Peripheral blood monocyte and T cell subsets in children with specific polysaccharide antibody deficiency (SPAD).

Specific polysaccharide antibody deficiency (SPAD) is a well reported immunodeficiency characterized by a failure to produce antibodies against polyvalent polysaccharide antigens, expressed by encapsulated microorganisms. The clinical presentation of these patients involves recurrent bacterial infections, being the most frequent agent Streptococcus (S.) pneumoniae. In SPAD patients few reports refer to cells other than B cells. Since the immune response to S. pneumoniae and other encapsulated bacteria was historically considered restricted to B cells, the antibody deficiency seemed enough to justify the repetitive infections in SPAD patients. Our purpose is to determine if the B cell defects reported in SPAD patients are accompanied by defects in other leukocyte subpopulations necessary for the development of a proper adaptive immune response against S. pneumoniae. We here report that age related changes observed in healthy children involving increased percentages of classical monocytes (CD14++ CD16- cells) and decreased intermediate monocytes (CD14++ CD16+ cells), are absent in SPAD patients. Alterations can also be observed in T cells, supporting that the immune deficiency in SPAD patients is more complex than what has been described up to now.

Follicular bronchiolitis as phenotype associated with CD25 deficiency.

Regulatory T cells [Tregs ; CD4(+) CD25(+) forkhead box protein 3 (FoxP3(+) )] are subsets of T cells involved in the maintenance of peripheral self-tolerance by actively suppressing the activation and expansion of autoreactive T cells. Signalling through the interleukin-2 receptor (IL-2R) contributes to T cell tolerance by controlling three important aspects of regulatory T cell (Treg ) biology. CD25 is the α-chain of the IL-2R that, in concert with the ß-chain and γ-chain, constitutes the complete IL-2R. CD25 contributes only to IL-2 binding affinity but not to the recruitment of signalling molecules. However, its importance in the development of a normal immune response is emphasized by the finding that a truncation mutant of CD25 results in an immunodeficiency in humans characterized by an increased susceptibility to viral, bacterial and fungal infections. In 1997, Sharfe et al. described an infant with severe bacterial, viral and fungal infections. Counts of autologous T lymphocytes were moderately low, T cells displayed a weak proliferative response to mitogens in vitro and the patient displayed no rejection of an allogeneic skin graft. However, unlike children with severe combined immunodeficiency (SCID), besides not having circulating T cells, the patient also developed peripheral lymphocytic proliferation and autoimmune primary biliary cirrhosis. We present the first female Argentine patient with mutation in CD25 associated with chronic and severe inflammatory lung disease (follicular bronchiolitis with lymphocyte hyperplasia), eczema and infections. She has no expression of CD25 on CD4(+) T cells and an extremely low amount of Tregs . The molecular study confirmed homozygous missense mutation in the alpha subunit of the IL-2 receptor (CD25αR) (c. 122 a > c; p. Y41S).

Immune response to Streptococcus pneumoniae in asthma patients: comparison between stable situation and exacerbation.

In Argentina, more than 3 million people suffer from asthma, with numbers rising. When asthma patients acquire viral infections which, in turn, trigger the asthmatic response, they may develop subsequent bacterial infections, mainly by Streptococcus (S.) pneumoniae. This encapsulated Gram(+) bacterium has been considered historically a T cell-independent antigen. Nevertheless, several papers describe the role of T cells in the immune response to S. pneumoniae. We evaluated the response to S. pneumoniae and compared it to the response to Mycobacterium (M.) tuberculosis, a different type of bacterium that requires a T helper type 1 (Th1) response, in cells from atopic asthmatic children, to compare parameters for the same individual under exacerbation and in a stable situation whenever possible. We studied asthma patients and a control group of age-matched children, evaluating cell populations, activation markers and cytokine production by flow cytometry, and cytokine concentration in serum and cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). No differences were observed in γδ T cells for the same patient in either situation, and a tendency to lower percentages of CD4(+) CD25(hi) T cells was observed under stability. A significantly lower production of tumour necrosis factor (TNF)-α and a significantly higher production of interleukin (IL)-5 was observed in asthma patients compared to healthy individuals, but no differences could be observed for IL-4, IL-13 or IL-10. A greater early activation response against M. tuberculosis, compared to S. pneumoniae, was observed in the asthmatic patients' cells. This may contribute to explaining why these patients frequently acquire infections caused by the latter bacterium and not the former.

