ALBERT

Description: Insulin-dependent type I diabetes mellitus (IDDM) is caused by autoimmune destruction of pancreatic β-islet cells mediated by inflammatory T cells. The pathogenic process evolves gradually for several years and becomes symptomatic when most Langerhans islands are destroyed. Antibodies against β-cell antigens (like anti-glutamic acid decarboxylase, GAD) are markers of the autoimmune reaction and levels of proinsulin C-peptide correlate with endogenous insulin secretion. Several immunosuppressive regimens have demonstrated clinical and laboratorial benefit in early onset IDDM, presumably sparing islets reserve, but most responses were transient and long term toxicity limited their continuous use. In view of durable remissions observed in various autoimmune diseases treated with high-dose immunosuppression and autologous hematopoietic stem cell transplantation (AHSCT), we started in December/03 a phase I/II trial of AHSCT in early-onset IDDM. Patients from 12–35 years old with 2 million/kg) after conditioning with 200 mg/kg cyclophosphamide and 4,5 mg/kg rabbit antithymocyte globulin- ATG (Thymoglobuline, SangStat). End points of the study are insulin needs (U/kg/d), glycosilated hemoglobin levels, anti-GAD titers and C-peptide levels. Four patients have been transplanted and the insulin usage of the first three patients is shown in the Figure. The first patient received high dose of steroids to prevent ATG hypersensitivity and showed increasing needs of insulin after mobilization. The other two patients received minimal (#2) or no (#3) steroid dose during conditioning and showed decreasing needs of insulin after mobilization (Figure). Patient #2 presented bilateral pneumonia while pancytopenic, recovered after treatment with antibiotics and Amphotericin-B but did not require insulin therapy. A fourth patient has just been discharged from the BMT Unit. Immunologic studies in the three patients with longer follow-up showed a progressive shift from Th1 to Th2 cytokine profile after transplantation which could provide a mechanism for the modulation of the autoimmune process by high dose immunosuppression and autologous HSC. In conclusion, the preliminary results are encouraging but must be validated with a larger number of patients (12 planned in this phase) and a longer followup (5 years). Figure Figure

Description: For patients with refractory or high risk leukemias, stem cell transplantation is still the only possible curative approach. Those patients, although, without a HLA identical matched sibling, unrelated stem cells are the only stem cell source. In patients with refractory disease this unrelated cells may be unavailable in a short time period. The use of HLA-mismatched familiy donors has been used before, by depleting in vitro T cells or selecting CD34+ cells. These methods are extremely expensive and patients suffer with delayed immune reconstitution. In an attempt to overcome the HLA barrier, Cyclophosphamide, a highly toxic agent against activated T cells, but not myeloablative, has been used after transplantation to ensure engraftment and avoid GVHD. We describe here two patients who were sequentially submitted to a citoreductive protocol described by Schmid et al (Blood 2006) followed after transplantation by Cyclophosphamide (50 mg/kg day +3 and day +4) as described by O’Donnel et al (BBMT 2002). We transplanted 2 boys (16 years-case 1 and 6 years-case 2) with secondary AML after MDS and AML relapse after second SCT respectively. Case 2 received two SCT from his HLA-id brother and was 8 months after the second transplant. Both had active disease at time of transplant and received full bone marrow from there HLA haploidentical mothers (3 loci mismatch). Engraftment was achieved on day +15 in both cases. Chimerism analysis on day +30 showed that they had 96% XX and 95% XX cells respectively in bone marrow. Case 1 never developed GVHD but relapsed after SCT. Case 2 are in complete remission and complete quimera day+90 after SCT. He developed GVHD (skin, liver and gut) were treated with prednisone and Basiliximab and we are now tapering imunossupression. Herby we demonstrated that haploidentical transplantation with the use of Cyclophosphamide after transplantation can be a useful transplant strategy in high risk patients without a matched sibling donor. Relapse and infections will always be the most frequent complications. In countries with limited health resources a protocol who avoid the expensive costs of cell depletion or selection can be a real alternative for patients without suitable donors.

