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Inborn errors of immunity are a group of genetic disorders caused by mutations that affect the development and/or function of several compartments of the immune system, predisposing patients to infections, autoimmunity, allergy and malignancies. In this regard, mutations that affect proteins involved in trafficking, priming, docking, or membrane fusion will impair the exocytosis of lytic granules of effector NK and cytotoxic T lymphocytes. This may predispose patients to hemophagocytic lymphohistiocytosis, a life-threatening immune disorder characterized by systemic lymphocyte and macrophage activation, and increased levels of cytokines, which lead to an uncontrolled hyperinflammation state and progressive multiorgan damage. In this review, we will describe a clinical case and recent advances in inborn errors of immunity predisposing to hemophagocytic lymphohistiocytosis. Summary sentence: Review of recent advances in inborn errors of immunity predisposing to hemophagocytic lymphohistiocytosis.

CD40 ligand (CD40L) deficiency is a rare inborn error of immunity presenting with heterogeneous clinical manifestations. While a detailed characterization of patients affected by CD40L deficiency is essential to an accurate diagnosis and management, information about this disorder in Latin American patients is limited. We retrospectively analyzed data from 50 patients collected by the Latin American Society for Immunodeficiencies registry or provided by affiliated physicians to characterize the clinical, laboratory, and molecular features of Latin American patients with CD40L deficiency. The median age at disease onset and diagnosis was 7 months and 17 months, respectively, with a median diagnosis delay of 1 year. Forty-seven patients were genetically characterized revealing 6 novel mutations in the CD40LG gene. Pneumonia was the most common first symptom reported (66%). Initial immunoglobulin levels were variable among patients. Pneumonia (86%), upper respiratory tract infections (70%), neutropenia (70%), and gastrointestinal manifestations (60%) were the most prevalent clinical symptoms throughout life. Thirty-five infectious agents were reported, five of which were not previously described in CD40L deficient patients, representing the largest number of pathogens reported to date in a cohort of CD40L deficient patients. The characterization of the largest cohort of Latin American patients with CD40L deficiency adds novel insights to the recognition of this disorder, helping to fulfill unmet needs and gaps in the diagnosis and management of patients with CD40L deficiency.

Germinal heterozygous activating STAT3 mutations represent a novel monogenic defect associated with multi-organ autoimmune disease and, in some cases, severe growth retardation. By using whole-exome sequencing, we identified two novel STAT3 mutations, p.E616del and p.C426R, in two unrelated pediatric patients with IGF-I deficiency and immune dysregulation. The functional analyses showed that both variants were gain-of-function (GOF), although they were not constitutively phosphorylated. They presented differences in their dephosphorylation kinetics and transcriptional activities under interleukin-6 stimulation. Both variants increased their transcriptional activities in response to growth hormone (GH) treatment. Nonetheless, STAT5b transcriptional activity was diminished in the presence of STAT3 GOF variants, suggesting a disruptive role of STAT3 GOF variants in the GH signaling pathway. This study highlights the broad clinical spectrum of patients presenting activating STAT3 mutations and explores the underlying molecular pathway responsible for this condition, suggesting that different mutations may drive increased activity by slightly different mechanisms. Fil: Gutiérrez, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Scaglia, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Keselman, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Martucci, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Karabatas, Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Domené, Sabina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Martin, Ayelen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Pennisi, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Blanco, Miguel. Hospital Universitario Austral, Endocrinología; Argentina Fil: Sanguineti, Nora. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Bezrodnik, Liliana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Area de Inmunología. Grupo de Inmunología; Argentina Fil: Di Giovanni, Daniela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Area de Inmunología. Grupo de Inmunología; Argentina Fil: Caldirola, María Soledad. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Area de Inmunología. Grupo de Inmunología; Argentina Fil: Esnaola Azcoiti, María. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Area de Inmunología. Grupo de Inmunología; Argentina Fil: Gaillard, María Isabel. Cincinnati Children's Hospital Medical Center, Gastroenterology, Hepatology and Nutrition ; Estados Unidos Fil: Denson, Lee A.. Cincinnati Children's Hospital Medical Center, Gastroenterology, Hepatology and Nutrition; Estados Unidos Fil: Zhang, Kejian. Cincinnati Children's Hospital Medical Center,Human Genetics ; Estados Unidos Fil: Husami, Ammar. Cincinnati Children's Hospital Medical Center,Human Genetics ; Estados Unidos Fil: Yayah Jones, Nana-Hawa. Cincinnati Children's Hospital Medical Center, Cincinnati Center for Growth Disorders, Division of Endocrinology; Estados Unidos Fil: Hwa, Vivian. Cincinnati Children's Hospital Medical Center, Cincinnati Center for Growth Disorders, Division of Endocrinology,; Estados Unidos Fil: Revale, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; Argentina Fil: Vázquez, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; Argentina Fil: Jasper, Héctor. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Kumar, Ashish. Cincinnati Children's Hospital Medical Center,Division of BM Transplantation and Immunodeficiency ; Estados Unidos Fil: Domene, Horacio Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina

Mendelian susceptibility to mycobacterial disease is a rare syndrome, which results in predisposition to clinical disease caused by poorly virulent mycobacterial species such as the bacille Calmette-Guerin vaccines and nontuberculous environmental mycobacteria. Such patients are also susceptible to virulent Mycobacterium tuberculosis but rarely present other unusual infectious diseases, with the exception of nontyphoid Salmonella, which can affect around half of them. We report a boy with a novel interleukin-12 receptor beta 1 deficiency, who developed BCG-osis and an inflammatory myofibroblastic tumor when he was 11 months. Clinically IL12 deficiency should be considered in genetic diagnosis of patients with an inherited IL12/INFγ axis and thymus affected. We do not know if the novel genetic diagnosis of our patient could be a factor of development of thymus lesion.

BACKGROUND Arthrographis kalrae is a hyaline fungus that grows forming arthroconidia. It is an opportunistic pathogen that causes infections in immunocompromised as in immunocompetent people and has been rarely isolated from human clinical samples. CASE REPORT We describe the case of a male child with primary immunodeficiency who initially presented unilateral pneumonia and progressed to bilateral involvement despite antibiotic, antifungal treatment. A. kalrae was diagnosed by pulmonary biopsy. He received posaconazole with resolution of disease. CONCLUSIONS This is the first case of A. kalrae pulmonary infection in a pediatric patient with chronic granulomatous disease in Argentina.

Arthrographis kalrae is a hyaline fungus that grows forming arthroconidia. It is an opportunistic pathogen that causes infections in immunocompromised as in immunocompetent people and has been rarely isolated from human clinical samples.We describe the case of a male child with primary immunodeficiency who initially presented unilateral pneumonia and progressed to bilateral involvement despite antibiotic, antifungal treatment. A. kalrae was diagnosed by pulmonary biopsy. He received posaconazole with resolution of disease.This is the first case of A. kalrae pulmonary infection in a pediatric patient with chronic granulomatous disease in Argentina.Arthrographis kalrae es un hongo hialino de crecimiento lento que, en su desarrollo, forma artroconidios. Es un patógeno oportunista que causa infecciones en personas inmunocomprometidas e inmunocompetentes, y ha sido aislado muy raramente en muestras clínicas de seres humanos.Se describe el caso de un paciente con inmunodeficiencia primaria y afectación pulmonar con evolución tórpida. Presentó compromiso de ambos pulmones a pesar del tratamiento antibiótico y antifúngico instaurado. Durante su seguimiento, se realizaron múltiples biopsias pulmonares y se aisló A. kalrae en el cultivo de tejido pulmonar. Recibió tratamiento con posaconazol, con buena respuesta y remisión de las lesiones.Este es el primer caso reportado de infección pulmonar por A. kalrae en un paciente pediátrico con enfermedad granulomatosa crónica en Argentina.

