Role of the draining lymph node in scrapie agent transmission from the skin

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases that affect humans and animals. Diseases include scrapie in sheep and Creutzfeldt-Jakob disease in humans. Following peripheral exposure, TSE agents usually accumulate on follicular dendritic cells (FDCs) in lymphoid tissues before neuroinvasion. Studies in mice have shown that TSE exposure through scarified skin is an effective means of transmission. Following inoculation by this route TSE agent accumulation upon FDCs is likewise essential for the subsequent transmission of disease to the brain. However, which lymphoid tissues are crucial for TSE pathogenesis following inoculation via the skin was not known. Mice were therefore created that lacked the draining inguinal lymph node (ILN), but had functional FDCs in remaining lymphoid tissues such as the spleen. These mice were inoculated with the scrapie agent by skin scarification to allow the role of draining ILN in scrapie pathogenesis to be determined. We show that following inoculation with the scrapie agent by skin scarification, disease susceptibility was dramatically reduced in mice lacking the draining ILN. These data demonstrate that following inoculation by skin scarification, scrapie agent accumulation upon FDCs in the draining lymph node is critical for the efficient transmission of disease to the brain.     

Follicular dendritic cell dedifferentiation reduces scrapie susceptibility following inoculation via the skin

Transmissible spongiform encephalopathies (TSEs) are a group of subacute infectious neurodegenerative diseases that are characterized by the accumulation in affected tissues of PrP(Sc), an abnormal isoform of the host prion protein (PrPc). Following peripheral exposure, TSE infectivity and PrP(Sc) usually accumulate in lymphoid tissues prior to neuroinvasion. Studies in mice have shown that exposure through scarified skin is an effective means of TSE transmission. Following inoculation via the skin, a functional immune system is critical for the transmission of TSEs to the brain, but until now, it has not been known which components of the immune system are required for efficient neuroinvasion. Temporary dedifferentiation of follicular dendritic cells (FDCs) by treatment with an inhibitor of the lymphotoxin-beta receptor signalling pathway (LTbetaR-Ig) 3 days before or 14 days after inoculation via the skin, blocked the early accumulation of PrP(Sc) and TSE infectivity within the draining lymph node. Furthermore, in the temporary absence of FDCs before inoculation, disease susceptibility was reduced and survival time significantly extended. Treatment with LTbetaR-Ig 14 days after TSE inoculation also significantly extended the disease incubation period. However, treatment 42 days after inoculation did not affect disease susceptibility or survival time, suggesting that the infection may have already have spread to the nervous system. Together these data show that FDCs are essential for the accumulation of PrP(Sc) and infectivity within lymphoid tissues and subsequent neuroinvasion following TSE exposure via the skin.     

Clusterin expression in follicular dendritic cells associated with prion protein accumulation

Peripheral accumulation of abnormal prion protein (PrP) in variant Creutzfeldt-Jakob disease and some animal models of transmissible spongiform encephalopathies (TSEs) may occur in the lymphoreticular system. Within the lymphoid tissues, abnormal PrP accumulation occurs on follicular dendritic cells (FDCs). Clusterin (apolipoprotein J) has been recognized as one of the molecules associated with PrP in TSEs, and clusterin expression is increased in the central nervous system where abnormal PrP deposition has occurred. We therefore examined peripheral clusterin expression in the context of PrP accumulation on FDCs in a range of human and experimental TSEs. PrP was detected immunohistochemically on tissue sections using a novel highly sensitive method involving detergent autoclaving pretreatment. A dendritic network pattern of clusterin immunoreactivity in lymphoid follicles was observed in association with the abnormal PrP on FDCs. The increased clusterin immunoreactivity appeared to correlate with the extent of PrP deposition, irrespective of the pathogen strains, host mouse strains or various immune modifications. The observed co-localization and correlative expression of these proteins suggested that clusterin might be directly associated with abnormal PrP. Indeed, clusterin immunoreactivity in association with PrP was retained after FDC depletion. Together these data suggest that clusterin may act as a chaperone-like molecule for PrP and play an important role in TSE pathogenesis.     

