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Immune cell types involved in early uptake and transport of recombinant mouse prion protein in Peyer’s patches of calves
Abstract
We have previously reported the early uptake and transport of foreign particles into Peyer's patches (PPs) of newborn and 2-month-old calves and shown that the peak uptake of particles occurs 6 h after inoculation, in addition to site- and size-related effects on particle uptake. We now report the distribution of immune cells within PPs of the distal ileum in newborn and 2-month-old calves inoculated with carbon black. The types of immune cells involved in the early uptake and transport of recombinant mouse prion protein (rMPrP) within PPs of newborn calf were investigated by using monoclonal antibodies CD11c, CD14, CD68, CD172a, and CD21. CD11c(+), CD14(+), CD68(+), CD172a(+), and CD21(+) immune cells were widely distributed in four tissue compartments (villi, dome, interfollicular region, and follicles) of PPs in the distal ileum of newborn and 2-month-old calves, whereas CD11c(+), CD14(+), CD172a(+), and CD21(+) immune cells were more prominently distributed in the dome areas of newborn calves than in 2-month-old calves. Moreover, CD11c(+) and CD14(+) dendritic cells, CD172a(+) and CD68(+) macrophages, and CD21(+) follicular dendritic cells containing rMPrP were primarily observed in the dome and inner follicular regions. The deposition of rMPrP within CD11c(+), CD14(+), CD172a(+), and CD68(+) cells, but not CD21(+) cells, was detected in villous regions. rMPrP-positive immune cells within the interfollicular regions included only CD11c(+) and CD172(+) cells. Although the particles used in this investigation do not include the infectious prion protein, PrP(Sc), our experimental setup provides a useful model for studying immune cells involved in the early uptake and transport of PrP(Sc).
... In the current study, epithelial CD11c+ and CD172a+ cells in NPALT were more commonly found in FAE than in non-FAE. CD11c and CD172a are markers of bovine dendritic cells and macrophages (Brooke et al, 1998;Miyazawa et al, 2006;Lwin et al, 2009). Previous studies of bovine pharyngeal and palatine tonsillar epithelium revealed a similar distribution of CD11c+ cells (Palmer et al, 2009(Palmer et al, , 2011. ...
... Substantial immunolabelling of CD11c and CD172a has previously been reported in the lymphoid follicles of ileal Peyer's patches of 2month-old calves (Lwin et al, 2009), which was not observed in these analyses of NPALT. The ileal Peyer's patch is a primary lymphoid organ in cattle and other ruminants, and is thus responsible for Bcell development and maturation (Yasuda et al, 2006a). ...
The mammalian nasopharynx is an anatomically complex region of the upper respiratory tract that directly communicates with the nasal cavity, laryngopharynx, oesophagus and trachea. The nasopharyngeal mucosa contains moderate quantities of mucosa-associated lymphoid tissue (MALT) that is appropriately located for immunological sampling but also creates vulnerability to pathogens. In recent years, the nasopharynx has been inculpated in the pathogenesis of important diseases of cattle (foot-and-mouth disease) and humans (COVID-19), yet the tissue has never been described in detail in any species. In order to characterize the morphology and cellular composition of the bovine nasopharynx, samples of mucosa were collected from the nasopharynx of five 8–13-month-old steers and examined using light microscopy, immunohistochemistry and multichannel immunofluorescence. Morphologically, the nasopharyngeal epithelium was highly heterogeneous, with a continuum ranging from stratified squamous epithelium to highly attenuated, follicle-associated epithelium (FAE). Distribution of MALT was similarly regionally variable ranging from absent to clusters of multiple lymphoid follicles. Phenotypic characterization demonstrated dense distributions of dendritic cells and T lymphocytes surrounding lymphoid follicles, which comprised mostly B lymphocytes. The FAE overlaying the lymphoid follicles also contained higher numbers of dendritic cells and lymphocytes compared with the adjacent non-lymphoid epithelium, although cytotoxic T cells were notably scarce in the FAE. The bovine nasopharyngeal lymphoid tissue had comparable elements to other MALTs with specific differences that may help to elucidate the pathogenesis of infectious agents that have specific tropism for this tissue.
... Together these data suggest that MNPs constitute the most clinically relevant target innate immune cell population both within and without the CNS during prion pathogenesis. C-X-C chemokine receptor type 4 Cxcr4 / Cmkar4, Lestr, Sdf1r, CD184 P70658 M [103] C-X-C chemokine receptor type 5 Cxcr5 / Blr1, Gpcr6, CD185 Q04683 L [109] EGF-like module-containing mucin-like hormone receptor-like 1 Emr1 / GpF4/80 Q61549 E [119] Eotaxin Ccl11 / Syca11 P48298 Forkhead box protein P3 Foxp3 Q99JB6 [120] Formyl peptide receptor-related sequence 1 Fpr-s1 / Fpr2, Fpr3, Fprl1, Lxa4r O08790 [121] Galectin-3 Lgals3 / MAC-2 P16110 E [106] Galectin-3-binding protein Lgals3bp / Cycap, Mama Q07797 E [99,101] Granulins Grn / PCDGF P28798 E [99,117,122] Granulocyte-macrophage colonystimulating factor Csf2 / GM-CSF P01587 M [105] Granzyme B Gzmb / Ctla1 P04187 Growth-regulated alpha protein Cxcl1 / Gro, Gro1, Mgsa, Scyb1 P12850 E [105] Guanylate-binding protein 4 Gbp4 / Gbp3 Q61107 M [99] H-2 class I histocompatibility antigen, D-37 alpha chain H2-T23 P06339 M [99] H-2 class I histocompatibility antigen, D-D alpha chain H2-D1 P01900 M [99] H-2 class I histocompatibility antigen, K-B alpha chain, H2-K1 Q7TN03 E [99] Transforming growth factor beta-1 Tgfb1 / TGFβ1 P04202 M [108,130, 146,147] Triggering receptor expressed on myeloid cells 2 Trem2 Q99NH8 M [98,99] Tumor necrosis factor Tnfa / TNFα P06804 E [106,108,130131132 Tumor necrosis factor ligand superfamily member 9 Tnfsf9 / Cd137l, Cd157l, Ly63l P41274 E Tumor necrosis factor receptor superfamily member 1A Tnfr1 / CD120a P25118 E [130,148,149] Tumor necrosis factor receptor superfamily member 1B Tnfr2 / CD120b P25119 E [130] Tumor necrosis factor receptor superfamily member 9 Tnfrsf9 / Ly63, CD137 P20334 E Tumor necrosis factor ligand superfamily member 11 Tnfsf11 / RANKL, CD254 O35235 E [29] TYRO protein tyrosine kinase-binding protein Tyrobp / DAP12, CD300d O54885 E [98,99,101] Tyrosine-protein phosphatase nonreceptor type substrate 1 Sirpa / Sirpα1, CD172a P97797 E [115] a ...
Prion diseases or transmissible spongiform encephalopathies are a unique category of infectious protein-misfolding neurodegenerative disorders. Hypothesized to be caused by misfolding of the cellular prion protein these disorders possess an infectious quality that thrives in immune-competent hosts. While much has been discovered about the routing and critical components involved in the peripheral pathogenesis of these agents there are still many aspects to be discovered. Research into this area has been extensive as it represents a major target for therapeutic intervention within this group of diseases. The main focus of pathological damage in these diseases occurs within the central nervous system. Cells of the innate immune system have been proven to be critical players in the initial pathogenesis of prion disease, and may have a role in the pathological progression of disease. Understanding how prions interact with the host innate immune system may provide us with natural pathways and mechanisms to combat these diseases prior to their neuroinvasive stage. We present here a review of the current knowledge regarding the role of the innate immune system in prion pathogenesis.
