Immune Network Modeling Predicts Specific Nasopharyngeal and Peripheral Immune Dysregulation in Otitis-Prone Children.

Acute otitis media (AOM) pathogenesis involves nasopharyngeal colonization by potential otopathogens and a viral co-infection. Stringently-defined otitis prone (sOP) children show characteristic patterns of immune dysfunction. We hypothesized that otitis proneness is largely a result of altered signaling between immune components that are otherwise competent, resulting in increased susceptibility to infection by bacterial otopathogens. To test this, we constructed a regulatory immune network model linking immune cells and signaling elements known to be involved in AOM and/or dysregulated in sOP children. The alignment of immune response mechanisms with data from in vivo and in vitro experimental observations produced 82 putative immune network models, each describing variants of immune regulatory networks consistent with available observations. Analysis of these models suggested that new measurements of serum levels of IL-4 and CXCL8 could refine competing models and resulted in the elimination of 38 of the models. Further analysis of the remaining 44 models suggested specific deviations in the predicted regulation of nasopharyngeal and peripheral immunity during response to AOM. Specifically, immune responses active in sOP children during AOM were characterized by early and constitutive activation of pro-inflammatory signaling in the nasopharynx and a Th2- and Treg-dominated profile in the periphery. We conclude that sOP children have altered regulation of key immune mediators during both health and pathogenesis. This altered regulation may be amenable to therapeutic intervention.

A mouse ear skin model to study the dynamics of innate immune responses against Staphylococcus aureus biofilms.

BACKGROUND: Staphylococcus aureus is a human pathogen that is a common cause of nosocomial infections and infections on indwelling medical devices, mainly due to its ability to shift between the planktonic and the biofilm/sessile lifestyle. Biofilm infections present a serious problem in human medicine as they often lead to bacterial persistence and thus to chronic infections. The immune responses elicited by biofilms have been described as specific and ineffective. In the few experiments performed in vivo, the importance of neutrophils and macrophages as a first line of defence against biofilm infections was clearly established. However, the bilateral interactions between biofilms and myeloid cells remain poorly studied and analysis of the dynamic processes at the cellular level in tissues inoculated with biofilm bacteria is still an unexplored field. It is urgent, therefore, to develop biologically sound experimental approaches in vivo designed to extract specific immune signatures from the planktonic and biofilm forms of bacteria. RESULTS: We propose an in vivo transgenic mouse model, used in conjunction with intravital confocal microscopy to study the dynamics of host inflammatory responses to bacteria. Culture conditions were created to prepare calibrated inocula of fluorescent planktonic and biofilm forms of bacteria. A confocal imaging acquisition and analysis protocol was then drawn up to study the recruitment of innate immune cells in the skin of LysM-EGFP transgenic mice. Using the mouse ear pinna model, we showed that inflammatory responses to S. aureus can be quantified over time and that the dynamics of innate immune cells after injection of either the planktonic or biofilm form can be characterized. First results showed that the ability of phagocytic cells to infiltrate the injection site and their motility is not the same in planktonic and biofilm forms of bacteria despite the cells being considerably recruited in both cases. CONCLUSION: We developed a mouse model of infection to compare the dynamics of the inflammatory responses to planktonic and biofilm bacteria at the tissue and cellular levels. The mouse ear pinna model is a powerful imaging system to analyse the mechanisms of biofilm tolerance to immune attacks.

Successful development of methodology for detection of hapten-specific contact hypersensitivity (CHS) memory in swine.

Hapten contact hypersensitivity (CHS) elicits a well-documented inflammation response that can be used to illustrate training of immune cells through hapten-specific CHS memory. The education of hapten-specific memory T cells has been well-established, recent research in mice has expanded the "adaptive" characteristic of a memory response from solely a function of the adaptive immune system, to innate cells as well. To test whether similar responses are seen in a non-rodent model, we used hapten-specific CHS to measure the ear inflammation response of outbred pigs to dinitrofluorobenzene (DNFB), oxazolone (OXA), or vehicle controls. We adapted mouse innate memory literature protocols to the domestic pig model. Animals were challenged up to 32 days post initial sensitization exposure to the hapten, and specific ear swelling responses to this challenge were significant for 7, 21, and 32 days post-sensitization. We established hapten-specific CHS memory exists in a non-rodent model. We also developed a successful protocol for demonstrating these CHS responses in a porcine system.

Evidence for a dynorphin-mediated inner ear immune/inflammatory response and glutamate-induced neural excitotoxicity: an updated analysis.