Advancing the management of primary immunodeficiency diseases in Latin America: Latin American Society for Immunodeficiencies (LASID) Initiatives.

Primary immunodeficiency diseases (PIDD) are associated with significant morbidity and mortality and result in a significant public health burden. This is in part due to the lack of appropriate diagnosis and treatment of these patients. It is critical that governments become aware of this problem and provide necessary resources to reduce this impact on health care systems. Leading physicians in their respective countries must be supported by their own governments in order to implement tools and provide education and thus improve the diagnosis and treatment of PIDD. The Latin American Society of Primary Immunodeficiencies (LASID) has initiated a large number of activities aimed at achieving these goals, including the establishment of a PIDD registry, development of educational programmes and guidelines, and the introduction of a PIDD fellowship programme. These initiatives are positively impacting the identification and appropriate treatment of patients with PIDD in Latin America. Nevertheless, much remains to be done to ensure that every person with PIDD receives proper therapy.

The role of cytokines in atopic asthma.

Atopic asthma results from airway inflammation triggered by an environmental allergen. Symptoms include wheezing, dyspnea and cough, airway narrowing and/or hyperresponsiveness to several inhaled stimuli. Inflammation develops in a two-phase fashion. The first phase after exposure to the allergen consists of degranulation and release of both histamine and other stored preformed inflammatory mediators as well as newly synthesized ones, including cytokines, all of which increase mucus secretion and smooth muscle contraction. The second phase occurs later and lasts longer; it is due to different molecules: several cytokines and chemokines, arachidonic acid derivatives, enzymes such as metalloproteinases and cell adhesion molecules. Cytokines are key players in the chronic inflammation in asthma patients, but details on their role and interactions still remain undetermined. Recent evidence suggests that allergic asthma is a multifaceted condition actively controlled by effector as well as regulatory T cells (Tregs). T helper (Th) 2 cells and Th17 cells increase airway inflammation, while Tregs are anti- inflammatory. Cytokines are involved in the development and activation of all T cell subpopulations. They are also involved directly or indirectly in most approaches to asthma treatment. Several cytokines have been tested as therapeutic targets and some of the currently used therapies like corticosteroids, beta agonists and allergen immunotherapy affect cytokine production. The increased knowledge on cytokine interplay and lymphocyte subsets should generate new therapeutic strategies in the near future.

Critical issues and needs in management of primary immunodeficiency diseases in Latin America.

Experts from six Latin American countries met to discuss critical issues and needs in the diagnosis and management of primary immunodeficiency diseases (PIDD). The diagnosis of PIDD is generally made following referral to an immunology centre located in a major city, but many paediatricians and general practitioners are not sufficiently trained to suspect PIDD in the first place. Access to laboratory testing is generally limited, and only some screening tests are typically covered by government health programmes. Specialised diagnostic tests are generally not reimbursed. Access to treatment varies by country reflecting differences in healthcare systems and reimbursement policies. An online PIDD Registry Programme for Latin America has been available since 2009, which will provide information about PIDD epidemiology in the region. Additional collaboration across countries appears feasible in at least two areas: a laboratory network to facilitate the diagnosis of PIDD, and educational programmes to improve PIDD awareness. In total, these collaborations should make it possible to advance the diagnosis and management of PIDD in Latin America.

A 475 years-old founder effect involving IL12RB1: a highly prevalent mutation conferring Mendelian Susceptibility to Mycobacterial Diseases in European descendants.

