Modulatory role of intravenous gammaglobulin (IgIV) on the in vitro antibody response to a pneumococcal polysaccharide antigen.

INTRODUCTION: Clinical observations in patients with specific antibody deficiency treated for periods of time with IgG infusion have suggested that IgG may have a positive immunoregulatory effect on the production of specific antibodies against pneumococcal polysaccharides. We developed an in vitro model to test the effect of an IgIV preparation on the antibody production in response to a pneumococcal polysaccharide serotype and on the antibody and cytokine production in response to both a protein antigen and a pneumococcal polysaccharide antigen. METHODS: We studied 37 consecutive patients referred to our clinics for evaluation of their recurrent respiratory infections. Subjects were divided into two groups: 22 patients without SAD and 15 patients with SAD. PBMCs were left unstimulated or were stimulated with tetanus toxoid or pneumococcal polysaccharide serotype 19, in the presence of human albumin or IgIV. IgG anti-Pn-19 antibody, IL-4 and IFN-γ concentrations in culture supernatants were determined by ELISA. RESULTS: An increase in IgG anti-Pn-19 antibodies, associated with an increase in IFN-γ and a decrease in IL-4 production was observed in cultures stimulated with pneumococcal polysaccharide in the presence of IgIV when patients were analyzed together. The enhancing effect of IgIV was more significant for both IgG anti-Pn19 antibodies and IFN-γ in patients without SAD. In contrast, IgIV caused a significant decrease in IL-4 secretion in patients with SAD, which was associated with an increase in IgG anti-Pn19 antibodies in 3 of 7 of these patients. CONCLUSIONS: These results suggest that IgIV has some immunomodulatory effect on the in vitro production of IgG anti-Pn19 antibodies and cytokine production in cell cultures stimulated with Pn-19 antigen and that this modulation may be associated with a Th1/Th2 regulatory mechanism. Further studies at a cellular and molecular level are in progress to examine if the differences in the in vitro modulatory response to IgIV in these two groups of patients may point to a functional defect in patients with SAD.

Attending to warning signs of primary immunodeficiency diseases across the range of clinical practice.

PURPOSE: Patients with primary immunodeficiency diseases (PIDD) may present with recurrent infections affecting different organs, organ-specific inflammation/autoimmunity, and also increased cancer risk, particularly hematopoietic malignancies. The diversity of PIDD and the wide age range over which these clinical occurrences become apparent often make the identification of patients difficult for physicians other than immunologists. The aim of this report is to develop a tool for educative programs targeted to specialists and applied by clinical immunologists. METHODS: Considering the data from national surveys and clinical reports of experiences with specific PIDD patients, an evidence-based list of symptoms, signs, and corresponding laboratory tests were elaborated to help physicians other than immunologists look for PIDD. RESULTS: Tables including main clinical manifestations, restricted immunological evaluation, and possible related diagnosis were organized for general practitioners and 5 specialties. Tables include information on specific warning signs of PIDD for pulmonologists, gastroenterologists, dermatologists, hematologists, and infectious disease specialists. CONCLUSIONS: This report provides clinical immunologists with an instrument they can use to introduce specialists in other areas of medicine to the warning signs of PIDD and increase early diagnosis. Educational programs should be developed attending the needs of each specialty.

Recurrent respiratory infections, specific antibody deficiencies, and memory B cells.

OBJECTIVES: To investigate the immunological phenotypes detected in children with recurrent upper and lower respiratory infections that have normal total immunoglobulin concentrations. METHODS: A cohort of over 60 children with recurrent respiration infections was evaluated for specific antibody deficiencies (SAD) and for memory B-cell abnormalities. A control group of children without recurrent infections was also evaluated. Evaluation included a detailed history of immunizations with pneumococcal vaccines; determination of IgM, IgG, IgA, and IgE concentrations; measurement of anti-pneumococcal polysaccharide antibody levels by ELISA and expression of CD27, IgD, and IgM on peripheral CD19(+)B cells by flow cytometry to determine the proportions of naive, IgM-memory B cells, and class-switched memory B cells. RESULTS: Patients were classified as having a SAD to either pure polysaccharides (PPV-SAD) or to conjugate polysaccharides (PCV-SAD) based on the number of polysaccharides to which they developed an adequate antibody response. A normal response to only 2 or fewer of 7 PCV or PPV serotypes was considered as evidence of SAD. Forty-one patients without SAD and 26 with SAD were identified. IgM-memory B cells were low in 3 of 41 patients without SAD; in 3 of 5 PPV-SAD patients; and in 10 of 21 PCV-SAD patients. Class-switched memory B cells were low in 19 of 41 patients without SAD; in all 5 patients with PPV-SAD; and in 11 of 21PCV-SAD patients. CONCLUSIONS: Patients with recurrent infection with or without SAD may have low IgM- and/or class-switched memory B cells. Ongoing research aims to determine the prognostic implications of these differences in patients with SAD.