Acoustic overstimulation (AOS) is defined as the stressful overexposure to high-intensity sounds. AOS is a precipitating factor that leads to a glutamate (GLU)-induced Type I auditory neural excitotoxicity and an activation of an immune/inflammatory/oxidative stress response within the inner ear, often resulting in cochlear hearing loss. The dendrites of the Type I auditory neural neurons that innervate the inner hair cells (IHCs), and respond to the IHC release of the excitatory neurotransmitter GLU, are themselves directly innervated by the dynorphin (DYN)-bearing axon terminals of the descending brain stem lateral olivocochlear (LOC) system. DYNs are known to increase GLU availability, potentiate GLU excitotoxicity, and induce superoxide production. DYNs also increase the production of proinflammatory cytokines by modulating immune/inflammatory signal transduction pathways. Evidence is provided supporting the possibility that the GLU-mediated Type I auditory neural dendritic swelling, inflammation, excitotoxicity, and cochlear hearing loss that follow AOS may be part of a brain stem-activated, DYN-mediated cascade of inflammatory events subsequent to a LOC release of DYNs into the cochlea. In support of a DYN-mediated cascade of events are established investigations linking DYNs to the immune/inflammatory/excitotoxic response in other neural systems.

Serum antibody response to Moraxella catarrhalis proteins in stringently defined otitis prone children.

BACKGROUND: Moraxella catarrhalis (Mcat) is a frequent pathogen of acute otitis media (AOM) in young children. Here we prospectively assessed naturally-induced serum antibodies to four Mcat vaccine candidate proteins in stringently defined otitis prone (sOP) and non-otitis prone (NOP) children age 6-36months old following nasopharyngeal (NP) colonization, at onset of AOM and convalescence from AOM. METHODS: Serum IgG and IgM antibody against recombinant Mcat proteins, oligopeptide permease A (OppA), outer membrane protein (OMP) CD, hemagglutinin (Hag), and PilA clade 2 (PilA2), were quantitated by ELISA. RESULTS: During NP colonization by Mcat all four antigens were immunogenic in both sOP and NOP children. However, sOP children had lower antibody responses than NOP children across age 6-36months, similar to our findings for protein vaccine candidates of Streptococcus pneumoniae (Spn) and Nontypeable Haemophilus influenzae (NTHi). sOP children displayed a later and lower peak of antibody rise than NOP children for all four antigens during NP colonization of Mcat. The age-dependent increase of antibody ranked as OppA>Hag5-9>OMP CD>PilA2 in both sOP and NOP children. Lower serum antibody levels to the Mcat antigens were measured in sOP compared to NOP children at the onset of AOM. We did not find a consistent significant increase of antibody at the convalescence phase after an AOM event. CONCLUSIONS: sOP children is a highly vulnerable population that mount lower serum antibody responses to Mcat candidate vaccine proteins compared to NOP children during asymptomatic NP carriage and at onset of AOM.

Vitamin D-binding protein is inversely associated with the incidence of gastrointestinal and ear infections in school-age children.

Circulating 25-hydroxy vitamin D (25(OH)D) is related to decreased rates of gastrointestinal and ear infections in school-age children. Vitamin D-binding protein (DBP) transports 25(OH)D and exerts immunological functions; however, it is unknown whether DBP is associated with infectious morbidity in children. We quantified plasma DBP concentrations in 540 school-age children at the time of recruitment into a cohort study in Bogotá, Colombia and obtained daily information on infectious morbidity symptoms and doctor visits during the school year. We compared the incidence rates of gastrointestinal and respiratory symptoms across quartiles of DBP concentration by estimating adjusted incidence rate ratios (IRRs) with 95% confidence interval (CI). We also estimated the per cent of the associations between DBP and morbidity that were mediated through 25(OH)D using a counterfactual frame. Mean ± s.d. DBP concentration was 2650 ± 1145 nmol/l. DBP was inversely associated with the rates of diarrhoea with vomiting (IRR for quartiles 2-4 vs. 1 = 0.48; 95% CI 0.25-0.92; P = 0.03) and earache/ear discharge with fever (IRR for quartiles 2-4 vs. 1 = 0.29; 95% CI 0.12-0.71; P = 0.006). The DBP-morbidity associations were not mediated through 25(OH)D. We conclude that plasma DBP predicts lower incidence of gastrointestinal and ear infections in school-age children independent of 25(OH)D.

Decreased TNF family receptor expression on B-cells is associated with reduced humoral responses to Streptococcus pneumoniae infections in young children.

