Antonella Isgrò

T-cell homeostasis alteration in HIV-1 infected subjects with low CD4 T-cell count despite undetectable virus load during HAART.

Objective:To investigate the pathogenesis of low CD4 T-cell count in subjects who are immunological non responders (InR) to HAART. Design:Thirty-five HIV-positive subjects on HAART for at least 1 year, all with undetectable HIV-1 RNA, were studied. Patients were defined as InR according to a CD4 cell increase < 20% from CD4 cell baseline or CD4 cell count < 200/μl; subjects with a CD4 T-cell increase > 20% from baseline and a CD4 cell count > 200/μl were defined as immunological responders (IR). We performed a comprehensive study to characterize the immune response of InR. Methods:The immunological phenotype of peripheral blood mononuclear cells, thymic naive T cells, T-cell receptor Vβ repertoire, serum concentration of interleukin (IL)-7, the expression of IL-7Rα on naive and memory CD4 and CD8 T cells, and regulatory T cells (Treg) were studied. Results:In InR a significant reduction (P < 0.0001) of naive and thymic naive CD4 T cells was associated with a reduced expression of IL-7Rα in both cell subsets, with an increased serum concentration of IL-7 was observed. Furthermore, an increased immune activation with a reduced Treg frequency and increased number of expansions of Vβ families was observed. Conclusions:The reduced expression of IL-7Rα associated with the persistent immune activation and the alteration of Treg frequencies in part explains the low level of CD4 T cells observed in InR.

Purified T-depleted, CD34+ peripheral blood and bone marrow cell transplantation from haploidentical mother to child with thalassemia.

Fetomaternal microchimerism suggests immunological tolerance between mother and fetus. Thus, we performed primary hematopoietic stem cell transplantation from a mismatched mother to thalassemic patient without an human leukocyte antigen-identical donor. Twenty-two patients with thalassemia major were conditioned with 60 mg/kg hydroxyurea and 3 mg/kg azathioprine from day -59 to -11; 30 mg/m(2) fludarabine from day -17 to -11; 14 mg/kg busulfan starting on day -10; and 200 mg/kg cyclophosphamide, 10 mg/kg thiotepa, and 12.5 mg/kg antithymocyte globulin daily from day -5 to -2. Fourteen patients received CD34(+)-mobilized peripheral blood and bone marrow progenitor cells; 8 patients received marrow graft-selected peripheral blood stem cells CD34(+) and bone marrow CD3/CD19-depleted cells. T-cell dose was adjusted to 2 x 10(5)/kg by fresh marrow cell addback at the time of transplantation. Both groups received cyclosporine for graft-versus-host disease prophylaxis for 2 months after transplantation. Two patients died (cerebral Epstein-Barr virus lymphoma or cytomegalovirus pneumonia), 6 patients reject their grafts, and 14 showed full chimerism with functioning grafts at a median follow-up of 40 months. None of the 14 patients who showed full chimerism developed acute or chronic graft-versus-host disease. These results suggest that maternal haploidentical hematopoietic stem cell transplantation is feasible in patients with thalassemia who lack a matched related donor.

Novel pharmacokinetic behavior of intravenous busulfan in children with thalassemia undergoing hematopoietic stem cell transplantation: a prospective evaluation of pharmacokinetic and pharmacodynamic profile with therapeutic drug monitoring

We prospectively studied the pharmacokinetics (PK) and clinical outcomes of intravenous busulfan (Bu) in 71 children with preexisting liver damage who underwent hematopoietic stem cell transplantation for thalassemia. Intravenous Bu was administered every 6 hours as part of a conditioning regimen with PK-based dose adjustment to target a conservative area under the concentration-versus-time curve (AUC) range (900-1350 microMol*min). The first-dose Bu clearance (CL) was significantly higher than the subsequent daily CL that remained unchanged in the ensuing days. One-third of patients required dose escalation based on dose 1 AUC, whereas dose reduction was needed in the subsequent days. At doses 5, 9, and 13, 78%, 81%, and 87% of patients, respectively, achieved the target range of AUC. A population PK analysis confirmed that the first-dose CL was 20% higher and that body weight was the most important covariate to explain PK variability. Patients with variant GSTA1*B had a 10% lower Bu CL than wild-type. These results suggest that the disease-specific behavior of intravenous Bu PK should be considered for PK-guided dose adjustment in patients with thalassemia, and the use of a conservative AUC range resulted in low toxicity, good engraftment, and good survival rate.

Hematopoietic Stem Cell Transplantation in Thalassemia and Sickle Cell Anemia

The globally widespread single-gene disorders β-thalassemia and sickle cell anemia (SCA) can only be cured by allogeneic hematopoietic stem cell transplantation (HSCT). HSCT treatment of thalassemia has substantially improved over the last two decades, with advancements in preventive strategies, control of transplant-related complications, and preparative regimens. A risk class-based transplantation approach results in disease-free survival probabilities of 90%, 84%, and 78% for class 1, 2, and 3 thalassemia patients, respectively. Because of disease advancement, adult thalassemia patients have a higher risk for transplant-related toxicity and a 65% cure rate. Patients without matched donors could benefit from haploidentical mother-to-child transplantation. There is a high cure rate for children with SCA who receive HSCT following myeloablative conditioning protocols. Novel non-myeloablative transplantation protocols could make HSCT available to adult SCA patients who were previously excluded from allogeneic stem cell transplantation.