Mutations in IFNGR1, IFNGR2, IL12RB1, IL12B, STAT1 and NEMO result in a common clinical phenotype known as Mendelian Susceptibility to Mycobacterial Diseases (MSMD). Interleukin-12 receptor beta1 (IL-12Rbeta1) deficiency is the most common genetic etiology for MSMD. Known mutations affecting IL12RB1 are recessively inherited and are associated with null response to both IL-12 and IL-23. Mutation IL12RB1 1623_1624delinsTT was originally described in 5 families from European origin (2 from Germany; 1 from Cyprus, France and Belgium). Interestingly, this same mutation was found in an unexpectedly high prevalence among IL-12Rbeta1 deficient patients in Argentina: 5-out-of-6 individuals born to unrelated families carried this particular change. To determine whether mutation 1623_1624delinsTT represents a DNA mutational hotspot or a founder effect, 34 polymorphic markers internal or proximal to IL12RB1 were studied in the Argentinean and the Belgian patients. A common haplotype spanning 1.45-3.51Mb was shared by all chromosomes carrying mutation 1623_1624delinsTT, and was not detected on 100 control chromosomes. Applying a modified likelihood-based method the age of the most recent common ancestor carrying mutation 1623_1624delinsTT was estimated in 475 years (95% CI, 175-1275), which is the time when the Spaniards initiated the colonization of the Americas. Mutation 1623_1624delinsTT represents the first founder effect described on IL-12Rbeta1, the most frequently affected gene in MSMD, and affecting patients with European ancestors. The reason(s) behind the persistency of this mutation across multiple generations, its relative high prevalence, and any potential selective advantage are yet to be established.

[Kawasaki disease. Immunological evaluation of 26 cases]. / Enfermedad de Kawasaki. Evaluación inmunológica de 26 casos.

Kawasaki disease (KD) is an acute febrile vasculitis of childhood, characterized by multiple clinical and biochemical features of inflammation with special involvement of the heart. The activation of lymphocytes and monocytes/macrophages and their secreted soluble products, cytokines, play a central role in the pathogenesis of the disease. In this study we performed immunologic studies in 26 patients with KD. No constant pattern of serum levels of IgG, IgA, IgM, C3 and C4 fractions of complement measured by Nephelometry and neither autoantibodies, FAN and ANCA performed by indirect immunofluorescence were found in 22 patients in the acute stage. Variable percentages of CD3, CD4, CD8, CD20, CD56 and DR in peripheral mononuclear cells specifically stained and analysed by flow cytometry were seen among 25 patients in the acute stage. CD25 was elevated in 17/25 cases. Serum levels of TNF alpha performed by ELISA in 12 patients in acute stage were low. Intracellular cytokines such as TNF alpha, IL1 beta, IL2 and IFN gamma were measured in peripheral mononuclear cells of 15 patients in acute stage, in 5th and 30th days after gammaglobulin treatment, utilizing specific staining and analysis by flow cytometry showing no sole characteristic profile. In 2 patients there was an elevated percentage of TNF alpha and IL1 beta in monocytes during the convalescent stage; both had coronary sequelae. More research on this question is needed. In conclusion, immunologic studies showed an heterogeneous profile and no laboratory finding was registered in the acute stage that could be used as predictive factor of cardiovascular involvement.