An underdeveloped or impaired immune response in young children is associated with increased susceptibility to Streptococcus pneumonia (Spn) infections. We determined serum antibody titers against 3 Spn vaccine candidate proteins and vaccine serotype polysaccharide antigens in a group of Spn infection prone 9-18months old and found lower IgG antibody titers to all tested antigens compared to age-matched non-infection-prone children. We also found the children had significantly reduced percentages of total memory B-cells, switched memory B-cells and plasma cells. We sought a mechanistic explanation for that result by examination of TNF family receptors (TNFRs) TACI, BCMA, and BAFFR receptor expression on B-cells and found significantly lower BAFFR and TACI expression; significantly lower proliferation of B-cells stimulated with exogenous BAFF; and diminished expression of co-stimulatory receptors B7-1 and B7-2 among infection prone vs. non-prone children. We conclude that lower expression of TNFRs, lower proliferation of B-cells in response to BAFF and lower expression of B7-1 and B7-2 by B-cells may contribute to reduced antibody responses to Spn and consequent infection proneness in young children.

Recent advances in knowledge regarding the head and neck manifestations of IgG4-related disease.

IgG4-related disease (IgG4-RD) is a chronic inflammatory disorder, characterized by elevated serum IgG4 levels as well as abundant infiltration of IgG4-positive plasmacytes and fibrosis in various organs, including the head and neck region. In particular, the salivary glands, orbit, and thyroid are common sites of disease involvement. IgG4-RD is diagnosed based on various clinical, serological, and histopathological findings, none of which are pathognomonic. Hence, various differential diagnoses, which exhibit elevated serum IgG4 levels and infiltration of IgG4-postive cells into tissues, need to be excluded, especially malignant diseases and mimicking disorders. Systemic corticosteroids are generally effective in inducing IgG4-RD remission; however, recurrent or refractory cases are common. In addition, although the pathogenic mechanisms of IgG4-RD remain unclear, an antigen-driven inflammatory condition is believed to be involved. Recent studies have indicated the important pathogenic role of B cell/T cell collaboration and innate immunity in this disease. Nevertheless, additional research and discussions are needed to resolve many remaining questions. In this review, we provide an overview of the recent insights on the history, clinical features, diagnosis, and treatment of IgG4-RD in the head and neck region. Furthermore, we have also addressed the pathogenesis of this disease.

Otitis-Prone Children Produce Functional Antibodies to Pneumolysin and Pneumococcal Polysaccharides.

The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group (P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children.

Peripheral blood antigen presenting cell responses in otitis-prone and non-otitis-prone infants.

Stringently defined otitis-prone (sOP) children represent a new classification of the otitis-prone condition. Previous studies showed dysfunction in Ab, B-cell memory and T-cell memory responses. We sought to determine whether there are defects in numbers, phenotype and/or function of professional APC in the peripheral blood of sOP infants. APC phenotypic counts, MHC II expression and intracellular cytokine levels were determined in response to TLR7/8 (R848) stimulation by flow cytometry. Innate immune mRNA expression was measured using RT-PCR and cytokines were measured using Luminex technology. Significant (P < 0.05) increases in the phenotypic counts of monocytes and conventional dendritic cells but not plasmacytoid DCs were observed in sOP compared with non-otitis-prone (NOP) age-matched infants. No significant differences in APC activation or function were observed. Expression of various TLRs, intracellular signaling molecules and downstream cytokines was also not found to be significantly different between sOP and NOP infants. Higher numbers of APCs in sOP infants suggest the possibility of a persistent mucosal inflammatory status. Transcriptional and cytokine profiles of PBMCs among sOP infants suggest their systemic innate responses are not different compared to NOP infants.

Characterization and inflammatory response of perivascular-resident macrophage-like melanocytes in the vestibular system.

