Peyer's Patch Development Research Articles

Disruption of the early-life microbiota alters Peyer’s patch development and germinal center formation in gastrointestinal-associated lymphoid tissue

During postnatal development, both the maturing microbiome and the host immune system are susceptible to environmental perturbations such as antibiotic use. The impact of timing in which antibiotic exposure occurs was investigated by treating mice from days 5-9 with amoxicillin or azithromycin, two of the most commonly prescribed medications in children. Both early-life antibiotic regimens disrupted Peyer's patch development and immune cell abundance, with a sustained decrease in germinal center formation and diminished intestinal immunoglobulin A (IgA) production. These effects were less pronounced in adult mice. Through comparative analysis of microbial taxa, Bifidobacterium longum abundance was found to be associated with germinal center frequency. When re-introduced to antibiotic-exposed mice, B. longum partially rescued the immunological deficits. These findings suggest that early-life antibiotic use affects the development of intestinal IgA-producing B cell functions and that probiotic strains could be used to restore normal development after antibiotic exposure.

Flt3 Ligand Regulates the Development of Innate Lymphoid Cells in Fetal and Adult Mice.

Flt3 ligand (Flt3L) promotes survival of lymphoid progenitors in the bone marrow and differentiation of dendritic cells (DCs), but its role in regulating innate lymphoid cells (ILCs) during fetal and adult life is not understood. By using Flt3L knockout and transgenic mice, we demonstrate that Flt3L controls ILC numbers by regulating the pool of α4β7(-) and α4β7(+) lymphoid tissue inducer cell progenitors in the fetal liver and common lymphoid progenitors in the bone marrow. Deletion of flt3l severely reduced the number of fetal liver progenitors and lymphoid tissue inducer cells in the neonatal intestine, resulting in impaired development of Peyer's patches. In the adult intestine, NK cells and group 2 and 3 ILCs were severely reduced. This effect occurred independently of DCs as ILC numbers were normal in mice in which DCs were constitutively deleted. Finally, we could show that administration of Flt3L increased the number of NKp46(-) group 3 ILCs in wild-type and even in Il7(-/-) mice, which generally have reduced numbers of ILCs. Taken together, Flt3L significantly contributes to ILC and Peyer's patches development by targeting lymphoid progenitor cells during fetal and adult life.

Weaning Markedly Affects Transcriptome Profiles and Peyer's Patch Development in Piglet Ileum.

Transcriptome analyses were conducted on the ileal mucosa of 14- to 35-day-old piglets to investigate postnatal gut development during suckling and postweaning. The transcriptome profiles of 14-day-old suckling piglets showed a considerably higher number of differentially expressed genes than did those of 21-, 28-, and 35-day olds, indicating an intensive gut development during the first 14–21 postnatal days. In addition, the analysis of biological pathways indicated that Chemotaxis Leucocyte chemotaxis was the most significantly affected pathway in suckling piglets between 14 and 21 days of age. Weaning negatively affected pathways associated with acquired immunity, but positively affected those associated with innate immunity. Interestingly, pathway Chemotaxis Leucocyte chemotaxis was found positively affected when comparing 14- and 21-day-old suckling piglets, but negatively affected in 28-day-old piglets weaned at 21 days of age, when compared with 28-day-old suckling piglets. Genes CXCL13, SLA-DOA (MHC class II), ICAM1, VAV1, and VCAM1 were involved in the pathway Chemotaxis Leukocyte chemotaxis and they were found to significantly change between 14- and 21-day-old suckling piglets and between groups of suckling and weaned piglets. The expression of these genes significantly declined after weaning at 14, 21, and 28 days of age. This decline indicated that CXCL13, SLA-DOA, ICAM1, VAV1, and VCAM1 may be involved in the development of Peyer’s patches (PP) because lower gene expression clearly corresponded with smaller areas of PP in the ileal mucosa of piglets. Moreover, weaning piglets prior to a period of intensive gut development, i.e., 14 days of age, caused significant adverse effects on the size of PP, which were not reverted even 14 days postweaning.

