Application of an enzyme‐labeled antigen method for visualizing plasma cells producing antibodies against Strep A,a carbohydrate antigen of Streptococcus pyogenes,in recurrent tonsillitis

Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme‐labeled antigen method is a novel histochemical technique that visualizes specific antibody‐producing cells in tissue sections by employing a biotin‐labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde‐fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme‐labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR‐detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti‐Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A‐reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.     

Nanoparticle Transport from Mouse Vagina to Adjacent Lymph Nodes

To test the feasibility of localized intravaginal therapy directed to neighboring lymph nodes, the transport of quantum dots across the vaginal wall was investigated. Quantum dots instilled into the mouse vagina were transported across the vaginal mucosa into draining lymph nodes, but not into distant nodes. Most of the particles were transported to the lumbar nodes; far fewer were transported to the inguinal nodes. A low level of transport was evident at 4 hr after intravaginal instillation, and transport peaked at about 36 hr after instillation. Transport was greatly enhanced by prior vaginal instillation of Nonoxynol-9. Hundreds of micrograms of nanoparticles/kg tissue (ppb) were found in the lumbar lymph nodes at 36 hr post-instillation. Our results imply that targeted transport of microbicides or immunogens from the vagina to local lymph organs is feasible. They also offer an in vivo model for assessing the toxicity of compounds intended for intravaginal use.     

Small B cell lymphocytic lymphoma presenting as obstructive sleep apnea

Background  

Most lymphomas that involve the tonsil are large B cell lymphomas. Large B-cell lymphoma is a high grade malignancy which progresses rapidly. Tonsillar lymphoma usually presents as either a unilaterally enlarged palatine tonsil or as an ulcerative and fungating lesion over the tonsillar area. Small lymphocytic lymphomas (SLL) of the Waldeyer's ring are uncommon.     

Mast cell activators: a new class of highly effective vaccine adjuvants

Mast cells (MCs) have recently received recognition as prominent effectors in the regulation of immune cell migration to draining lymph nodes and lymphocyte activation. However, their role in the development of humoral immune responses is not clear. Here, we demonstrate that subcutaneous or nasal administration of small-molecule MC activators with vaccine antigens evokes large increases in antigen-specific serum immunoglobulin G (IgG) responses. These responses were MC dependent and correlated with increased dendritic cell and lymphocyte recruitment to draining lymph nodes. Nasal instillation of these formulations also evoked antigen-specific secretory IgA and provided protection against anthrax lethal toxin challenge in vitro and against vaccinia virus infection in vivo. Collectively, these results define the MC as an integral sensory arm of the adaptive immune system. Moreover, they highlight MC activators as a new class of vaccine adjuvants, capable of inducing protective antigen-specific immune responses through needle-free routes of administration.     

Different subclass distribution of IgA-producing cells in human lymphoid organs and various secretory tissues

A highly reproducible paired immunofluorescence staining method was used to map the relative distribution of IgA1- and IgA2-producing cells in peripheral lymphoid organs and various secretory tissues. Spleen, peripheral lymph nodes, and tonsils all contained a marked predominance (91 to 95%) of IgA1 immunocytes. However, striking variations were demonstrated among the secretory tissues with regard to the median proportion of IgA1-producing cells: nasal mucosa, 96%; lacrimal glands, 81%; major salivary glands, 66%; mammary glands, 63%; gastric and proximal small intestinal mucosa, 84 to 77%; ileum, 55%; and large bowel, 41%. Thus, IgA2 production is relatively enhanced mainly in the distal gut and in mammary and salivary glands, in that order.     

Development of a model of natural infection with Mycobacterium bovis in white-tailed deer.