Enfermedad de Kawasaki. Evaluación inmunológica de 26 casos. / [Kawasaki disease. Immunological evaluation of 26 cases]

Kawasaki disease (KD) is an acute febrile vasculitis of childhood, characterized by multiple clinical and biochemical features of inflammation with special involvement of the heart. The activation of lymphocytes and monocytes/macrophages and their secreted soluble products, cytokines, play a central role in the pathogenesis of the disease. In this study we performed immunologic studies in 26 patients with KD. No constant pattern of serum levels of IgG, IgA, IgM, C3 and C4 fractions of complement measured by Nephelometry and neither autoantibodies, FAN and ANCA performed by indirect immunofluorescence were found in 22 patients in the acute stage. Variable percentages of CD3, CD4, CD8, CD20, CD56 and DR in peripheral mononuclear cells specifically stained and analysed by flow cytometry were seen among 25 patients in the acute stage. CD25 was elevated in 17/25 cases. Serum levels of TNF alpha performed by ELISA in 12 patients in acute stage were low. Intracellular cytokines such as TNF alpha, IL1 beta, IL2 and IFN gamma were measured in peripheral mononuclear cells of 15 patients in acute stage, in 5th and 30th days after gammaglobulin treatment, utilizing specific staining and analysis by flow cytometry showing no sole characteristic profile. In 2 patients there was an elevated percentage of TNF alpha and IL1 beta in monocytes during the convalescent stage; both had coronary sequelae. More research on this question is needed. In conclusion, immunologic studies showed an heterogeneous profile and no laboratory finding was registered in the acute stage that could be used as predictive factor of cardiovascular involvement.

Progressive multifocal leukoencephalopathy in a patient with hypogammaglobulinemia.

We describe a child with congenital hypogammaglobulinemia that was diagnosed at 13 months of age. When he was 4 years old, gait disturbances began. The main neurological manifestations were progressive spastic tetraparesis and intellectual and speech deterioration. No infectious agent was identified. A magnetic resonance imaging scan of the central nervous system revealed periventricular demyelinating areas in the frontal, temporal, and parietal lobes with cortical atrophy. Stereotactic brain biopsy confirmed the diagnosis of progressive multifocal leukoencephalopathy caused by JC virus. He was treated with intravenous and intraventricular cytarabine and interferon-alpha, and there was clinical improvement. We emphasize the need for brain biopsy as soon as a neurological complication is suspected in patients with congenital hypogammaglobulinemia for whom cerebrospinal cultures or polymerase chain reaction analyses are negative.

[Phenotypic changes in the mononuclear cells of patients with primary immunodeficiencies]. / Alteraciones fenotípicas en células mononucleares de pacientes con inmunodeficiencias primarias.

The study of differentiation antigens of circulating mononuclear cells in 70 patients with primary immunodeficiency (PID) using monoclonal antibodies allowed us to define phenotypic profiles that are characteristic of the different described syndromes. In common variable immunodeficiency we found percentages of lymphocytes within normal ranges, and an altered CD4/CD8 ratio. In sex-linked agammaglobulinemia, absence of B lymphocytes with normal distribution of regulatory populations (CD4/CD8) were found. These results allow us to distinguish two clinically and infectologically similar conditions. In selective IgA deficiency, distribution of lymphocytic populations was normal. In immunodeficiency with hyper IgM, considered up to date as an abnormal maturation of B lymphocytes, we observed a deficiency in cellular immune response, and a phenotypic profile characterized by: decreased number of CD3 cells, inverted CD4/CD8 ratio, and increased CD38 population; this profile being similar to the one that we found in predominantly cellular immunodeficiency. In predominantly cell-mediated immunodeficiency and in those immunodeficiencies associated to other defects (such as: hyper IgE syndrome, Di George syndrome), the most important finding was a significative increase in CD38 population. Although it's not possible to consider on this basis that there is a defect at the thymic level of T-cells maturation, the high levels of circulating CD38 cells were a clear indication of altered cellular immune response in our series of patients. Patients with predominantly cell-mediated immunodeficiency showed the lowest levels of CD4 cells and the corresponding inversion of CD4/CD8 ratio. In Di George syndrome we found a markedly diminished CD8 population that differentiates this entity from the rest of the studied syndromes. In chronic mucocutaneous candidosis distribution of lymphocytic populations was normal, but a significative increase in the percentages of CD11b+ cells was observed. In patients with antibodies deficiency that received substitutive treatment with gammaglobulin we found no variations in lymphocytic populations distribution. In the group of patients with altered cellular immunity treated with thymic hormones, observed phenotypic changes (increase in T-cells population, trend to normalization in CD4/CD8 ratio, and decrease in CD38 population) were transient, and lasted only during the treatment period. We considered that describing these phenotypic profiles is a useful diagnosis tool when evaluating patients with PID, since these profiles are characteristic and very stable.