... In some cases, they are believed to carry the prion protein to the nervous system, and in others, they may function to slow down the infection by destroying the prion protein. 19 In any case, diseased macrophages are unpredictable. Macrophages are the most affected cells in adult patients with Gaucher disease. ...
Mutant GBA was found recently to be the most prevalent risk factor for familial parkinsonism. The two diseases do not share common symptoms and there is no direct pathway to explain the mechanism by which GBA mutations can confer the risk. Increased burden on the degradative pathway caused by defective glucocerebrosidase, or toxic side effects of glycosylated lipids accumulation were proposed to explain brain damage. Both hypotheses are not sufficient to explain the linkage. In order to develop a more inclusive theory we introduced into the model the prion theory and the second hit. Other possibilities are also brought into consideration.
After oral challenge of the pathological prion protein, the pathogen was first detected in the distal ileum and then deposited in the brain. The present study aims determining the possible neuronal transport pathways from the small intestine to the brain in the cattle and sheep using a tracer protein. After horseradish peroxidase was injected into the wall in the duodenum of the calf and lamb and in the ileum of the lamb, the greater part of labeled neurons was detected in the celiac and cranial mesenteric ganglion complex. In the dorsal root ganglia T3 to L4 of both animals, some sensory neurons were always found to be labeled. Some parasympathetic preganglionic neurons were labeled in the dorsal motor nucleus of the vagus nerve after injections into the duodenum of the cattle and sheep, but extremely a small number of them were labeled after ileal application. The number of labeled sensory neurons in the nodose ganglion after duodenal injections of the sheep was much greater than that after duodenal application of the cattle. After ileal injections in the sheep, practically no labeled sensory neurons were found in the nodose ganglion. These results suggest that the pathological prion protein is mainly transported to the spinal cord and brain via the sympathetic nervous system and partially via the sensory neurons in the dorsal root ganglia. The vagus nerve seems to contribute to the transport of the pathogen not from the ileum, but from the duodenum.
Newborn calves lack a mature immune system. The immune system develops with age, but the role of the expression of cytokine receptors in the development of immune cells of Peyer's patches (PPs) in the intestines of calves in the first 2 months has not yet been elucidated. In this study, the distribution of immune cells and the expression of interleukin (IL) receptors (R) in the ileal PPs of newborn and 2-month-old calves were investigated immunohistochemically with monoclonal antibodies against bovine CD4, CD8, IgM, γδTCR, T19, WC3, WC5, and WC6 antigens. The expression of ILRs was examined with antibodies against CD25 (IL-2Rα), IL-2Rγ, IL-4R, IL-6R, IL-10R, and IL-13R antigens. CD4(+), CD8(+), γδTCR(+), T19(+), and WC6(+) cells were found to be more widely distributed in the ileal PPs of 2-month-old calves than in those of newborn calves. Moreover, the expression of CD25 (IL-2Rα), IL-4R, and IL-13R in the ileal PPs of 2-month-old calves was more prominent than that in newborn calves. These data suggest that the immune system of calves at 2 months of age is developed by reactions to foreign antigens and aging.
Penetration of the gut mucosa by pathogens expressing invasion genes is believed to occur mainly through specialized epithelial cells, called M cells, that are located in Peyer's patches. However, Salmonella typhimurium that are deficient in invasion genes encoded by Salmonella pathogenicity island 1 (SPI1) are still able to reach the spleen after oral administration. This suggests the existence of an alternative route for bacterial invasion, one that is independent of M cells. We report here a new mechanism for bacterial uptake in the mucosa tissues that is mediated by dendritic cells (DCs). DCs open the tight junctions between epithelial cells, send dendrites outside the epithelium and directly sample bacteria. In addition, because DCs express tight-junction proteins such as occludin, claudin 1 and zonula occludens 1, the integrity of the epithelial barrier is preserved.
Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrP(Sc) accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 microg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.
Ligated ileal loops of calves were inoculated with live and heat-killed Mycobacterium paratuberculosis and were examined by light and electron microscopy. At 5 hours after inoculation, acid-fast bacilli were in subepithelial macrophages, but not in M cells covering domes. At 20 hours, more than 50 acid-fast bacilli per cross section were in subepithelial macrophages in domes. Both living and heat-killed bacilli passed into domes. Addition of anti-M. paratuberculosis bovine serum to the inoculum enhanced entry of bacteria into domes. By electron microscopy, intact bacilli with electron-transparent zones (peribacillary spaces) were in the supranuclear cytoplasm of M cells at 20 hours. M cells also contained vacuoles, including electron-dense material interpreted as degraded bacilli. Subepithelial and intraepithelial macrophages contained bacilli and degraded bacterial material in phagosomes. These results suggest that calf ileal M cells take up bacilli, and that subepithelial and intraepithelial macrophages secondarily accept bacilli or bacterial debris which are expelled from M cells.
Significant differences were found between the follicle-associated epithelial cells (FAE) covering the domes of the jejunal and ileal Peyer's patches (PP) in calves. Whereas the FAE of the jejunal PP had scattered membraneous (M) cells among absorptive epithelial cells, the FAE of the ileal PP constituted a homogeneous population of cells different from both absorptive cells and M cells. The FAE of the ileal PP had short microvilli or folds, cytoplasmic vesicles and vacuoles containing acid phosphatase. Fifty nanometer membrane-bounded particles were found in lacunae extending from the lateral cell border. These particles appeared to have been shed from the FAE. Finger-like projections, rich in microfilaments, extended into the lacunae. M cells had short, sparse microvilli, many vesicles, few lysosomal structures, and they enfolded groups of mononuclear leucocytes. Transcytosis of macromolecular tracers occurred in both M cells and in the FAE of the ileal PP, though the uptake was greater in the latter. The 50 nm particles were also found between cells in the lymphoid tissue underneath the FAE of the ileal PP. The FAE of PP in pre- and post-natal lambs from 115 days of gestation and onwards, and goat kids, showed similar structural features.
Despite the fact that the Peyer's patch (PP) is the primary site for antigen uptake in the intestine, the cellular basis of antigen handling after transport into the PP is poorly understood. We performed immunohistology of murine PPs using the dendritic cell (DC)-reactive monoclonal antibodies N418, NLDC-145, M342, and 2A1, as well as antibodies to other T cell, B cell, and macrophage markers. N418+, 2A1+, NLDC-145-, M342- cells form a dense layer of cells in the subepithelial dome (SED), just beneath the follicle epithelium, and are scattered throughout the follicle, sparing the germinal center. In contrast, N418+, 2A1+, NLDC-145+, and M342+ DCs are present in the interfollicular T cell regions (IFR). CD3+ and CD4+, but no CD8+ T cells were present in the SED and the follicle, including the germinal center, while CD3+, CD4+, and CD8+ T cells were present in the IFR. B cells and macrophages were poorly represented in the SED as no B220+ cells, only few Mac-1lo cells, and no F4/80+ cells were present at this site. In contrast, Mac-1hi cells were found in the IFR and lamina propria of intestinal villi, while F4/80+ cells were found only in the latter. In further phenotypic studies, we analyzed surface molecules of PP and spleen DCs by flow cytometry and found that these cells had similar fluorescence profiles when stained with N418, NLDC-145, and 33D1 DC-reactive antibodies, and antibodies to the costimulatory molecules B7-1 (1G10) and B7-2 (GL1). In contrast, PP DCs expressed 5-10-fold higher levels of major histocompatibility complex class II antigens (IEk) than spleen DCs. Finally, in functional studies, we demonstrated that both PP and spleen DCs process soluble protein antigens during overnight culture and induce similar levels of proliferation in CD3+ T cells, and CD4+/Mel 14hi T cells from T cell receptor transgenic mice. The in vivo relevance of such presentation was shown by the fact that PP DCs isolated from Balb/c mice after being fed ovalbumin stimulated proliferation in ovalbumin T cell receptor T cells. Taken together, our data suggest that DCs in the SED of the PP are uniquely positioned for the processing of antigens passed into the PP from the overlying M cell, and that PP DCs are effective at processing and presenting oral antigens to naive T cells.