[An immunoenzymatic test for IgG antibody levels against 10 serotypes of Streptococcus pneumoniae]. / Estandarización en Colombia de una prueba ELISA para la evaluación de los niveles séricos de anticuerpos IgG contra diez serotipos de Streptococcus pneumoniae.

INTRODUCTION: Streptococcus pneumoniae is a major cause of morbi-mortality in early childhood and elderly. However, a test to measure the antibody responses after specific vaccination is not available in Colombia. OBJECTIVE: An immunoenzymatic test was standardized for the measurement of serum IgG levels against 10 serotypes of S. pneumoniae in response to the specific vaccination. MATERIAL AND METHODS: Capsular polysaccharides 1, 3, 4, 5, 6B, 9V, 14, 18, 19F, 23F of S. pneumoniae were used as antigens in a solid-phase ELISA. These responses were characterized in a randomized selected healthy individuals from a Colombian population. RESULTS: The reference and control sera showed great reactivity against all the polysaccharides evaluated, especially against polysaccharide 14 and 19F. The lowest reactivity in these two sera was observed against polysaccharide 3 and 4. Among the children evaluated, polysaccharide 5/19F showed the highes pre-vaccination reactivity, and polysaccharide 14/19F showed the highest post-vaccination reactivity. Among the adults, polysaccharides 14 and 19F showed the greatest reactivity pre- and post-vaccination. For all the polysaccharides (excepting polysaccharide 5), an inverse association among high polysaccharide-specific pre-vaccination- and the increase of post-vaccination-IgG levels was observed. CONCLUSION: This ELISA test reliably quantifies the serum levels of specific IgG against 10 serotypes of S. pneumoniae. According to the responses by healthy individuals, the current study validates parameters used internationally as an adequate the response to the 23-valent pneumococcal vaccine.

A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia.

Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells. However, not all leukemia cells are sensitive to GC, and no assay to stratify patients is available. In the GC-sensitive T-cell ALL cell line CEM-C7, auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response. This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations. The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay. There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples, but not for a probe set that detects a specific, low abundance GR transcript (exon 1A3). Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and, with further development, may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.

Estandarización en Colombia de una prueba ELISA para la evaluación de los niveles séricos de anticuerpos IgG contra diez serotipos de Streptococcus pneumoniae / An immunoenzymatic test for IgG antibody levels against 10 serotypes of Streptococcus pneumoniae

Introducción. Streptococcus pneumoniae es causante de gran morbimortalidad en niños pequeños y ancianos. Sin embargo, en Colombia no está disponible una prueba que evalúe la respuesta humoral a la vacunación específica contra este microorganismo Objetivo. Estandarizar en Colombia un ensayo inmunoenzimático para evaluar los niveles séricos de anticuerpos IgG contra diez serotipos de S. pneumoniae en respuesta a la vacunación específica y caracterizar esta respuesta en individuos sanos de nuestra población. Materiales y métodos. Se hizo un ELISA en fase sólida utilizando como antígenos los polisacáridos capsulares 1, 3, 4, 5, 6B, 9V, 14, 18, 19F y 23F de S. pneumoniae. Resultados. Los sueros de referencia y control reaccionaron fuertemente contra los polisacáridos evaluados, especialmente contra 14 y 19F. En los cinco niños sanos evaluados, los polisacáridos 5 y 19F presentaron los mayores títulos antes de la vacunación. Antes de la vacunación en los niños, y antes y después de la vacunación en los adultos, los polisacáridos 14 y 19F reaccionaron fuertemente. Para todos los polisacáridos, excepto para el 5, existe una relación inversa entre títulos altos de anticuerpos IgG antes de la vacunación y la razón de incremento de los títulos después de la misma. Conclusión. Esta prueba ELISA cuantifica de forma confiable los niveles de IgG sérica contra diez serotipos de S. pneumoniae y, de acuerdo con los resultados obtenidos en individuos sanos de nuestra población, en este trabajo se validan los parámetros internacionales para considerar adecuada la respuesta a la vacuna 23-valente contra este microorganismo.