Description: Introduction: Multiple sclerosis (MS) is a chronic, inflammatory and demyelinating disease of the central nervous system (CNS) leading to gradual axonal degeneration. It is a classical auto-immune disease, thus high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (AHSCT) could “reset” the immune disorder by controlling autoreactive clones and by restoring self-tolerance after immunological recovery. Objectives: Describe the experience of the Brazilian Cooperative Group of AHSCT for Autoimmune Diseases in 41 MS patients followed-up to five years. Methods: Stem cells were mobilized from the bone marrow with Cy (2g/m2) and Filgrastim (10μg/kg/d SC), collected by leukapheresis and cryopreserved. Patients were conditioned with BEAM (BCNU 300 mg/m2, cytarabine 200 mg/m2, etoposide 200 mg/m2 ×4, melphalan 140 mg/m2) and horse antilymphocyte globulin (ATG) 90 mg/kg) or cyclophosphamide (CY) 50 mg/kg ×4 and rabbit ATG (rATG) 4.5 mg/kg, followed by stem cell infusion. Quality of life was assessed by the Short Form-36 Health Survey Questionnaire (SF-36). Results: Between January 2001 and June 2006, 21 patients received BEAM/ATG and 20 received CY/ATG. Medium age was 42 years (27–53 years), 24 (58.5%) were females, 33 (80.4%) had secondary progressive MS form, 80.95% had EDSS (Expanded Disability Status Scale) 6.0 or higher. No significant difference was seen between the groups regarding age, sex, disease presentation and EDSS. The mean number of CD34+ infused cells was 8.8×106/kg (2,5– 25.13×106/kg). Mean engraftment was 9,3 days (7–10 days) for neutrophils and 13 days (7–24 days) for platelets. 18 patients (46%) had neutropenic fever, 8 (20.0%) had pneumonia and 5 (12.5%) had allergic reactions to lymphoglobulin. Three patients (14.3%) died in the BEAM group due to conditioning regiment toxicity, sepsis and alveolar hemorrhage. Mean hospital stay was 35.47 days (20–168 days) in the BEAM group and 20.15 days (14–32 days) in the CY group (p

Description: T-cell prolymphocytic leukemia (T-PLL) is a malignant proliferation of lymphoid cells with a mature postthymic phenotype. The disease is characterized by lymphadenopathy, splenomegaly, skin lesions, and elevated white blood cell count, and it is often resistant to conventional therapy. Classic cytogenetic studies have revealed the presence of complex karyotypes and some recurrent chromosomal abnormalities, of which the most frequent are t(14;14)(q11;q32), inv(14)(q11q32), t(X;14)(q28;q11), i(8)(q10), and t(8;8)(p12;q11). Conventional cytogenetic techniques are insufficient to fully characterize chromosomal abnormalities, especially complex and cryptic aberrations. In this report, we describe a case of T-PLL with t(Y;14)(q12;q11) and a ring chromosome r[i(8)(q10)] studied by classic cytogenetics and spectral karyotyping (SKY). These abnormalities have not been previously described in T-PLL. A 41-year-old man was admitted with night sweats, weight loss, lymphadenopathy and hepatosplenomegaly without skin rash. Peripheral blood counts were: platelets 128 x 109/L and leukocytes 93.6 x 109/L. Hemoglobin levels were 12.5 g/dL. Immunophenotypic studies showed the following results: CD2+, CD3+, CD5+, CD7+, CD4−, CD8+, T-cell receptor (TCR) a/b+, CD1-, and terminal deoxynucleotidyl transferase-negative (TdT-). The patient was treated with fludarabine and CHOP therapy, neither of which resulted in a response. The patient was then treated with alemtuzumab (Campath®) and complete resolution of lymphadenopathy and normalization of blood counts was achieved. Classic cytogenetic and SKY analyses were performed on a 3-day phytohemagglutinin-stimulated culture of peripheral blood lymphocytes. The observed karyotype was abnormal in 18 of the metaphases analyzed: 46, t(X;14)(q28;q11), t(Y;14)(q12;q11), r[i(8)(q10)]. The co-existence of 2 translocations involving both sex chromosomes in P-TLL is a rare occurrence; however, abnormalities involving band Xq28 (the site for the MCTP-1B1 gene which has homology to TCL-1 on 14q32.1) are common in T-PLL. In addition, translocations involving the 14q11-q13 region (TCRa and TCRd) result in juxtaposition of enhancer elements responsible for the expression of TCR genes next to different oncogene loci. This leads to disruption of transcriptional pathways involved in normal T cell development, and ultimately, leukemic transformation. The fibroblast growth factor receptor-1 (FGFR-1) gene, in 8p11, has been implicated in a recurrent breakpoint in a myeloproliferative syndrome associated with T-cell lymphoma and the monocytic leukemia zinc finger (MOZ) gene in acute myelogenous leukemia. The amplification of the q arm of chromosome 8 accompanied by deletion of 8p sequences distal to the breakpoints could mean that two events act synergistically to contribute to the malignant phenotype in T-PLL. It is possible to suggest that the loss of a tumor suppressor gene or activation of an oncogene on 8p cooperates with amplification of the q arm and/or the expression of TCL-1/MTCP-1B1 in T-PLL. Secondary abnormalities of chromosome 8 may play an important role in the development of T-PLL. In this case of T-PLL with rare cytogenetic abnormalities, treatment with alemtuzumab resulted in a clinical response.

