The immune response in adenoids and tonsils

The adenoid and tonsils are lymphoid tissues located in the pharynx that play an important role in host defense against invading antigens of the upper respiratory tract. Histologically, these structures consist of four well-defined microcompartments which all participate in the immune response: the cryptepithelium, the follicular germinal center with the mantle zone and interfollicular area. With the uptake of antigen by M-cells present in the cryptepithelium a process is initiated which ultimately results in the generation and dissemination of antigen-specific memory and mainly dimeric IgA-producing effector B-lymphocytes. This process requires successful cognate interactions between antigen-presenting cells and lymphocytes and mutually between lymphocytes, which depend not only on antigen-specific signals but also on the expression of various complementary adhesion and costimulatory molecules.     

On the isolation of mast cells from human adenoids and tonsils

Human adenoids and tonsils were disintegrated mechanically and the cells dispersed by passage through a stainless-steel screen in EDTA-containing buffer. Collagenase digestion did not increase the yield of adenoidal cells. The mast cell content of the cell suspensions was in the range of 1–10 mast cells/10⁴ cells with an estimated mean of 1–2 mast cells/10⁴ cells, a value considerably below previous reports on adenoidal cell suspensions. The mast cell content was determined by staining with toluidine blue at low pH (to prevent interference by phagocytes). The mast cell count as assessed by alcian blue staining and by fluorescence microscopy after FITC-anti-human IgE binding was similar. Various attempts to enrich the cell suspension (i.e. by differential centrifugation, by gradient centrifugation on Ficoll or Ficoll-Hypaque and by velocity sedimentation at unit gravity) all gave negative results.     

Human adenovirus replication and persistence in hypertrophic adenoids and palatine tonsils in children

The role of human adenovirus (HAdV) infection in different acute diseases, such as febrile exudative tonsillitis, conjunctivitis, and pharyngoconjunctival fever is well established. However, the relationships, if any, of HAdV persistence and reactivation in the development of the chronic adenotonsillar disease is not fully understood. The present paper reports a 3-year cross-sectional hospital-based study aimed at detecting and quantifying HAdV DNA and mRNA of the HAdV hexon gene in adenoid and palatine tonsil tissues and nasopharyngeal secretions (NPS) from patients with adenotonsillar hypertrophy or recurrent adenotonsillitis. HAdV C, B, and E were detectable in nearly 50% of the patients, with no association with the severity of airway obstruction, nor with the presence of recurrent tonsillitis, sleep apnea or otitis media with effusion (OME). Despite the higher rates of respiratory viral coinfections in patients with HAdV, the presence of other viruses, including DNA and RNA viruses, had no association with HAdV replication or shedding in secretions. Higher HAdV loads in adenoids showed a significant positive correlation with the presence of sleep apnea and the absence of OME. Although this study indicates that a significant proportion (~85%) of individuals with chronic adenotonsillar diseases have persistent nonproductive HAdV infection, including those by HAdV C, B, and E, epithelial and subepithelial cells in tonsils seem to be critical for HAdV C production and shedding in NPS in some patients, since viral antigen was detected in these regions by immunohistochemistry in four patients, all of which were also positive for HAdV mRNA detection.     

Detection of respiratory viruses by molecular methods

SUMMARY: Clinical laboratories historically diagnose seven or eight respiratory virus infections using a combination of techniques including enzyme immunoassay, direct fluorescent antibody staining, cell culture, and nucleic acid amplification tests. With the discovery of six new respiratory viruses since 2000, laboratories are faced with the challenge of detecting up to 19 different viruses that cause acute respiratory disease of both the upper and lower respiratory tracts. The application of nucleic acid amplification technology, particularly multiplex PCR coupled with fluidic or fixed microarrays, provides an important new approach for the detection of multiple respiratory viruses in a single test. These multiplex amplification tests provide a sensitive and comprehensive approach for the diagnosis of respiratory tract infections in individual hospitalized patients and the identification of the etiological agent in outbreaks of respiratory tract infection in the community. This review describes the molecular methods used to detect respiratory viruses and discusses the contribution that molecular testing, especially multiplex PCR, has made to our ability to detect respiratory viruses and to increase our understanding of the roles of various viral agents in acute respiratory disease.     