Introduction. Streptococcus pneumoniae is a major cause of morbi-mortality in early childhood and elderly. However, a test to measure the antibody responses after specific vaccination is not available in Colombia. Objective. An immunoenzymatic test was standardized for the measurement of serum IgG levels against 10 serotypes of S. pneumoniae in response to the specific vaccination. Material and methods. Capsular polysaccharides 1, 3, 4, 5, 6B, 9V, 14, 18, 19F, 23F of S. pneumoniae were used as antigens in a solid-phase ELISA. These responses were characterized in a randomized selected healthy individuals from a Colombian population. Results. The reference and control sera showed great reactivity against all the polysaccharides evaluated, especially against polysaccharide 14 and 19F. The lowest reactivity in these two sera was observed against polysaccharide 3 and 4. Among the children evaluated, polysaccharide 5/19F showed the highes pre-vaccination reactivity, and polysaccharide 14/19F showed the highest post-vaccination reactivity. Among the adults, polysaccharides 14 and 19F showed the greatest reactivity pre- and post-vaccination. For all the polysaccharides (excepting polysaccharide 5), an inverse association among high polysaccharide-specific pre-vaccination- and the increase of post-vaccination-IgG levels was observed. Conclusion. This ELISA test reliably quantifies the serum levels of specific IgG against 10 serotypes of S. pneumoniae. According to the responses by healthy individuals, the current study validates parameters used internationally as an adequate the response to the 23-valent pneumococcal vaccine.

Efficacy of gene therapy for X-linked severe combined immunodeficiency.

BACKGROUND: The outcomes of gene therapy to correct congenital immunodeficiencies are unknown. We reviewed long-term outcomes after gene therapy in nine patients with X-linked severe combined immunodeficiency (SCID-X1), which is characterized by the absence of the cytokine receptor common gamma chain. METHODS: The nine patients, who lacked an HLA-identical donor, underwent ex vivo retrovirus-mediated transfer of gamma chain to autologous CD34+ bone marrow cells between 1999 and 2002. We assessed clinical events and immune function on long-term follow-up. RESULTS: Eight patients were alive after a median follow-up period of 9 years (range, 8 to 11). Gene therapy was initially successful at correcting immune dysfunction in eight of the nine patients. However, acute leukemia developed in four patients, and one died. Transduced T cells were detected for up to 10.7 years after gene therapy. Seven patients, including the three survivors of leukemia, had sustained immune reconstitution; three patients required immunoglobulin-replacement therapy. Sustained thymopoiesis was established by the persistent presence of naive T cells, even after chemotherapy in three patients. The T-cell-receptor repertoire was diverse in all patients. Transduced B cells were not detected. Correction of the immunodeficiency improved the patients' health. CONCLUSIONS: After nearly 10 years of follow-up, gene therapy was shown to have corrected the immunodeficiency associated with SCID-X1. Gene therapy may be an option for patients who do not have an HLA-identical donor for hematopoietic stem-cell transplantation and for whom the risks are deemed acceptable. This treatment is associated with a risk of acute leukemia. (Funded by INSERM and others.)

Effect of L-arginine supplementation on immune responsiveness in patients with sickle cell disease.