A large number of perivascular cells expressing both macrophage and melanocyte characteristics (named perivascular-resident macrophage-like melanocytes, PVM/Ms), previously found in the intra-strial fluid-blood barrier, are also found in the blood-labyrinth barrier area of the vestibular system in normal adult cochlea, including in the three ampullae of the semicircular canals (posterior, superior, and horizontal), utricle, and saccule. The cells were identified as PVM/Ms, positive for the macrophage and melanocyte marker proteins F4/80 and GSTα4. Similar to PVM/Ms present in the stria vascularis, the PVM/Ms in the vestibular system are closely associated with microvessels and structurally intertwined with endothelial cells and pericytes, with a density in normal (unstimulated) utricle of 225 ± 43/mm(2); saccule 191 ± 25/mm(2); horizontal ampullae 212 ± 36/mm(2); anterior ampullae 238 ± 36/mm(2); and posterior ampullae 223 ± 64/mm(2). Injection of bacterial lipopolysaccharide into the middle ear through the tympanic membrane causes the PVM/Ms to activate and arrange in an irregular pattern along capillary walls in all regions within a 48-h period. The inflammatory response significantly increases vascular permeability and leakage. The results underscore the morphological complexity of the blood barrier in the vestibular system, with its surrounding basal lamina, pericytes, as well as second line of defense in PVM/Ms. PVM/Ms may be important to maintain blood barrier integrity and initiating local inflammatory response in the vestibular system.

Role of the alternative and classical complement activation pathway in complement mediated killing against Streptococcus pneumoniae colony opacity variants during acute pneumococcal otitis media in mice.

There is considerable evidence that phase variation among transparent and opaque colony phenotypes of Streptococcus pneumoniae (Spn) plays an important role in the pneumococcal adherence and invasion. The current study was designed to investigate the interactions of the opacity phenotype variants of Spn with specific complement pathway activation in a mouse model of acute otitis media (AOM). Although the opaque colony phenotype was expected to be more resistant to complement mediated killing compared to the transparent Spn variant, we discovered that C3b deposition on the transparent Spn is, in large part, dependent on the alternative pathway activation. There were no significant differences in resistance to complement mediated opsonophagocytosis between the two variants in factor B deficient mice. In addition, an in vitro study demonstrated that significantly more C4b-binding protein (C4BP) (the classical pathway inhibitor) and factor H (FH) (the alternative pathway inhibitor) bound to the transparent strain compared with the opaque one. Our data suggest that the difference in the relative virulence of Spn opacity phenotypes is associated with its ability to evade complement-mediated opsonophagocytosis in a mouse model of pneumococcal AOM.

Distinct signalling mechanisms mediate neutrophil attraction to bacterial infection and tissue injury.

The signals that guide neutrophils to sites of tissue injury or infection remain elusive. H(2)O(2) has been implicated in neutrophil sensing of tissue injury and transformed cells; however, its role in neutrophil recruitment to infection has not been explored. Here, using a pharmacological inhibitor of NADPH oxidases, diphenyleneiodonium (DPI), and genetic depletion of an epithelial-specific NADPH oxidase, we show that H(2)O(2) is not required for neutrophil detection of localized infection with the Gram-negative bacterium Pseudomonas aeruginosa. In contrast, PI3K signalling is required for neutrophil responses to both wounding and infection. In vivo imaging using a H(2)O(2) probe detects dynamic H(2)O(2) generation at wounds but not at infected tissue. Moreover, DPI no longer inhibits neutrophil wound attraction when P. aeruginosa is present in the media. Finally, DPI also fails to inhibit neutrophil recruitment to localized infection with the Gram-positive bacterium, Streptococcus iniae. Our findings demonstrate that different signals are involved in sensitizing neutrophils to pathogen versus non-pathogen induced tissue damage, providing a potential target to preferentially suppress non-specific immune damage without affecting the response to infection.

[An exceptional component C3 deficiency revealed by serum protein electrophoresis]. / Un exceptionnel déficit héréditaire en fraction C3 du complément dépisté par électrophorèse des protéines sériques.

We report the case of a 5-years old child referred to the pediatric clinic due to a prolonged history of recurrent otitis. Initial immunologic investigation was normal but a severe C3 complement deficiency was detected by the absence of beta 2-globulin protein fraction using serum protein capillary electrophoresis. C3 was not detected in serum and total complement haemolytic activity was decreased. His mother and father had half of the C3 normal plasma level and a heterozygous mutation of the C3 gene. The diagnosis of hereditary deficiency of the third complement component (C3) with compound heterozygous mutation of the gene was made. This defect in complement protein C3, described to date in only 20 families in the world, is associated with repeated infections. The child is treated with oracillin with relatively good control of symptoms.

[Lymphocyte subsets in patients with recurrent upper airway infections]. / Limfocitu populiaciju pokyciai sergant pasikartojanciomis virsutiniu kvepavimo taku infekcinemis ligomis.