Appearance and phenotype of murine follicular dendritic cells expressing VCAM-1

The architecture of lymphoid follicles is determined by a series of interactions between lymphoid and follicular stromal cells. A cardinal population in the non-lymphoid compartment is the follicular dendritic cell (FDC), whose communication with resting and activated B cells involves various adhesive interactions. The FDC phenotype variably includes the display of vascular cell adhesion molecule (VCAM-1). In this report we investigated the appearance and follicular tissue distribution of VCAM-1 in murine peripheral lymphoid tissues, and compared VCAM-1 with other FDC markers using immunohistochemistry. Correlating the appearance of VCAM-1 with other murine FDC-associated markers (CR1.2 [complement receptor 1.2 or CD35/21] and FDC-M1) revealed that the display of VCAM-1 is restricted to a subset of CR1.2-positive FDCs. We found that the expression of VCAM-1 antigen in the spleen or peripheral lymph nodes on FDCs requires antigenic stimulus, and that it coincides with germinal center formation. The VCAM-1 expression is associated with the appearance of mucosal addressin cell adhesion molecule (MAdCAM-1), with some slight differences in occurrence. The appearance of VCAM-1 and MAdCAM-1 antigens on FDCs may serve as indicators of FDC activation.     

Rapid screening and confirmatory methods for biochemical diagnosis of human prion disease

Transmissible spongiform encephalopathies (TSEs) are characterised by accumulation of an abnormal isoform of prion protein (PrP^sc), mainly in the brain but also in various peripheral tissues. Home-made assays consisting of non-standardised protocols are used currently for laboratory diagnosis of human TSE. The purpose of the present study was to test the ability of two commercial assays, TeSeE™ CJD ELISA and TeSeE™ Western blot, to detect PrPsc in cerebral and lymphoid tissues of TSE patients. Both tests detected a PrPsc-significant signal in the brains of 54 affected patients and not in 51 controls, yielding 100% specificity and 100% sensitivity. Furthermore, three post-mortem spleens and two pre-mortem tonsils from three patients with variant Creutzfeldt-Jakob disease (vCJD) were detected correctly. The expected PrPsc molecular patterns were found in TSE patient brain tissue and in the tonsils and spleens of the three vCJD patients. In conclusion, these rapid and robust in vitro tools were suitable for routine human TSE diagnosis and characterisation. CJD could also be diagnosed during the patient's lifetime by detection of PrPsc in the tonsil. A diagnostic strategy associating TeSeE™ CJD ELISA screening to biochemical confirmation by TeSeE™ Western blot is proposed.     

Tissue localization and retention of antigen in relation to the immune response

Antigen persists for months or even years in lymphoid tissues of immune animals and this antigen is believed to participate in the induction and maintenance of B-cell memory as well as in the maintenance of serum antibody levels. In the present report we describe the phenomenon of antigen localization and long-term retention on mouse follicular dendritic cells (FDCs). The antigens used were injected in the hind footpads of immune mice and the popliteal lymph nodes were the lymphoid organs generally studied. In addition to presenting the morphological features of mouse FDCs, we report the results of a study of the mechanism of antigen migration from the site of initial localization in the lymph node subcapsular sinus to the regions of follicular retention in the cortex. The migration was followed by light and electron microscopy. The results support the concepts that immune complexes are trapped in the subcapsular sinus and are transported by a group of nonphagocytic cells to follicular regions. The mechanism of transport may involve either migration of pre-FDCs with a concomitant maturation into FDCs, or cell-to-cell transport utilizing dendritic cell processes and membrane fluidity; or a combination of the two mechanisms may be in operation.     

