PubMed Health⌕ Search

Biomedical subjects

F Candotti

Publications and source records attributed to F Candotti.

51 records · Page 3Linked to original sources
PubMed1997

Interleukin-4 signaling in B lymphocytes from patients with X-linked severe combined immunodeficiency.

Interleukin-4 (IL-4) is an important cytokine for B and T lymphocyte function and mediates its effects via a receptor that contains gammac. B cells derived from patients with X-linked severe combined immunodeficiency (X-SCID) are deficient in gammac and provide a useful model in which to dissect the role of this subunit in IL-4-mediated signaling. We found that although IL-4 stimulation of X-SCID B cells did not result in Janus tyrosine kinase-3 (JAK3) phosphorylation, other IL-4 substrates including JAK1 and IRS-1 were phosphorylated. Additionally, we detected signal transducers and activators of transcription 6 (STAT6) tyrosine phosphorylation and DNA binding activity in X-SCID B cells with a wide range of gammac mutations. However, reconstitution of these X-SCID B cells with gammac enhanced IL-4-mediated responses including STAT6 phosphorylation and DNA binding activity and resulted in increased CD23 expression. Thus, gammac is not necessary to trigger IL-4-mediated responses in B cells, but its presence is important for optimal IL-4-signaling. These results suggest that two distinct IL-4 signaling pathways exist.

B-Lymphocytes↗
PubMed1997

Advances in the understanding of cytokine signal transduction: the role of Jaks and STATs in immunoregulation and the pathogenesis of immunodeficiency.

Cytokines are of great importance in the growth and differentiation of hematopoietic and other cells. Moreover, they are also crucial in immunoregulation and in host defense. Although our understanding of the molecular basis of cytokine action is far from complete, recent advances have substantially improved our knowledge of cytokine-dependent signal transduction. The delineation of the structure of cytokine receptors and the signaling pathways they utilize has provided clues as to how the strikingly specific effects of cytokines are achieved. Additionally, the basis of some of the pleiotropic and redundant effects of cytokines has also become clear. The discovery of the Janus family of protein tyrosine kinases (Jaks) and the STATs (signal transducers and activators of transcription) has also provided key insights into the mechanism by which intracellular signals are transduced. The following paradigm has emerged: cytokines induce dimerization of receptor subunits that are constitutively associated with Jaks. This activates the Jaks, which then phosphorylate the receptors. The phosphorylated receptors are bound by SH2-containing proteins, one class of which is the STATs. Activated STATs, then, translocate to the nucleus to effect gene transcription. Though the Jaks do not explain much in terms of specificity in signaling, the function of the STATs does. The discovery of patients with autosomal recessive severe combined immunodeficiency due to mutations of a particular Jak, Jak3, and the phenotype of knockout mice lacking Jak3 and various STATs demonstrate the specific and critical roles of these molecules in the development and function of the immune system.

Adjuvants, Immunologic↗
PubMed1996

In vitro correction of JAK3-deficient severe combined immunodeficiency by retroviral-mediated gene transduction.

Mutations affecting the expression of the Janus family kinase JAK3 were recently shown to be responsible for autosomal recessive severe combined immunodeficiency (SCID). JAK3-deficient patients present with a clinical phenotype virtually indistinguishable from boys affected by X-linked SCID, a disease caused by genetic defects of the common gamma chain (gamma c) that is a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. The specific interaction of JAK3 and gamma c represents the biochemical basis for the similarities between these two immunodeficiencies. Both forms of SCID are characterized by recurrent, severe infections leading to death in infancy unless successfully treated by allogeneic bone marrow transplantation. Because of the potentially lethal complications associated with allogeneic bone marrow transplantation and the frequent lack of suitable marrow donors, the development of alternative forms of therapy is highly desirable. To this end, we investigated a retroviral-mediated gene correction approach for JAK3-deficiency. A vector carrying a copy of JAK3 cDNA was constructed and used to transduce B cell lines derived from patients with JAK3-deficient SCID. We demonstrate restoration of JAK3 expression and phosphorylation upon IL-2 and IL-4 stimulation. Furthermore, patients' cells transduced with JAK3 acquired the ability to proliferate normally in response to IL-2. These data indicate that the biological defects of JAK3-deficient cells can be efficiently corrected in vitro by retroviral-mediated gene transfer, thus providing the basis for future investigation of gene therapy as treatment for JAK3-deficient SCID.