Dendritic cells (DC) are thought to initiate Ab synthesis by activation of T cells, which then provide cytokine and cell-bound "help" to B cells. Here, we provide evidence that DC can capture and retain unprocessed Ag in vitro and in vivo, and can transfer this Ag to naive B cells to initiate a specific Ab response. The response is skewed with 4- to 13-fold higher titers of IgG than IgM, and the predominant subclasses of Ab produced in naive animals are those associated with Th2-type responses. Ag retention and the skew in class switching is a physiologic phenomenon because DC loaded with Ag in vivo and isolated 24 h later initiated a class-switched, Ag-specific Ab response in naive animals. In vitro studies confirmed that DC provide naive B cells with signals that are essential for the synthesis of class-switched Ab. Taken together, these observations show that DC have an important role in the initiation of Ab synthesis by direct interaction with B cells.
We describe the anatomical localization of three distinct dendritic cell (DC) subsets in the murine Peyer's patch (PP) and explore the role of chemokines in their recruitment. By two-color in situ immunofluorescence, CD11b(+) myeloid DCs were determined to be present in the subepithelial dome (SED) region, whereas CD8alpha(+) lymphoid DCs are present in the T cell-rich interfollicular region (IFR). DCs that lack expression of CD8alpha or CD11b (double negative) are present in both the SED and IFR. By in situ hybridization, macrophage inflammatory protein (MIP)-3alpha mRNA was dramatically expressed only by the follicle-associated epithelium overlying the SED, while its receptor, CCR6, was concentrated in the SED. In contrast, CCR7 was expressed predominantly in the IFR. Consistent with these findings, reverse transcriptase polymerase chain reaction analysis and in vitro chemotaxis assays using freshly isolated DCs revealed that CCR6 was functionally expressed only by DC subsets present in the SED, while all subsets expressed functional CCR7. Moreover, none of the splenic DC subsets migrated toward MIP-3alpha. These data support a distinct role for MIP-3alpha/CCR6 in recruitment of CD11b(+) DCs toward the mucosal surfaces and for MIP-3beta/CCR7 in attraction of CD8alpha(+) DCs to the T cell regions. Finally, we demonstrated that all DC subsets expressed an immature phenotype when freshly isolated and maintained expression of subset markers upon maturation in vitro. In contrast, CCR7 expression by myeloid PP DCs was enhanced with maturation in vitro. In addition, this subset disappeared from the SED and appeared in the IFR after microbial stimulation in vivo, suggesting that immature myeloid SED DCs capture antigens and migrate to IFR to initiate T cell responses after mucosal microbial infections.
Although the ultimate target of infection is the central nervous system (CNS), there is evidence that the enteric nervous system (ENS) and the peripheral nervous system (PNS) are involved in the pathogenesis of orally communicated transmissible spongiform encephalopathies. In several peripherally challenged rodent models of scrapie, spread of infectious agent to the brain and spinal cord shows a pattern consistent with propagation along nerves supplying the viscera. We used immunocytochemistry (ICC) and paraffin-embedded tissue (PET) blotting to identify the location and temporal sequence of pathological accumulation of a host protein, PrP, in the CNS, PNS, and ENS of hamsters orally infected with the 263K scrapie strain. Enteric ganglia and components of splanchnic and vagus nerve circuitry were examined along with the brain and spinal cord. Bioassays were carried out with selected PNS constituents. Deposition of pathological PrP detected by ICC was consistent with immunostaining of a partially protease-resistant form of PrP (PrP(Sc)) in PET blots. PrP(Sc) could be observed from approximately one-third of the way through the incubation period in enteric ganglia and autonomic ganglia of splanchnic or vagus circuitry prior to sensory ganglia. PrP(Sc) accumulated, in a defined temporal sequence, in sites that accurately reflected known autonomic and sensory relays. Scrapie agent infectivity was present in the PNS at low or moderate levels. The data suggest that, in this scrapie model, the infectious agent primarily uses synaptically linked autonomic ganglia and efferent fibers of the vagus and splanchnic nerves to invade initial target sites in the brain and spinal cord.
Bovine spongiform encephalopathy, variant Creutzfeldt-Jakob disease (vCJD) and possibly also sheep scrapie are orally acquired transmissible spongiform encephalopathies (TSEs). TSE agents usually replicate in lymphoid tissues before they spread into the central nervous system. In mouse TSE models PrP(c)-expressing follicular dendritic cells (FDCs) resident in lymphoid germinal centres are essential for replication, and in their absence neuroinvasion is impaired. Disease-associated forms of PrP (PrP(Sc)), a biochemical marker for TSE infection, also accumulate on FDCs in the lymphoid tissues of patients with vCJD and sheep with natural scrapie. TSE transport mechanisms between gut lumen and germinal centres are unknown. Migratory bone marrow-derived dendritic cells (DCs), entering the intestinal wall from blood, sample antigens from the gut lumen and carry them to mesenteric lymph nodes. Here we show that DCs acquire PrP(Sc) in vitro, and transport intestinally administered PrP(Sc) directly into lymphoid tissues in vivo. These studies suggest that DCs are a cellular bridge between the gut lumen and the lymphoid TSE replicative machinery.
Variant Creutzfeldt-Jakob disease and scrapie are typically initiated by extracerebral exposure to prions, and exhibit early prion accumulation in germinal centers. Follicular dendritic cells (FDCs), whose development and maintenance in germinal centers depends on tumor necrosis factor (TNF) and lymphotoxin (LT) signaling, are thought to be indispensable for extraneural prion pathogenesis. Here, we administered prions intraperitoneally to mice deficient for TNF and LT signaling components. LT alpha(-/-), LT beta(-/-), LT betaR(-/-), and LT alpha(-/-) x TNFalpha(-/-) mice resisted infection and contained no infectivity in spleens and lymph nodes (when present). However, TNFR1(-/-), TNFR2(-/-), and some TNFalpha(-/-) mice developed scrapie similarly to wild-type mice. High prion titers were detected in lymph nodes, but not spleens, of TNFR1(-/-) and TNF alpha(-/-) mice despite absence of FDCs and germinal centers. Transfer of TNFR1(-/-) fetal liver cells into lethally irradiated Prnp(0/0) mice restored infectivity mainly in lymph nodes. Prion protein (PrP) colocalized with a minority of macrophages in tumor necrosis factor receptor (TNFR) 1(-/-) lymph nodes. Therefore, prion pathogenesis can be restricted to lymphoreticular subcompartments, and mature follicular dendritic cells are dispensable for this process. Macrophage subsets are plausible candidates for lymphoreticular prion pathogenesis and neuroinvasion in the absence of FDCs, and may represent a novel target for postexposure prophylaxis.