Description: Background. High dose chemotherapy (HDCT) followed by autologous PBSC rescue has been increasingly used for the treatment of several human diseases. However, little is known on the extent of this therapy on the marrow mesenchymal stem cells (MSCs). Aims. To evaluate the feasibility of expansion and multipotencial differentiation of MSCs isolated from patients after HDCT. Patients and Methods. Twelve lymphoma’s patients (LP) free of disease in bone marrow (BM) were enrolled in the study. They were submitted to BEAM’s protocol with autologous PBSC rescue 28 to 1836 days before the sample collection. Six normal bone marrow donors (ND) were used as controls. The LP and ND median age were 37.5 (range 22–49) and 31.5 years old (range 23–42), respectively. MSCs were isolated by plastic adherence and expanded ex vivo by cultivation in flasks with α-MEM with 15% fetal bovine serum. Media was changed every 3–4 days. At 90% confluence, the cells were re-plated and expanded. The isolation efficiency, colony-forming unit-fibroblast (CFU-F) frequency, growth kinetics, phenotypic characteristics, cell cycle status, multi-lineage differentiation capacity as well as hematopoiesis-supportive function were determined and compared with those of ND-MSCs. This study protocol and the consent form were approved by the institution ethics committees. Results. The results were analyzed by Mann-Whitney test and are expressed as median (range) to LP and ND, respectively. MSCs were successful isolated from all BM samples collected for this study. The cell population showed typical fibroblast-like morphology, appearing as an adherent, spindle shaped cell layer and growing to confluence after a few weeks of culture. The number of CFU-F found at 14 days of culture were 0.94 (0.00–3.75) and 1.25 (0.13–9.25) x10−5 nucleated cells (p = 0.4421). The doubling time between the 1st and 2nd passages was 80.66 (34.08–195.35) and 46.30 (36.36–270.59) hours (p = 0.1025). The cell clones proliferated extensively until 8.17 (1.81–28.27) and 18.11 (11.85–27.48) population doublings (p = 0.0668) in 71.50 (46–88) and 81 (57–103) cultivation days (p = 0.1505). Immunophenotypically, these cells were positive for the CD73, CD105, CD90, CD29, CD13, CD44, CD49e, CD54, HLA-class 1 and Stro-1 markers and negative for CD34, CD45, CD14, CD51/61, HLA-DR and KDR. Regarding the cell cycle status, 85.63 (63.19–92.17) and 82.41 (82.19–87.02) % were in GO-G1 phase (p = 1,000), while only 12.17 (3.33–36.81) and 10.67 (6.59–12.05) % were in S phase (p = 0,6828). All samples tested were capable of differentiating along adipogenic, osteogenic and chondrogenic lineages in vitro, demonstrated by morphology, cyto- and imunohistochemistry or RT-PCR reaction (PPARg and osteopontin genes expression). After co-culture with CD34+ cord blood cells for 1 and 4 weeks, no significant difference CD34+ expansion or colony-forming cells (BFU-E or CFU-GM) were observed between the CD34+ cells/LP-MSCs and CD34+ cells/ND-MSCs co-cultures with cytokines or not. Interpretation and Conclusions. Our results demonstrate that is possible to cultivate and expand MSCs with multipotential differentiation capabilities and hematopoiesis-supportive function from patients after HDCT. Despite there were no significant differences in the median values between LP and ND, the comparative study indicates a possible damage in MSCs by HDCT.