ACETYL CHOLINESTERASE IS EXPRESSED IN THE FOLLICULAR DENDRITIC CELLS OF GERMINAL CENTRES: DIFFERENCES BETWEEN NORMAL AND NEOPLASTIC FOLLICLES

Acetyl cholinesterase (AcChE) was demonstrated by histochemistry in the follicular dendritic cells (FDCs) of the germinal centres of lymph nodes, tonsils, and bowel lymphoid tissue. Its presence in the FDCs was confirmed by double immunostaining for CD21 or DRC-1. AcChE-positive FDCs are concentrated in the inner portion of the light zone of the germinal centre, being absent from the dark zone. In the lymphoid tissue surrounding the germinal centres are AcChE-positive blood vessels; double staining shows that the AcChE is present in the pericytes surrounding the endothelium of the blood vessels. In contrast to the reactive follicle, the AcChE reactivity in FDCs of follicle centre lymphoma is absent or minimally expressed, although the dense FDC mesh is well stained with CD21 or DRC-1. This suggests that the AcChE is not constitutively expressed in FDCs but that its expression is influenced by the state and activity of the lymphoid cells in the germinal centre. The reduced level of AcChE staining can be profitably employed in the diagnosis of follicle centre lymphoma.     

Appearance and phenotype of murine follicular dendritic cells expressing VCAM‐1

The architecture of lymphoid follicles is determined by a series of interactions between lymphoid and follicular stromal cells. A cardinal population in the non‐lymphoid compartment is the follicular dendritic cell (FDC), whose communication with resting and activated B cells involves various adhesive interactions. The FDC phenotype variably includes the display of vascular cell adhesion molecule (VCAM‐1). In this report we investigated the appearance and follicular tissue distribution of VCAM‐1 in murine peripheral lymphoid tissues, and compared VCAM‐1 with other FDC markers using immunohistochemistry. Correlating the appearance of VCAM‐1 with other murine FDC‐associated markers (CR1.2 [complement receptor 1.2 or CD35/21] and FDC‐M1) revealed that the display of VCAM‐1 is restricted to a subset of CR1.2‐positive FDCs. We found that the expression of VCAM‐1 antigen in the spleen or peripheral lymph nodes on FDCs requires antigenic stimulus, and that it coincides with germinal center formation. The VCAM‐1 expression is associated with the appearance of mucosal addressin cell adhesion molecule (MAdCAM‐1), with some slight differences in occurrence. The appearance of VCAM‐1 and MAdCAM‐1 antigens on FDCs may serve as indicators of FDC activation. Anat Rec 268:160–168, 2002. © 2002 Wiley‐Liss, Inc.     

滤泡树突状细胞与生发中心反应研究的新进展

生发中心是在T细胞依赖性抗体应答过程中于外周淋巴组织内形成的一个特殊的结构。在GC内,受抗原刺激而活化的B细胞进行克隆扩增、IgV区基因的体细胞高度突变、亲和力成熟以及同类型转换,最终形成记忆性B细胞或是产生Ig的浆细胞。在GC内B细胞增殖的同时,也启动了凋亡机制,以确保最终形成的记忆B细胞或浆细胞对抗原的高度特异性。FDCs是参与再次免疫应答的重要细胞,它主要是通过表面的FcR和CR将免疫复合物结合在细胞膜上,并选择性的将抗原递呈给表达高亲和力BCR的B细胞,使之激活并产生抗体或形成记忆B细胞。因此,FDCs在生发中心反应、免疫记忆的维持、B细胞的分化、成熟以及记忆B细胞的形成具有极其重要的作用。但最近的研究对FDCs及其结合的免疫复合物的重要性提出了质疑,认为FDCs在生发中心反应、B细胞的分化、成熟以及记忆B细胞的形成中的作用很可能是非特异性的,并对驻留在FDCs表面的免疫复合物的其它潜在功能进行了讨论。     

Protease-resistant prion protein in brain and lymphoid organs of sheep within a naturally scrapie-infected flock

