Quantitative and functional evaluation of innate immune responses in patients with common variable immunodeficiency.

OBJECTIVES: We evaluate the frequency and functional response of innate immune cells in peripheral blood (PB) from patients with common variable immunodeficiency (CVID) and healthy controls upon activation with agonists of the Toll-like receptors (TLR) TLR2, TLR4, and TLR9. In addition, several nonsynonymous single nucleotide polymorphisms (SNPs) within these TLR genes were examined. METHODS: Flow cytometry was used to perform immunophenotyping and evaluate the expression of cell surface markers. Levels of cytokines in the culture supernatants were evaluated using cytometric bead array technology. SNPs in the TLR genes were evaluated from genomic DNA using different sequencing techniques. RESULTS: Our results demonstrate that the frequency of CD1d-restricted TCR invariant natural killer T cells in PB was significantly reduced in the patients with CVID. A marked, though not significant, reduction in absolute numbers of plasmacytoid dendritic cells and natural killer cells was also observed in these patients. Interestingly, CD80 and CD86 expression on innate cells upon stimulation with TLR ligands was not altered in the patients although 3 of them exhibited low baseline levels of these surface molecules on monocytes compared to healthy controls. We also observed a significant increase in TNF-alpha levels in supernatants of PB mononuclear cells from CVID patients after stimulation with lipopolysaccharide. Finally, no association was found between the presence of nonsynonymous SNPs within the TLR genes and the clinical presentation of CVID. CONCLUSIONS: Taken together, our study demonstrates than innate immune responses are disturbed in some CVID patients and prompts the evaluation of innate immunity genes as candidates to explain the CVID clinical phenotype.

Apoptosis as a mechanism of natural resistance to HIV-1 infection in an exposed but uninfected population.

BACKGROUND: Apoptosis, also known as programmed cell death, has been reported not only as a pathogenic mechanism, but also as a mechanism of resistance and control of a variety of infections. Particularly during HIV-1 infection, apoptosis is the main mechanism by which infected and uninfected CD4+ lymphocytes are eliminated. However, apoptosis as a mechanism of natural resistance to HIV infection has this far not been explored. OBJECTIVE: To determine whether apoptosis could explain, at least in part, the natural resistance to HIV infection observed in some exposed but uninfected individuals (ESN). RESULTS: Our data shows that peripheral blood monocytes in the ESN group has a predisposition to undergo spontaneous apoptosis, as well as apoptosis induced by HIV infection in vitro, compared with monocyte population from the control group at low risk of HIV infection. CONCLUSIONS: These findings suggest that, in some ESN individuals, monocytes could play an important role in the control of HIV infection by undergoing apoptosis. However, since the variability among individuals is large, studies with larger cohorts focusing in monocyte apoptosis as pathogenic mechanisms are required.

Normal expression of IFN-gammaR in four patients with uncommon mycobacterial infection phenotypes

Several primary immunodeficiency diseases affecting the interleukin 12/interferon gamma (IFN-gamma) pathway have been identified, most of them characterized by recurrent and protracted infections produced by intracellular microorganisms, particularly by several species of mycobacteria. In the present study we analyzed the expression of IFN-gamma receptor (IFN-gammaR) and signal transducer and activator of transcription 1 (STAT-1) in 4 children with Mycobacterium tuberculosis infection of uncommon clinical presentation. These molecules were evaluated by flow cytometry and Western blotting in B cells transformed with Epstein-Barr virus and mutations were scanned by single-strand conformational polymorphisms and DNA sequencing. The expression of IFN-gammaR1 was normal in all 4 patients. The genetic analysis of IFN-gammaR1 and IFN-gammaR2 coding sequences did not reveal any mutation. The expression of the STAT-1 molecule was similar in patients and healthy controls; however, when the phosphorylation of this transcription factor in response to IFN-gamma activation was evaluated by Western blot, a significant lower signal was evident in one patient. These data indicate that there are no alterations in the expression or function of the IFN-gammaR chains in these patients. However, the low level of STAT-1 phosphorylation found in one of these patients might be explained by a defect in one of the molecules involved in the signal transduction pathway after IFN-gamma interacts with its receptor. In the other three patients the inability to eliminate the mycobacteria may be due to a defect in another effector mechanism of the mononuclear phagocytes.

