the period of remission lasting over 5 years in some patients.

Independence of exogenous insulin following immunoablation and stem cell reconstitution in newly diagnosed diabetes type I.

Type I diabetes mellitus is a metabolic disease caused by chronic immune attack against the insulin-producing cells of the pancreas. It has recently been shown that the clinical course of this disease can be interrupted by immune ablation and PBSCT. In this report, we describe our experience with this treatment modality in a series of eight cases. Patients with newly diagnosed type I diabetes were received treatment consisting of two to three plasmaphereses, hematopoietic stem cell mobilization with CY and G-CSF, collection of at least 3 × 10(6) per kg of CD34+ cells, and conditioning with CY and anti-thymocyte globulin followed by stem cell infusion. All patients became independent of exogenous insulin after the transplantation. One patient resumed low-dose insulin 7 months after transplantation. Six out of eight patients were given acarbose for better glycemic control after transplantation. All patients exhibited good glycemic control: the average HbA1c concentrations were 12.3% at diagnosis, and 5.6 and 6.2% at 3 and 6 months after transplantation, respectively. We conclude that at least temporary independence of exogenous insulin can be achieved in type I diabetes patients following immunoablation and reconstitution of the immune system with autologous PBSCs.

Evaluation of relative anti-myeloma activity of high-dose melphalan followed by the first peripheral blood stem cell transplantation, as compared with the second transplantation, and to VAD chemotherapy.

Current treatment in multiple myeloma consists of three courses of chemotherapy in low doses with subsequent hematopoietic stem cell mobilization to the peripheral blood using high-dose cyclophosphamide, collection and conditioning with high-dose chemotherapy (melphalan) followed by retransplantation of autologous peripheral blood stem cells (PBSCT). Only a few studies compare the effects of different phases of therapy on parameters, such as monoclonal immunoglobulin level and the presence of malignant CD38(+) and CD56(+) cells in blood and marrow. The aim of this study was to compare the effects of these two major phases of treatment (conventional and high dose) in the same patients, and furthermore, to compare the effects of the second course of high-dose therapy followed by PBSCT with the effects of the first one. Fifteen patients were included in the study. On average, conventional chemotherapy only slightly reduced the values of all disease markers. In contrast, high-dose therapy resulted in a dramatic effect, rapidly normalizing the values of all parameters. The effects of second PBSCT were only modest compared to the first. These data suggest that high-dose therapy is an efficient method to reduce tumor load in multiple myeloma. Conventional-dose chemotherapy may be simply a waste of time for some patients and may be either omitted or administered after high-dose therapy to consolidate remission.

Triple transplantation of autologous peripheral blood stem cells each time following conditioning with 100 mg/m2 of melphalan for multiple myeloma patients in poor performance status.

Approximately one third of multiple myeloma patients (below 60 years) are diagnosed either in advanced disease or with significant comorbidities. Many other patients referred to transplant centers have already been heavily pretreated with multiple courses of various conventional chemotherapies. These patients are frequently in bad or even grave clinical condition; they are unlikely to survive standard high-dose melphalan (200 mg/m(3)) chemotherapy and autologous hematopoietic stem cell transplantation. Palumbo et al reported a protocol for elderly patients that utilized reduced conditioning (melphalan 100 mg/m(2) three times at 2-month intervals, each time supported by autologous hematopoietic rescue). We have used this protocol as a start to develop a method to induce a remission in the aforementioned subgroup of myeloma patients. Patients with stage III disease and WHO performance status 2 or higher are treated with one or two cycles of cyclophosphamide (2 to 4 g/m(2)) and undergo peripheral blood stem cells collection. Subsequently, they are treated with three to four doses of melphalan (100 mg/m(2)) at 8- to 12-weeks intervals each time supported by infusion of peripheral blood stem cells. To date 13 patients have been entered into the protocol. With one exception of transiently stable disease, the remaining patients obtained at least partial remission and three, complete remission. The compliance was good and better with each subsequent course. For half of the patients the problem was a short duration of response. This method when developed may offer a new treatment alternative for a subgroup of high-risk multiple myeloma patients.

Fourier and fractal analysis of maxillary alveolar ridge repair using platelet rich plasma (PRP) and inorganic bovine bone.

This report concerns the regeneration of the maxillary alveolar process in a 17-year-old patient who had lost the upper central incisors together with alveolar bone as a result of a car accident. Three months later, GBR (guided bone regeneration) was started with the use of autogenic platelet rich plasma (PRP) and inorganic bovine bone. The regenerated bone was analysed after 10 months and compared with intact bone using Fourier analysis of radiograms. The radial and spatial distribution of Fourier transforms showed that the original trabecular pattern existing in the intact bone on both sides of the defect was replicated in an evident way in the regenerated bone. Fractal analysis of intact and regenerated bone showed a higher fractal dimension for intact bone in comparison with regenerated bone, confirming a lower complexity of the newly formed trabecular structures. Replication of the original trabecular pattern in regenerated bone allows us to conclude that genetic mechanisms are influencing the organization of the trabecular pattern of regenerated bone tissue, probably under the influence of the growth factors contained in autologous PRP.