Description: Background. Type 1 diabetes mellitus (T1DM) is an autoimmune disease. A safe induction of an autoimmunity-free status may become a promising therapy. We hypothesize that intense immuno- and myelosuppression followed by autologous hematopoietic stem cell (HSC) rescue is an option to establish a lasting immunetolerance by eliminating auto-reactive lymphocytes and thus facilitate a possible recovery of autologous insulin production. Aims. Evaluate the HSC mobilization, infusion and engraftment in T1DM patients. Patients and Methods. Between January of 2004 and July of 2006 15 T1DM patients (12 male/3 female), with no more than 6 weeks after the first episode of hyperglycemia, were selected and enrolled. Their median age was 17 years old (range 14–31). HSC were mobilized into the peripheral blood (PB) by cyclophosphamide (Cy) 2g/m2 and filgrastim (G-CSF) 10 μg/kg/day. Peripheral blood stem cells (PBSC) were collected by leukapheresis using COBE Spectra (Gambro BCT, Lakewood, CO) blood cell separator. Collected PBSC were mixed with a cryoprotectant solution (autologous plasma and 10% dimethyl-sulfoxide (DMSO)), then frozen and stored in a mechanical freezer (−80°C). The CD34+ cell were counted using the ISHAGE protocol by a FACSort flow cytometer and CellQuest software packages (Becton Dickinson, San Jose, CA, USA). The conditioning regimen was Cy 200mg/kg and anti-lymphocyte globulin (4.5mg/kg). The protocol and the consent form were approved by the institution and the national ethics committees. Results. All results are expressed as median (range), except when specified. The collection was done on the 7th day (7–9) after the chemotherapy. The pre-apheresis values were: WBC (×103/μL) 7.9 (2.6–56.0); Hct (%) 37.4 (30.9–44.7); platelets (×103/μL) 177 (87–295); CD34+ (μL) 80.9 (36.5–167.6). The blood volemia processed and apheresis duration (min) were 2.8 (2.0–3.1) and 235 (177–280), respectively. Five patients (33.3%) experienced mild adverse reactions related to G-CSF administration (osteo-muscular pain and headache) and six (40%) related to citrate infusion (paresthesia and tremors), although thirteen (86%) had received prophylactic intravenous calcium infusion. The final yield values were: volume (mL) 210 (170–273); WBC (×108/Kg) 6.0 (3.0–14.9); Hct (%) 5.0 (3.3–9.0); CD34+ (×106/kg) 9.6 (5.8–22.5). Fourteen patients have already been transplanted. The time between cryopreservation and infusion was 21 days (13–35). The dose of DMSO infused was 0.4 mL/kg (0.0–0.5) (one patient received washed PBSC). All patients presented mild complications related to infusion: 13 (93%) nausea or vomiting, 8 (57%) flushing, 5 (36%) abdominal pain, 4 (29%) headache and 3 (21%) dyspnea, but only one needed oxygen supplementation. None of the patients presented complications like renal failure, hepatic insufficiency or cardiac arrhythmias. The time to reach granulocytes recovery (500/μL) was 9 days (7–10). Conclusions. The present data suggest that HSC mobilization in T1DM patients are very effetive, precocious and provide a good collection of CD34+ cells. The infusion of PBSC is a safe and reliable procedure with a low incidence of severe side effects and early engraftment. These results could be explained by a good bone marrow function as these patients had never been submitted to any kind of myelo or immunosuppressive therapy.