Segmented Filamentous Bacterium Uses Secondary and Tertiary Lymphoid Tissues to Induce Gut IgA and Specific T Helper 17 Cell Responses

Segmented filamentous bacterium (SFB) is a symbiont that drives postnatal maturation of gut adaptive immune responses. In contrast to nonpathogenic E. coli, SFB stimulated vigorous development of Peyer's patches germinal centers but paradoxically induced only a low frequency of specific immunoglobulin A (IgA)-secreting cells with delayed accumulation of somatic mutations. Moreover, blocking Peyer's patch development abolished IgA responses to E. coli, but not to SFB. Indeed, SFB stimulated the postnatal development of isolated lymphoid follicles and tertiary lymphoid tissue, which substituted for Peyer's patches as inductive sites for intestinal IgA and SFB-specific T helper 17 (Th17) cell responses. Strikingly, in mice depleted of gut organized lymphoid tissue, SFB still induced a substantial but nonspecific intestinal Th17 cell response. These results demonstrate that SFB has the remarkable capacity to induce and stimulate multiple types of intestinal lymphoid tissues that cooperate to generate potent IgA and Th17 cell responses displaying only limited target specificity.

Mucosal Addressin Cell-Adhesion Molecule-1 Controls Plasma-Cell Migration and Function in the Small Intestine of Mice

Immunoglobulin (Ig) A secretion into the intestinal lumen is an important immune mechanism of the gastrointestinal (GI) tract. B cells proliferate and differentiate into IgA-secreting plasma cells (PC) within lymphoid organs then migrate directly into the intestinal lamina propria. We aimed to elucidate the in vivo role of the mucosal addressin cell-adhesion molecule-1 (MAdCAM-1), which is constitutively expressed in the GI-associated lymphoid tissue, in B-cell migration. We generated MAdCAM-1-deficient mice (MAdCAM(Delta)) and evaluated the B-cell compartment of the GI-associated lymphoid tissue. We also assessed PC migration to the small intestine and the intestinal immune response after oral immunization. In MAdCAM(Delta) mice, the size of Peyer's patches was drastically reduced, compared with that of wild-type mice; this difference was detectable by 3 days after birth, indicating that MAdCAM-1 is dispensable for embryonic Peyer's patch development but mediates recruitment of lymphocytes into this lymphoid organ at later stages. Moreover, antigen-specific, IgA-positive PC were severely compromised in their migration to the small intestine; accordingly, there was a reduced number of IgA-secreting PC in the lamina propria of the small intestine. The MAdCAM(Delta) mice were unable to mount a normal intestinal IgA response after oral immunization with cholera toxin. These data provide in vivo evidence that MAdCAM-1 is required for the localization and function of IgA-secreting PC in the intestine.

The Critical Role of Iκbα Dependent Nuclear Export of NF-κb in B-Cell Development.

Activity of the nuclear factor-κB (NF-κB) family of transcription factors is tightly regulated by its inhibitor, IκBα, through cytoplasmic localization of latent NF-κB: IκBα complexes. This arrangement is essential for efficient signal-inducible activation and regulation of biologic functions. Maintenance of cytoplasmic localization of latent NF-κB: IκBα complex requires continuous nuclear export that is dependent on the N-terminal nuclear export sequence (N-NES) of IκBα. While these mechanisms have been elucidated through in vitro studies, the biological significance of this “nucleocytoplasmic shuttling” has yet to be evaluated in vivo. To address this, we derived mice harboring germ-line M45A, L48A, and I52A amino acid substitutions in the N-NES of IκBα. In splenic B-cells, the disrupted N-NES caused constitutive nuclear accumulation of IκBα and inactive c-Rel containing complexes but surprisingly not IκBα: p65 complexes. Since p65 contains a NES sequence and c-Rel does not, nuclear export of N-NES mutant IκBα:NF-κB complexes appear to be NF-κB family member dependent. Functionally, NF-κB activity in splenic B-cells after stimulation with IgM or LPS was clearly reduced in the mutants compared to wild-type by electrophoretic mobility shift assay. B-cell development in the bone marrow of mice harboring the mutation was impaired, showing a preponderance of pro/pre B-cells and few mature B-cells compared to their wild type littermates (p < 0.001). Concordantly, there were significantly fewer B-cells in the spleen (p < 0.05) and lymph nodes (p < 0.01) of the mutant mice. Additionally, populations of T2, follicular (FO), and marginal zone (MZ) B-cells, which represent mature B-cells in the spleen, were also reduced in the mutant mice (p < 0.001). To demonstrate that this B-cell maturation defect in IκBα mutant mice was B-cell intrinsic, sublethally irradiated Jak3-deficient mice were transplanted with BM from either wild-type or mutant mice. B-cell development in mice transplanted with mutant donors was impaired relative to those with wild-type donors in a fashion identical to that of the primary mutants described above. Finally, severe phenotypes in inguinal lymph nodes and Peyer's patch development were present, with mutant mice frequently lacking these secondary organs/tissues, the underlying mechanisms of which are currently being investigated. In conclusion, our findings uncover an in vivo mechanism controlling NF-κB localization and its essential role in the generation of mature B-cells and certain secondary lymphoid organs.