To better understand the role of dendritic cells (DCs) in human immunodeficiency virus (HIV) transmission at mucosal surfaces, we examined the expressions of the HIV adhesion molecule, dendritic-cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN), its closely related homologue DC-SIGNR, and HIV coreceptors by distinct DC populations in the intestinal and genital tracts of humans and rhesus macaques. We also developed monoclonal antibodies (MAbs) specific for DC-SIGN or DC-SIGNR. In the Peyer's patches, DC-SIGN expression was detected in the interfollicular regions and in clusters of cells in the subepithelial dome regions. DC-SIGN expression was not found on plasmacytoid DCs. DC-SIGNR expression was restricted to endothelial cells in approximately one-third of the capillaries in the terminal ileum. In the vaginal epithelium, Langerhans' cells did not express DC-SIGN, whereas subepithelial DCs in the lamina propria expressed moderate levels of DC-SIGN. Finally, the rectum contained cells that expressed high levels of DC-SIGN throughout the entire thickness of the mucosa, while solitary lymphoid nodules within the rectum showed very little staining for DC-SIGN. Triple-color analysis of rectal tissue indicated that CCR5(+) CD4(+) DC-SIGN(+) DCs were localized just beneath the luminal epithelium. These findings suggest that DC-SIGN(+) DCs could play a role in the transmission of primate lentiviruses in the ileum and the rectum whereas accessibility to DC-SIGN(+) cells is limited in an intact vaginal mucosa. Finally, we identified a MAb that blocked simian immunodeficiency virus interactions with rhesus macaque DC-SIGN. This and other specific MAbs may be used to assess the relevance of DC-SIGN in virus transmission in vivo.
Transmissible spongiform encephalopathy or prion diseases are fatal neurodegenerative disorders of humans and animals often
initiated by oral intake of an infectious agent. Current evidence suggests that infection occurs initially in the lymphoid
tissues and subsequently in the central nervous system (CNS). The identity of infected lymphoid cells remains controversial,
but recent studies point to the involvement of both follicular dendritic cells (FDC) and CD11c+ lymphoid dendritic cells. FDC generation and maintenance in germinal centers is dependent on lymphotoxin alpha (LT-α) and
LT-β signaling components. We report here that by the oral route, LT-α −/− mice developed scrapie while LT-β −/− mice did
not. Furthermore, LT-α −/− mice had a higher incidence and shorter incubation period for developing disease following inoculation
than did LT-β −/− mice. Transplantation of lymphoid tissues from LT-β −/− mice, which have cervical and mesenteric lymph nodes,
into LT-α −/− mice, which do not, did not alter the incidence of CNS scrapie. In other studies, a virus that is tropic for
and alters functions of CD11c+ cells did not alter the kinetics of neuroinvasion of scrapie. Our results suggest that neither FDC nor CD11c+ cells are essential for neuroinvasion after high doses of RML scrapie. Further, it is possible that an as yet unidentified
cell found more abundantly in LT-α −/− than in LT-β −/− mice may assist in the amplification of scrapie infection in the periphery
and favor susceptibility to CNS disease following peripheral routes of infection.
Natural sheep scrapie is a prion disease characterized by the accumulation of PrP(Sc) in brain and lymphoid tissues. Previous studies suggested that lymph node macrophages and follicular dendritic cells (FDC) accumulate PrP(Sc). In this study, lymph nodes were analyzed for the presence of PrP(Sc) and macrophage or FDC markers using dual immunohistochemistry. A monoclonal antibody (mAb) to the C-terminus of PrP reacted with CD172a+ macrophages and CD21+ FDC processes in secondary follicles. However, a PrP N-terminus-specific mAb reacted with CD21+ FDC processes but not CD172a+ macrophages in secondary follicles. Neither the PrP N-terminus nor C-terminus-specific mAb reacted with CD172a+ macrophages in the medulla. These results indicate that lymph node follicular macrophages acquire PrP(Sc) by phagocytosis of CD21+ FDC processes. The results also suggest that follicular macrophages have proteases that process full-length PrP(Sc) to N-terminally truncated PrP(Sc).
The concept that circulating dendritic cells mediate neuroinvasion in transmissible spongiform encephalopathies received strong
support from recent observations that prion protein is expressed in myeloid dendritic cells. We observed that prion protein
fragment 106-126 is a chemoattractant for monocyte-derived immature but not mature dendritic cells. Signaling events in chemotaxis
involved enzymes downstream of Gq protein and were inhibited by blockade of sphingosine kinase, suggesting transactivation of sphingosine-1-phosphate-dependent
cell motility by prion protein.
The immunohistochemical localisation of the disease-specific protein, PrP(Sc), was examined in the distal ileum of cattle up to 40 months after they had been exposed orally to the agent of bovine spongiform encephalopathy (BSE), in the intestines and mesenteric lymph nodes of an additional group of cattle, killed six months after a similar exposure, and in the distal ileum of naturally occurring clinical cases of BSE. PrP(Sc) was detected, mainly in macrophages, in a small proportion of the follicles of Peyer's patches in the distal ileum of the experimentally exposed cattle throughout much of the course of the disease. The observations are in agreement with the infectivity data derived from mouse bioassays of the distal ileum. At the later stages of the disease, the proportion of immunostained follicles increased as the total number of follicles decreased with age. In the additional experimental group of cattle, PrP(Sc) was confined to the Peyer's patches in the distal ileum. No immunostaining was detected in the lymphoid tissue of the distal ileum of naturally occurring clinical cases of BSE. In some of the clinically affected experimentally induced and naturally occurring cases of BSE there was sparse immunostaining of the neurons of the distal ileal myenteric plexus.
The control of damaging inflammation by the mucosal immune system in response to commensal and harmful ingested bacteria is unknown. Here we show epithelial cells conditioned mucosal dendritic cells through the constitutive release of thymic stromal lymphopoietin and other mediators, resulting in the induction of 'noninflammatory' dendritic cells. Epithelial cell-conditioned dendritic cells released interleukins 10 and 6 but not interleukin 12, and they promoted the polarization of T cells toward a 'classical' noninflammatory T helper type 2 response, even after exposure to a T helper type 1-inducing pathogen. This control of immune responses seemed to be lost in patients with Crohn disease. Thus, the intimate interplay between intestinal epithelial cells and dendritic cells may help to maintain gut immune homeostasis.
Three 80- to 95-month-old Holstein dairy cattle infected naturally with the agent of bovine spongiform encephalopathy (BSE) and slaughtered at abattoirs in Japan were examined for the distribution of disease-specific and protease-resistant prion protein (PrP(Sc)) by immunohistochemistry (IHC) and Western blot (WB) analyses. The cattle showed no clinical signs or symptoms relevant to BSE but were screened as positive by enzyme-linked immunosorbent assay, a rapid test for BSE. This positive result was confirmed by IHC or WB in a specimen of the medulla oblongata. Histopathologically, these cattle showed no vacuolation in tissue sections from the central nervous system except for the medulla oblongata. Both IHC and WB analyses revealed PrP(Sc) accumulation in the brain, spinal cord, satellite and ganglionic cells of the dorsal root ganglia, and the myenteric plexus of the distal ileum. In addition, small amounts of PrP(Sc) were detected in the peripheral nerves of 2 cattle by WB. No PrP(Sc) was demonstrated by either method in the Peyer's patches of the distal ileum; lymphoid tissues including the palatine tonsils, lymph nodes, and spleen; or other tissues. The distribution of PrP(Sc) accumulation in the preclinical stage was different between naturally infected cattle and cattle inoculated experimentally with the BSE agent.