Altered Clonogenic Capability and Stromal Cell Function Characterize Bone Marrow of HIV-Infected Subjects with Low CD4 + T Cell Counts Despite Viral Suppression during HAART

BACKGROUND Inflammatory cytokines in bone marrow may impair hematolymphopoiesis in human immunodeficiency virus (HIV)-infected subjects who do not experience reconstitution of CD4(+) T cells despite suppression of virus replication while receiving highly active antiretroviral therapy (HAART) (immunological nonresponders). METHODS Bone marrow samples from 12 immunological nonresponders receiving HAART were studied and compared with samples from 11 immunological responders. The mean CD4(+) T cell count (+/- standard deviation) was 174 +/- 68 cells/mm(3) and plasma HIV RNA levels had been <50 copies/mL for at least 1 year for individuals enrolled in the study. The clonogenic capability of bone marrow samples was evaluated using the colony forming cell assay and the long-term culture-initiating cell assay. CD34(+) cells from the colony forming cell assay were pooled for real-time polymerase chain reaction analysis of Fas and Fas ligand. Bone marrow cytokine production (interleukin-2 and tumor necrosis factor-alpha) and stromal interleukin-7 levels were analyzed by enzyme-linked immunosorbent assay in both groups. Flow cytometric analysis of CD4(+) and CD8(+) T cell subsets was performed. RESULTS A reduced clonogenic capability and a decrease in the level of more primitive progenitor cells were observed in parallel with lower production of interleukin-2 and increased tumor necrosis factor-alpha levels. A significant upregulation of Fas and Fas ligand on CD34(+) cells and a higher stromal interleukin-7 production were observed. Impairment of the naive T cell compartment and persistent T cell activation were observed in peripheral blood. CONCLUSIONS Samples from immunological nonresponders show reduced growth of in vitro colonies and an altered cytokine production in bone marrow. The cytokine pattern observed and the altered Fas and Fas ligand pathway may determine stem cell apoptosis and low CD4(+) cell recovery. These features, which are similar to those observed in HIV-infected subjects before starting therapy, persist despite treatment.

Late-Onset Hemorrhagic Cystitis in Children after Hematopoietic Stem Cell Transplantation for Thalassemia and Sickle Cell Anemia: A Prospective Evaluation of Polyoma (BK) Virus Infection and Treatment with Cidofovir

Little is known about late-onset hemorrhagic cystitis (HC) in children, its relationship to BK virus, and treatment with cidofovir (CDV) following hematopoietic stem cell transplantation (HSCT). We prospectively investigated BK virus reactivation in children who underwent HSCT from a matched related donor for thalassemia or sickle cell anemia following busulfan-cyclophosphamide-based conditioning regimens and analyzed risk factors for development of HC and its treatment with CDV. Grade 2-4 HC occurred in 30 patients with a cumulative incidence of 26% (95% confidence interval [CI] = 18%-34%). The cumulative incidences of BK viruria and viremia were 81% (95% CI = 69%-89%) and 28% (95% CI = 18%-40%), respectively. Multivariate analysis revealed that use of antithymocyte globulin (ATG) (hazard ratio [HR] = 10.5; P = .001), peak BK viruria >100,000 copies/mL (HR = 6.2; P = .004), and grade II-IV acute graft-versus-host disease (HR = 5.3; P = .007) were predictive factors for HC. Nineteen patients with HC were given CDV at 1.5 mg/kg/day 3 times a week, or 5 mg/kg/week. The median duration of therapy was 27 days (range, 21-180 days), and a median of 9 doses were given (range, 6-22). All patients had a complete clinical response (CCR), and 69% had a microbiological response at 4 weeks. Eleven patients with BK virus-related HC receiving supportive care also had CCR. The median duration of HC in these patients was similar to that in patients treated with CDV. None of the patients with HC cleared BK viruria when CCR was achieved. We conclude that late-onset HC is more prevalent in children with sustained high BK viruria who are treated with ATG or who develop graft-versus-host disease. Randomized clinical trials are urgently needed to better define the role of CDV in treating BK virus-related HC.

Functional interleukin-7/interleukin-7Rα, and SDF-1α/CXCR4 are expressed by human periodontal ligament derived mesenchymal stem cells