The hallmark of transmissible spongiform encephalopathies (TSE), such as scrapie in sheep, is the accumulation in tissues of an insoluble and protease resistant form (PrPres) of the cellular prion protein. In this study, we evaluated whether the diversity in both the clinical pattern and the PrP genotypes of scrapied sheep from the same flock was connected with different levels and/or glycoform patterns of the PrPres in the brain and lymphoid organs of the animals. Whereas the PrPres levels in spleen, lymph nodes and tonsils from sheep of different PrP genotypes and clinical status appeared comparable, they were highly variable in brain, particularly in the brain stem and the cerebellum. PrPres was only detected in sheep bearing at least one VRQ allele, including three asymptomatic sheep and the highest PrPres load was found in the cerebellum of VRQ/VRQ animals. All together, levels of PrPres in brain did not necessarily correlate with the severity of the clinical disease but might depend on the PrP genotype of the animals. Different brain regions from a given sheep displayed a similar glycopattern of PrPres, whereas the apparent molecular sizes of the unglycosylated and diglycosylated forms of the protein differed between brain and lymphoid tissues. We did not find any notifiable differences in the glycopattern of PrPres in brain from sheep of different PrP genotypes or different clinical status and this PrPres glycotype was also similar to that found in brain from four cattle BSE.     

Differential expression of Ca(2+)-binding proteins on follicular dendritic cells in non-neoplastic and neoplastic lymphoid follicles.

We studied the Ca(2+)-capture ability of follicular dendritic cells (FDCs) in tonsillar secondary lymphoid follicles (LFs) and the expression of six Ca(2+)-binding proteins (CBPs), caldesmon, S-100 protein, calcineurin, calbindin-D, calmodulin, and annexin VI in LFs of various lymphoid tissues and caldesmon and S-100 protein in neoplastic follicles of follicular lymphomas. First, Ca(2+)-capture cytochemistry revealed extensive Ca(2+) capture in the nuclei and cytoplasm of FDCs, but little or none in follicular lymphocytes. All six CBPs were localized immunohistochemically in the LFs and were always present in the basal light zone. Immunoelectron microscopic staining of FDCs was classified into two patterns: caldesmon was distributed in the peripheral cytoplasm like a belt; S-100 protein, calcineurin, calbindin-D, and calmodulin were distributed diffusely in the cytosol. Annexin VI was, however, negative on FDCs. Immunocytochemistry also demonstrated CBP-positive FDCs within FDC-associated clusters isolated from germinal centers. In situ hybridization revealed diffuse calmodulin mRNA expression throughout the secondary LFs. These data indicate that the CBPs examined may regulate Ca(2+) in the different subcellular sites of FDCs, and the roles of CBPs may be heterogeneous. We also investigated the distribution of caldesmon and S-100 protein in follicular lymphomas on paraffin-embedded tissue sections. FDCs within grades I and II neoplastic follicles clearly expressed caldesmon, but not S-100 protein, except a part of grade II neoplastic follicles. FDCs within grade III follicles showed no caldesmon, but frequently expressed S-100 protein. These results demonstrate that the caldesmon and S-100 protein staining patterns of grade I follicular lymphomas are different from those of grade III follicular lymphomas and suggest that FDC networks in grade I neoplastic follicles may be similar to those in the light zone within non-neoplastic follicles, FDC networks in grade III neoplastic follicles may be similar to those in dark and basal light zones within non-neoplastic follicles, and grade II follicles may be intermediate between grade I and grade III follicles.     

Human follicular dendritic cells: function,origin and development

Follicular dendritic cells (FDCs) have important functions in the selection of memory B lymphocytes during germinal center reactions (GCR). They present native antigens to potential memory cells, of which only B cells with high affinity B cell receptors (BCR) can bind. These B lymphocytes survive, whereas nonbinding B cells undergo apoptotic cell death. FDCs are present in follicles of any secondary lymphoid organ and belong to the stromal cells of these organs. Ectopic FDC-formation can be found in a number of autoimmune diseases and/or chronic inflammatory situations. This indicates that the development of FDCs is not restricted to secondary lymphoid organs, but that it is rather a matter of local conditions that drives a precursor cell type into FDC-maturation. A precursor of FDCs has presently not been identified, but phenotypic marker studies, in vitro experiments with fibroblast-like cell lines, and recent data on mesenchymal precursor cells from the peripheral blood suggest a close relation to fibroblast-like cells.     