Detection and quantification of IgM+ lymphocytes in fetal lamb spleen, liver and lymph nodes by flow cytometry

The first stage in Peyer's patch development in the fetal lamb is characterized by the colonization of the rudimentary Peyer's patches by precursor cells expressing the IgM surface receptor. In the fetal lamb, the spleen has been implicated as the source of gene-rearranged IgM(+) B lymphocytes. This study was intended to quantitate IgM(+) lymphocytes in the spleen, lymph nodes and liver of fetal lambs at various gestational ages between 63 and 110 days using flow cytometry. Flow-cytometric analysis revealed that IgM(+) lymphocytes were rare in the liver being consistently less than 1% at every gestational age examined. IgM(+) lymphocytes were detected in the spleen (mean 9.18%) and prescapular lymph nodes (mean 11.89%) as early as 63 days. In both spleen and lymph nodes, the highest representation of IgM(+) lymphocytes occurred between 70 and 86 days gestation. The highest mean percentage of IgM(+) lymphocytes was observed in the spleen (22.63%) and lymph nodes (17.02%) at 75 days gestation. From 98 days onwards, B-lymphocyte density gradually decreased in both spleen and prescapular lymph nodes. This study indicates that substantial populations of IgM(+) lymphocytes were present in both the spleen and prescapular lymph nodes from 70 days gestation and implies that both of these locations could be potential sources for the normal colonization of the ileal Peyer's patches.

Tyrosine kinase receptor RET is a key regulator of Peyer’s Patch organogenesis

Normal organogenesis requires co-ordinate development and interaction of multiple cell types, and is seemingly governed by tissue specific factors. Lymphoid organogenesis during embryonic life is dependent on molecules the temporal expression of which is tightly regulated. During this process, haematopoietic 'inducer' cells interact with stromal 'organizer' cells, giving rise to the lymphoid organ primordia. Here we show that the haematopoietic cells in the gut exhibit a random pattern of motility before aggregation into the primordia of Peyer's patches, a major component of the gut-associated lymphoid tissue. We further show that a CD45+CD4-CD3-Il7Ralpha-c-Kit+CD11c+ haematopoietic population expressing lymphotoxin has an important role in the formation of Peyer's patches. A subset of these cells expresses the receptor tyrosine kinase RET, which is essential for mammalian enteric nervous system formation. We demonstrate that RET signalling is also crucial for Peyer's patch formation. Functional genetic analysis revealed that Gfra3-deficiency results in impairment of Peyer's patch development, suggesting that the signalling axis RET/GFRalpha3/ARTN is involved in this process. To support this hypothesis, we show that the RET ligand ARTN is a strong attractant of gut haematopoietic cells, inducing the formation of ectopic Peyer's patch-like structures. Our work strongly suggests that the RET signalling pathway, by regulating the development of both the nervous and lymphoid system in the gut, has a key role in the molecular mechanisms that orchestrate intestine organogenesis.

Mammalian Polycomb complexes are required for Peyer's patch development by regulating lymphoid cell proliferation

The vertebrate Polycomb Group (PcG) genes encode proteins that form large multimeric and chromatin-associated complexes implicated in the stable repression of developmentally essential genes. Rnf110 and Phc2 are shown to be components of mammalian PcG multimeric complexes in HeLa cells. Here we report defects in Peyer's patch (PP) development in Rnf110 mutant mice, which is synergically exaggerated by Phc2 mutation. PP development involves a series of inductive interactions and subsequent differentiation and proliferation between lymphoid and mesenchymal cells in late gestational stage. Rnf110 and Phc2 mutations impair development of PP anlagen by affecting proliferation of lymphoid lineage cells populated in PP anlagen in gene-dosage dependent manner. We suggest that PcG complexes may act to mediate certain inductive signals maybe through IL-7Rα to allow sufficient proliferation of lymphoid inducer cells during PP organogenesis.