Hematopoiesis in the bone marrow (BM) is maintained by specific interactions between both hematopoietic and non‐hematopoietic stromal cells, which are mesenchymal stem cells (MSCs) capable of giving rise to several cell types. The human periodontal ligament (PDL), a tissue of ectomesenchymal origin, has been shown to also be a source of MSCs. We have investigated whether MSCs expanded from the PDL of healthy volunteers express characteristics similar to BM‐derived stem cells using structural, immunocytochemical and molecular approaches. Their ability to support the growth of hematopoietic progenitors was also analyzed. The PDL‐MSCs exhibited a fibroblast‐like morphology and their chromatin was dispersed, indicating active gene transcription. The mesenchymal‐related antigens CD90, CD29, CD166, CD105, and CD44 were homogeneously detected by cytofluorimetric analysis, whereas membrane CXCR4 was expressed only by a minority of cells. The PDL‐MSCs differentiated in vitro into osteogenic and adipogenic cells. Immunolocalization of IL‐7, IL‐7Rα, SDF‐1α, and CXCR4 resulted in a diffuse but specific labeling. RT‐PCR analysis confirmed the expression of the above‐mentioned transcripts. The cells spontaneously produced high levels of IL‐7 and SDF‐1α and were able to support the development and long‐term maintenance of BM precursor cells more efficiently than murine stromal cells and similarly to normal BM human stromal cells. We examined IL‐7 and SDF‐1α secretion pathway during adipogenic and osteogenic differentiation. IL‐7 increased during osteogenic and adipogenic differentiation, while the SDF‐1α secretion was downregulated during osteogenic differentiation but increased during adipogenic induction. Our study provides evidence that in human PDL there is an accessible niche of MSCs showing the features of BM‐derived MSCs. J. Cell. Physiol. 214: 706–713, 2008.

Bone Marrow Clonogenic Capability, Cytokine Production, and Thymic Output in Patients with Common Variable Immunodeficiency

In patients with primary Ab deficiencies, hematological and immunological abnormalities are frequently observed. A regenerative failure of hemopoietic stem/progenitor cells has been hypothesized. We evaluated in the bone marrow (BM) of 11 patients with common variable immunodeficiency, the phenotype of BM progenitors and their in vitro growth by colony-forming cell (CFC) and long-term culture (LTC) assays. A significant decrease in erythroid and mixed CFC and, to a greater extent, in primitive LTC-CFC progenitors was observed in patients compared with healthy controls. The frequency of BM pre-B and pro-B cells correlated directly with the absolute number of CD19+ lymphocytes. BM cells cultured in vitro produced spontaneously lower amounts of IL-2 and elevated levels of TNF-α compared with controls, indicating a skewing toward a proapoptotic cytokine pattern. In addition, stromal cells generated after BM LTC secreted less IL-7 and displayed by immunohistochemistry an altered phenotype. These findings were associated with a significant decrease in naive Th cells coexpressing CD31 in the peripheral blood. These results indicate an impaired growth and differentiation capacity of progenitor cells in patients with common variable immunodeficiency.

Recovery of Hematopoietic Activity in Bone Marrow from Human Immunodeficiency Virus Type 1-Infected Patients during Highly Active Antiretroviral Therapy

The mechanisms responsible for the hematopoietic failure in human immunodeficiency virus type 1 (HIV-1)-infected patients are still unknown. Several findings indicate that the in vitro proliferative potential of precursor cells from AIDS patients is reduced. The changes seen in bone marrow (BM) morphology and the defective BM functions associated with cytopenias have both been proposed as potential explanations. In patients treated with highly active antiretroviral therapy (HAART) an immune reconstitution associated with increased whole blood cell counts has been described. We have investigated the effects of HAART on the number of colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs), using long-term BM cell cultures (LTBMC) in a group of subjects with HIV-1 infection enrolled in an open study to evaluate the mechanisms of immune reconstitution during HAART. In each patient, the increase in colony growth was homogeneous, regardless of the type of hematopoietic progenitor cells assayed; in four subjects an increase in the most primitive progenitor cells (LTC-ICs) was observed. These findings were associated with the in vivo data showing increased numbers of BM mononuclear cells (BMMCs) after HAART and with a rise in peripheral CD4(+) T cell counts and decreased levels of plasma HIV-1 RNA. A decreased number of hematopoietic progenitor cells and/or a defective modulation of progenitor cell growth might be the cause of the hematological abnormalities in AIDS patients. Controlling HIV-1 replication by HAART could determine a restoration of stem cell activity, probably because of the suppression of factors that inhibit normal hematopoiesis.

Second hematopoietic SCT in patients with thalassemia recurrence following rejection of the first graft

There is a substantial incidence of graft failure in patients with thalassemia after myeloablative conditioning regimens especially in class 3 patients in whom its incidence could be as high as 8–38.5%. Most patients with graft failure have recurrence of thalassemic marrow. Historically, results of second transplants for thalassemia were poor because of a high rejection rate and/or increased TRM. Sixteen patients with thalassemia recurrence following rejection of the first graft and with a median age of 9 years (range, 4–20) were given second transplants using BM (n=7) or PBSC (n=9) after preparation with a new treatment protocol. All but two patients received stem cells from the same donor. The median interval between two transplants was 28 months (range, 8–204). The sustained engraftment rate was high (94%) with only one patient having primary graft failure. The probability of overall survival, event-free survival, TRM and graft failure were 79, 79, 16 and 6%, respectively. There were three transplant-related deaths. Thirteen patients are alive with Lansky/Karnofsky score of 100. This intensified treatment protocol was well tolerated with no significant increase in toxicity. The excellent results obtained with this new preparative regimen allow us to recommend it for second transplantation for patients with thalassemia recurrence.