Isolated lymphoid follicle maturation induces the development of follicular dendritic cells

Isolated lymphoid follicles (ILFs) are recently identified lymphoid structures in the small intestine with features similar to Peyer's patches (PPs). Using immunohistochemistry we characterized the composition of ILFs in the small intestines of immunocompetent mice and of mice that lacked PPs as a result of either genetic deficiency of lymphotoxin or temporary in utero lymphotoxin-beta receptor-signalling blockade. We showed that although both immature and mature ILFs were present in the intestines of immunocompetent mice, PP-deficiency induced a significantly greater number of mature ILFs. We found that in addition to B-lymphocyte-containing germinal centres, mature ILFs also possessed large networks of follicular dendritic cells (FDCs). These features were not detected within immature ILFs. Indeed, the presence of FDCs could be used to reliably distinguish ILF maturity. Further analysis revealed that the area occupied by the FDCs within mature ILFs was substantial. The total area occupied by FDCs in all the mature ILFs in mice lacking PPs was equivalent to the total area occupied by FDCs in all the PPs and the few mature ILFs in immunocompetent mice. Based on these data we reasoned that in the absence of PPs, mature ILFs are important inductive sites for intestinal immune responses. Indeed, in mice that lacked PPs, ILF maturation coincided with a restoration of faecal immunoglobulin A levels to values that were comparable to those found in immunocompetent mice. Taken together, these data imply that the induction of germinal centres and FDC networks within mature ILFs in response to PP deficiency provides an important compensatory mechanism.     

Expression of CD137 (4-1BB) on human follicular dendritic cells

Follicular dendritic cells (FDCs) are the antigen (Ag)-trapping accessory cells of the germinal centres (GCs), essential for the development of humoral immune responses and memory. FDCs reside in the microenvironment of secondary lymphoid tissue where Ag-activated B cells expand, and undergo isotype switching and affinity maturation prior to becoming memory B cells. In addition to delivering Ag, FDCs also provide potent nonspecific accessory signals to the B cells, which are important for the GC reaction. In this report, we show that human tonsilar FDCs express the costimulatory molecule CD137. Surface expression of CD137 on FDCs was confirmed by immunofluorescent labelling and fluorescence-activated cell sorter analysis. CD137 was also highly expressed by the human cell line HK, which displays many characteristics of in vivo FDCs. The interaction between B cells and FDCs is essential for the GC reactions, and our finding suggests that CD137 plays a role in FDC-regulated B-cell responses.     

Metal microcrystal pollutants; the heat resistant,transmissible nucleating agents that initiate the pathogenesis of TSEs?

This paper exposes the flaws in the conventional consensus on the origins of transmissible spongiform encephalopathies (TSEs) which decrees that the protein-only misfolded 'prion' represents the primary aetiological transmissible agent, and then reviews/presents the emerging data which indicates that environmental exposure to metal microcrystal pollutants (sourced from munitions, etc.) represents the heat resistant, transmissible nucleating agents which seed the metal-prion protein (PrP)-ferritin fibril crystals that cause TSE. Fresh analytical data is presented on the levels of metals in ecosystems which support populations affected by clusters of variant Creutzfeldt-Jacob disease (vCJD), sporadic/familial CJD, and the scrapie types of TSE that have emerged in the UK, Sicily, Sardinia, Calabria and Japan. This data further substantiates the abnormal geochemical template (e.g., elevated strontium (Sr), barium (Ba) and silver (Ag)) which was observed as a common hallmark of the TSE cluster ecosystems across North America, thereby supporting the hypothesis that these microcrystals serve as the piezoelectrion nucleators which seed the growth/multireplication of the aberrant metal-PrP-ferritin fibril features which characterise the neuropathology of the TSE diseased brain. A secondary pathogenic mechanism entails the inactivation of the sulphated proteoglycans which normally regulate the mineralisation process. This can be induced by a rogue metal mediated chelation of free sulphur, or by contamination with organo-sulphur pollutants that substitute at natural sulphur bonds, or via a mutation to the S-proteoglycan cell line; thereby enabling the aberrant overgrowth of rogue fibril crystal formations that possess a piezoelectric capacity which compromises the ability of the contaminated individual to process incoming acoustic/tactile pressure waves in the normal way. The crystals transduce incoming sonic energy into electrical energy, which, in turn, generates magnetic fields on the crystal surfaces that initiate chain reactions of free radical mediated spongiform neurodegeneration. Metal microcrystal nucleating agents provide a group of plausible aetiological candidates that explain the unique properties of the TSE causal agent - such as heat resistance, transmissibility, etc. - which the protein-only prion model fails to fulfill. This paper also discusses the possible nutritional measures that could best be adopted by populations living in high risk TSE ecosystems; as a means of preventing the successful implantation of these rogue microcrystals and their consequent hypermineralisation of the soft tissues within the CNS.     