Comparative evidence for a link between Peyer's patch development and susceptibility to transmissible spongiform encephalopathies

BackgroundEpidemiological analyses indicate that the age distribution of natural cases of transmissible spongiform encephalopathies (TSEs) reflect age-related risk of infection, however, the underlying mechanisms remain poorly understood. Using a comparative approach, we tested the hypothesis that, there is a significant correlation between risk of infection for scrapie, bovine spongiform encephalopathy (BSE) and variant CJD (vCJD), and the development of lymphoid tissue in the gut.MethodsUsing anatomical data and estimates of risk of infection in mathematical models (which included results from previously published studies) for sheep, cattle and humans, we calculated the Spearman's rank correlation coefficient, rs, between available measures of Peyer's patch (PP) development and the estimated risk of infection for an individual of the corresponding age.ResultsThere was a significant correlation between the measures of PP development and the estimated risk of TSE infection; the two age-related distributions peaked in the same age groups. This result was obtained for each of the three host species: for sheep, surface area of ileal PP tissue vs risk of infection, rs = 0.913 (n = 19, P < 0.001), and lymphoid follicle density vs risk of infection, rs = 0.933 (n = 19, P < 0.001); for cattle, weight of PP tissue vs risk of infection, rs = 0.693 (n = 94, P < 0.001); and for humans, number of PPs vs risk of infection, rs = 0.384 (n = 46, P = 0.008). In addition, when changes in exposure associated with BSE-contaminated meat were accounted for, the two age-related patterns for humans remained concordant: rs = 0.360 (n = 46, P = 0.014).ConclusionOur findings suggest that, for sheep, cattle and humans alike there is an association between PP development (or a correlate of PP development) and susceptibility to natural TSE infection. This association may explain changes in susceptibility with host age, and differences in the age-susceptibility relationship between host species.

Inducible lymphoid tissues in the adult gut: recapitulation of a fetal developmental pathway?

The intestinal immune system faces an extraordinary challenge from the large numbers of commensal bacteria and potential pathogens that are restrained by only a single layer of epithelial cells. Here, I discuss evidence that the intestinal immune system develops an extensive network of inducible, reversible lymphoid tissues that contributes to the vital equilibrium between the gut and the bacterial flora. I propose that this network is induced by cryptopatches, which are small clusters of dendritic cells and lymphoid cells that are identical to fetal inducers of lymph-node and Peyer's-patch development.

NALT-genesis is induced by Id2 gene-dependent CD3 −CD4 +CD45 + cells

CD3 −CD4 +CD45 + cells are thought to be an inducer cell population for Peyer's patch (PP) and lymph node development. Nasopharyngeal-associated lymphoid tissue (NALT) is an organized lymphoid structure found on both sides of the nasopharyngeal duct dorsal to the cartilaginous soft palate. Although NALT is considered to be functionally analogous to PP in the intestinal tract, the association of CD3 −CD4 +CD45 + cells with the initiation of NALT development has not yet been clarified. To examine this issue further, we isolated CD3 −CD4 +CD45 + cells from the fetal intestines of wild-type mice. Id2 −/− mice genetically deficient in NALT were adoptively transferred with purified CD3 −CD4 +CD45 + cells or fetal liver cells. After the adoptive transfer, we employed immunohistochemical analysis to examine the expression of PNAd, an important addressin for the recruitment of lymphocytes, in the nasal tissue of Id2 −/− mice transferred with fetal liver cells. Though NALT-like structures were indeed induced in Id2 −/− mice adoptively transferred with CD3 −CD4 +CD45 + cells or fetal liver cells, those structures were not observed to express PNAd. These findings indicate that CD3 −CD4 +CD45 + cells and Id2 genes are key to the initiation of NALT. However, they also suggest that an additional element is required for the induction of PNAd.

RelB is required for Peyer's patch development: differential regulation of p52-RelB by lymphotoxin and TNF.