B-Lymphocytes↗
PubMed1996

Retroviral-mediated gene correction for X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency (XSCID) is a lethal disease caused by a defect in the gene encoding the common gamma chain (gamma-c) of the receptor for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. Allogeneic bone marrow transplantation, the current therapy of choice for this defect, is often complicated by graft-versus-host disease and/or incomplete reconstitution of B-lymphocyte functions. Correction of the gene defect at the level of the autologous lymphohematopoietic progenitors could therefore represent an improvement in the medical management of these patients. To study the feasibility of a gene therapy approach for XSCID, a retroviral vector expressing gamma-c was used to transduce Epstein-Barr virus-transformed B-cell lines derived from patients with XSCID. After transduction, XSCID cells newly expressed gamma-c on the cell surface at levels comparable to those observed on B-cell lines obtained from normal donors. Moreover, the reconstituted gamma-c restored function to the IL-2 and IL-4 receptors as shown by signal transduction mediated by phosphorylation of the JAK1 and JAK3 members of the Janus family of tyrosine kinases and by restoration of cellular proliferation in response to IL-2.

B-Lymphocytes↗
PubMed1996

Signaling via IL-2 and IL-4 in JAK3-deficient severe combined immunodeficiency lymphocytes: JAK3-dependent and independent pathways.

Both IL-2 and IL-4 bind to receptors containing the common gamma chain and JAK3. Although JAK3 is required for proper lymphoid development, the precise roles of this kinase in IL-2 and IL-4 signaling in lymphocytes have not been defined. Here, we have studied IL-2 and IL-4 signaling in B cell lines lacking JAK3. Although IL-2-induced phosphorylation of IL-2R beta, JAK1, and STAT5 all required the presence of JAK3, IL-4-mediated phosphorylation of JAK1, STAT6, and insulin receptor substrates 1 and 2 did not. However, IL-4-induced effects were clearly improved following JAK3 expression. These data indicate that IL-4 signaling occurs in the absence of of JAK3, but is comparatively inefficient. These findings may help in understanding the pathogenesis of the immunodeficiency that occurs with mutations of JAK3 and may suggest a mechanism for the pleiotropic effects of IL-4.

B-Lymphocytes↗
PubMed1995

Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID).

Severe combined immune deficiency (SCID) represents a heterogenous group of hereditary diseases. Mutations in the common gamma-chain (gamma c), which is part of several cytokine receptors including those for interleukin (IL)-2, IL-4, IL-7, IL-9 and IL-15, are responsible for X-linked SCID, which is usually associated with a lack of circulating T cells and the presence of B lymphocytes (T- B+ SCID). The gene(s) responsible for autosomal recessive T- B+ SCID is still unknown. The Jak-3 protein kinase has been found to associate with the gamma c-chain-containing cytokine receptors. Therefore Jak-3 or other STAT proteins with which it interacts are candidate genes for autosomal recessive T- B+ SCID. Here we investigate two unrelated T- B+ SCID patients (both from consanguineous parents) who have homozygous mutations in the gene for Jak-3. One patient carries a mutation (Tyr100-->Cys) in a conserved tyrosine residue in the JH7 domain of Jak-3 which is absent in more than 150 investigated chromosomes. The other patient carries a homozygous 151-base-pair deletion in the kinase-like domain, leading to a frameshift and premature termination. Both mutations resulted in markedly reduced levels of Jak-3. These findings show that abnormalities in the Jak/STAT signalling pathway can account for SCID in humans.

B-Lymphocytes↗
PubMed1993

Application of molecular analysis to genetic counseling in the Wiskott-Aldrich syndrome (WAS).

The Wiskott-Aldrich syndrome (WAS) is a severe X-linked, recessive disorder, with a high mortality rate at early age due to hemorrhages, infections, and lymphoid malignancies. The molecular pathogenesis of the disease is unknown. Carrier females of WAS are clinically and immunologically normal, thus precluding carrier detection by simple laboratory tests. Major advances in molecular genetics have allowed mapping of the WAS gene to the pericentromeric short arm of the X chromosome, and have made carrier detection and prenatal diagnosis feasible by segregation analysis with closely linked polymorphic DNA markers. Furthermore, the observation that carriers of WAS exhibit a unilateral inactivation of the X chromosome in hematopoietic cells has provided a new tool for carrier detection. However, critical interpretation of molecular analysis data is essential to provide accurate genetic counseling to WAS families.

Dosage Compensation, Genetic↗
PubMedDate not supplied

Expansion of hepatic and hematopoietic stem cells utilizing mouse embryonic liver explants.

