Miniero R

Gene Therapy for immunodeficiency due to Adenosine Deaminase Deficiency

BACKGROUND We investigated the long-term outcome of gene therapy for severe combined immunodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency. METHODS We infused autologous CD34+ bone marrow cells transduced with a retroviral vector containing the ADA gene into 10 children with SCID due to ADA deficiency who lacked an HLA-identical sibling donor, after nonmyeloablative conditioning with busulfan. Enzyme-replacement therapy was not given after infusion of the cells. RESULTS All patients are alive after a median follow-up of 4.0 years (range, 1.8 to 8.0). Transduced hematopoietic stem cells have stably engrafted and differentiated into myeloid cells containing ADA (mean range at 1 year in bone marrow lineages, 3.5 to 8.9%) and lymphoid cells (mean range in peripheral blood, 52.4 to 88.0%). Eight patients do not require enzyme-replacement therapy, their blood cells continue to express ADA, and they have no signs of defective detoxification of purine metabolites. Nine patients had immune reconstitution with increases in T-cell counts (median count at 3 years, 1.07x10(9) per liter) and normalization of T-cell function. In the five patients in whom intravenous immune globulin replacement was discontinued, antigen-specific antibody responses were elicited after exposure to vaccines or viral antigens. Effective protection against infections and improvement in physical development made a normal lifestyle possible. Serious adverse events included prolonged neutropenia (in two patients), hypertension (in one), central-venous-catheter-related infections (in two), Epstein-Barr virus reactivation (in one), and autoimmune hepatitis (in one). CONCLUSIONS Gene therapy, combined with reduced-intensity conditioning, is a safe and effective treatment for SCID in patients with ADA deficiency. (ClinicalTrials.gov numbers, NCT00598481 and NCT00599781.)

Lactobacillus reuteri (American Type Culture Collection Strain 55730) Versus Simethicone in the Treatment of Infantile Colic: A Prospective Randomized Study

OBJECTIVE. The goal was to test the hypothesis that oral administration of Lactobacillus reuteri in a prospective randomized study would improve symptoms of infantile colic. METHODS. Ninety breastfed colicky infants were assigned randomly to receive either the probiotic L reuteri (108 live bacteria per day) or simethicone (60 mg/day) each day for 28 days. The mothers avoided cows milk in their diet. Parents monitored daily crying times and adverse effects by using a questionnaire. RESULTS. Eighty-three infants completed the trial: 41 in the probiotic group and 42 in the simethicone group. The infants were similar regarding gestational age, birth weight, gender, and crying time at baseline. Daily median crying times in the probiotic and simethicone groups were 159 minutes/day and 177 minutes/day, respectively, on the seventh day and 51 minutes/day and 145 minutes/day on the 28th day. On day 28, 39 patients (95%) were responders in the probiotic group and 3 patients (7%) were responders in the simethicone group. No adverse effects were reported. CONCLUSIONS. In our cohort, L reuteri improved colicky symptoms in breastfed infants within 1 week of treatment, compared with simethicone, which suggests that probiotics may have a role in the treatment of infantile colic.

Selective growth response to IL‐3 of a human leukaemic cell line with megakaryoblastic features

A new human leukaemic cell line (M‐O7) with the phenotypic characteristics of CFU‐mega is described. Its cells are positive for T200 leucocyte common antigen (LCA) and negative with MAbs recognizing T and B cells and mature myelomonocytic antigens. In contrast, they react with MAbs recognizing antigenic determinants common to multilineage (CD13, CD33, CD34) and to bipotent erythromegakaryoblastic (CD36, H25) haemopoietic precursors, and with MAbs specific for platelet glycoproteins (CD41w, CD42w). A small proportion (10%) of the cells were large and multinucleated, and on electron microscopy examination showed peripheral splitting of platelet‐like cytoplasm particles. When transferred to a serum‐free Iscove modified Dulbeccos medium supplemented with human insulin and transferrin, M‐O7 cells stop proliferating. Of the haemopoietic growth factors tested for their ability to restore the proliferative activity of this quiescent population, only rH IL‐3 proved effective. Moreover, it also increased the cloning efficiency in methylcellulose more than any other CSFs. The M‐O7 cell line may provide a valuable tool for the biological assay of IL‐3, and a model for biochemical studies of the megakaryocytic lineage.