Human respiratory syncytial virus in children with lower respiratory tract infections or influenza-like illness and its co-infection characteristics with viruses and atypical bacteria in Hangzhou,China

BackgroundHuman respiratory syncytial virus (RSV) is the most important viral pathogen in children. However, its epidemic patterns and co-infection characteristics are not fully understood.ObjectivesWe attempted to determine the level of genetic variation of RSV, and describe the prevalence and co-infection characteristics of RSV in Hangzhou during two epidemic seasons.Study designSingle respiratory samples from 1820 pediatric patients were screened for RSV and genotyped by RT-PCR and sequencing. In all RSV positive specimens, we screened for viruses and atypical bacteria. Demographic and clinical information was recorded and analyzed.ResultsA total of 34.5% and 3.8% of samples from acute lower respiratory tract infections (ALRI) and influenza-like illness (ILI) were positive for RSV, respectively. Phylogenetic analysis revealed that 61.1% of the selected 167 RSV strains were NA1, 31.1% were BA, 3.6% were ON1, 2.4% were CB1, and 1.8% were NA3. A new genotype, BA11 was identified, which comprised 98.1% of BA strains in this study, while the rest were BA10. A total of 36.4% and 9.1% of RSV-positive children with ALRI and ILI respectively were found to be co-infected. Rhinovirus was the most common additional respiratory virus, followed by human metapneumovirus. Except for fever, no significant differences in other clinical presentation between the RSV mono-infection and co-infection groups were observed.ConclusionsThe circulating RSV strains had high genetic variability with RSV-B showing a more local pattern. In ALRI cases, co-infection of RSV with other viruses or atypical bacteria has no significant effect on the clinical presentation except fever.     

Identification and immunolocalization of the innate immune receptor CD14 in hypertrophic adenoids and tonsils

The purpose of this study is to determine the expression of CD14 as a marker of the innate immunity in hypertrophic adenoids and tonsils. Twenty-four pediatric patients (age <12 years) with obstructive adenotonsillar hypertrophy, confirmed by sleep study were included in this study. Intensity and expression of positive CD14 infiltrating cells was assessed by immunohistochemistry in specific histologic areas. In tonsils, CD14 immunoreactivity was demonstrated in intraepithelial lymphocytes located in the basal layer of the stratified squamous mucoepithelium. CD14 expression was significantly higher in mucosal layers and inter-follicular areas of tonsils than adenoid tissues [(p < 0.001), (p = 0.021), respectively]. CD14 expression was significantly higher in the submucosal layers of adenoids than tonsil tissues (p = 0.002). Hypertrophic adenoids and tonsils from children with OSA are prominent sites of innate defense, with over expression of CD14. The enhanced expressions of CD14 cells in adenoids and tonsils may be an important factor for the development and persistence of adenoids and tonsils enlargement causing OSA in children. CD14 expression in adenoids and tonsils illustrates an important immunological sentinel function of the innate immunity of the upper airway.     

多重PCR检测儿童呼吸道感染常见病毒

目的 对深圳、粤东地区儿童呼吸道病毒感染进行监测、分析,探讨其流行规律.方法 2006年6月-2008年6月,于汕头大学附属医院、深圳市第四人民医院、揭阳市人民医院采集毛细支气管炎、支气管肺炎、喘息支气管炎等的患儿咽拭子或鼻咽分泌物标本686份,多重PCR进行9种呼吸道病毒检测.结果 在686例标本中病毒阳性362例(52.77%),其中呼吸道感染患儿中呼吸道合胞病毒(RSV)感染占首位,达到31.22%(113/362),其次是鼻病毒(RHV)为16.85%(61/362),最低是流感病毒B型(IVB)占0.83%(3/362),流感病毒A型(IVA)占14.36%(52/362),副流感病毒1型(PIV1)和副流感病毒3型(PIV3)分别占7.73%(28/362)和8.29%(30/362),腺病毒(AdV)达到9.67%(35/362),而人博卡病毒(hBOV)和人偏肺病毒(hMPV)分别占6.08%(22/362)和4.97%(18/362).结论 RSV、RHV及IVA是华南地区儿童呼吸道感染的主要病毒病原,混合感染以RSV、IVA联合其他病毒感染为主,诊治时应根据所感染病原体制定有针对性的措施.     