Ex vivo embryonic liver explant culture is a novel and attractive approach to obtain abundant hepatic and hematopoietic stem cells. Gene therapy of autologous hepatic and hematopoietic stem cells represents an alternative therapeutic approach to liver transplantation for genetic and metabolic disorders. In this study we characterize the growth and differentiation of hepatic stem cells utilizing embryonic liver cultures. Day 9.5 liver buds are microdissected and cultured under specific conditions. Modulation of growth conditions by addition of hepatocyte growth factor, Flt-3 ligand, and stem cell factor leads to enrichment of hepatic progenitor cells in embryonic liver explants. Under these conditions, we also demonstrate the role of a novel marker PRAJA-1 to identify hepatic stem cells and transitional hepatocytes. Utilization of dexamethasone enhanced pseudolobule formation with increased hepatocytic and biliary differentiation. Transforming growth factor-beta leads to enrichment of biliary cells in the culture. Gut formation is enhanced in the presence of interleukin-3 and blood formation by increasing the mesodermal tissue in these cultures. We also show increased retroviral-mediated expression of the green fluorescent protein expression in the expanded hepatic and hematopoietic stem cells under different culture conditions. Thus, the embryonic liver explant culture is an attractive source for hepatic progenitors and is a possible step towards generating nontumorigenic immortalized hepatocytes with possible transplantation applications.

Animals↗
PubMedDate not supplied

Functional depletion of T cells by vincristine and methylprednisolone as an in vitro model for the prevention of graft versus host disease.

BACKGROUND AND METHODS. The outcome of mismatched bone marrow transplantation is still severely hampered by graft versus host disease (GVHD) and graft rejection. Procedures for the recognition and selective elimination of T cells are still unsatisfactory due to the increased incidence of graft failure and late rejection. Lymphocyte proliferation and generation of cytotoxic T cells (CTL) in response to allogeneic cells are considered good in vitro correlates of GVHD and have been used in the present study to asses the capacity of two drugs (vincristine, VCR, and methylprednisolone, MP) to affect the T cells involved in these reactions. RESULTS AND CONCLUSION. Treatment in vitro with VCR and MP has been shown to inhibit the functional capacity of peripheral blood lymphocytes to proliferate (mean reduction 95.8%) and to generate CTL in response to haploidentical stimulator cells. Responsiveness to antigens and mitogens was affected to a minor extent (mean reduction 40% and 65.5%, respectively), and the method allowed recovery of hemopoietic precursors. The results suggest that treatment of donor bone marrow with VCR and MP is worth studying as a new approach to the prevention of GVHD in haploidentical bone marrow transplantation.

Bone Marrow Cells↗
PubMedDate not supplied

[Cellular and molecular therapy in severe combined immunodeficiencies].

Severe combined immunodeficiencies are usually fatal diseases unless affected children are admitted to protective isolation unit or unless the underlying immunological defect is treated by transplanting bone marrow from an healthy donor. The patients present, with early onset, life-threatening infections from fungal, viral or bacterial agents. Since only a minority of patients has an HLA-identical donor, recently other strategies have been devised including bone marrow transplantation from donors other than HLA-identical within the family or from HLA-non identical family members or from HLA-matched unrelated donors included in the International Bone Marrow Volunteers' Registry. In these cases, in order to realize this approach the "purging" of T-cells of the HLA-non identical donor bone marrow is necessary. Overall survival after HLA-identical BMT is 76%, when all BMT of the European multicenter analysis are considered, while in BMT from non-identical donors is 56%. Recently particular cases of SCID caused by enzyme deficiency, such as adenosine-deaminase (ADA), have been treated by molecular therapy with administration of polyethylene glycol (PEG) conjugated ADA: PEG protects from degradation and inhibits clearance of the enzyme. This approach, already realized in 15 children, allows reconstitution of cellular and humoral immunity, as demonstrated by one case treated by our group.

Adenosine Deaminase↗
PubMedDate not supplied

[Bone marrow transplantation in congenital defects of immunity].

BMT can cure several congenital immunological defects: if in these disease the engrafting is easier, the GVH reactions are more frequent and severe. The possibility to deplete from T lymphocyte the marrow before infusion, has overcame this difficulty. From 1968 183 BMT have been performed in Europe on patients with SCID (70 from HLA-identical donor, 113 from HLA-nonidentical donor). The survival after 2 years is 76% in the first group, and 56% in the second group (100 marrows have been T-depleted with different techniques). Strict isolation procedures before the transplant are very important to achieve good results. The possibility to treat different immunodeficiency With BMT are also discussed.

Bone Marrow Transplantation↗