Analysis of immune reconstitution in children undergoing cord blood transplantation

OBJECTIVE The aim of this study was to investigate and compare immune reconstitution in allogeneic cord blood transplantation (CBT) and bone marrow transplantation (BMT) recipients. MATERIALS AND METHODS Twenty-three children underwent CBT from either human leukocyte antigen-identical siblings (11 cases) or unrelated donors (12 cases) were enrolled in the study, together with 23 matched children receiving BMT. Patients were analyzed 2-3 and 12-15 months after transplant. Recovery of T-, B-, and NK-lymphocyte subsets, proliferative in vitro response to mitogens, as well as cytotoxic activities, were investigated. RESULTS CBT recipients showed a marked increase in the number of B lymphocytes as compared with patients who underwent BMT (p < 0.001). The absolute number of CD3(+) and CD8(+) T cells, as well as the proliferative response to T-cell mitogens, recovered with time after transplantation, irrespective of the source of stem cells used. Recipients of unrelated CBT had a better recovery of CD4(+) T lymphocytes (p < 0.01). Among patients experiencing acute graft-versus-host disease (GVHD), children given CBT had a much greater production of CD4(+) CD45RA(+) T cells than BMT recipients (p < 0.005). Recovery of NK cell number and innate cytotoxic activities was fast, irrespective of the source of stem cells used. CONCLUSIONS Despite the much lower number of lymphocytes transferred with the graft, recovery of lymphocyte number and function toward normal in CBT recipients was rapid and comparable to that observed after transplantation of bone marrow progenitors. This prompt immune recovery possibly was favored by the reduced incidence and severity of GVHD observed in children who underwent CBT.

Severe thrombotic microangiopathy: an infrequent complication of bone marrow transplantation. Gruppo Italiano Trapianto Midollo Osseo (GITMO).

Thrombotic microangiopathy (TMA) usually occurs during the first weeks following transplantation in the setting of systemic infections or graft-versus-host reaction. However, some cases without any evidence of other complications or after autologous transplantation have been reported. Transplant-associated TMA (BMT-TMA) incidence ranges from 0% to 74%, possibly due to different diagnostic criteria. The GITMO Group provided the opportunity to retrospectively study 4334 consecutive Italian patients who received bone marrow transplants (1759 allogeneic and 2575 autologous BMT), during the 1985–1995 period. The present report focuses on patients with severe TMA requiring specific treatment. We identified nine cases of TMA as a complication of allogeneic BMT (0.51%), whereas three patients developed the syndrome after ABMT (0.13%); four of the 12 patients were not receiving CsA at the time of TMA onset. Finally, it is noteworthy that TMA occurred in seven patients as a late complication (up to 90 days after BMT). Despite intensive treatment, five of the seven patients with thrombotic thrombocytopenic purpura died. One death was observed among the five cases with hemolytic uremic syndrome.

The role of thalidomide in the treatment of refractory chronic graft-versus-host disease following bone marrow transplantation in children.

Chronic graft-versus-host disease (cGVHD) is a frequent complication of allogeneic bone marrow transplantation (BMT). Thalidomide was found to have immunosuppressive properties and it has been used in a limited number of children with cGVHD. We report our experience with refractory and/or high-risk cGVHD in 14 children. Six children showed complete clinical response to thalidomide in a median time of 2 months. Four children had partial responses and four failed. Side-effects were usually mild (somnolence, constipation) and only two patients developed sensory peripheral neuropathy. An increased incidence of infectious complications attributable to thalidomide was not observed. Nine out of 10 responding patients are alive 49–111 months post-BMT. Thalidomide can be effective particularly in children with prevailing mucocutaneous cGVHD. All patients should be carefully monitored to detect peripheral neuropathy early.

Mycophenolate mofetil (MMF) as therapy for refractory chronic GVHD (cGVHD) in children receiving bone marrow transplantation.

Mycophenolate mofetil (MMF) is an alternative immunosuppressant which inhibits the proliferation of T and B lymphocytes. The purpose of the present study was to evaluate the safety and efficacy of MMF as salvage therapy for chronic GVHD (cGVHD) in children receiving allogeneic bone marrow transplantation. Fifteen children, 3–16 years of age, who had received grafts from HLA-compatible siblings (n = 8), partially matched related donors (n = 2) or matched unrelated donors (n = 5), developed extensive cGVHD which had proved unresponsive to standard immunosuppressive therapy. Patients were treated with MMF at the dose of 15–40 mg/kg/day in combination with other immunosuppressive therapy for a median of 4 months (range 1–15 months). The overall response rate (complete or partial response) was 60%. Thirteen percent had only minor responses, whereas 27% of patients had progressive disease. Best responses were seen in patients with GI tract (60% of complete responses) or mouth (33% of complete responses) cGVHD and skin involvement (43% of complete responses) that did not include sclerodermatous manifestations. Once MMF was started, improvements in the clinical manifestations of cGVHD allowed a significant reduction of steroids in 45% of patients and discontinuation in 27% of cases. Six patients (40%) experienced adverse events, with gastrointestinal symptoms predominating. Five patients experienced opportunistic infections. MMF was discontinued after 35–180 days in six patients for the following reasons: parents choice (n = 2), liver toxicity (n = 1), poor compliance (n = 2), and no response (n = 1). In conclusion, these preliminary results suggest that MMF in combination with other immunosuppressive agents may have a role to play in patients with cGVHD. Prospective clinical trials are needed to establish exact indications for therapy and dosage scheduling. Bone Marrow Transplantation (2000) 25, 1067–1071.