[Changes in distribution of T subpopulations in the peripheral blood of patients with severe idiopathic anemia]. / Alteración de la distribución de subpoblaciones T en la sangre periferica de pacientes con anemia severa idiopática.

The following immunological studies were performed in circulating mononuclear cells of 17 patients with severe aplastic anemia (SAA): a) lymphocytic phenotypes; b) proliferative response to PHA; c) determination of interleukin 2 (IL2) production and d) expression of Tac CD25. Fifteen of the seventeen patients showed altered CD4/CD8 regulatory populations, expressed as a significantly diminished CD4/CD8 ratio (0.72 +/- 0.19, NV: 1.8 +/- 0.6) (Table 1). The proliferative response to PHA was normal in 80% of the cases; only 2 of the patients showed a diminished response to the mitogen (Fig. 1). IL2 production by PHA-stimulated mononuclear cells was significantly increased (56.6 +/- 9.8; NV: 11 +/- 7.69) (Fig. 1), and a deficient expression to CD25 antigen was also recorded (Table 2). In the other two patients, we observed a normal CD4/CD8 ratio (Table 1, patients 1 and 2) with absence of proliferative response to PHA and hypo-production of IL2 (Fig. 1). These results suggest that in these two cases the hematopoietic defect could be associated to a primary deficiency of cellular immunity. Our results support the current concept of diversity of pathogenetic mechanisms implicated in SAA, and suggest that there are groups of patients with variable degrees of immunological defect that can be delineated through laboratory assays. On the other hand, the altered distribution of regulatory populations mostly due to an absolute decrease of the CD4 subpopulation, associated to the hyperproduction of IL2 and deficient expression of the Tac antigen in most of our patients, suggest the existence of functional alterations.

Alteración de la distribución de subpoblaciones T en la sangre periferica de pacientes con anemia severa idiopática. / [Changes in distribution of T subpopulations in the peripheral blood of patients with severe idiopathic anemia]

The following immunological studies were performed in circulating mononuclear cells of 17 patients with severe aplastic anemia (SAA): a) lymphocytic phenotypes; b) proliferative response to PHA; c) determination of interleukin 2 (IL2) production and d) expression of Tac CD25. Fifteen of the seventeen patients showed altered CD4/CD8 regulatory populations, expressed as a significantly diminished CD4/CD8 ratio (0.72 +/- 0.19, NV: 1.8 +/- 0.6) (Table 1). The proliferative response to PHA was normal in 80

of the cases; only 2 of the patients showed a diminished response to the mitogen (Fig. 1). IL2 production by PHA-stimulated mononuclear cells was significantly increased (56.6 +/- 9.8; NV: 11 +/- 7.69) (Fig. 1), and a deficient expression to CD25 antigen was also recorded (Table 2). In the other two patients, we observed a normal CD4/CD8 ratio (Table 1, patients 1 and 2) with absence of proliferative response to PHA and hypo-production of IL2 (Fig. 1). These results suggest that in these two cases the hematopoietic defect could be associated to a primary deficiency of cellular immunity. Our results support the current concept of diversity of pathogenetic mechanisms implicated in SAA, and suggest that there are groups of patients with variable degrees of immunological defect that can be delineated through laboratory assays. On the other hand, the altered distribution of regulatory populations mostly due to an absolute decrease of the CD4 subpopulation, associated to the hyperproduction of IL2 and deficient expression of the Tac antigen in most of our patients, suggest the existence of functional alterations.

