Lymphoid and non-lymphoid cells in nasal-associated lymphoid tissue (NALT) in the rat

Summary Lymphocyte and macrophage subpopulations and the stroma of mucosa-associated lymphoid tissue in the nasal cavity of the rat were examined by application of immunohistochemical and enzyme histochemical methods to cryostat sections. Nasal-associated lymphoid tissue was composed of a loose reticular network with lymphocytes and macrophages, covered by epithelium. The epithelium was infiltrated with B cells, T helper (W3/13-positive) and T suppressor/cytotoxic or large granular cells (OX8-positive), ED1-positive macrophages and Ia-positive cells. The B cell areas were populated by B cells, immunopositive for surface IgM or IgG. B cells with surface IgA or IgE were rare. Germinal centres were found infrequently. T helper cells were scattered throughout the B cell area. A few ED1-positive macrophages and ED5-positive follicular dendritic cells were observed. Strong Ia staining (mostly of B cells) was found in this area. The T cell areas contained T helper and T suppressor/cytotoxic cells in about equal amounts, and numerous ED1-positive macrophages. ED1 staining was also found in the subepithelial area. Numerous ED1-, ED2- and ED3-positive macrophages were found in the border between the lymphoid mass and the surrounding connective tissue. A few non-lymphoid cells showed weak acid phosphatase or non-specific esterase activity. The morphological observations suggest that nasal-associated lymphoid tissue plays an important role in the first contact with inhaled antigens.     

Establishment of an In Vitro System Representing the Chicken Gut-Associated Lymphoid Tissue

The bursa of Fabricius is critical for B cell development and differentiation in chick embryos. This study describes the production in vitro, from dissociated cell suspensions, of cellular agglomerates with functional similarities to the chicken bursa. Co-cultivation of epithelial and lymphoid cells obtained from embryos at the appropriate developmental stage regularly led to agglomerate formation within 48 hours. These agglomerates resembled bursal tissue in having lymphoid clusters overlaid by well organized epithelium. Whereas lymphocytes within agglomerates were predominantly Bu-1a⁺, a majority of those emigrating onto the supporting membrane were Bu-1a⁻ and IgM⁺. Both agglomerates and emigrant cells expressed activation-induced deaminase with levels increasing after 24 hours. Emigrating cells were actively proliferating at a rate in excess of both the starting cell population and the population of cells remaining in agglomerates. The potential usefulness of this system for investigating the response of bursal tissue to avian Newcastle disease virus (strain AF2240) was examined.     

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses^1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system^4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells^4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages^9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens⁷. While NALT disappear in humans by the age of two years, the Waldeyer''s Ring and similarly structured lymphatic organs continue to persist throughout life⁶. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses¹¹.Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-α knockout mice have atrophied NALT, the Peyer''s patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice^12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines^1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.     

Bronchus-Associated Lymphoid Tissue (BALT) and Survival in a Vaccine Mouse Model of Tularemia

Background

Francisella tularensis causes severe pulmonary disease, and nasal vaccination could be the ideal measure to effectively prevent it. Nevertheless, the efficacy of this type of vaccine is influenced by the lack of an effective mucosal adjuvant.

Methodology/Principal Findings

Mice were immunized via the nasal route with lipopolysaccharide isolated from F. tularensis and neisserial recombinant PorB as an adjuvant candidate. Then, mice were challenged via the same route with the F. tularensis attenuated live vaccine strain (LVS). Mouse survival and analysis of a number of immune parameters were conducted following intranasal challenge. Vaccination induced a systemic antibody response and 70% of mice were protected from challenge as showed by their improved survival and weight regain. Lungs from mice recovering from infection presented prominent lymphoid aggregates in peribronchial and perivascular areas, consistent with the location of bronchus-associated lymphoid tissue (BALT). BALT areas contained proliferating B and T cells, germinal centers, T cell infiltrates, dendritic cells (DCs). We also observed local production of antibody generating cells and homeostatic chemokines in BALT areas.