Normal expression of IFN-gammaR in four patients with uncommon mycobacterial infection phenotypes.

Several primary immunodeficiency diseases affecting the interleukin 12/interferon gamma (IFN-gamma) pathway have been identified, most of them characterized by recurrent and protracted infections produced by intracellular microorganisms, particularly by several species of mycobacteria. In the present study we analyzed the expression of IFN-gamma receptor (IFN-gammaR) and signal transducer and activator of transcription 1 (STAT-1) in 4 children with Mycobacterium tuberculosis infection of uncommon clinical presentation. These molecules were evaluated by flow cytometry and Western blotting in B cells transformed with Epstein-Barr virus and mutations were scanned by single-strand conformational polymorphisms and DNA sequencing. The expression of IFN-gammaR1 was normal in all 4 patients. The genetic analysis of IFN-gammaR1 and IFN-gammaR2 coding sequences did not reveal any mutation. The expression of the STAT-1 molecule was similar in patients and healthy controls; however, when the phosphorylation of this transcription factor in response to IFN-gamma activation was evaluated by Western blot, a significant lower signal was evident in one patient. These data indicate that there are no alterations in the expression or function of the IFN-gammaR chains in these patients. However, the low level of STAT-1 phosphorylation found in one of these patients might be explained by a defect in one of the molecules involved in the signal transduction pathway after IFN-gamma interacts with its receptor. In the other three patients the inability to eliminate the mycobacteria may be due to a defect in another effector mechanism of the mononuclear phagocytes.

Molecular characterization of the CCR 5 gene in seronegative individuals exposed to human immunodeficiency virus (HIV).

BACKGROUND: Both clinical and laboratory evidence in exposed seronegative (ESN) individuals to human HIV-1 has suggested the existence of mechanisms of natural resistance to the infection. A 32 base-pair deletion in the gene that codes for the CCR5, which is the main coreceptor for HIV-1, confers a high degree of resistance to HIV-1 infection. However, the genotype Delta32/Delta32 is present only in 2-4% of Caucasoid ESN individuals suggesting the existence of other mechanisms of protection. Mutations different from Delta32 have also been proposed as playing a role in resistance/susceptibility to this infection. OBJECTIVE: To screen for different mutations along the entire coding region of the ccr5 gene that can potentially explain the persistent seronegativity in a group of ESN individuals. STUDY DESIGN: Of a total of 86 individuals analyzed for Delta32 mutation by the PCR technique, 36 scored HIV seropositive (SP) and 50 were ESN. The entire group of ESN individuals was screened for other mutations in the ccr5 gene by single strand conformational polymorphism (SSCP) and DNA sequencing. RESULTS: The frequency of the mutant allele Delta32 was 4% (4/100) for ESN individuals and 4.2% (3/72) for SP individuals. The homozygous mutant genotype (Delta32/Delta32) was found in only 2% (1/50) of ESN individuals, but in no SP individuals. The heterozygous genotype was found in 8.3% (3/36) of SP individuals and in 4% (2/50) of ESN individuals. The differences in the allelic and genotypic frequencies among the groups were not statistically significant. A comparison between the observed and the expected genotypic frequencies showed that they were significantly different for the ESN group, suggesting a protective, yet indirect effect of the mutant genotype. CONCLUSIONS: The screening of the entire coding region of the ccr5 gene in all ESN did not revealed no other mutations that could account for resistance to HIV-1 infection. Although the CCR5 molecule is the most important coreceptor for HIV-1, mutations in this gene do not account for most of the cases of natural resistance to this virus that have so far been reported.

Endemically exposed asymptomatic individuals show no increase in the specific Leishmania (Viannia) panamensis-Th1 immune response in comparison to patients with localized cutaneous leishmaniasis.