Colony-stimulating factor 1-dependent resident macrophages play a regulatory role in fighting Escherichia coli fecal peritonitis.

Osteopetrotic op/op mice have less than 5% of the normal number of macrophages in the peritoneal cavity (W. Wiktor-Jedrzejczak, A. Ahmed, C. Szczylik, and R.R. Skelly, J. Exp. Med. 156:1516-1527, 1982). Fecal peritonitis was induced by intraperitoneal injection of 0.5 ml of 5% autoclaved feces in saline along with Escherichia coli grown from feces of mice of the same colony and added in doses ranging between 10 and 10(6) CFU. Such infection led to a septic shock and either was lethal within 24 h or became cured without additional treatment of the mice. The op/op mice survived administration of 30-times-smaller doses of bacteria compared with their normal littermates. Analysis of the kinetics of cellular changes in the peritoneal cavity associated with such infection revealed that this increased susceptibility of macrophage-deficient mice cannot be explained by a direct role of macrophages in combating the infection. Instead, it appeared that the increased susceptibility to fatal fecal peritonitis was most likely due to delayed and impaired recruitment of neutrophils to the site of infection in mutant mice. The increased susceptibility of the op/op mice to E. coli fecal peritonitis was not due to their possible increased sensitivity to endotoxin, since the mutant mice tolerated lipopolysaccharide doses more than twice those tolerated by control littermates. On the other hand, their susceptibility to exogenous tumor necrosis factor alpha and interleukin-1 alpha was increased. Both mutant op/op and control mice were able to survive secondary challenge with 10(6) E. coli (administered along with feces) lethal for both types of mice on primary challenge. These data suggest that colony-stimulating factor 1-dependent resident peritoneal macrophages play a role in controlling primary infection by recruiting neutrophils and are not required for efficient response to secondary infection.

Granulocyte-macrophage colony-stimulating factor corrects macrophage deficiencies, but not osteopetrosis, in the colony-stimulating factor-1-deficient op/op mouse.

The op mutation in the mouse is in the coding region of the colony-stimulating factor-1 (CSF-1) gene, prevents formation of biologically active factor, and, thus, results in generalized macrophage deficiency and, in osteopetrosis, secondary to deficiency of osteoclasts. Although a few macrophages and osteoclasts are present in these mutants, it was not clear whether the inability of endogenous granulocyte-macrophage CSF (GM-CSF) to compensate for the absence of CSF-1 was due to the limitations of biological activity of this molecule or to its inability to reach respective target populations. In this study, we examined whether sc GM-CSF in large doses (20-40 micrograms/mouse.day) for 3 weeks would correct some or all of the deficiencies observed in mutant mice. All organ macrophage populations tested (liver, spleen, thymus, marrow, pleural, and peritoneal cavity) were significantly increased, reaching levels exceeding those observed in normal mice. Restoration of peritoneal and pleural macrophage populations by sc GM-CSF is of particular interest, because it was not previously observed in op/op mice treated with sc CSF-1. In contrast, there was no indication of increased bone resorption, no appearance of osteoclasts, and no tooth eruption in response to GM-CSF treatment. These data suggest that GM-CSF is able to compensate for the absence of CSF-1 during macrophage formation, but is unable to play a similar role in osteoclast differentiation.

Distinct in vivo functions of two macrophage subpopulations as evidenced by studies using macrophage-deficient op/op mouse.

The op/op mice totally lack macrophage growth factor colony-stimulating factor (CSF)-1 and thus, by definition are completely depleted of CSF-1-dependent functions of the macrophage cell lineage. Moreover, they possess a severe and generalized macrophage deficiency. However, residual macrophages of these mice should still have normal CSF-1-independent functions. Studies designed to elucidate this issue have revealed that op/op mice are capable of normal in vivo phagocytic function and demonstrate normal humoral and cellular response postimmunization with sheep red blood cells. However, release of monokines such as tumor necrosis factor and granulocyte CSF following administration of endotoxin is severely impaired in op/op mice as compared with littermate controls. These studies suggest that the CSF-1-dependent macrophage population (absent in the op/op mouse) is primarily responsible for regulatory functions of these cells mediated by monokines, while the CSF-1-independent macrophage population (present in the op/op mouse) is primarily responsible for the classical macrophage functions in immunity such as phagocytosis, antigen processing and presentation.

Administration of colony stimulating factor-1 corrects some macrophage, dental, and skeletal defects in an osteopetrotic mutation (toothless, tl) in the rat.