The objective of this study was to develop a suitable experimental model of natural Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus), describe the distribution and character of tuberculous lesions, and to examine possible routes of disease transmission. In October 1997, 10 mature female white-tailed deer were inoculated by intratonsilar instillation of 2 x 10(3) (low dose) or 2 x 10(5) (high dose) colony forming units (CFU) of M. bovis. In January 1998, deer were euthanatized, examined, and tissues were collected 84 to 87 days post inoculation. Possible routes of disease transmission were evaluated by culture of nasal, oral, tonsilar, and rectal swabs at various times during the study. Gross and microscopic lesions consistent with tuberculosis were most commonly seen in medial retropharyngeal lymph nodes and lung in both dosage groups. Other tissues containing tuberculous lesions included tonsil, trachea, liver, and kidney as well as lateral retropharyngeal, mandibular, parotid, tracheobronchial, mediastinal, hepatic, mesenteric, superficial cervical, and iliac lymph nodes. Mycobacterium bovis was isolated from tonsilar swabs from 8 of 9 deer from both dosage groups at least once 14 to 87 days after inoculation. Mycobacterium bovis was isolated from oral swabs 63 and 80 days after inoculation from one of three deer in the low dose group and none of four deer in the high dose group. Similarly, M. bovis was isolated from nasal swabs 80 and 85 days after inoculation in one of three deer from the low dose group and 63 and 80 days after inoculation from two of four deer in the high dose group. Intratonsilar inoculation with M. bovis results in lesions similar to those seen in naturally infected white-tailed deer; therefore, it represents a suitable model of natural infection. These results also indicate that M. bovis persists in tonsilar crypts for prolonged periods and can be shed in saliva and nasal secretions. These infected fluids represent a likely route of disease transmission to other animals or humans.     

Persistence of infection in mice inoculated intranasally with Cryptococcus neoformans

Cryptococcus neoformans was instilled intranasally into mice which were periodically sacrificed to determine the course of infection. Cryptococci persisted within the nasal passages throughout the 90 day study. Extranasal dissemination began 14–28 days after instillation and was still demonstrable 90 days post-exposure. Ten percent mortality was observed in mice receiving 10⁶ cryptococci, while no mortality was observed in mice exposed to 10³ or 10⁴ cryptococci. Our research suggests that nasal colonization with C. neoformans can precede pulmonary and systemic cryptococcosis by weeks or months.     

Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

Background

There have been few reports on the role of Fc receptors (FcRs) and immunoglobulin G (IgG) in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs) in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa.

Methods

In FcγRIIB deficient (KO) and C57BL/6 (WT) mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA). Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c⁺ MHC class II⁺ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c⁺ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs) in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs) differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL).

Results

In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c⁺ MHC class II⁺ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c⁺ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously.

Conclusion

Antigen-specific IgG ameliorates allergic airway inflammation via FcγRIIB on DCs.     

Activated Human Nasal Epithelial Cells Modulate Specific Antibody Response against Bacterial or Viral Antigens

Nasal mucosa is an immune responsive organ evidenced by eliciting both specific local secretory IgA and systemic IgG antibody responses with intra-nasal administration of antigens. Nevertheless, the role of nasal epithelial cells in modulating such responses is unclear. Human nasal epithelial cells (hNECs) obtained from sinus mucosa of patients with chronic rhinosinusitis were cultured in vitro and firstly were stimulated by Lactococcus lactis bacterium-like particles (BLPs) in order to examine their role on antibody production. Secondly, both antigens of immunodominant protein IDG60 from oral Streptococcus mutans and hemagglutinin (HA) from influenza virus were tested to evaluate the specific antibody response. Stimulated hNECs by BLPs exhibited a significant increase in the production of interleukin-6 (IL-6), and thymic stromal lymphopoietin (TSLP). Conditioned medium of stimulated hNECs has effects on enhancing the proliferation of CD4+ T cells together with interferon-γ and IL-5 production, increasing the costimulatory molecules on dendritic cells and augmenting the production of IDG60 specific IgA, HA specific IgG, IgA by human peripheral blood lymphocytes. Such production of antigen specific IgG and IgA is significantly counteracted in the presence of IL-6 and TSLP neutralizing antibodies. In conclusion, properly stimulated hNECs may impart immuno-modulatory effects on the antigen-specific antibody response at least through the production of IL-6 and TSLP.     