In Colombia, most cases of human cutaneous leishmaniasis are caused by Leishmania (Viannia) panamensis. Interestingly, up to 30% of the exposed population do not suffer from clinical leishmaniasis although it is likely that they are continuously infected with Leishmania parasites. Since it is believed that the induction of efficient Th1 immune responses protects against Leishmania infections both in humans and in animal models, we determined if endemically exposed asymptomatics showed stronger Leishmania-specific Th1 immune responses than patients with active localized cutaneous leishmaniasis (LCL). We found that Montenegro skin test responses were slightly higher among asymptomatic individuals compared to patients suffering from LCL. However, PBMC from patients with LCL showed similar Leishmania-specific proliferative responses compared to PBMC from asymptomatic individuals. Furthermore, PBMC from both groups also secreted similar amounts of IFN-gamma, IL-12p40 and IL-10 after in vitro exposure to L. panamensis. No IL-4 was detected in the supernatants. Taken together our results suggest that lack of LCL development in endemically exposed asymptomatics cannot be explained by stronger systemic anti-Leishmania Th1 immune responses or decreased Th2 responses in these individuals in comparison to individuals who develop LCL. It may be possible that other mechanisms are responsible for resistance to cutaneous leishmaniasis in Colombia in endemically exposed asymptomatics.

[Clinical and molecular characterization of autosomal recessive chronic granulomatous disease caused by p47-phox deficiency]. / Caracterización clínico--molecular de la enfermedad granulomatosa crónica autosómica recesiva causada por déficit de p47-phox.

BACKGROUND: The cytosolic protein p47-phox (phagocyte oxidase) is one of the essential components of the superoxide generating system in phagocytes and its defect causes approximately 30% of the chronic granulomatous disease (CGD) cases. AIM: Two patients were studied, belonging to the same family, without a consanguinous background, in which deficiency or absence of superoxide generation was found together with recurrent and severe infections in one case and benign infections in the second. METHODS: The presence of gp91-, p67- and p47-phox in patients and controls was determined by Western Blot analysis of granulocytes. Sequencing of PCR amplified DNA was performed by an enzymatic method. RESULTS: Western Blot analysis showed normal expression of gp91 and p67 and absence of p47-phox. The molecular genetic study demonstrated a homocygotic dinucleotide GT (GT) deletion at the beginning of exon 2 of the p47-phox gene. The same mutation has been found in European, American and Japanese patients. CONCLUSIONS: The molecular characterization of this pathology done for the first time in Chile is important for diagnostic classification, patient prognosis, and adequate genetic advice and a possible future therapy.

Frequency of CCR5 delta-32 mutation in human immunodeficiency virus (HIV)-seropositive and HIV-exposed seronegative individuals and in general population of Medellin, Colombia.

Repeated exposure to human immunodeficiency virus (HIV) does not always result in seroconversion. Modifications in coreceptors for HIV entrance to target cells are one of the factors that block the infection. We studied the frequency of Delta-32 mutation in ccr5 gene in Medellin, Colombia. Two hundred and eighteen individuals distributed in three different groups were analyzed for Delta-32 mutation in ccr5 gene by polymerase chain reaction (PCR): 29 HIV seropositive (SP), 39 exposed seronegative (ESN) and 150 individuals as a general population sample (GPS). The frequency of the Delta-32 mutant allele was 3.8% for ESN, 2.7% for GPS and 1.7% for SP. Only one homozygous mutant genotype (Delta-32/Delta-32) was found among the ESN (2.6%). The heterozygous genotype (ccr5/Delta-32) was found in eight GPS (5.3%), in one SP (3.4%) and in one ESN (2.6%). The differences in the allelic and genotypic frequencies among the three groups were not statistically significant. A comparison between the expected and the observed genotypic frequencies showed that these frequencies were significantly different for the ESN group, which indirectly suggests a protective effect of the mutant genotype (Delta-32/Delta-32). Since this mutant genotype explained the resistance of infection in only one of our ESN persons, different mechanisms of protection must be playing a more important role in this population.