Detection of respiratory viruses in gargle specimens of healthy children

BackgroundRespiratory tract viral infection is one of the most common and important diseases in children. Polymerase chain reaction (PCR) tests are often used to detect viruses in samples, it is difficult to interpret the clinical significance of PCR positivity, which may reflect a past, imminent or active asymptomatic infection due to their high sensitivity. Although single respiratory viruses have been detected in samples from children with symptoms, other respiratory viruses can also be detected simultaneously. However, the clinical importance of these findings for the symptoms is not known.ObjectivesTo investigate the prevalence of respiratory viruses among children without any symptoms such as acute respiratory illness and/or fever.Study designFrom week twenty-five 2013 to week twenty-six 2014, gargle samples were collected from children once a week and these samples were subjected to real-time PCR to detect respiratory viruses. On each sampling day, we asked the parents about their children’s health condition.ResultsAmong the 286 samples collected, 200 were from asymptomatic children. In the asymptomatic condition, human parechovirus, adenovirus, enterovirus, rhinovirus, coronavirus 229E and HKU1 were observed in 45 episodes. In samples from symptomatic children, parainfluenza viruses, respiratory syncytial virus and coronavirus OC43 were detected in addition to those mentioned above.ConclusionsVarious viruses of different species were detected in the specimens from the children regardless of their health status. It might be speculated that host factors such as the function of the immune system influence the clinical outcome of the infection. However, this needs to be studied further.     

Epidemiology of bacteria and viruses in the respiratory tract of humans and domestic pigs

Bacteria and viruses were analysed in the upper respiratory tract of symptomatic pig farmers and their domestic pigs. Eighty six human nasal and 495 (50 pools) porcine snout swabs were collected in Schleswig-Holstein, Germany. Staphylococcus (S.) aureus (62.8%, 54/86), human rhino- and coronaviruses (HRV, 29.1%, 25/86; HCoV, 16.3%, 14/86) were frequently detected in humans, while Haemophilus parasuis (90.0%, 45/50), Mycoplasma hyorhinis (78.6%, 11/14), Enterovirus G (EV-G, 56.0%, 28/50) and S. aureus (36.0%, 18/50), respectively, were highly prevalent in pigs. The detection of S. aureus in human follow-up samples indicates a carrier status. The methicillin-resistant phenotype (MRSA) was identified in 33.3% (18/54) of nasal swabs and in one of 18 (5.6%) pooled snout swabs that were tested positive for S. aureus. Strains were indicative of the livestock-associated clonal complex CC398, with t011 being the most common staphylococcal protein A type. Enterobacterales and non-fermenters were frequently isolated from swabs. Their detection in follow-up samples suggests a carrier status. All were classified as being non-multiresistant. There was no example for cross-species transmission of viruses. In contrast, transmission of S. aureus through occupational contact to pigs seems possible. The study contributes to the ‘One Health’ approach.     

Detection of respiratory viruses and subtype identification of influenza A viruses by GreeneChipResp oligonucleotide microarray

Acute respiratory infections are significant causes of morbidity, mortality, and economic burden worldwide. An accurate, early differential diagnosis may alter individual clinical management as well as facilitate the recognition of outbreaks that have implications for public health. Here we report on the establishment and validation of a comprehensive and sensitive microarray system for detection of respiratory viruses and subtyping of influenza viruses in clinical materials. Implementation of a set of influenza virus enrichment primers facilitated subtyping of influenza A viruses through the differential recognition of hemagglutinins 1 through 16 and neuraminidases 1 through 9. Twenty-one different respiratory virus species were accurately characterized, including a recently identified novel genetic clade of rhinovirus.     