Dendritic cells (DC) are suspected to be involved in transmissible spongiform encephalopathies, including bovine spongiform encephalopathy (BSE). We detected the disease-specific, protease-resistant prion protein (PrP(bse)) in splenic DC purified by magnetic cell sorting 45 days after intraperitoneal inoculation of BSE prions in immunocompetent mice. We showed that bone marrow-derived DC (BMDC) from wild-type or PrP-null mice acquired both PrP(bse) and prion infectivity within 2 h of in vitro culture with a BSE inoculum. BMDC cleared PrP(bse) within 2 to 3 days of culture, while BMDC infectivity was only 10-fold diminished between days 1 and 6 of culture, suggesting that the infectious unit in BMDC is not removed at the same rate as PrP(bse) is removed from these cells. Bone marrow-derived plasmacytoid DC and bone marrow-derived macrophages (BMM) also acquired and degraded PrP(bse) when incubated with a BSE inoculum, with kinetics very similar to those of BMDC. PrP(bse) capture is probably specific to antigen-presenting cells since no uptake of PrP(bse) was observed when splenic B or T lymphocytes were incubated with a BSE inoculum in vitro. Lipopolysaccharide activation of BMDC or BMM prior to BSE infection resulted in an accelerated breakdown of PrP(bse). Injected by the intraperitoneal route, BMDC were not infectious for alymphoid recombination-activated gene 2(0)/common cytokine gamma chain-deficient mice, suggesting that these cells are not capable of directly propagating BSE infectivity to nerve endings.
Controversial results have been observed in mouse models regarding the role of lymphoid tissues in prion pathogenesis. To
investigate the role of dendritic cells (DC), we used a transgenic mouse model. In this model (CD11c-N17Rac1), a significant
reduction of CD8+ CD11chi DC has been described, and the remaining CD8+ DC demonstrate a reduced capacity for the uptake of apoptotic cells. After intraperitoneal prion infection, significantly
longer incubation times were observed in CD11c-N17Rac1 mice than in controls, indicating that a defect in CD8+ CD11chi DC significantly impedes neuroinvasion after intraperitoneal infection. In contrast, no distinct differences were observed
between CD11c-N17Rac1 mice and controls after oral infection. This provides evidence that oral and intraperitoneal prion infections
differ in lymphoreticular requirements.
The presence of BSE prion infectivity in asymptomatic cattle and its tissue distribution are important concerns for both human and veterinary health and food safety. In this work, a collection of tissues from asymptomatic cattle challenged orally with BSE and culled at 20, 24, 27, 30 and 33 months have been used to inoculate intracerebrally BoPrP-Tg110 mice expressing bovine PrP to assess their infectivity. Results demonstrate that BSE infectivity in asymptomatic cattle is essentially restricted to the nervous system, Peyer's patches and tonsils, as reported previously for terminally BSE-diseased cattle. BSE infectivity was detectable in Peyer's patches and tonsils at all time points analysed, but infectivity in nervous tissues (brainstem and sciatic nerve) was only detectable after 27 months from inoculation. Infectivity in brainstem increased markedly at 33 months after inoculation. All other investigated tissues or fluids (spleen, skeletal muscle, blood and urine) revealed no detectable infectivity throughout the time course studied.
Natural sheep scrapie is a prion disease characterized by the accumulation of PrPSc in brain and lymphoid tissues. Previous studies suggested that lymph node macrophages and follicular dendritic cells (FDC) accumulate PrPSc. In this study, lymph nodes were analyzed for the presence of PrPSc and macrophage or FDC markers using dual immunohistochemistry. A monoclonal antibody (mAb) to the C-terminus of PrP reacted with CD172a+ macrophages and CD21+ FDC processes in secondary follicles. However, a PrP N-terminus-specific mAb reacted with CD21+ FDC processes but not CD172a+ macrophages in secondary follicles. Neither the PrP N-terminus nor C-terminus-specific mAb reacted with CD172a+ macrophages in the medulla. These results indicate that lymph node follicular macrophages acquire PrPSc by phagocytosis of CD21+ FDC processes. The results also suggest that follicular macrophages have proteases that process full-length PrPSc to N-terminally truncated PrPSc.
The use of biodegradable particles as oral delivery vehicles for macromolecular drugs was investigated. We evaluated the binding, uptake and absorption of poly-dl-lactide (PLA) micro- and nanoparticles in Caco-2 monolayers and in ileal tissue and gut associated lymphoid tissue (GALT) of anaesthetised rats and rabbits. Using a range of experimental techniques, we found that approximately 10% of administered micro- and nanoparticles were adsorbed to the apical membranes of each of the five intestinal models. Nanoparticles were found to be absorbed better than microparticles. Overall, little discrimination in uptake patterns was evident between Peyer's patch (PP) and non-PP tissue while rat ileum showed a greater uptake capacity than rabbit. Our results show that uptake of PLA particles was low capacity, size-dependent and predominantly transcellular in all systems. A low proportion of the apically-bound particles was absorbed, with uptake exclusion evident for particles >4μm. The affinity of PLA particles for intestinal epithelia and GALT needs to be greatly enhanced in order to achieve improved oral bioavailability of macromolecules.
Dendritic cells (DCs) play a key role in activating and orientating immune responses. Little is currently known about DC recruitment during Cryptosporidium parvum infection. In the intestine, epithelial cells act as sensors, providing the first signals in response to infection by enteric pathogens. We analyzed the contribution of these cells to the recruitment of DCs during cryptosporidiosis. We found that intestinal epithelial cells produced a broad range of DC-attracting chemokines in vitro in response to C. parvum infection. The supernatant of the infected cells induced the migration of both bone marrow-derived DCs (BMDC) and the SRDC lymphoid dendritic cell line. Chemokine neutralization abolished DC migration in these assays. We next analyzed chemokine mRNA expression in the mucosa of C. parvum-infected neonatal mice and recruitment of the various subsets of DCs. Myeloid (CD11c+ CD11b+) and double-negative DCs (CD11c+ CD11b- CD8alpha-) were the main subsets recruited in the ileum during C. parvum infection, via a mechanism involving IFNgamma. DCs were also recruited and activated in the draining lymph nodes during C. parvum infection, as shown by the upregulation of expression of MHC II and of the costimulation molecules CD40 and CD86.
To investigate the uptake and transport patterns of variously sized particles in Peyer's patches (PPs) of calves, intestinal loops were created in four newborn and two 2-month-old calves, and the loops were inoculated with various particles, including carbon black, fluorescein isothiocyanate (FITC)-labeled latex, FITC-labeled dextran, bovine serum, and recombinant mouse prion protein (rMPrP). The intestinal loops were recovered at 3, 6, 9, and 24 h in newborn calves and at 24 h in 2-month-old calves after inoculation, and the transport of the particles was examined by histological and immunohistochemical means. The uptake of the particles was quantified by estimation of signal intensities. A greater intensity was found in newborn calves compared with the 2-month-old calves. The peak uptake of carbon black, FITC-labeled latex, and rMPrP in the PPs of the distal ileum occurred at 6 h after inoculation in newborn calves and then progressively decreased with time. Uptake was also dependent on the site within the small intestine and the size of the particle studied. The transport of carbon black, FITC-labeled latex, and FITC-labeled dextran occurred via the bloodstream, the mesenteric lymph nodes, and the liver of newborn calves. rMPrP was found primarily in the interfollicular regions of the submucosa of the distal ileum of newborn calves. Thus, distal ileal PPs are probably more effective at particle absorption than the jejunal PPs, and the peak uptake of the PPs within the newborn calf occurs at 6 h after inoculation.