Increase in granulocyte-macrophage-colony-stimulating factor secretion and the respiratory burst with decreased L-selectin expression in hyper-IgE syndrome patients.

BACKGROUND: The hyper-IgE syndrome is a primary immunodeficiency characterized by severe recurrent abscesses, pneumonia with pneumatocele formation, and elevated serum IgE. Eosinophilia, neutrophil chemotactic defects, and marked tissue damage are frequently present in this syndrome. OBJECTIVE: To study whether functional changes in cytokines, adhesion molecules, and neutrophils might help explain these clinical observations. METHODS: The following functions were analyzed in patients with the hyper-IgE syndrome and in controls: (1) production of granulocyte-macrophage-colony-stimulating factor by peripheral blood mononuclear cells by ELISA; (2) respiratory burst and reactive oxygen intermediates production by peripheral neutrophils using the luminol-enhanced chemiluminescense technique; and (3) expression of L-selectin on granulocytes and lymphocytes by flow cytometry. RESULTS: Patients with hyper-IgE syndrome had significantly increased production of granulocyte-macrophage-colony-stimulating factor by resting or stimulated mononuclear cells, increased generation of reactive oxygen intermediates by neutrophils treated with opsonized zymosan, and reduced L-selectin expression on quiescent and activated granulocytes and lymphocytes. CONCLUSIONS: Our results suggest that an important feature of the hyper-IgE syndrome is the increased production of granulocyte-macrophage-colony-stimulating factor, which may explain the reduced L-selectin expression, decreased chemotaxis, and increased oxygen radical production and tissue damage in this disease.

Molecular characterization of autosomal recessive chronic granulomatous disease caused by a defect of the nicotinamide adenine dinucleotide phosphate (reduced form) oxidase component p67-phox.

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytes in which defective production of microbicidal oxidants leads to severe recurrent infections. CGD is caused by mutations in any of 4 genes encoding components of nicotinamide adenine dinucleotide phosphate (reduced form; NADPH) oxidase, the multisubunit enzyme that produces the precursor of these oxidants, superoxide. Approximately 5% of CGD patients have an autosomal recessive form of disease caused by a severe deficiency of p67-phox, a 526-amino acid subunit of the oxidase that appears to regulate electron transport within the enzyme. Here we report the biochemical and molecular characterization of 6 unrelated kindreds with p67-phox deficiency. These studies show that, as in gp91-phox and p22-phox deficiencies, the p67-phox CGD patients show a high degree of heterogeneity in the genetic defects that underlie their disease. Five different mutant alleles were identified: (1) a nonsense mutation in exon 4 (C(304) --> T); (2) a 5-nucleotide (nt) deletion in exon 13 (nts 1169-1173); (3) a splice mutation in the first nucleotide of intron 4 (G --> A); (4) a deletion of 1 nt in exon 9 (A(728)); and (5) a 9-nt in-frame deletion in exon 2 (nts 55-63). The splice mutation was seen in 3 unrelated kindreds, while the 5-nt deletion was seen in 2 apparently unrelated families (both of Palestinian origin). Homozygosity was present in 4 of the kindreds, 2 of which had consanguineous parentage. In the isolated neutrophils of each of the affected patients in the 6 kindreds, there was no measurable respiratory burst activity and no p67-phox protein detected by immunoblot analysis. The level of 67-phox mRNA was less than 10% of normal in the mononuclear leukocytes from 3 of the 4 patients analyzed by Northern blot studies. Thus, this heterogeneous group of mutations in p67-phox all lead to marked instability of mRNA or protein (or both) that results in the complete loss of NADPH oxidase activity.

Molecular analysis of chronic granulomatous disease caused by defects in gp91-phox.