Isolation of follicular dendritic cells from human tonsils and adenoids. II. Immunocytochemical characterization

Follicular dendritic cells (FDC) are specialized cells found only within lymphoid follicles. They bind immune complexes and play a role in the presentation of antigen to follicular B cells and in the generation of B cell memory. In the present report the isolation of FDC from human tonsils and adenoids is described. These isolated cells have an unusual spherical arrangement and enclose lymphocytes within extensions of their membranes. Their ultrastructural features are similar to those observed in situ. The reactivity of isolated FDC with a number of monoclonal antibodies was analyzed by immunofluorescence and by immunostaining (at the electron microscopic level) with colloidal gold. In keeping with the results of previous investigations on tissue sections IgM, IgG and IgA (but not IgD) can be detected on the surface of isolated FDC, as can C3b receptors and the FDC-associated antigen detected by monoclonal antibody R4/23. The immunoglobulins associated with FDC are mostly embedded in an electron-dense material. The majority of the lymphoid cells enclosed within the membrane extensions of FDC are of B cell type. These results suggest that isolated FDC may be suitable for further in vitro investigation of their role in the humoral immune response.     

The adequacy of gross pathological examination of routine tonsils and adenoids in patients 21 years old and younger

Most hospitals microscopically examine all routine tonsil and adenoid specimens from healthy pediatric patients with recurrent infections or obstructive sleep apnea. Concern over missing the rare unsuspected, significant diagnosis propagates this practice. Careful gross examination for asymmetry and clinical findings should obviate the need for routine microscopic examination of tonsil and adenoid specimens in patients age 21 years and younger. A retrospective study was conducted using the SNOMED database of 4070 patients age 21 years or younger who underwent tonsillectomy and/or adenoidectomy between 1970 and July 2001 at the University of Florida. The age distribution of the study group was 0 to 5 years (52%), 6 to 12 years (37%), and 13 to 21 years (11%). Specimens consisted of tonsils only (15%), tonsils and adenoids (40%), and adenoids only (45%). Clinically significant diagnoses were diagnoses that impacted the care of patients and included malignancies and some infections. Non-clinically significant diagnoses included normal, acute or chronic tonsillitis, and tonsillar hyperplasia. Clinically significant pathological processes were seen in the tonsil or adenoid specimens of 3 of the 4070 patients. These 3 cases included a 2-year-old male with Burkitt's lymphoma, a 19-year-old male with non-Hodgkin's lymphoma (small noncleaved cell, non-Burkitt's type), and an 11-year-old male with a probable viral process but in whom a lymphoma could not be absolutely excluded. All 3 of these patients had signs and symptoms, including significant cervical lymphadenopathy, meriting microscopic analysis of the specimens. In conclusion, microscopic examination of all routine tonsils and adenoids for individuals 21 years or younger is not indicated. Gross examination is still recommended. Clinical suspicion and specimen asymmetry should be used to determine when thorough histological examination is merited.     

Detection of herpes viruses in respiratory secretions of patients undergoing artificial ventilation

The significance of detection of herpes viruses in respiratory secretions of critically ill patients is controversial. The study aim was to determine the prevalence of herpes virus DNA in respiratory secretions in patients on artificial ventilation. Respiratory secretions taken thrice weekly from 174 patients in a tertiary center intensive therapy unit (ITU) were tested for herpes simplex virus (HSV) by nested PCR. Samples from 61 patients in ITU for 4 days or more were also tested for Epstein Barr Virus (EBV) and cytomegalovirus (CMV) using real‐time PCR. HSV positivity increased with ITU stay with 18.6% admission samples positive, 32.5% day 2–5 samples, and 65.9% day 6–39 samples. Being HSV positive on admission did not influence mortality (9/27, 33.3% vs. 38/118, 32.2%) however, subsequently, mortality of those negative but becoming positive was higher than in those remaining negative (10/35, 29% vs. 5/24 21%). At least one sample was EBV positive in 61% and CMV positive in 19% of patients tested. Of 63 patients tested for all three viruses, 4 were positive for three viruses, 23 patients for two viruses, 24 for one virus and 12 were negative for all the above viruses. Detection of HSV, EBV and CMV is common in ITU patients. Becoming HSV positive while in ITU may increase mortality. J. Med. Virol. 82:1406–1409, 2010. © 2010 Wiley‐Liss, Inc.     