Description: This is a retrospective analysis of 1084 patients who received an allogeneic HSCT in 10 Brazilian Centers, from February 1983 to March 2003, aiming to validate the EBMT risk score. Data from transplanted patients and donors regarding patients age, disease stage at transplantation, HLA full-match sibling donor or full-match unrelated donors, donor-recipient gender match and the interval from diagnosis to transplantation, were used as variables to calculate the EBMT risk score. This score was analyzed using Cox Proportional Hazards Model. The OS, DFS, TRM and relapse were analyzed using Kaplan-Meier and cumulative incidence, whenever appropriate. In all there were 647 (60%) males and 437 (40%) females, the median age was 32 years old (range 1 – 59); 898 (83%) were in chronic phase, 146 (13%) were in accelerated phase and 40 (4%) were in blast crisis; 151 (14%) were younger than 20 years old, 620 (57%) were between 20 and 40 and 313 (29%) were older than 40; 1025 (95%) received HLA full-match sibling transplant and only 59 (5%) received an unrelated transplant. Female donor to male recipient occurred in 283 (26%) transplants. The interval from diagnosis to transplantation was less than 12 months in 223 (21%) cases and greater in 861 (79%). The OS, DFS, TRM and, relapse were 49%, 50%, 45%, 25%, respectively. The risk score 0–1 occurred in 179 (17%), score 2 in 397 (37%), score 3 in 345 (32%), score 4 in 135 (12%), and score 5–6 in 28 (2%). The risk scores 0–1 and 2 did not show any difference in terms of OS (58% and 55%, respectively) but they were significantly better than scores 3 or more (score 3 – 44%, 4 – 36 % and, 5-6 - 27%, respectively) (P

Description: Background: Zygomycosis is an uncommon, severe, life-threatening fungal infection in the immunocompromised host. The most common clinical presentation is rhinocerebral, primary pulmonary and disseminated disease. Myocardial involvement has been described in several case reports, mostly associated with pulmonary symptoms. Cardiac manifestations may, although, dominate the clinical picture of disseminated mucormycosis. These include myocardial infarction, congestive heart failure, conduction system disease, valvular incompetence and pericarditis. Diagnosis is based on histopathology. Objectives: we describe a 46-year-old man, (refractory follicular lymphoma), submitted to non-myeloablative SCT. Six months after SCT he developed cough, weight loss and skin lesions. Biopsies confirmed the diagnosis of cGVHD, and prednisone and CsA was started on D+180. Day+206 he developed fever and headache, uveitis, vitreous hemorrhage and rapid deterioration of consciousness. The MRI of the brain showed multifocal rounded white matter abnormalities with no gadolinium enhancement over the temporal, frontal and parietal lobes bilaterally as well as the periventricular region. Some lesions showed restriction on the diffusion sequence. The lesions did not show vascular territories distribution. CSF samples were tested for the presence of viral and fungal infection by polymerase chain reaction (PCR). Herpesvirus infections (CMV, HSV1, HSV2, VZV, EBV, HHV-6, HHV-7 and HHV-8 were investigated by a panherpes PCR with two pairs of primers targeting the DNA polymerase region. Polyomavirus (JC and BK) and picornavirus were also investigated. No virus was identified by PCR. Panfungal 18S rDNA directed PCR tested negative in 2 CSF samples taken with one week interval. Without any confirmed diagnosis we treated him with broad spectrum antibiotics and antifungals (initially with amphotericin and afterwards with Voriconazole). On day 212 he developed a cardiac arrhythmia (ventricular bigeminy and premature ventricular beats) promptly reverted by Amiodarone. Echocardiogram showed no alteration. D+214 he had recovered consciousness, but developed uncontrolled seizures and cardiac arrest. At that time electrocardiogram showed left bundle branch block. At autopsy no macroscopic alterations could be found in the brain but in the heart multiple white lesions were seen (fig 1) and in the kidneys. In myocardium, CNS and kidney large, non septate hyphae could be seen. In the brain thrombi occluding vessels could be found. Conclusions: Zygomycosis is increasingly reported in immunosuppressed patients. Diagnosis is difficult unless extensive radiologic examinations and invasive procedures (surgery and biopsy) can be performed. Unfortunately we focused our diagnostic approach to the SNC because the predominant manifestation was neurologic. Zygomycosis remains a highly lethal infection especially in imunossupressed patients unable to discontinue immunosuppressant drugs. Early diagnosis and aggressive treatment are the only possibilities to reach a successful outcome. Figure Figure