Purpose Several studies have shown that subcutaneous immunoglobulin (SCIG) infusions demonstrate similar efficacy to intravenous Ig (IVIG) in preventing infections in patients with primary immunodeficiency diseases (PID), and are safe and well tolerated in this population. This open, prospective/retrospective, multicenter study was designed to compare the effectiveness, safety and tolerability of a 16 % liquid human IgG preparation (Beriglobina P), administered SC, with previous IVIG treatment in PID pediatric patients in Argentina. Methods Fifteen subjects were enrolled in the study, and a total of 13 subjects (aged 6–18 years) completed the 36-week SCIG treatment period. All children had previously received IVIG treatment. The dose of SCIG equaled the previous IVIG dose and subjects received an average weekly dose of 139 mg/kg (range 105–181) during the SCIG period. Results Significantly higher serum IgG trough levels were recorded on SCIG treatment at 16, 24, and 36 weeks, when compared with previous IgG trough levels on steady-state IVIG treatment. The annualized infection rate was 1.4 infections/subject/year during the IVIG administration period compared with 0.4 infections/subject/year during the SCIG period. All subjects who completed the study chose to continue administering SCIG at home after the study had ended. Conclusions These data confirm that self-administered SCIG therapy is a well-tolerated and effective alternative to IVIG therapy for children with PID. Fil: Bezrodnik, Liliana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gómez Raccio, Andrea. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina Fil: Belardinelli, Gabriela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina Fil: Regairaz, Lorena. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina. Provincia de Buenos Aires. Ministerio de Salud. Hospital de Niños "Sor Maria Ludovica" de la Plata; Argentina Fil: Díaz Ballve, Damacia. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina Fil: Seminario, Gisela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina Fil: Moreira, Ileana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina Fil: Riganti, Carlos. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Pedro Elizalde" (ex Casa Cuna); Argentina Fil: Cantisano, Claudio. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Pedro Elizalde" (ex Casa Cuna); Argentina Fil: Díaz, Héctor. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Pedro Elizalde" (ex Casa Cuna); Argentina Fil: Di Giovanni, Daniela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina

The signal transducer and activator of transcription (STAT) family of proteins regulate gene transcription in response to a variety of cytokines. STAT5B, in particular, plays an important role in T cells, where it is a key mediator of interleukin-2 (IL-2) induced responses. STAT5B deficiency causes a rare autosomal recessive disorder reported in only a handful of individuals. There are currently ten published cases of STAT5B deficiency, four of which are Argentinians. Fil: Bezrodnik, Liliana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Di Giovanni, Daniela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina Fil: Caldirola, María Soledad. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina Fil: Esnaola Azcoiti, María. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina Fil: Torgerson, Troy. University of Washington; Estados Unidos Fil: Gaillard, María Isabel. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina

Hyper-IgM (HIGM) syndrome is a heterogeneous group of disorders characterized by normal or elevated serum IgM levels associated with absent or decreased IgG, IgA and IgE. Here we summarize data from the HIGM syndrome Registry of the Latin American Society for Immunodeficiencies (LASID). Of the 58 patients from 51 families reported to the registry with the clinical phenotype of HIGM syndrome, molecular defects were identified in 37 patients thus far. We retrospectively analyzed the clinical, immunological and molecular data from these 37 patients. CD40 ligand (CD40L) deficiency was found in 35 patients from 25 families and activation-induced cytidine deaminase (AID) deficiency in 2 unrelated patients. Five previously unreported mutations were identified in the CD40L gene (CD40LG). Respiratory tract infections, mainly pneumonia, were the most frequent clinical manifestation. Previously undescribed fungal and opportunistic infections were observed in CD40L-deficient patients but not in the two patients with AID deficiency. These include the first cases of pneumonia caused by Mycoplasma pneumoniae, Serratia marcescens orAspergillus sp. and diarrhea caused by Microsporidium sp. or Isospora belli. Except for four CD40L-deficient patients who died from complications of presumptive central nervous system infections or sepsis, all patients reported in this study are alive. Four CD40L-deficient patients underwent successful bone marrow transplantation. This report characterizes the clinical and genetic spectrum of HIGM syndrome in Latin America and expands the understanding of the genotype and phenotype of this syndrome in tropical areas. Fil: Cabral Marques, Otavio. Universidade de Sao Paulo; Brasil Fil: Klaver, Stefanie. Universidade de Sao Paulo; Brasil Fil: Schimke, Lena F. Universidade de Sao Paulo; Brasil Fil: Ascendino, Évelyn H. Universidade de Sao Paulo; Brasil Fil: Khan, Taj Ali. Universidade de Sao Paulo; Brasil Fil: Pereira, Paulo Vítor Soeiro. Universidade de Sao Paulo; Brasil Fil: Falcai, Angela. Universidade de Sao Paulo; Brasil Fil: Vargas Hernández, Alexander. Instituto Politécnico de México; México Fil: Santos-Argumedo, Leopoldo. Instituto Politécnico de México; México Fil: Bezrodnik, Liliana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Moreira, Ileana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños ; Argentina Fil: Seminario, Gisela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños ; Argentina Fil: Di Giovanni, Daniela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños ; Argentina Fil: Raccio, Andrea Gómez. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños ; Argentina Fil: Porras, Oscar. Hospital Nacional de Niños, “Dr. Carlos Sáenz Herrera”; Costa Rica Fil: Weber, Cristina Worm. Pediatric Allergy & Immunology Clinic; Brasil Fil: Ferreira, Janaíra Fernandes. Albert Sabin Hospital; Brasil Fil: Tavares, Fabiola Scancetti. Hospital de Base do Distrito Federal; Brasil Fil: de Carvalho, Elisa. Hospital de Base do Distrito Federal; Brasil Fil: Valente, Claudia França Cavalcante. Hospital de Base do Distrito Federal; Brasil Fil: Kuntze, Gisele. Integrated Center of Pediatric Specialties; Brasil Fil: Galicchio, Miguel. Hospital de Niños ; Argentina Fil: King, Alejandra. Hospital ; Chile Fil: Rosário Filho, Nelson Augusto. Universidade Federal do Paraná; Brasil Fil: Grota, Milena Baptistella. Universidade Estadual de Campinas; Brasil Fil: dos Santos Vilela, Maria Marluce. Universidade Estadual de Campinas; Brasil Fil: Di Gesu, Regina Sumiko Watanabe. Conceição Children’s Hospital; Brasil Fil: Lima, Simone. Childrens Hospital ; Brasil Fil: de Souza Moura, Leiva. Childrens Hospital ; Brasil Fil: Talesnik, Eduardo. Pontificia Universidad Católica de Chile; Chile Fil: Mansour, Eli. Universidade Estadual de Campinas; Brasil Fil: Roxo Junior, Pérsio. Universidade de Sao Paulo; Brasil Fil: Aldave, Juan Carlos. Hospital Nacional ; Perú Fil: Goudouris, Ekaterine. Universidade Federal do Rio de Janeiro; Brasil Fil: Pinto Mariz, Fernanda. Universidade Federal do Rio de Janeiro; Brasil Fil: Berrón Ruiz, Laura. Instituto Nacional de Pediatría; México Fil: Staines Boone, Tamara. Unidad Médica de Alta Especialidad #25; México Fil: Calderón, Wilmer O. Córdova. Clínica Montefiori; Perú Fil: del Carmen Zarate Hernández, María. Universidad Autónoma de Nuevo León; México Fil: Grumach, Anete S.. Universidade Federal do ABC; Brasil Fil: Sorensen, Ricardo. Children’s Hospital of New Orleans; Estados Unidos Fil: Durandy, Anne. Inserm; Francia Fil: Torgerson, Troy R.. University of Washington; Estados Unidos Fil: Carvalho, Beatriz Tavares Costa. Universidade de Sao Paulo; Brasil Fil: Espinosa Rosales, Francisco. Instituto Nacional de Pediatría; México Fil: Ochs, Hans D.. University of Washington; Estados Unidos Fil: Condino Neto, Antonio. Universidade de Sao Paulo; Brasil

Chronic granulomatous disease (CGD) is a primary immunodeficiency that affects the oxidative mechanism of microbial killing of phagocytic cells. The defect is characterized by a lack or severely reduced superoxide anion (O2−) production by phagocytes. Seventy percent of CGD cases are X-linked (X-CGD) and they are caused by mutations in the gene encoding for gp91phox, one of the two subunits of the flavocytochrome b558 of the NADPH oxidase. We identified an abnormal transcript arising from a novel splice site mutation within the gene encoding gp91phox, which suggested that the mutation affected normal mRNA splicing. Thus, the effect of this mutation leads to the complete absence of the flavocytochrome b558 in neutrophil membranes, which caused the biochemical phenotype X91°-CGD in this family. These molecular findings help to explain the early onset and severe phenotype in this X-CGD kindred. © 2004 Wiley-Liss, Inc.