Conclusions

These data indicate that PorB might be an optimal adjuvant candidate for improving the protective effect of F. tularensis antigens. The presence of BALT induced after intranasal challenge in vaccinated mice might play a role in regulation of local immunity and long-term protection, but more work is needed to elucidate mechanisms that lead to its formation.     

Phylogeny of Gut-Associated Lymphoid Tissue

Gastrointestinal (GI) tracts from four anuran and four urodelespecies were examined macroscopically and histologically fromthe esophagus to the cloaca. Anatomic observations were facilitatedby treating the GI tracts with acetic acid, a procedure whichremoves gut epithelium and exposes underlying structures. Histologicanalysis involved routine as well as specific histochemicaltechniques. Detailed observations reveal that anuran amphibianspossess nodular, well-organized gut-associated lymphoid tissue(GALT) throughout their GI tracts. In striking contrast, nosuch patterns of lymphoid tissue have been noted in the Urodela. Similar techniques have revealed the location of lymphoid tissuesin the urogenital system (e.g., cloaca and bladder) of advancedanurans. These nodules appear to be lacking in the primitiveanuran Xenopus and in all the urodele species we examined. Preliminary observations have confirmed GALT and urogenital-associatedlymphoid tissues in the snapping turtle.     

Distribution of T-cell subsets and immunoglobulin-containing cells in nasal-associated lymphoid tissue (NALT) of chickens

The present study demonstrated the localization of the T-cell subsets (CD4+ and CD8+) and immunoglobulin (Ig)-containing cells (IgA, IgM, and IgG) in the nasal mucosa and its accessory structures. These lymphoid structures may be compared with nasal-associated lymphoid tissue (NALT) of rats and mice. In the chicken NALT, T-cell subsets were more widely distributed than Ig-containing cells, especially in large lymphoid accumulations restricted to the respiratory mucosa in the nasal cavity and the nasolacrimal duct. These lymphoid accumulations in the mucosa of the nasal cavity and nasolacrimal duct consisted of widely distributed CD8+ cells and deeply aggregated CD4+ cells adjacent to large germinal centers. In these lymphoid accumulations, IgG-containing cells were more frequently observed than IgM- and IgA-containing cells. T-cell subsets, predominantly CD8+ cells were more widely distributed in the duct epithelium of the lateral nasal glands than Ig-containing cells. Moreover, numerous CD8+ cells and a few Ig-containing cells were found in the chicken salivary glands, especially around the orifice of their ducts into the oral cavity. Therefore, it seems likely that the chicken NALT plays an important part in the upper respiratory tract, with a close relationship to the paraocular immune system.     

Electron Tomography of HIV-1 Infection in Gut-Associated Lymphoid Tissue

Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET) to image HIV-1 in gut-associated lymphoid tissue (GALT) of HIV-1–infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1–infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural understanding of HIV-1 transmission within lymphoid tissue.     

家鸡G蛋白偶联受体139基因的结构、序列及表达特征分析

G蛋白偶联受体139(GPR139)是一种孤儿受体。在脊椎动物中,针对其结构、表达和功能的研究相对缺乏。本文以家鸡大脑c DNA为模板,采用PCR方法,扩增并克隆得到家鸡GPR139(c GPR139)基因。结果显示:家鸡GPR139基因c DNA由2个外显子组成;ORF区域长1056 bp,编码351个氨基酸的前体蛋白,在其第三跨膜区末端包含D-R-Y基序,属于G蛋白偶联受体家族视紫红质亚家族。将家鸡GPR139前体蛋白与人、大鼠、小鼠GPR139前体蛋白的氨基酸序列进行比对,分别具有91.78%、89.17%、89.17%的相似度。采用RT-PCR方法,本研究也检测了家鸡GPR139的组织表达情况。结果显示:家鸡GPR139基因在大脑、中脑、小脑、后脑、下丘脑及垂体中表达量较高,而在卵巢、精巢、肺、肾脏、肌肉组织中,GPR139基因仅有微弱表达。这些结果首次揭示了GPR139基因在非哺乳动物(家鸡)中的结构特征和组织表达特点,为探究其在家鸡中的生理功能奠定一些基础。     