The ovine nasal mucosa: an alternative tissue site for mucosal immunization

The ovine nasal mucosal environment has histological and ultrastructural features that resemble well-known inductive sites of mucosa-associated lymphoid tissue. In the present study, the nasal mucosa was assessed as a potential mucosal tissue site for delivering vaccines to sheep. Sheep were immunized by either injection with the model antigen, Keyhole Limpet Haemocyanin (KLH), and aluminium hydroxide gel (alum) or by aerosol spray with KLH with and without cholera toxin (CT). Sheep immunized by injection with KLH/alum and aerosol spray with KLH/CT induced strong anti-KLH IgG and IgA serum antibody responses as well as specific T cell memory. Anti-KLH IgG1 responses were significantly higher following immunization by injection and no significant differences in anti-KLH IgG2 responses were detected between groups. Sheep immunized with KLH by aerosol spray without CT did not produce serum antibody and T cell memory responses. Antibody-secreting cells were present in the parotid lymph nodes (draining lymph nodes) of sheep immunized with KLH/alum and KLH/CT, but secreted only Ag-specific IgG1, and not IgG2 or IgA. These results suggest that aerosolization of soluble antigen formulations with CT may provide an alternative method of delivering nasal vaccines to sheep and other large animal species, and that further improvements in antigen penetration of nasal tissues may dramatically improve the strength of the immune response.     

Comparison of lymphocyte production in lymphoid organs and their compartments using the metaphase-arrest technique

Summary Lymphocyte proliferation was studied in normal young anesthetized pigs by the metaphase-arrest technique using vincristine (VCR). In each animal biopsies were taken simultaneously from the thymus, mesenteric lymph nodes, spleen, palatine tonsil and Peyer's patches from the ileum and jejunum. After taking the first samples, 0.25 mg VCR/ kg body weight was injected i.v. and then four more biopsies were excised for up to 3.5 h after VCR. Imprints of the lymphoid organs were evaluated as an overall index for each organ, and histological sections were used to determine the mitotic index in typical B-and T-lymphocyte areas in these organs. In follicles of mesenteric lymph nodes, tonsils and the two types of Peyer's patches a comparable increase in the mitotic index was found, 3.62% per hour. In the corona the increase was also comparable but much lower, 0.43% per hour and in the interfollicular area similarly 0.38% per hour. In the spleen the mitotic rate was 0.69% for the white pulp and 0.42% per hour for the red pulp. In the thymic cortex the mitotic index increased by 0.49% and in the medulla by a surprisingly high value of 0.32% per hour. The metaphase-arrest technique in larger animals enables a comparison of lymphocyte production among organs and their different compartments, and demonstrates the important contribution of peripheral lymphoid organs to the renewal of the lymphocyte pools.List of Abbreviations DNA deoxyribonucleic acid - ¹⁴ C-TdR thymidine labelled with ¹⁴C - ³ H-TdR tritiated thymidine - r _m rate of entry of cells in mitosis per hour - VCR vincristine sulphate Partly presented at the XII Int. Anat. Congress in London, August 1985     

Involvement of human mucous saliva and salivary mucins in the aggregation of the oral bacteria Streptococcus sanguis,Streptococcus oralis,and Streptococcus rattus

The contribution of human parotid (Par) and submandibular/sublingual (SM/SL) saliva and of the human whole salivary mucin fraction (HWSM) to saliva-induced bacterial aggregation was studied for S. sanguis C476, S. oralis I581, and S. rattus HG 59. The mucous SM/SL saliva showed a much higher aggregation potency towards the S. sanguis and S. oralis strain than did the serous Par saliva. The SM/SL saliva-induced aggregation was observed after 30 min, at 60 min followed by the Par saliva-induced aggregation, and showed a 4-fold higher aggregation titer of 128 for S. sanguis, and an 8-fold higher titer of 516 for S. oralis. In contrast, the Par saliva showed a slightly higher aggregation activity than the SM/SL saliva towards S. rattus as judged by a twofold higher titer of 64. Morphologically, however, the SM/SL saliva-induced aggregation of S. rattus was far more pronounced as was also found for S. sanguis. Finally, the HWSM-induced aggregation showed a 4 to 8-fold higher titer than the originating salivary source, measuring 2048 for S. oralis and 128 for S. rattus. Moreover, no difference was observed in aggregation activity between the HWSM from whole saliva of a blood group O donor and the HWSM from SM/SL saliva of a blood group A donor. All the data point to an important, though not exclusive role of the human salivary mucin fraction in the saliva-induced aggregation of these strains.     