[Lymphocytic, phenotypic and functional studies in primary immunodeficiencies]. / Estudios linfocitarios, fenotípicos y funcionales en inmunodeficiencias primarias.

In this report we present the leukocyte phenotypic analysis of 64 cases of primary immune deficiencies (PID). Functional studies related to lymphocyte activation (CD25 (Tac) antigen expression and response to exogenous IL2) as well as immunoregulatory pathways (spontaneous suppressor activities and suppression by soluble factors) were also considered taking immunodeficiency with hyper-IgM (IDHM) as model. The study of mononuclear cell populations with monoclonal antibodies allowed the characterization of defined phenotypes. In common variable immunodeficiency, B cells were present in normal percentages. In sex-linked agammaglobulinemia there was a lack of B lymphocytes and normal distribution of regulatory populations. These results point out the difference between these two entities despite their clinical and infective similarities. Excess of cells expressing CD38 antigen (NV: 4 +/- 2) were found in: predominantly cell mediated immunodeficiency (PCMI): 38 +/- 20; ataxia telangiectasia: 25 +/- 8, hyper-IgE syndrome: 24 +/- 13; Di George syndrome (DGS): 24 +/- 9, chronic mucocutaneous candidiasis: 15 +/- 7. The increased expression of this antigen was correlated with the presence of compromised cellular immunity. The DGS presented the lowest level of CD8 cells (6 +/- 5; NV: 21 +/- 7). In two patients with IDHM, the phenotypic profile was similar to that found in PCMI (low CD3 cells, low CD4/CD8 ratio and elevated CD38 cells). The depressed proliferative response to PHA demonstrates a cellular immune defect. In both patients we found a low expression of CD25 antigen in stimulated cells. Moreover, the addition of exogenous IL2 decreased the proliferative response to PHA in a dose-dependent fashion, suggesting that the cells expressing the CD25 antigen have suppressor capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Estudios linfocitarios, fenotípicos y funcionales en inmunodeficiencias primarias. / [Lymphocytic, phenotypic and functional studies in primary immunodeficiencies]

In this report we present the leukocyte phenotypic analysis of 64 cases of primary immune deficiencies (PID). Functional studies related to lymphocyte activation (CD25 (Tac) antigen expression and response to exogenous IL2) as well as immunoregulatory pathways (spontaneous suppressor activities and suppression by soluble factors) were also considered taking immunodeficiency with hyper-IgM (IDHM) as model. The study of mononuclear cell populations with monoclonal antibodies allowed the characterization of defined phenotypes. In common variable immunodeficiency, B cells were present in normal percentages. In sex-linked agammaglobulinemia there was a lack of B lymphocytes and normal distribution of regulatory populations. These results point out the difference between these two entities despite their clinical and infective similarities. Excess of cells expressing CD38 antigen (NV: 4 +/- 2) were found in: predominantly cell mediated immunodeficiency (PCMI): 38 +/- 20; ataxia telangiectasia: 25 +/- 8, hyper-IgE syndrome: 24 +/- 13; Di George syndrome (DGS): 24 +/- 9, chronic mucocutaneous candidiasis: 15 +/- 7. The increased expression of this antigen was correlated with the presence of compromised cellular immunity. The DGS presented the lowest level of CD8 cells (6 +/- 5; NV: 21 +/- 7). In two patients with IDHM, the phenotypic profile was similar to that found in PCMI (low CD3 cells, low CD4/CD8 ratio and elevated CD38 cells). The depressed proliferative response to PHA demonstrates a cellular immune defect. In both patients we found a low expression of CD25 antigen in stimulated cells. Moreover, the addition of exogenous IL2 decreased the proliferative response to PHA in a dose-dependent fashion, suggesting that the cells expressing the CD25 antigen have suppressor capacity.(ABSTRACT TRUNCATED AT 250 WORDS)