Reticulum cells in the ontogeny of nasal-associated lymphoid tissue (NALT) in the rat

This study concerns the ontogeny of reticulum cells (RC) in the nasal-associated lymphoid tissue (NALT) of Wistar and Brown-Norway rats. A panel of monoclonal antibodies (mAb) directed against RC in peripheral lymphoid organs (antibodies ED10ED15) was used, together with a recently developed antibody ED17, which recognizes macrophages and Langerhans cells. Early in embryogenesis, staining with common connective tissue markers, ED14 and ED15, was found. ED17-positive cells were present before cells positive to ED1, a pan-macrophage marker, or Ia glycoproteins were observed. The first differentiation of reticulum was seen at the day of birth, when ED10 recognized a distinct area in the nasal mucosa. The first T-lymphocytes were found at the same time. Two days after birth, B-cells and ED11-positive cells were present in the NALT area. Fourteen days after birth, T- and B-cell compartments were recognizable. ED10 was found predominantly in the T-cell area and ED11 was mainly confined to the B-cell compartment. We conclude that the development of the NALT is closely accompanied by the phenotypic specialization of the reticulum. This suggests that the reticulum plays an important role in the compartmentalization of NALT tissue and in the retention of lymphocyte subsets within these compartments.     

Development,Alteration and Real Time Dynamics of Conjunctiva-Associated Lymphoid Tissue

Purpose

Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model.

Methods

Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy.

Results

Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min.

Conclusions

CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.     

淋巴细胞发育相关基因在鼻腔NK/T细胞淋巴瘤中表达的研究

探索鼻腔NK/T细胞淋巴瘤(N-NK/T-L)中淋巴细胞发育相关基因的表达及意义。方法:通过对EOMEs,ETS-1和MEF基因进行克隆、探针制备和组织芯片制备,对25例N-NK/T-L以及同部位11例B细胞淋巴瘤(BCL)、6例T细胞淋巴瘤(TCL)、3例正常脾组织和5例慢性炎性鼻黏膜组织进行了原位杂交检测。结果:EOMES、ETS-1、MEF基因在N-NL/T-L、BCL、TCL、脾和鼻粘膜中均有表达,但阳性率和表达强度不同。EOMES基因在N-NK/T-L的表达强度与BCL和TCL间有差异,MEF基因在N-NK/T-L的表达阳性率和强度与TCL间有差异,而ETS-1基因在N-NK/T-L与对照组间的表达无差异。T-bet、EOMES和MEF基因在N-NK/T-L的表达具有相关性。结论EOMES和MEF基因在N-NK/T-L中高表达,可能在该肿瘤发生和组织坏死中起重要作用,而ETS-1因在N-NK/T-L和对照组中普遍表达,提示其可能在淋巴造血系统的发育和功能中起基础性的共同通路。     

Zicam-Induced Damage to Mouse and Human Nasal Tissue

Intranasal medications are used to treat various nasal disorders. However, their effects on olfaction remain unknown. Zicam (zinc gluconate; Matrixx Initiatives, Inc), a homeopathic substance marketed to alleviate cold symptoms, has been implicated in olfactory dysfunction. Here, we investigated Zicam and several common intranasal agents for their effects on olfactory function. Zicam was the only substance that showed significant cytotoxicity in both mouse and human nasal tissue. Specifically, Zicam-treated mice had disrupted sensitivity of olfactory sensory neurons to odorant stimulation and were unable to detect novel odorants in behavioral testing. These findings were long-term as no recovery of function was observed after two months. Finally, human nasal explants treated with Zicam displayed significantly elevated extracellular lactate dehydrogenase levels compared to saline-treated controls, suggesting severe necrosis that was confirmed on histology. Our results demonstrate that Zicam use could irreversibly damage mouse and human nasal tissue and may lead to significant smell dysfunction.