Potential Roles of CCR5+ CCR6+ Dendritic Cells Induced by Nasal Ovalbumin plus Flt3 Ligand Expressing Adenovirus for Mucosal IgA Responses

We assessed the role of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ dendritic cells (DCs) for induction of ovalbumin (OVA)-specific antibody (Ab) responses following mucosal immunization. Mice given nasal OVA plus an adenovirus expressing Flt3 ligand (Ad-FL) showed early expansion of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ DCs in nasopharyngeal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs). Subsequently, this DC subset became resident in submandibular glands (SMGs) and nasal passages (NPs) in response to high levels of CCR-ligands produced in these tissues. CD11b⁺/CD11c⁺ DCs were markedly decreased in both CCR5^−/− and CCR6^−/− mice. Chimera mice reconstituted with bone marrow cells from CD11c-diphtheria toxin receptor (CD11c-DTR) and CCR5^−/− or CD11c-DTR and CCR6^−/− mice given nasal OVA plus Ad-FL had elevated plasma IgG, but reduced IgA as well as low anti-OVA secretory IgA (SIgA )Ab responses in saliva and nasal washes. These results suggest that CCR5⁺CCR6⁺ DCs play an important role in the induction of Ag-specific SIgA Ab responses.     

Cellular and humoral IgA responses after single and multiple local injections of antigen

IgA responses in submandibular salivary glands, cervical lymph nodes, and saliva of rats were studied. Immunoglobulin-containing cells of the IgA isotype were examined by immunofluorescence of mononuclear cells isolated from the submandibular salivary glands and cervical lymph nodes after primary and multiple local injections of Streptococcus mutans. Also, salivary and serum antibodies to S. mutans were determined using an ELISA. The results support immunologic memory for the secretory (salivary) IgA system at both the cellular and humoral levels. Comparison of the dynamics of the IgAICC responses among the tissues and secretions after the injection regimes suggests that the cervical lymph nodes may provide an enriched tissue source for secretory IgA responses in the oral cavity.     

Prevalence of oral colonization with Candida albicans and anti-C. albicans IgA in the saliva of normal children and children with acute lymphoblastic leukaemia

The prevalence of oral colonization with C. albicans was studied in 40 children with acute lymphoblastic leukaemia (ALL) and 40 matched normal children. Colonization was more common in the ALL group (25% colonized) than the normal group (7.5% colonized). Total saliva IgA levels were lower in the ALL group, but low levels did not correlate with oral colonization. The three normal children with oral colonization all had anti-C. albicans IgA in their saliva, as had seven of the ten colonized ALL patients.     

Expression and function of tight junctions in the crypt epithelium of human palatine tonsils.

The human palatine tonsils have surface and crypt stratified epithelium and may be initiated via the epithelium to mount immune responses to various presenting antigens. Here we investigated the expression and function of tight junctions in the epithelium of human palatine tonsils from patients with tonsillar hypertrophy or recurrent tonsillitis. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, -7, -8, and -14 mRNAs were detected in tonsillar hypertrophy. Occludin and claudin-14 were expressed in the uppermost layer of the tonsil surface epithelium, whereas ZO-1, JAM-1, and claudin-1, -4, and -7 were found throughout the epithelium. In the crypt epithelium, claudin-4 was preferentially expressed in the upper layers. In freeze-fracture replicas, short fragments of continuous tight junction strands were observed but never formed networks. In the crypt epithelium of recurrent tonsillitis, the tracer was leaked from the surface regions where occludin and claudin-4 disappeared. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, and -14, but not claudin-7, mRNAs were decreased in recurrent tonsillitis compared with those of tonsillar hypertrophy. These studies suggest unique expression of tight junctions in human palatine tonsillar epithelium, and the crypt epithelium may possess an epithelial barrier different from that of the surface epithelium.     