Targeted disruption of the Rel/NF-kappaB family members NF-kappaB2, encoding p100/p52, and RelB in mice results in anatomical defects of secondary lymphoid tissues. Here, we report that development of Peyer's patch (PP)-organizing centers is impaired in both NF-kappaB2- and RelB-deficient animals. IL-7-induced expression of lymphotoxin (LT) in intestinal cells, a crucial step in PP development, is not impaired in RelB-deficient embryos. LTbeta receptor (LTbetaR)-deficient mice also lack PPs, and we demonstrate that LTbetaR signaling induces p52-RelB and classical p50-RelA heterodimers, while tumor necrosis factor (TNF) activates only RelA. LTbetaR-induced binding of p52-RelB requires the degradation of the inhibitory p52 precursor, p100, which is mediated by the NF-kappaB-inducing kinase (NIK) and the IkappaB kinase (IKK) complex subunit IKKalpha, but not IKKbeta or IKKgamma. Activation of RelA requires all three IKK subunits, but is independent of NIK. Finally, we show that TNF increases p100 levels, resulting in the specific inhibition of RelB DNA binding via the C-terminus of p100. Our data indicate an important role of p52-RelB heterodimers in lymphoid organ development downstream of LTbetaR, NIK and IKKalpha.

Development of Peyer's patches, follicle-associated epithelium and M cell: Lessons from immunodeficient and knockout mice

Studies with immunodeficient and knockout mice have re- vealed that the development of mucosa-associated lymphoid tissues (MALT) and peripheral lymphoid nodes share com- mon mechanisms, but also require distinct signals. Gene disruption of lymphotoxins or their cognate receptors affects both Peyer's patch and lymph node organogenesis. Disrup- tion of the osteoprotegerin TNF-family member gene does not impair Peyer's patch development, but prevents formation of peripheral lymph nodes. Peyer's patch do not form in mice with a deleted gene encoding a B lymphocyte-specific chemokine receptor, while most peripheral lymph nodes, ex- cept inguinal, are normal in numbers and architecture. In B or T lymphocyte-deficient mice, Peyer's patches, with their overlying follicle-associated epithelium (FAE), are present although reduced in number and size. No Peyer's patches develop in RAG deficient mice. Formation of FAE with typical M cells has not been analyzed in these mice. M cell formation requires the close association of immune cells with differentiated enterocytes and their conversion appears to be transcriptionally regulated. The development of MALT, FAE and probably M cells is a multistep process that requires signalling pathways common to all secondary lymphoid tissues, but also MALT-specific factors.

Emerging concepts in M cell function and Peyer's patch development

Emerging concepts in M cell function and Peyer's patch development

Effects of tumor necrosis factor and lymphotoxin on peripheral lymphoid tissue development.

Previously, we have reported that neutralization of surface lymphotoxin (LT-alphabeta) in mice which expressed an LT-beta receptor-Fc fusion protein, driven by the cytomegalovirus promoter, resulted in an array of anatomic abnormalities. We now report that mice which express a tumor necrosis factor (TNF) receptor p60-Fc fusion protein (which neutralizes TNF and soluble LT-alpha3 activity) develop unique lymphoid abnormalities. Our data demonstrate that some aspects of peripheral lymphoid organ development require both surface LT-alphabeta and TNF interacting with their specific receptors. However, these related cytokines are also capable of signaling distinct developmental events. Splenic MAdCAM-1 expression, follicular dendritic cell localization and normal Peyer's patch development all require both surface LT-alphabeta and TNF activity. Marginal zone formation and splenic B cell localization primarily require surface LT-alphabeta-LT-beta receptor interactions. Primary follicle formation was dependent upon TNF receptor(s) engagement. Interestingly spleen, lymph nodes and Peyer's patches from TNF receptor p60-Fc-expressing mice all develop different abnormalities, suggesting distinct pathways of development in these lymphoid organs. Thymus development appears to be independent of these signaling pathways. These results demonstrate that TNF and LT are crucial for normal peripheral, but not central lymphoid organ development.

Lymphotoxin-alpha-deficient and TNF receptor-I-deficient mice define developmental and functional characteristics of germinal centers.