UNLABELLED: The aim of our study was to evaluate the digressions of lymphocyte subsets in patients with recurrent upper airway infectious diseases. METHODS: We studied 35 patients (mean of age 11.1+/-2.1 years) with recurrent upper airway infections. The first group consisted of patients, who had acute upper airway infections: rhinitis, pharyngitis, laryngitis and tracheitis more than 6 times per last year, sinusitis or otitis more than 4 times per last year. The control group comprised of 9 healthy subjects. Subsets of lymphocytes (CD3+, CD4+, CD8+, CD4+/CD8+, CD16+/56+ and CD19+) were detected by FACS Calibur cytometer. RESULTS: We found a significantly lower count of CD4+ lymphocytes in the patients' group compared to the control group (37.5+/-1.2 vs 45.7+/-3.1% of total lymphocytes, p<0.01). We did not find any significant differences of other lymphocyte subsets between patients and control groups. CONCLUSION: We propose that patients with recurrent upper airway infections have alterations of the cellular immunity -- decreased amount of CD4+ lymphocytes.

[Ear, nose, and throat manifestations in patients with primary immunodeficiencies]. / Manifestaciones otorrinolaringológicas de los pacientes con immunodeficiencias primarias.

OBJECTIVE: To determine the flowgram for the timely diagnosis of the immunodeficiencies congenital primary, based on the demonstrations otorrinolaringol recurrent chronicles to difficult control, with or without associated systemic diseases. MATERIAL AND METHOD: Retrospective study, descriptive and observational that included patient with diagnostic of primary known immunodeficiency. They were checked the clinical files to know their evolution and medical treatment-surgical. RESULTS: They were found 10 patient with diagnostic of some primary immunodeficiency; this was made based on the infectious symptoms otolaryngal chronic recurrent to difficult control; they were six men and four women between the four and 36 years old. They were found the following immunodeficiencies: one of hyper-IgM, three by IgG, variable common three, a deficit with C3 of the complement, two of IgA more atopia. CONCLUSIONS: The chronicle sinusitis or recurrent and the otitis mean chronicle or recurrent can be the only demonstrations of a patient with primary immunodeficiency. The immunology evaluation must include, at least: hematic biometric completes with differential, seric levels of immunoglobulins, the same as subclass of IgG, C4 and C3 of the complement, response to immunization with proteins (diphtheria and tetanus), and polysaccharide antigens (pneumovax).

Manifestaciones otorrinolaringológicas de los pacientes con inmunodeficiencias primarias / Otolaringol symptoms chronic in patients with deficiencies congenital primary

Objetivo. Determinar el flujograma para el diagnóstico oportuno de las inmunodeficiencias congénitas primarias, basado en las manifestaciones otorrinolaringológicas crónicas recurrentes de difícil control, con o sin enfermedades sistémicas asociadas. Material y método. Estudio retrospectivo, descriptivo y observacional que incluyó a pacientes con diagnóstico de inmunodeficiencia primaria conocida. Se revisaron los expedientes clínicos para conocer su evolución y tratamiento médico-quirúrgico. Resultados. Se encontraron 10 pacientes con diagnóstico de algunas inmunodeficiencias primarias; esto se hizo con base en los síntomas infecciosos otorrinolaringológicos crónicos recurrentes de díficil control; fueron seis hombres y cuatro mujeres entre los cuatro y 36 años de edad. Se encontraron las siguientes inmunodeficiencias: una de hiper IgM, tres por IgG, tres comunes variables, un déficit con C3 del complemento, dos de IgA más atopia. Conclusiones. La sinusitis crónica o recurrente y la otitis media crónica o recurrente pueden ser las únicas manifestaciones de un paciente con inmunodeficiencia primaria. La evaluación inmunológica debe incluir, por lo menos: biometría hemática completa con diferencial, niveles séricos de inmunoglobulinas, al igual que subclases de IgG, C4 y C3 del complemento, respuesta a inmunización con proteínas (difteria y tétanos), y antígenos polisacáridos (pneumovax)

On the function of the mastoid cavity with emphasis on its role in middle ear defence.

The function of the mastoid cavity is still unknown. In the present article a theory of its main function is presented. The mastoid cavity is of vital importance in the non-immunological defence of the middle-ear. It is the most important site for the production of secretions that "wash" the middle-ear free of bacteria and viruses. The mucociliary system and the gravitation force, if the body is in erect position, transports the secretions through the two openings of the ear, the aditus and antrum and the ostia of the Eustachian tube, to be expelled into the epipharynx. Based on this theory pathological disorders of the ear are discussed as well as the effect of ventilation tubes on otosalpingitis and recurrent otitis media. Other plausible mastoid cavity functions are also discussed.