A quantitative, light microscopic morphometric model for uptake of particulates by Peyer's patch M cells was developed. Rabbit intestinal loops containing Peyer's patches were inoculated with fluorescent, non-degradable polystyrene microparticles (600-750 nm), and their localization in Peyer's patches was traced after varying time periods. The particles were localized sequentially at the FAE cell surface, spanning the entire width of FAE cells, and within the subepithelial dome as a function of time. The particles were associated with 5D9+ or 1D9+ M cells, but were not taken up or transported by villus epithelia. The kinetics suggested a synchronous wave of uptake and transepithelial transport. Quantitative analysis revealed a considerably greater uptake efficiency of polystyrene microspheres in comparison to other biological particles.
A mAb EBM11, raised against human macrophages [M phi] was found to detect a bovine M phi diameter-subpopulation. The Ag was strictly intracellular and was expressed in M phi only at a certain state of maturation. Its expression was regulated independently of the activation state of the cells, as revealed by treating M phi in vitro with bovine rIFN-alpha I1, IFN-gamma, or TNF-alpha, all potent M luminal diameter activators. Such treatment had no apparent effect on Ag expression. The Ag was present in 1 to 5% of peripheral blood leukocytes, i.e., up to 20% of circulating blood monocytes, in both normal noninfected cattle and in cattle persistently infected with bovine viral diarrhoea virus. Blood monocytes of the latter group were activated in vivo, but apparently did not reach a more mature state than found in noninfected animals. After an acute infection with bovine herpes virus type 1, the frequency and total number of EBM11+ cells decreased dramatically in inverse relationship to an equally significant increase in frequency and total number of circulating monocytes. In cryostat sections of normal tissues, the EBM11 mAb reacted with sinus M phi and M phi in germinal centers of lymphoid tissues, with alveolar M phi and liver Kupffer cells. In skin it reacted with few scattered M phi in the dermis, but not with epidermal Langerhans cells. This latter feature distinguishes the bovine system from the human. In virus-induced inflammatory processes in skin and keratinized epithelia EBM11+ cells constituted a subpopulation of the infiltrating M phi. The data obtained suggest that EBM11 mAb could be useful both for the elucidation of differentiation/maturation pathways of cells of the monocyte-macrophage lineage as well as for studies of M phi-virus interactions in virus infections. In either aspect cattle could provide a useful comparative model.
Mature mixed breed ewes were used to study sites of maximum amino acid uptake and kinetic properties of neutral and basic amino acid transport systems in sheep small intestine. Uptake of 5 mM lysine increased with distance distal to the pylorus such that maximum uptake was obtained in the ileum. Distribution ratios (DR) (cpm per ml intracellular fluid/cpm per ml medium) for methionine and lysine, determined in an atmosphere of nitrogen or oxygen, indicated that intestinal transport was largely dependent on respiration derived energy. Methionine DR determined after 60 min of incubation under nitrogen suggested that the methionine system(s) had some ability to utilize energy derived from substrate phosphorylation. Kinetic constants (Km and Vmax) were determined for methionine, glycine and lysine transport. The relative order of affinity by the amino acids for the transport site(s) (based on Km data) was lysine > methionine > glycine. The relative order for rate of transport (based on Km and Vmax data) was methionine > lysine > glycine. These results are similar to data published for the rat small intestine. Competition for binding between leucine and lysine was observed in sheep intestine. Saturating concentrations of leucine decreased lysine uptake by approximately 50%.
Five groups of eight newborn calves were used to study absorption of colostral immunoglobulin G. One feeding of 2 liters of pooled colostrum was given at one of 6, 12, 24, 36, or 48 h after birth. Concentrations of immunoglobulin G in blood plasma and feces were measured by an immunodiffusion technique. Plasma volume and fecal excretion also were measured. When colostrum was given 6 h after birth, 65.8% of the ingested immunoglobulin G appeared in the plasma. This percentage declined rapidly to reach 46.9%, 11.5%, 6.7%, and 6.0% when colostrum was given at the ages of 12, 24, 36, and 48 h. Total fecal immunoglobulin G increased linearly with age. The quantities not recovered from plasma and feces reached a maximum when colostrum was given at 24 or 36 h after birth. Immunoglobulin G can be "lost" to a great extent via routes other than plasma and feces during this time. Quantities of immunoglobulin G measured in plasma represent apparent rather than true absorption.
The intestinal transit of large (micro-) particles to other sites of the body remains a controversial issue of relevance in various fields of study. In this report fluorescent polystyrene latex microparticles in the size range of 2 microns were used as models for nonspecifically absorbed nonbiodegradable particulates. They were administered to young adult rats as a single oral dose of 1.65 x 10(9) particles; Peyer's patches and surrounding normal absorptive small intestinal tissue were collected at various time points. Quantification of solubilized tissue samples and fluorescence (epi- and confocal) qualitative and quantitative microscopy showed uptake of latex microparticles in all parts of the intestine sampled, but with the proximal segment the preferential site of absorption. The maximum uptake of particles occurred 0.5 hr after dosing in all three segments of the small intestine; there were progressively smaller numbers with distance from the pylorus and with time. Translocation of small numbers of particles to the mesenteric lymph nodes was also detected at 0.5 hr. Transmucosal passage of particles occurred primarily in the villous tissues adjacent to the Peyer's patch regions. These studies give confirmatory evidence for the uptake and translocation of microparticulates across the mucosal barrier and provide new information regarding site- and time-related effects on particle uptake and the involvement of the villous epithelium in particle translocation.
M cell transport is an important factor in induction of mucosal immune responses and is exploited by pathogenic microorganisms for invasion of the intestinal mucosa. The specific molecular recognition systems and nonspecific adherence mechanisms that determine the efficiency of the M cell transport pathway are largely unknown. Future studies on the interactions of micoorganisms with this highly specialized epithelial cell will enhance our understanding of microbial pathogenesis and will lead to more effective strategies for targeting of vaccines and live microbial vaccine vectors to the mucosal immune system.
The present study examines the relationship between size and particle transit across the mucosal barrier of the gastrointestinal tract to other sites of the body. The extent of particle uptake with increasing size, the tissue distribution and cut-off points for 2-20 microns particles is investigated.
An established fluorescent latex particle-young adult rat model is used and particle numbers in small intestine and mesenteric lymph nodes, 0.5 h post administration, counted by fluorescence microscopy in bulk tissue specimens and cryosections.
Bulk tissue analysis provides evidence for the presence of particles of all sizes in the Peyer's patch regions, but only for 2 microns particles in the nodal tissues. Microscopy establishes uptake of both 2 and 6 microns particles in most intestinal and nodal tissue sites and compartments. By contrast, uptake of the larger particles is much reduced.
Although more of the smaller (2 microns) particles are taken up, particularly by epithelial tissues, the 6 microns size appears more efficient in terms of volume translocated to lymph nodes. This could have implications in the therapeutic use of particles for drug and vaccine delivery and for radiation safety.