BACKGROUND: L-arginine (L-Arg) is deficient in sickle cell disease (SSD) during vasoocclusion. We investigated possible causal relationship between L-Arg deficiency and immune dysfunction in SSD in steady-state. PROCEDURE: Fifteen patients with SSD in steady-state and 13 controls were studied. Plasma L-Arg levels were measured using liquid chromatography. T cell subsets and CD3zeta (CD3zeta) chain expression were analyzed using flow cytometry. Lymphocyte proliferative response to phytohemagglutinin (PHA) and production of IL-6 and interferon-gamma (IFN-gamma) were evaluated with and without L-Arg. RESULTS: SSD patients had significantly lower L-Arg levels than controls. CD3 and CD19 cell populations were comparable for both groups, but SSD patients had above normal numbers of natural killer cells (P = 0.06). Patients and controls exhibited significantly increased lymphocyte blastogenesis to PHA after introduction of L-Arg to cultures; response of patients was significantly greater than values for control individuals. Proliferative response to candida in SSD patients was significantly lower than in controls; L-Arg supplementation did not increase this response. L-Arg had no effect on blastogenic response to PPD and candida albicans. No effect was likewise seen in production of IL-6 and IFN-gamma after addition of L-Arg. CD3zeta chain expression increased after addition of L-Arg in both groups; differences were insignificant. CONCLUSION: L-Arg levels in steady-state SSD are significantly lower than in controls. L-Arg supplementation enhanced lymphocyte blastogenesis to PHA for both controls and patients, but not in response to antigen. There were no significant differences in CD3zeta chain expression although upregulation of expression occurred after L-Arg supplementation for both groups.

Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.

Previously, several individuals with X-linked SCID (SCID-X1) were treated by gene therapy to restore the missing IL-2 receptor gamma (IL2RG) gene to CD34+ BM precursor cells using gammaretroviral vectors. While 9 of 10 patients were successfully treated, 4 of the 9 developed T cell leukemia 31-68 months after gene therapy. In 2 of these cases, blast cells contained activating vector insertions near the LIM domain-only 2 (LMO2) proto-oncogene. Here, we report data on the 2 most recent adverse events, which occurred in patients 7 and 10. In patient 10, blast cells contained an integrated vector near LMO2 and a second integrated vector near the proto-oncogene BMI1. In patient 7, blast cells contained an integrated vector near a third proto-oncogene,CCND2. Additional genetic abnormalities in the patients' blast cells included chromosomal translocations, gain-of-function mutations activating NOTCH1, and copy number changes, including deletion of tumor suppressor gene CDKN2A, 6q interstitial losses, and SIL-TAL1 rearrangement. These findings functionally specify a genetic network that controls growth in T cell progenitors. Chemotherapy led to sustained remission in 3 of the 4 cases of T cell leukemia, but failed in the fourth. Successful chemotherapy was associated with restoration of polyclonal transduced T cell populations. As a result, the treated patients continued to benefit from therapeutic gene transfer.

Primary immunodeficiency diseases in Latin America: the second report of the LAGID registry.

This is the second report on the continuing efforts of LAGID to increase the recognition and registration of patients with primary immunodeficiency diseases in 12 Latin American countries: Argentina, Brazil, Chile, Colombia, Costa Rica, Honduras, Mexico, Panama, Paraguay, Peru, Uruguay, and Venezuela. This report reveals that from a total of 3321 patients registered, the most common form of primary immunodeficiency disease was predominantly antibody deficiency (53.2%) with IgA deficiency reported as the most frequent phenotype. This category was followed by 22.6% other well-defined ID syndromes, 9.5% combined T- and B-cell inmunodeficiency, 8.6% phagocytic disorders, 3.3% diseases of immune dysregulation, and 2.8% complement deficiencies. All countries that participated in the first publication in 1998 reported an increase in registered primary immunodeficiency cases, ranging between 10 and 80%. A comparison of the estimated minimal incidence of X-linked agammaglobulinemia, chronic granulomatous disease, and severe combined immunodeficiency between the first report and the present one shows an increase in the reporting of these diseases in all countries. In this report, the estimated minimal incidence of chronic granulomatous disease was between 0.72 and 1.26 cases per 100,000 births in Argentina, Chile, Costa Rica, and Uruguay and the incidence of severe combined immunodeficiency was 1.28 and 3.79 per 100,000 births in Chile and Costa Rica, respectively. However, these diseases are underreported in other participating countries. In addition to a better diagnosis of primary immunodeficiency diseases, more work on improving the registration of patients by each participating country and by countries that have not yet joined LAGID is still needed.