Chronic granulomatous disease (CGD) is an uncommon inherited disorder of phagocytic cells in which a defective respiratory burst leads to severe recurrent bacterial and fungal infections. The disease is a consequence of mutations in one of the four molecules that constitute the NADPH oxidase system of electron transport, whose most critical component is an unusual flavocytochrome b localized in the plasma and specific granule membranes. Mutations in the CYBB gene (localized in the short arm of the X chromosome) encoding the beta-subunit of this flavocytochrome (gp91-phox), which is are responsible for 60-65% of all cases of CGD. In this paper, we report the molecular characterization of seven unrelated kindreds native from Colombia and Brazil with CGD caused by gp91-phox deficiency. The exons with the possible mutation were identified by single-strand conformational polymorphism (SSCP) of genomic DNA and then confirmed by DNA sequencing. In one patient we found a substitution of A to G in the penultimate nucleotide of intron 12 (IVS12-2A-->G). In four other cases, four different nonsense mutations were detected: R91X, W106X, R157X, and R290X and the other two patients showed missense substitutions: E225V and C244Y. In six of these kindreds, all mothers were carriers but one that did not present any change in the gp91-phox gene, which indicates a de novo mutation in this kindred. Then, these family-specific mutations in gp91-phox produce different structural defects that alter the expression or function of an essential component of phagocyte oxidase.

Interleukin 4 and interferon-gamma secretion by antigen and mitogen-stimulated mononuclear cells in the hyper-IgE syndrome: no TH-2 cytokine pattern.

BACKGROUND: Enhanced production of TH-2 cytokines plays a key role in increased IgE production in allergic diseases. Reports about the cytokine profile secreted by peripheral blood mononuclear cells of patients with hyper-IgE syndrome, however, are controversial, suggesting alternative causes for increased IgE production in this syndrome. OBJECTIVE: We wished to determine whether mononuclear cells from patients with hyper-IgE syndrome have a pattern of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) production characteristic of a predominance of TH-2 cells and whether the cytokine production pattern is constant over time. METHODS: IL-4 and IFN-gamma secretion by peripheral blood mononuclear cells stimulated with phytohemagglutinin and D. pteronyssinus was measured by ELISA in culture supernatants. Patients with the hyper-IgE syndrome were evaluated 3 times at 4-week intervals and compared with asthmatic patients and normal subjects. RESULTS: In PHA-stimulated cultures, patients with hyper-IgE syndrome had an IL-4 and IFN-gamma secretion similar to that of controls, while asthmatic patients had increased IL-4 and decreased IFN-gamma production. Cultures stimulated with D. pteronyssinus showed a variable pattern of secretion for both cytokines. CONCLUSIONS: In allergic diseases, increased serum IgE level is the result of a TH-2 pattern of cytokine production, with high IL-4 and decreased IFN-gamma protein secretion. The increased serum IgE concentration typical of the hyper-IgE syndrome is likely the result of a different immunoregulatory process.

A study of granulocyte respiratory burst in patients with allergic bronchial asthma.

The respiratory burst of phagocytes plays an important role in the tissue damage that accompanies the inflammatory response. One of these conditions is allergic bronchial asthma, therefore, to evaluate the activation state of peripheral granulocytes the generation of reactive oxygen metabolites was evaluated using Luminol-enhanced chemiluminescence (LCL) and reduction of cytochrome C by superoxide. The resting granulocytes of the asthmatic patients under crisis showed a higher LCL compared to the noncrisis patients and control subjects. The granulocytes stimulated with PMA presented a significant increase in the respiratory burst in both groups of asthmatics. The granulocytes of noncrisis asthmatics challenged with Ops-Zym and with fMLP + Ops-Zym showed a higher metabolic activity, whereas the asthmatics under crisis presented no difference between reactive oxygen generation and that of the control group. The quantitative analysis of superoxide generation by granulocytes of the same patients did not show differences among the groups. Our findings suggest that the granulocytes of crisis and noncrisis asthmatics seem to be in a hyperreactive state and with a higher metabolic response when compared to the control group. However, the patients present a different behavior depending on stimulus used to activate cells. This could indicate that in peripheral blood exist different granulocyte populations depending on the inflammatory response taking place in the respiratory tract.