Signal transducer and activator of transcription 5b (STAT5b) deficiency, first reported in a patient who carried a p.Ala630Pro missense mutation in the Src homology 2 (SH2) domain, results in a rare clinical condition of GH insensitivity (GHI), IGF-I deficiency (IGFD), and severe immune dysregulation manifesting as progressive worsening of pulmonary function.The new patient presented with severe cutaneous eczema, episodic infections in the first years of life, and autoimmune thyroiditis. Immunological evaluation revealed T lymphopenia, but severe pulmonary symptoms were notably absent. She concomitantly exhibited pronounced growth failure, reaching an adult height of 124.7 cm [-5.90 SD score (SDS)]. Endocrine evaluations (normal provocative GH tests; low serum IGF-I, -3.7 SDS, and IGF-binding protein-3, -4.5 SDS) were consistent with GHI and IGFD.Analysis of the STAT5B gene revealed a novel homozygous missense mutation, p.Phe646Ser, located within the βD' strand of the SH2 domain. Reconstitution studies demonstrated expression of the p.Phe646Ser variant was less robust than wild type but, in contrast to the previously described STAT5B p.Ala630Pro SH2 mutation, could be phosphorylated in response to GH and interferon-γ. The phosphorylated p.Phe646Ser, however, could not drive transcription.A novel STAT5B p.Phe646Ser mutation has been identified in a patient with clinical characteristics of STAT5b deficiency. Only the second STAT5B missense mutation identified, its lack of transcriptional activities despite GH-induced phosphorylation, confirms the crucial role of STAT5b for regulating the expression of IGF1 and provides insights into the importance of the SH2 βD' strand for full STAT5b transcriptional activities. Whether the phosphorylated p.Phe646Ser variant retained functions that prevented pulmonary distress remains unresolved.

The Wilms’ tumor gene is overexpressed in many types of haematological malignancies including acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML) and Ph negative myeloproliferative disorders. WT1 holds great promise for immunotherapy of leukaemia since it is expressed at high levels in blast cells but not in normal tissues, it is involved in the maintenance of malignant phenotype and it is spontaneously immunogenic. Many clinical trials using MHC class I-restricted WT1 peptide, have been recently performed in patients affected by AML, MDS, lung and breast cancer with satisfactory clinical results and without relevant toxicity. The first aim of the study was to analyze the natural immunity against WT1 in different types of haematological malignancies and the second was to set up a vaccination approach in the mouse model using the WT1 full length protein and to test the safety and efficacy of this vaccine. Using a dot blot technique, we analyzed for the presence of WT1 specific antibodies 107 patients including: 18 AML, 20 MDS, 22 Multiple Myeloma, 14 CML, 12 Idiopatic Myelofibrosis (IM), 6 CMML, and 15 ALL and 30 healthy subjects as control. For the second aim, we purified the WT1 full length protein. Complete coding sequence was cloned in a pGEX bacterial expression vector for the production of the fusion protein GST-WT1 that was subsequently purified by glutathione conjugated beads. Twenty C57BL7/6 mice were immunized with 50 μg of purified WT1 protein and 50 μL of Freund adjuvant every 15 days, for a total of 3 immunizations, 20 mice were injected with GST and adjuvant and 20 with PBS alone as control. 2 weeks after the last administration, 10 vaccinated mice and 10 controls have been injected with 200.000 TRAMP-C2 cells. Ten mice for each group were sacrificed and lymphocytes, cultured in presence of IL-2 to perform a cytotoxicity assay. Antibodies against WT1 have been evaluated. Toxicity on normal tissues has been rouled out using the histochemical analysis of different tissues. Results: Dot blot analysis demonstrated the presence of significant levels of WT1 IgG antibodies in 81% and IgM in 76% of the patients but not in normal subjects so demonstrating that WT1 is highly immunogenic. Particularly 88% of AML, 85% of MDS, 63% of MM, 100% of CML, 83% of IF and 66% of CMML presented high levels of antibodies. After mice vaccination, no organ toxicity has been observed. Importantly, immunized mice showed high levels of both, IgM and IgG antibodies with a mean value of luminescence of 690000 in WT1 injected mice vs 7500 in control mice and high levels of CTLs against WT1 as compared to mice injected with PBS or GST alone. Importantly, in control mice the mean size of the tumor was 1,6±0,4 cm, by contrast in vaccinated mice the tumor was unable to develop. Data obtained from this study clearly demonstrated that WT1 is immunogenic. The full length protein vaccination approach is able to elicit both, umoral and cytotoxic response and it results in a significant antitumor effect in vivo. Although further studies are required to compare the efficacy of this approach to the peptide based vaccine, WT1 full length protein could represent a valid vaccination strategy allowing to overcome the HLA restriction limit of the peptide approach.