The toothless (tl/tl) mutation in the rat results in a paucity of osteoclasts and osteopetrosis that cannot be corrected by bone marrow transplantation. In the present study we demonstrate that tl/tl rats also have profound deficiencies of femoral, peritoneal, and pleural cavity macrophages. Furthermore, the macrophage colony stimulating activity of post-endotoxin sera from tl/tl rats is substantially reduced, suggesting that, as in the case of the op mutation in mice, the basis of the tl mutation is a deficiency of the macrophage growth factor, colony stimulating factor-1 (CSF-1). Consistent with this suggestion, treatment of tl/tl rats from birth for up to six weeks with CSF-1 reduced the osteopetrosis, increased body weight, and permitted tooth eruption. In addition, CSF-1 treatment induced large numbers of osteoclasts in tl/tl bones and macrophages in the peritoneal cavity and bone marrow. Persistence of metaphyseal sclerosis, however, indicated that the disease was not totally corrected by this treatment. These studies indicate that the basis of the tl mutation is most likely another CSF-1 deficiency, and further emphasize the role of this growth factor in osteoclast differentiation.

Correction by CSF-1 of defects in the osteopetrotic op/op mouse suggests local, developmental, and humoral requirements for this growth factor.

Mice that are mutant at the op locus have a severe deficiency of mononuclear phagocytes due to an inactivating mutation in the CSF-1 (macrophage colony-stimulating factor, M-CSF) gene. op/op mice are toothless, possessing skeletal abnormalities, a low body weight, and compromised fertility; they are osteopetrotic due to a deficiency of osteoclasts. The congenital osteopetrosis, toothless phenotype, osteoclast deficit, and the defects in splenic and femoral macrophages were corrected by routes of administration of human recombinant CSF-1 that maintained normal circulating CSF-1 concentrations. Early restoration of circulating CSF-1 was required for rescue of the toothless phenotype, but only partially restored body weight. In contrast, the deficiencies of pleural and peritoneal cavity macrophages and the reduced female fertility were not corrected by restoration of circulating CSF-1. These results suggest that although circulating CSF-1 is required for osteoclast and macrophage production, local synthesis and action of the growth factor are important for certain target cell populations.

[Effect of hemodialysis fluids with different pH values and buffer types used in peritoneal dialysis on the phagocyte system in the peritoneal cavity. The experimental part]. / Wplyw plynów dializacyjnych o róznych wartosciach pH i rodzajach buforów stosowanych w dializie otrzewnowej na uklad fagocytów jamy otrzewnowej. Czesc doswiadczalna.

In a four-hour exchange of dialysing fluid in 120 healthy mice it was checked whether and in what degree changes of the pH of the dialysing fluid in the range from 5.09 to 7.02 and change of the buffer in the dialysing fluid (acetate, lactate) have an effect on the phagocytic and bactericidal ability of the phagocytic cells in the peritoneal dialysate. The study of the count of phagocytic cells, their bactericidal ability (NBT test) and phagocytosis ability (latex test) showed that neither the type of the used buffer nor pH changes of the dialysing fluid in the range 5.09-6.18 had no effect on the defensive properties of the phagocytes in healthy peritoneal cavity. The observation of a statistically significant increase in the proportion of cells of low phagocytic ability in the group of mice receiving acetate buffer of pH 7.02 requires further studies.

Kinetics of destruction and regeneration of the haemopoietic system after administration of busulphan and cyclophosphamide followed by bone marrow transplantation. Peripheral blood parameters.

The kinetics of restoration of haemopoiesis was studied in 10 patients prepared for allogenic bone marrow transplantation with busulphan combined with cyclophosphamide. The morphology of peripheral blood after administration of these drugs and transplantation of allogenic bone marrow was similar to that reported elsewhere after irradiation, cyclophosphamide administration and bone marrow transplantation, the cell counts falling almost to zero within several days after the end of the pharmacological preparation, and later rising to normal values within the period from several weeks to several months after transplantation.

Hemopoiesis in mouse heterozygous for the W trait. Difference in postirradiation recovery related to the defect in formation of transient endogenous spleen colonies.

The hemopoietic regeneration following midlethal irradiation in Wv/+ mice had similarly biphasic kinetics as in their hematologically normal +/+ littermates. The first abortive phase of regeneration was either severely reduced (formation of transient endogenous spleen colonies, reticulocyte count, granulocyte count) or absent (spleen and femur cellularity, platelet count, PCV) in Wv/+ mice, when compared to +/+ mice. The second phase leading to permanent recovery of hemopoiesis was in Wv/+ mice delayed in time. Moreover, although to a lesser extent the values of spleen and femur cellularity, PCV and platelet count were decreased in Wv/+ mice. Postirradiation bleeding, which stimulated particularly the 1st phase of regeneration both in Wv/+ and +/+ mice did not lead to the minimization of differences between above two genotypes. It is suggested that the observed differences in the abortive regeneration between Wv/+ and +/+ mice are primarily dependent on the presence in Wv/+ mice of selective defect of transient endogenous colony forming units (TE-CFUs). Moreover, it is possible that the differences in the second phase of regeneration leading to permanent recovery are secondarily dependent on the TE-CFUs defect, as most probably the TE-CFU is the step in stem cell differentiation to mature cells.