Identification of CXCL13 as a new marker for follicular dendritic cell sarcoma

The homeostatic chemokine CXCL13 is preferentially produced in B-follicles and is crucial in the lymphoid organ development by attracting B-lymphocytes that express its selective receptor CXCR5. Follicular dendritic cells (FDCs) have been identified as the main cellular source of this chemokine in lymphoid organs. Recently, genome-wide approaches have suggested follicular CD4 T-helper cells (T(H)F) as additional CXCL13 producers in the germinal centre and the neoplastic counterpart of T(H)F (CD4+ tumour T-cells in angioimmunoblastic T-cell lymphoma) retains the capability of producing this chemokine. In contrast, no data are available on CXCL13 expression on FDC sarcoma (FDC-S) cells. By using multiple approaches, we investigated the expression of CXCL13 at mRNA and protein level in reactive and neoplastic FDCs. In reactive lymph nodes and tonsils, CXCL13 protein is mainly expressed by a subset of FDCs in B-cell follicles. CXCL13 is maintained during FDC transformation, since both dysplastic FDCs from 13 cases of Castleman's disease and neoplastic FDCs from ten cases of FDC-S strongly and diffusely express this chemokine. This observation was confirmed at mRNA level by using RT-PCR and in situ hybridization. Of note, no CXCL13 reactivity was observed in a cohort of epithelial and mesenchymal neoplasms potentially mimicking FDC-S. FDC-S are commonly associated with a dense intratumoural inflammatory infiltrate and immunohistochemistry showed that these lymphocytes express the CXCL13 receptor CXCR5 and are mainly of mantle zone B-cell derivation (IgD+ and TCL1+). In conclusion, this study demonstrates that CXCL13 is produced by dysplastic and neoplastic FDCs and can be instrumental in recruiting intratumoural CXCR5+ lymphocytes. In addition to the potential biological relevance of this expression, the use of reagents directed against CXCL13 can be useful to properly identify the origin of spindle cell and epithelioid neoplasms.     

Follicle lysis in HIV-free lymphoid tissues

Follicle lysis (FL), the 'moth-eaten' degeneration of follicular dendritic cells (FDCs), previously regarded as specific for HIV-infected lymph follicles, was later claimed to exist in HIV-free patients. This study was performed to clarify the differences between HIV-free and HIV-induced FL. HIV-free and HIV-infected lymphoid tissues were analyzed immunohistologically. Most of HIV-free FLs were shown to lack the dark zones, the areas in germinal center composed mostly of much larger and clumped noncleaved cells, whereas HIV-induced FL in persistent generalized lymphadenopathy (PGL) lymph nodes included highly proliferative dark zones. Thus, FL in HIV-free lymphoid organs was suggested to be a physiological degenerative process with poor proliferative activity, while FL in PGL nodes includes two processes, FDC degeneration and B lymphocyte proliferation.     