Spred proteins are inducible inhibitors of signalling induced by receptor tyrosine kinases. They are implicated in negative feedback interactions that regulate intracellular pathways. The repressive function of Spred proteins targets several TK receptors so resulting in a variety of biological effects. Spred proteins, after growth factor stimulation, translocate to the plasma membrane, become tyrosine phosphorilated and interact with components of the Ras/MAPK and Ras/Raf/Erk pathways. Abnormal activation of Ras/MAPK pathway has been demonstrated in many haematological disorders including AML. In order to assess whether the activation of this pathway, leading to the disruption of many biological process, could be ascribed not only to the constitutive activation of TKs but also to defective signalling inhibition, we studied the function of Spred1 in AML. Using a Real Time PCR we studied the expression level of Spred1 in 80 AML samples (15 PB and 65 BM), 26 normal controls (16 PB and 10 BM) and 12 CD34+ samples sorted from AML patients and 12 from healthy subjects. We found that Spred1 transcript amount is significant reduced in AML samples when compare to normal controls (2−DeltaDeltaCt =0,084 vs 2,4 p=0,0000001) (range in AML 0,3–0,003). This difference is even more sound in AML CD34+ cells where the Spred1 transcript is 3 logs lower compared to normal CD34+ (2−DeltaDeltaCt =0,001 vs 2,27 p=0,0000001). Western blot using an antibody against Spred1 demonstrated the reduction or the absence of Spred1 protein in AML cells. Sequence analysis allowed to exclude the presence of mutations in the coding and promoter regions. In order to better understand the mechanism leading to the abrogation of Spred1 we analyzed the factors responsible for Spred1 transcription. We demonstrated that the transcription factor WT1 binds to and activates the promoter region of Spred1. WT1 KTS- is the isoform with transcriptional activity, while the isoform KTS+ is mainly localized at the cytoplasmatic level as confirmed by immunofluorescence analysis. Immunofluorescence assay and western blot carried out in NIH3T3 cells transfected with plasmids containing KTS+ and KTS- isoforms demonstrated that Spred1 protein is present at high levels in WT1 KTS- cells but not in KTS+ transfected cells. Finally we demonstrated, using capillary electrophoresis, that in AML patients the WT1 KTS- isoform was reduced or completely absent (ratio KTS+/KTS- ranging from 2 to 9,6 compared to a normal ratio ranging from 1, to 1,4 in cells from healthy subjects. Finally, in patients who achieved a complete remission after chemotherapy, WT1 KTS+/KTS- ratio returned within the normal range and Spred1 transcript and protein were significantly upregulated. This study clearly demonstrates that WT1 transcription activity is altered in AML cells in which WT1 may probably play a role of oncogene rather than of tumor suppressor gene. This leads to the absence of Spred1 transcript and protein which is a physiological inhibitor of RTK mediated signalling.