Scrapie in sheep has recently become again a target of control measures and eradication programs. Crucial for the effectiveness of these measures is the detection of infected sheep during the long and potentially hazardous incubation period. However, routine-diagnosis is mostly limited to clinical examinations when disease becomes apparent, and to postmortem investigations. Through the detection of the scrapie-specific isoform of the prion protein (PrPSc) by Western blot in the spleen and lymph nodes from scrapie-infected mice and sheep, we have shown previously that diagnosis during the preclinical stage is possible. We introduce here an improved method for the diagnosis of mouse scrapie shortly after infection. Through a homogenization procedure that includes a collagenase digestion step, and through extraction and salting-out of PrPSc by Sarkosyl and NaCl, respectively, we were able to detect PrPSc in spleen tissue of intraperitoneally infected mice seven days postinfection. Moreover, the new protocol makes sample-handling easier and reduces the hands-on time. We also successfully enriched PrPSc from spleen tissue through immobilized metal affinity chromatography (IMAC); however, for the diagnosis at the earliest stage of infection, extraction of PrPSc by Sarkosyl and NaCl was more effective.
The involvement of spleen macrophages in the early stages of scrapie pathogenesis was studied by applying the 'macrophage-suicide technique' to scrapie-infected mice. This method comprises critically the intravenous administration to mice of dichloromethylene disphosphonate encapsulated into liposomes. Depletion of spleen macrophages before scrapie infection induced an increased amount of scrapie inoculum in the spleen, consequently leading to accelerated scrapie agent replication in the early phase of pathogenesis, as followed by PrPres accumulation, a specific hallmark of scrapie. The same effect was observed when spleen macrophages were depleted just before the beginning of scrapie agent replication. These findings suggest that macrophages may partly control scrapie infection in peripheral tissues by sequestration of the scrapie inoculum and may thus impair early scrapie agent replication in the spleen. In addition to macrophages, most follicular dendritic cells and B lymphocytes, which are thought to support scrapie agent replication, were also transiently depleted by dichloromethylene disphosphonate administration. This suggests that a compensatory mechanism is sufficient to ensure the persistence of infection in these early stages of pathogenesis.
In scrapie-infected mice, prions are found associated with splenic but not circulating B and T lymphocytes and in the stroma, which contains follicular dendritic cells (FDCs). Formation and maintenance of mature FDCs require the presence of B cells expressing membrane-bound lymphotoxin-alpha/beta. Treatment of mice with soluble lymphotoxin-beta receptor results in the disappearance of mature FDCs from the spleen. We show that this treatment abolishes splenic prion accumulation and retards neuroinvasion after intraperitoneal scrapie inoculation. These data provide evidence that FDCs are the principal sites for prion replication in the spleen.
Although the transmissible spongiform encephalopathies (TSEs) are neurological diseases, TSE agents usually replicate in lymphoid tissues long before infection spreads to the brain. Studies of a mouse scrapie TSE model have demonstrated that mature follicular dendritic cells (FDCs) expressing the host prion protein (PrPc) are essential for replication of infection in lymphoid tissues1 and subsequent spread of infection to the nervous system2. Abnormal forms of PrP (PrPSc) accumulate on FDCs in scrapie-infected mice2, 3, and in patients with variant Creutzfeldt-Jakob disease (ref. 4). Here we confirm that, as predicted, treatment that interferes with the integrity of FDCs also interferes with TSE pathogenesis.
The gastrointestinal uptake of micro- and nanoparticles has been the subject of recent efforts to develop effective carriers that enhance the oral uptake of drugs and vaccines. Here, we used correlative instrumental neutron activation analysis and electron microscopy to quantitatively and qualitatively study the gastrointestinal uptake and subsequent tissue/organ distribution of 4, 10, 28, and 58 nm diameter metallic colloidal gold particles following oral administration to mice. In our quantitative studies we found that colloidal gold uptake is dependent on particle size: smaller particles cross the gastrointestinal tract more readily. Electron microscopic studies showed that particle uptake occurred in the small intestine by persorption through single, degrading enterocytes in the process of being extruded from a villus. To our knowledge this is the first report, at the ultrastructural level, of gastrointestinal uptake of particulates by persorption through holes created by extruding enterocytes.
Transmissible spongiform encephalopathies (TSEs) are often acquired peripherally, for example by ingestion or iatrogenic exposure. After entry, TSE agents, as identified by disease-specific protein accumulation, usually accumulate on follicular dendritic cells (FDCs) in lymphoid tissues long before infection spreads to the brain. Neuroinvasion of TSE agents is significantly impaired in the absence of mature FDCs. Treatments that interfere with the integrity or function of FDCs extend survival time by blocking replication in lymphoid tissues and spread to the brain. The identification of FDCs as critical for TSE pathogenesis provides a cellular target to which therapies can be specifically directed.
In most documented infectious forms of transmissible spongiform encephalopathies, prions must transit through the lymphoreticular compartment before invading the central nervous system. A major goal has been to identify the cell susbsets that support replication and propagation of prions from sites of penetration to sites of neuroinvasion. The conclusions, still fragmentary and confusing, point at a few candidates: follicular dendritic cells (FDCs) and more recently, dendritic cells (DCs). It is clear, however, that lymphoinvasion does not depend on a single-cell type but needs a coordinated network of cells. Discrepancies between models suggest that the actors may vary according to prion strains. A second center of interest has emerged following reports that anti-prion protein (PrP) antibodies blocked in vitro cell conversion of normal PrP into pathological PrP and cured infected cell lines. As isoform conversion is a critical event in prion propagation and formation of lesions, the identification of immune agents capable of inhibiting the reaction is of major importance. In vivo experiments suggest that antibodies produced in transgenic mice or an ongoing immune reaction induced by peptides can prevent PrP conversion and retard disease progression. These results do not say whether clinical disease can be durably delayed and if immunological tolerance to PrP can be easily broken in infected individuals. Altogether, these results suggest that the unconventional relationship between prions and the immune system is on the eve of new and fascinating developments. Whether they will provide innovative strategies for early diagnosis and preventive treatments is still an open question.
We established a panel of monoclonal antibodies (mAbs) against prion protein (PrP) by immunizing PrP gene-ablated mice with the pathogenic isoform of prion protein (PrPSc) or recombinant prion protein (rPrP). The mAbs could be divided into at least 10 groups by fine epitope analyses using mutant rPrPs and pepspot analysis. Seven linear epitopes, lying within residues 56-90, 119-127, 137-143, 143-149, 147-151, 163-169, and 219-229, were defined by seven groups of mAbs, although the remaining three groups of mAbs recognized discontinuous epitopes. We attempted to examine whether any of these epitopes are located on the accessible surface of PrPSc. However, no mAbs reacted with protease-treated PrPSc purified from scrapie-affected mice, even when PrPSc was dispersed into a detergent-lipid protein complex, to reduce the size of PrPSc aggregates. In contrast, denaturation of PrPSc by guanidine hydrochloride efficiently exposed all of the epitopes. This suggests that any epitope recognized by this panel of mAbs is buried within the PrPSc aggregates. Alternatively, if the corresponding region(s) are on the surface of PrPSc, the region(s) may be folded into conformations to which the mAbs cannot bind. The reactivity of a panel of mAb also showed that the state of PrPSc aggregation influenced the denaturation process, and the sensitivity to denaturation appeared to vary between epitopes. Our results demonstrate that this new panel of well-characterized mAbs will be valuable for studying the biochemistry and biophysics of PrP molecules as well as for the immuno-diagnosis of prion diseases.
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The objective of this study was to evaluate the distribution and phagocytotic function of macrophages in bovine caruncles during pregnancy. Pregnant Holstein caruncles obtained at a slaughterhouse and by Caesarean section just before the onset of parturition were used. Macrophages in these caruncles were observed immunohistochemically and histochemically. Macrophages were present in the parenchyma of caruncles throughout pregnancy. The macrophages were irregular in shape, and exhibited thin, long processes along the main and intermediate septa of caruncles. Macrophages increased as the gestation period progressed. Acid phosphatase activity of the macrophages appeared at 5–6 months of gestation and remained until the end of gestation period. In particular, macrophages showed strong acid phosphatase activity at the peripheral region of the caruncles just facing the chorionic villi. In addition, they were conglomerated in the hyperplastic prominences of caruncles. These results indicate that caruncle macrophages maintain the appropriate size and shape of each caruncle during pregnancy.