Pulmonary cell response in mice following intranasal instillation with Cryptococcus neoformans

Cryptococcus neoformans was instilled intranasally into mice which were sacrificed to determine the fate of instilled cryptococci and the pulmonary cellular response. During the first 12 h, instilled cryptococci decreased significantly in numbers and did not disseminate to extrapulmonary sites. There was a transient increase in neutrophils which may have been due to an influx of bacteria which accompanied the process of intranasal instillation.     

Cathepsin E in follicle associated epithelium of intestine and tonsils: localization to M cells and possible role in antigen processing

A specific rabbit anti-human serum was used selectively to localize the aspartic proteinase cathepsin E to follicle associated epithelium (FAE) of human and rat intestine, including jejunum, ileum, appendix, colon and rectum, as well as of human palatine, pharyngeal and lingual tonsils. Coexpression of class II histocompatibility antigen HLA-DR antigen has been observed in some of the cathepsin E-positive epithelial cells. In addition, cathepsin E has been detected in a few mononuclear cells of intestinal lymphoid structures and tonsils resembling interdigitating reticulum cells of lymph nodes. Another aspartic proteinase, cathepsin D, has been found to be poorly represented in FAE and intensely expressed by macrophages. Electron immunocytochemistry localized cathepsin E to endosomal vesicles and endoplasmic reticulum of M cells in rat and human ileum as well as of M-like cells in human palatine tonsil. The results suggest a possible role of endosomal cathepsin E in the processing of macromolecules and microorganisms transported by M cells and related epithelial cells to mucosal associated lymphoid tissue (MALT).     

Trichinella spiralis: the influence of short chain fatty acids on the proliferation of lymphocytes, the goblet cell count and apoptosis in the mouse intestine

This study was carried out to determine the influence of short chain fatty acids (SCFA) on spleen and mesenteric lymph node lymphocyte proliferation, goblet cells and apoptosis in the mouse small intestine during invasion by Trichinella spiralis. BALB/c mice were infected with 250 larvae of T. spiralis. An SCFA water solution containing acetic, propionic and butyric acids (30:15:20 mM) was administered orally starting 5 days before infection and ending 20 days post infection (dpi). Fragments of the jejunum were collected by dissection 7 and 10 dpi, and were examined for apoptotic cells in the lamina propria of the intestinal mucosa, and for goblet cells. The proliferation index of the cultured spleen and mesenteric lymph node lymphocytes with MTT test was also determined. The orally administered SCFA solution decreased the proliferation of mesenteric lymph node lymphocytes in the mice infected with T. spiralis at both examination times, but did not influence the proliferative activity of the spleen cells. Seven dpi, both in the spleen and mesenteric lymph nodes, the highest proliferation index of concanavalin A (Con A)-stimulated lymphocytes was found in the group of uninfected animals receiving SCFA animals. This tendency could still be seen 10 dpi in the mesenteric lymph nodes but not in the spleen, where the proliferation index in this group had significantly decreased. In vitro studies revealed, that butyric and propionic acids added to the cell cultures suppressed the proliferation of Con A-stimulated mesenteric lymph nodes and spleen lymphocytes taken from uninfected and T. spiralis-infected mice. Acetic acid stimulated proliferation of splenocytes taken from uninfected mice but did not affect lymphocyte proliferation in mesenteric lymph nodes from uninfected or infected mice. Orally administered SCFA increased the number of goblet cells found in the epithelium of the jejunum 7 dpi, but this number had decreased 10 dpi. The number of apoptotic cells in the lamina propria of the intestinal mucosa of animals infected with the T. spiralis and receiving SCFA was also lower, particularly 10 dpi. The above results show that SCFA can participate in the immune response during the course of trichinellosis in mice.