Second allogeneic bone marrow transplantation in acute leukemia : a multicenter study from the Gruppo Italiano Trapianto Di Midollo Osseo (GITMO)

Thirty-eight second allogeneic bone marrow transplants (BMT) for acute leukemia relapsed after first BMT were performed in 13 Italian centers between 1987 and 1994. Twenty-one patients had acute myelogenous leukemia (AML), 17 acute lymphoblastic leukemia (ALL); at second BMT 24 patients were in complete remission (CR) and 14 in relapse. The median time to relapse after first BMT was 10 months (range 1–70). Grade II or greater acute graft-versus-host disease (GVHD) after second transplant occurred in 34.2% of patients and a chronic GVHD in 31.5% of patients. Twenty-four patients died: seven from early transplant-related mortality (TRM), 13 from relapse and four from late toxicity. As of 31 July 1996, at a median follow-up of 47 months (range 22–85), there are 14 survivors. The three-year probability of TRM, relapse and event-free survival (EFS) is 28%, 40% and 42% respectively. In 20 of 27 evaluable patients, remission duration after second BMT was longer than after the first BMT. A diagnosis of AML was correlated with a better outcome. These data support the usefulness of second allograft in selected patients with AML relapsing after a first BMT.

Impact of thrombotic thrombocytopenic purpura on leukemic children undergoing bone marrow transplantation

Thrombotic thrombocytopenic purpura (TTP) has emerged as one of the main transplant-related complications over the last 15 years. The current study defines the incidence and the risk factors for the occurrence of TTP in 131 consecutive leukemic children who were transplanted between January 1994 and December 1997 at four Italian pediatric centers. Patients with ALL (101), AML (21), MDS (9), underwent an HLA-identical sibling BMT (82) or an HLA-identical unrelated BMT (49), receiving a conditioning regimen consisting of high-dose chemotherapy in 24 patients and of F-TBI combined with high-dose chemotherapy in 107 patients. The diagnosis of TTP was retrospectively evaluated on the basis of parallel criteria. TTP treatment varied according to the protocol of each treatment center. Twenty-eight of 131 patients (21.4%) developed TTP at a median of 46 days (range 21–80) after BMT. Multivariate analysis demonstrated that the risk of TTP was higher in patients who underwent unrelated BMT (P value = 0.02). Acute GVHD, stage of disease at BMT, conditioning with TBI, gender, age, did not appear to be associated with the occurrence of TTP. As to the outcome, TTP resolved in 19 patients while in nine it was the principal cause of death (32.1%). In patients with TTP, LDH peak value was the only statistically significant factor (P = 0.001) related to severe TTP. In conclusion, our experience demonstrates that leukemic children undergoing BMT, especially from an unrelated donor, should be carefully assessed for TTP which appears to be a severe and relatively common transplant-related complication when strict diagnostic criteria are applied. Bone Marrow Transplantation (2000) 26, 1005–1009.

High Faecal Calprotectin Levels in Healthy, Exclusively Breast-Fed Infants

Background: Faecal calprotectin has been proposed as a sensitive marker for gastrointestinal inflammation in children and adults. High levels have been reported in healthy newborns and during the first months of life; the effect of the kind of feeding on the calprotectin concentration in stools is controversial. Objective: To evaluate faecal calprotectin values in healthy, exclusively breast-fed (BF) or formula-fed (FF) infants. Methods: Stool samples were obtained from 74 healthy infants (39 exclusively BF and 35 exclusively FF) with a median age of 51 days (range 13–90). Exclusion criteria were acute infections and treatment with anti-inflammatory drugs. Stool samples were stored at –20°C until they were analysed, and the faecal calprotectin concentration was detected using a commercial quantitative enzyme-linked immunoassay (Calprest; Eurospital SpA, Trieste, Italy). Results: The median faecal calprotectin concentration was significantly higher in BF infants (555.00 µg/g, range 122.5–2,000.0 µg/g) than in FF ones (206.60 µg/g, range 31.2–797.6 µg/g) (p < 0.001). We observed a significantly higher median stool frequency in BF infants than in FF ones (p < 0.001), but multiple regression analysis (independent variables: kind of feeding and stool frequency; dependent variable: calprotectin) showed a significant coefficient for the kind of feeding, but not for stool frequency (p = 0.937). Conclusions: Our findings show that the kind of feeding influences the faecal calprotectin concentration, with higher values in healthy exclusively BF infants than in FF ones. Our study does not allow us to clearly identify the reason for our finding; this could be due to hormones (such as ghrelin and leptin), cytokines and other immunostimulating and growth factors (such as epidermal growth factor and granulocyte colony-stimulating factor) in human milk, which contribute to the development of the gastrointestinal immune system. Further investigations are needed to better clarify the mechanism underlying the relationship between feeding and faecal calprotectin levels in young infants.