Human nasopharyngeal-associated lymphoreticular tissues. Functional analysis of subepithelial and intraepithelial B and T cells from adenoids and tonsils

Subepithelial and intraepithelial lymphocytes of human adenoids and tonsils were characterized and directly compared to determine the potential contribution of these tissues to mucosal and systemic immune responses. The distribution of T and B cell subsets, cytokine patterns, and antibody (Ab) isotype profiles were similar for adenoids and tonsils. Both tissues contained predominantly B cells ( approximately 65%), approximately 5% macrophages, and 30% CD3(+) T cells. The T cells were primarily of the CD4(+) subset ( approximately 80%). Tonsillar intraepithelial lymphocytes were also enriched in B cells. The analysis of dispersed cells revealed a higher frequency of cells secreting IgG than IgA and the predominant Ig subclass profiles were IgG1 > IgG3 and IgA1 > IgA2, respectively. In situ analysis also revealed higher numbers of IgG- than IgA-positive cells. These IgG-positive cells were present in the epithelium and in the subepithelial zones of both tonsils and adenoids. Mitogen-triggered T cells from tonsils and adenoids produced both Th1- and Th2-type cytokines, clearly exhibiting their pluripotentiality for support of cell-mediated and Ab responses. Interestingly, antigen-specific T cells produced interferon-gamma and lower levels of interleukin-5. These results suggest that adenoids and tonsils of the nasopharyngeal-associated lymphoreticular tissues represent a distinct component of the mucosal-associated lymphoreticular tissues with features of both systemic and mucosal compartments.     

Isolation of follicular dendritic cells from human tonsils and adenoids. V. Effect on lymphocyte proliferation and differentiation

Follicular dendritic cells (FDC) are located only in lymph follicles and are characterized by their capacity to retain high amounts of immune complexes on their plasma membranes. As their functions in germinal centres are unknown, we isolated them from human tonsils and cultured them with autologous lymphoid cells. Cultures of lymphoid cells alone or with added macrophages were used as controls. Lymphoid cells incorporated tritiated thymidine only when FDC and lectins were added; this could be shown after several periods of time. However, the Ig secretion by lymphoid cell populations was inhibited by FDC after several days in vitro. In contrast, the supernatants of lymphocytes cultured alone or with macrophages only for the same periods of time contained increasing amounts of immunoglobulins. This inhibitory effect of FDC on immunoglobulin production was observed for all considered isotypes. Our data suggest that FDC stimulate lymphoid cell proliferation but reduce B-cell differentiation. This is the first accessory cell activity definitely shown for FDC in cultures.     

A longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion

We analyzed data from a 14-year longitudinal study of respiratory infections in young children to determine the relative importance of viral respiratory infection and nasopharyngeal colonization with Streptococcus pneumoniae and Haemophilus influenzae as factors influencing the occurrence of acute otitis media with effusion. The incidence of this disorder was increased in children with viral respiratory infections (average relative risk, 3.2; P less than 0.0001). Infection with respiratory syncytial virus, influenza virus (type A or B), and adenovirus conferred a greater risk of otitis media than did infection with parainfluenza virus, enterovirus, or rhinovirus. Colonization of the nasopharynx with Str. pneumoniae or H. influenzae had a lesser effect on the incidence of the disease (average relative risk; 1.5; P less than 0.01). Infections with the viruses more closely associated with acute otitis media (respiratory syncytial virus, adenovirus, and influenza A or B) were correlated with an increased risk of recurrent disease. Prevention of selected otitis-associated viral infections should reduce the incidence of this disease.