Mice deficient in LT alpha (LT alpha-/-) lack lymph nodes and Peyer's patches. This action of LT alpha in lymph node organogenesis appears to be mediated by the membrane form of LT using a mechanism independent of TNF receptor I (TNFR-I) or II (TNFR-II). In contrast, normal Peyer's patch development appears to require both LT alpha and TNFR-I, with TNFR-I-/- mice showing hypoplastic Peyer's patch structures. LT alpha-/- mice also fail to support the normal segregation of T-cell and B-cell zones within the splenic white pulp. Again, this occurs via a mechanism independent of TNFR-I or TNFR-II. Additionally, follicular dendritic cell (FDC) clusters or germinal centers fail to develop in the spleen of LT alpha-/- animals. Mice deficient in either TNF alpha or TNFR-I also fail to develop splenic FDC clusters and germinal centers, indicating that signaling by both LT alpha and TNF alpha is required for development of these specialized lymphoid tissue structures. Finally, the splenic white pulp areas in LT alpha-/- mice lack the marginal zone of monoclonal antibody MOMA-1-staining metallophilic macrophages, whereas TNFR-I-deficient mice have preserved MOMA-1 staining. Thus, certain actions of LT alpha to regulate spleen white pulp architecture are mediated by receptors other than TNFR-I, most likely by the LT beta R or a closely related receptor. We tested whether germinal centers are essential for maturation of T-cell-dependent antibody responses. When LT alpha-/- mice were immunized with low doses of NP-ovalbumin (NP-OVA) adsorbed to alum, there was dramatically impaired production of high affinity anti NP IgG; however, after immunization with high doses of NP-OVA adsorbed to alum, LT alpha-/- mice mounted a high affinity NP-specific serum IgG response similar to wild-type mice, all in the absence of germinal centers or clustered FDC. Thus, although germinal centers enhance the processes required for maturation of the humoral immune response, the mechanisms responsible for affinity maturation are not absolutely dependent on the presence of germinal centers.

Anti-μ treatment suppresses immunoglobulin light-chain gene expression and Peyer's patch development

Anti-μ treated mice have been used extensively as a model for suppressed B-cell development [Murgita R.A., Mattioli C.A. and Tomasi T.B., Jr. (1973) J. exp. Med. 138, 209; Manning D.D. (1975) J. Reticuloendothel. Soc. 18, 63; Manning D.D. and Jutila J.W. (1972) J. Immun. 108, 282; Janeway C. A., Jr., Murgita R.A., Weinbaum F.I., Asofsky R. and Wigzell H. (1977) Proc. natn. Acad. Sci. U.S.A. 74, 4582; Hayglass K.T., Naides S.J., Benacerraf B. and Sy M.-S. (1985) J. Molec. Cell. Immun. 2, 107; Manning D.D. (1972) J. Immun. 109, 1152; Cooper M.D., Kearney J.F., Gathings W.E. and Lawton A. R. (1980) Immun. Rev. 52, 29; Burrows P.D., Kearney J.F., Lawton A.R. and Cooper M.D. (1978) J. Immun. 120, 1526]. However, little molecular evaluation has been performed on these animals to determine the level at which B-lineage cells are arrested. Experiments reported here were designed to determine the effects of anti-μ treatment of newborn mice on Ig-specific mRNA expression in lymphocyte populations. Newborn CBA/J mice received i.p. injections of goat anti-μ IgG or non-immune goat IgG, every 2 days, from birth until age 4 weeks. The degree of B-cell suppression in anti-μ treated mice was evident by low serum Ig levels and lack of surface Ig + cells in splenic lymphocytes. Morphologically, spleens of B-cell depleted mice were slightly reduced or normal size, while the total area of Peyer's patches (PP) was three-fold less than control mice. Spleen cells from anti-μ suppressed mice contained high levels of μ-mRNA, but markedly reduced levels of mRNA specific for other Ig heavy-chain isotypes, as determined by DNA excess dot blot and Northern blot hybridizations. RNA specific for other sequences (actin or IL-2 receptor) was not affected and hybridization to parent plasmid (pACYC) was not detected. In addition, suppression of κ- and λ-mRNA accumulation was evident. This was surprising, since the target for anti-μ treatment appears to be a B-cell population expressing intact surface IgM, a stage in B-cell development in which both μ- and light-chain-specific mRNA accumulation should be detected. Our results suggest one of the following models: (1) anti-μ treatment deletes all Ig + cells from the animal, so that only μ expressing pre-B-cells remain; or (2) anti-μ suppresses B-cell development by inhibiting κ and λ transcription, perhaps by some feedback mechanism in which the presence of surface Ig is required to maintain light-chain transcription.