Orally Administered Prion Protein Is Incorporated by M Cells and Spreads into Lymphoid Tissues with Macrophages in Prion Protein Knockout Mice

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases. Infection by the oral route is assumed to be important, although its pathogenesis is not understood. Using prion protein (PrP) knockout mice, we investigated the sequence of events during the invasion of orally administered PrPs through the intestinal mucosa and the spread into lymphoid tissues and the peripheral nervous system. Orally administered PrPs were incorporated by intestinal epitheliocytes in the follicle-associated epithelium and villi within 1 hour. PrP-positive cells accumulated in the subfollicle region of Peyer''s patches a few hours thereafter. PrP-positive cells spread toward the mesenteric lymph nodes and spleen after the accumulation of PrPs in the Peyer''s patches. The number of PrP molecules in the mesenteric lymph nodes and spleen peaked at 2 days and 6 days after inoculation, respectively. The epitheliocytes in the follicle-associated epithelium incorporating PrPs were annexin V-positive microfold cells and PrP-positive cells in Peyer''s patches and spleen were CD11b-positive and CD14-positive macrophages. Additionally, PrP-positive cells in Peyer''s patches and spleen were detected in the vicinity of peripheral nerve fibers in the early stages of infection. These results indicate that orally delivered PrPs were incorporated by microfold cells promptly after challenge and that macrophages might act as a transporter of incorporated PrPs from the Peyer''s patches to other lymphoid tissues and the peripheral nervous system.Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative diseases that infect humans and both wild and domestic animals. They include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle.¹ The common neuropathological features within the central nervous system (CNS) of TSEs are seen as a spongiform pathology, neuronal loss,² glial activation,³ and the accumulation of an abnormal and protease-resistant conformer of the scrapie-associated prion proteins (PrP-res or PrP^Sc),⁴ which are closely associated with the infection.⁵It has been reported that variant CJD in humans is most likely to have occurred because of the transmission of BSE after the consumption of beef contaminated with the BSE agent.⁶ Therefore, the oral route of TSE infection is widely assumed to be important under natural conditions. Many of the infectious agents accumulate in the gut-associated lymphoid tissues (GALT) after oral infection, such as the Peyer''s patches and mesenteric lymph nodes (MLN) before spreading to the CNS.⁷ It is necessary for the infectious agents to cross the intestinal epithelium before they can accumulate in the GALT. In addition, there are microfold cells (M cells) within the follicle-associated epithelium (FAE) that are specialized for the transepithelial transport of macromolecules and particles.⁸ One in vitro study has demonstrated that M cells actively transcytose the scrapie agents into the basolateral side of the epithelium.⁹ However, it is still a matter of controversy as to whether M cells may be involved in the in vivo transport of the infectious agents across the intestinal epithelium. After alimentary uptake of the infectious agents, they accumulate in the GALT and the lymphoreticular systems (eg, the spleen and other peripheral lymph nodes) long before they are detected in the CNS.¹⁰ As the GALT and the lymphoreticular systems are highly innervated, they are believed to be important sites for the infectious agents to gain contact with the nervous system (ie, neuroinvasion).¹¹ Once neuroinvasion occurs, the infectious agents reach their initial CNS target sites by spreading in a retrograde direction along efferent nerve fibers.¹²In the lymphoid tissues, it is believed that the macrophages, dendritic cells (DCs), and follicular dendritic cells (FDCs) are involved in the transportation and replication of the infectious agents. Macrophages are prevalent candidates for both spread¹³ and clearance¹⁴ of the infectious agents. DCs can capture and retain protein antigens in a nondegraded state.^15,16 These characteristics suggest that the macrophages and DCs may act as a transporter of the infectious agents from the gut to lymphoid tissues. FDCs express high levels of cellular PrPs (PrP^c), and therefore an early accumulation of PrP^Sc is seen in them.^17,18 Many studies of the alimentary pathogenesis of TSEs have been conducted to elucidate how infectious agents spread from the GALT to the CNS, although this has not been clearly determined yet. Therefore, the aim of the present study was to reveal the cells involved in the early stages of the pathogenesis of oral TSE infection, such as the sites of entry, spread, and neuroinvasion.