An important aspect of the bovine spongiform encephalopathy (BSE) epidemic has been an apparent age-dependent risk of infection, with younger cattle being more likely to become infected than older cattle. Our objective was to determine the age-dependent risk of infection of dairy cattle. We first reviewed unpublished data on the feeding patterns of proprietary concentrates for dairy-replacement cattle. These data showed that autumn- and spring-born cattle would receive different feeding patterns of proprietary concentrates, and so age-dependent risk of infection profiles were obtained separately for autumn- and spring-born cattle. We used back-calculation methods to analyse BSE-epidemic data collected in Great Britain between 1984 and 1996. Dairy cattle were most at risk in the first 6 months of life; adult cattle were at relatively low risk of infection. Between 6 and 24 months of age, risk profiles reflected feeding patterns of proprietary concentrates in each of the autumn- and spring-born cohorts.
While there is a growing consensus on the understanding of the propagation pathways after oral infection of transmissible spongiform encephalopathy (TSE) agents and even if the central role of follicular dendritic cells is identified, little is known about the key players in the first steps of the infection and about the site of the disease development. We investigated the role of gut macrophages, which are capable of capturing aggregates of the prion protein. PLGA particles containing clodronate were designed in order to be orally administered and to target Peyer's patches for inducing gut-associated macrophages suicide in mice. Mice were subsequently infected with scrapie or BSE by the oral route. It was found that the efficacy of macrophage suppression in the Peyer's patches correlated well with an earlier appearance of PrPres in these formations and with a higher amount of PrPres at a later stage of the infection. Thus, the capture of infectious particles that have crossed the epithelial gut barrier and their elimination by macrophages seems to be a key event to restrict the amount of agent initiating the infection.
The comprehension of the immune system and the role of DC in the pathological diseases may contribute to their use in veterinary medicine in the prevention and treatment of many diseases. Currently, most dendritic cell (DC) research occurs in the human and murine model systems on the generation of cells from the bone marrow or peripheral blood mononuclear cells (PBMC) cultured in vitro. Despite the lack of available immunological reagents such as antibodies and cytokines, analogous cells have been generated and identified in many different species and reviewed in this study.
Dendritic cells (DCs) are professional antigen presenting cells, which initiate primary immune responses and also play an important role in the generation of peripheral tolerance. There is no reliable method established for the isolation of bovine peripheral blood DCs, and furthermore, the phenotypes and the functions of bovine DCs are still not fully clear. In the present study, we have attempted to identify bovine peripheral blood DCs by negative-selection. In bovine peripheral blood mononuclear cells (PBMC), we have newly characterized the phenotype of DCs, which is CD11c+/CD172a+. These cells display features of myeloid type DCs. In the thymic medulla, CD11c+/CD172a+ cells were also present and CD1+/CD172a+ cells were additionally detected as a population of DCs. The data suggest that one of the bovine DCs phenotypes from PBMC is derived from myeloid lineages lacking a CD1 molecule, which then drift to several tissues, and that they then may express a CD1 molecule upon their functional differentiation.
To determine the mechanisms of intestinal transport of infection, and early pathogenesis, of sheep scrapie, isolated gut-loops were inoculated to ensure that significant concentrations of scrapie agent would come into direct contact with the relevant ileal structures (epithelial, lymphoreticular, and nervous). Gut loops were inoculated with a scrapie brain pool homogenate or normal brain or sucrose solution. After surgery, animals were necropsied at time points ranging from 15 min to 1 month and at clinical end point. Inoculum-associated prion protein (PrP) was detected by immunohistochemistry in villous lacteals and in sub-mucosal lymphatics from 15 min to 3.5 h post-challenge. It was also detected in association with dendritic-like cells in the draining lymph nodes at up to 24 h post-challenge. Replication of infection, as demonstrated by the accumulation of disease-associated forms of PrP in Peyer's patches, was detected at 30 days and sheep developed clinical signs of scrapie at 18-22 months post-challenge. These results indicate discrepancies between the routes of transportation of PrP from the inoculum and sites of de novo-generated disease-associated PrP subsequent to scrapie agent replication. When samples of homogenized inoculum were incubated with alimentary tract fluids in vitro, only trace amounts of protease-resistant PrP could be detected by western blotting, suggesting that the majority of both normal and abnormal PrP within the inoculum is readily digested by alimentary fluids.
To elucidate the still-unknown pathogenesis of bovine spongiform encephalopathy (BSE), an oral BSE challenge and sequential kill study was carried out on 56 calves. Relevant tissues belonging to the peripheral and central nervous system, as well as to the lymphoreticular tract, from necropsied animals were analysed by highly sensitive immunohistochemistry and immunoblotting techniques to reveal the presence of BSE-associated pathological prion protein (PrPSc) depositions. Our results demonstrate two routes involving the autonomic nervous system through which BSE prions spread by anterograde pathways from the gastrointestinal tract (GIT) to the central nervous system (CNS): (i) via the coeliac and mesenteric ganglion complex, splanchnic nerves and the lumbal/caudal thoracic spinal cord (representing the sympathetic GIT innervation); and (ii) via the Nervus vagus (parasympathetic GIT innervation). The dorsal root ganglia seem to be subsequently affected, so it is likely that BSE prion invasion of the non-autonomic peripheral nervous system (e.g. sciatic nerve) is a secondary retrograde event following prion replication in the CNS. Moreover, BSE-associated PrPSc was already detected in the brainstem of an animal 24 months post-infection, which is 8 months earlier than reported previously. These findings are important for the understanding of BSE pathogenesis and for the development of new diagnostic strategies for this infectious disease.
During preclinical stages of cattle orally infected with bovine spongiform encephalopathy (BSE), the responsible agent is confined to ileal Peyer's patches (IPP), namely in nerve fibers and in lymph follicles, before reaching the peripheral and central nervous systems. No infectivity has been reported in other bovine lymphoid organs, including jejunal Peyer's patches (JPP). To determine the potential sites for prion neuroinvasion in IPP, we analyzed the mucosal innervation and the interface between nerve fibers and follicular dendritic cells (FDC), two dramatic influences on neuroinvasion. Bovine IPP were studied at three ages, viz., newborn calves, calves less than 12 months old, and bovines older than 24 months, and the parameters obtained were compared with those of JPP. No differences in innervation patterns between IPP and JPP were found. The major difference observed was that, in calves of less than 12 months, IPP were the major mucosal-associated lymphoid organ that possessed a large number of follicles with extended FDC networks. Using a panel of antibodies, we showed that PP in 24-month-old bovines were highly innervated at various strategic sites assumed to be involved in the invasion and replication of the BSE pathogen: the suprafollicular dome, T cell area, and germinal centers. In PP in calves of less than 12 months old, no nerve fibers positive for the neurofilament markers NF-L (70 kDa) and NF-H (200 kDa) were observed in contact with FDC. Thus, in view of the proportion of these protein subunits present in neurofilaments, the innervation of the germinal centers can be said to be an age-dependent dynamic process. This variation in innervation might influence the path of neuroinvasion and, thus, the susceptibility of bovines to the BSE agent.