Experimental infection of rhesus macaques with Streptococcus pneumoniae: a possible model for vaccine assessment.

BACKGROUND: We explored the possibility of using normal adult rhesus macaques for the preclinical assessment of safety, immunogenicity, and efficacy of newly developed vaccines against Streptococcus pneumoniae infection of the lung. METHODS: Our primary objective was to determine whether an intra-bronchial inoculum of at least 10(6)S. pneumoniae colony-forming units, or one as high as 10(8)-10(9) organisms, could detectably survive in rhesus macaques for a period longer than 1-2 weeks. If so, we hypothesized, it would be possible to observe signs of pneumonia commonly observed in humans, and discriminate between vaccinated/protected animals and controls. Infection was detectable in bronchoalveolar lavage fluids 3-5 weeks post-inoculation. RESULTS: The clinical course of disease mimicked aspects of that of human pneumococcal pneumonia. Signs of inflammation typical of the disease in humans, such as elevated concentrations of neutrophils and of pro-inflammatory cytokines in bronchoalveolar lavage fluids were also observed. CONCLUSIONS: These findings underscore the utility of this model to assess the safety, immunogenicity, and efficacy of newly developed S. pneumoniae vaccines.

Immunological evaluation of allergic respiratory children with recurrent sinusitis.

The objective of this study was to evaluate humoral immunity of allergic respiratory children with chronic/recurrent sinusitis. Twenty-seven allergic respiratory (persistent mild/moderate asthma and persistent allergic rhinitis) children (7-15-year old) with chronic or recurrent sinusitis were evaluated. Patients had symptoms and abnormal computer tomography scan even after two adequate treatments (long-lasting antibiotics, decongestants, and short-term oral corticosteroids). clinical examination, sweat test, total blood cell count, measurement of serum levels of: total and specific IgE, immunoglobulins (G, M, A), IgG subclasses, antibodies to Haemophilus influenza type b (IgG anti-Ps Hib) and pneumococcal serotypes (IgG anti-Ps 1, 3, 5, 6B, 9V, and 14) before and after active immunization (Act-Hib and Pneumo23, Aventis Pasteur SA, Lyon, France), Rubella neutralizing antibody titers and human immunodeficiency virus antibodies. Specific IgE to inhalant allergens higher than class III were observed in 24/27 patients. One patient had IgA plus IgG2 deficiency and other an IgG3 deficiency. Eight and 12 of 27 patients had IgG2 and IgG3 serum levels below 2.5th percentile, respectively. Immunological responses to protein and polysaccharide antigens were normal in all patients. Although our patients have been appropriately treated of their allergic diseases, they persisted with chronic/recurrent sinusitis and 60% of them had a documented osteomeatal complex blockade. In spite of the diagnosis of IgA plus IgG2 deficiency and an isolated IgG3 deficiency, in all patients an adequate response to Ps antigens was observed. Primary and/or secondary humoral immunodeficiency seems not to be the main cause of chronic/recurrent sinusitis in patients with respiratory allergic disease.

Hemopoietic function after use of IL-1 with chemotherapy or irradiation.

IL-1 has putative chemo- and radioprotective properties, but its effects on primitive hemopoietic stem cell (PHSC) and early multilineage precursor function when given with these modalities is unknown. C57BL6/J (B6) mice, given IL-1 20 h before cyclophosphamide (200 mg/kg for four biweekly doses) or before irradiation (500 cGy), were sacrificed after 4 wk. Their marrow was used as donor cells, and that from B6-Hbb(dGpi1a) (B6-GPI) mice was used as competitor cells in competitive repopulation. Percentages of B6 cells were measured at 30 and 150 days. Stem cell numbers were estimated using binomial statistics. IL-1 alone did not affect stem cell function. As expected, significant declines in early multilineage precursor and PHSC function occurred with chemotherapy and radiation alone. IL-1 with chemotherapy led to exacerbation of these losses in function and numbers (p < 0.05). A similar reduction in function occurred using IL-1 before irradiation. In summary, IL-1 with chemotherapy or radiation worsened chemotherapy- and radiation-induced functional damage to PHSC and other hemopoietic precursors, suggesting that improvements in survival do not necessarily translate into preservation of hemopoietic function.