PSX-B-26 Mitotic activity of immune cells in the intestines of piglets in the treatment of gastrointestinal pathology

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 271-272
Author(s):  
Pavel Parshin ◽  
Larisa Sashnina ◽  
Evgenii Mikhailov ◽  
Boris Shabunin ◽  
Svetlana Vorotnikova
Keyword(s):  
Mitotic Activity ◽  
Immune Cells ◽  
Cell Counting ◽  
Intestinal Immunity ◽  
Cell Hyperplasia ◽  
Ki 67 ◽  
Gm Csf ◽  
Vascular Walls ◽  
Gastrointestinal Pathology ◽  
Imagej Software

Abstract The study aimed to evaluate the effect of a drug based on the granulocyte-macrophage colony-stimulating factor (GM-CSF) on intestinal immune cells’ mitotic activity in treating gastrointestinal pathology. Two groups of 40 sick piglets with diarrhea at the age of 5–7 days were formed. The 1st group was the control. Animals of the 2nd group received an intramuscular injection of “Quinokol”(enrofloxacin) at a dose of 0.5 ml / 10 kg once a day for 5 days. The 3d - “Quinokol” at the same dose combined with GM-CSF at a dose of 0.1 ml/kg twice with an interval of 48 hours. From forcedly slaughtered piglets (by Directive 93/119 / EC) with the diarrheal syndrome (n = 4) and from animals of the experimental groups (n = 4), duodenum sites were taken for research. Immunohistochemical study of Ki-67 expression with additional staining with hematoxylin Karratsi was carried out according to the method described by V. Stafford. Cell counting and morphometric changes were performed using the ImageJ software. In preparations from sick piglets and piglets, which were used “Quinokol”, the main mitotic activity was 0.0036 ± 0.00047 and 0.0041 ± 0.00054 mitoses / μm2, respectively (fig 1 - A). Single Ki-67-positive cells were found in the stroma of the villi, vascular walls, and submucosa. In some areas, the bean or rod-shaped nucleus of Ki-67 positive cells was distinguished. At the samples from the 3d group (fig 1 - C, D), a sharp increase in the mitotic activity of immune cells in the stroma of the villi was noted - 0.0067 ± 0.00072 mitoses/μm2 and significantly more than in the 1st and 2nd groups by 64% and 86%, respectively (P < 0.001). The combined use of “Quinokol” with GMC-SF increased cell hyperplasia in the villous stroma, which indicates the activation of local intestinal immunity.

scholarly journals Molecular Pathology of Well-Differentiated Pulmonary and Thymic Neuroendocrine Tumors: What Do Pathologists Need to Know?

2021 ◽  
Vol 32 (1) ◽  
pp. 154-168 ◽  
Author(s):  
Marco Volante ◽  
Ozgur Mete ◽  
Giuseppe Pelosi ◽  
Anja C. Roden ◽  
Ernst Jan M. Speel ◽  
...  
Keyword(s):  
Neuroendocrine Tumors ◽  
Young Patients ◽  
Carcinoid Tumors ◽  
Neuroendocrine Neoplasms ◽  
Grey Zone ◽  
Cell Hyperplasia ◽  
Ki 67 ◽  
Familial Form ◽  
Well Differentiated ◽  
Neuroendocrine Carcinomas

AbstractThoracic (pulmonary and thymic) neuroendocrine tumors are well-differentiated epithelial neuroendocrine neoplasms that are classified into typical and atypical carcinoid tumors based on mitotic index cut offs and presence or absence of necrosis. This classification scheme is of great prognostic value but designed for surgical specimens, only. Deep molecular characterization of thoracic neuroendocrine tumors highlighted their difference with neuroendocrine carcinomas. Neuroendocrine tumors of the lung are characterized by a low mutational burden, and a high prevalence of mutations in chromatin remodeling and histone modification-related genes, whereas mutations in genes frequently altered in neuroendocrine carcinomas are rare. Molecular profiling divided thymic neuroendocrine tumors into three clusters with distinct clinical outcomes and characterized by a different average of copy number instability. Moreover, integrated histopathological, molecular and clinical evidence supports the existence of a grey zone category between neuroendocrine tumors (carcinoid tumors) and neuroendocrine carcinomas. Indeed, cases with well differentiated morphology but mitotic/Ki-67 indexes close to neuroendocrine carcinomas have been increasingly recognized. These are characterized by specific molecular profiles and have an aggressive clinical behavior. Finally, thoracic neuroendocrine tumors may arise in the background of genetic susceptibility, being MEN1 syndrome the well-defined familial form. However, pathologists should be aware of rarer germline variants that are associated with the concurrence of neuroendocrine tumors of the lung or their precursors (such as DIPNECH) with other neoplasms, including but not limited to breast carcinomas. Therefore, genetic counseling for all young patients with thoracic neuroendocrine neoplasia and/or any patient with pathological evidence of neuroendocrine cell hyperplasia-to-neoplasia progression sequence or multifocal disease should be considered.

scholarly journals Gut symbiotic microbes imprint intestinal immune cells with the innate receptor SLAMF4 which contributes to gut immune protection against enteric pathogens

Gut ◽  
2017 ◽  
Vol 67 (5) ◽  
pp. 847-859 ◽  
Author(s):  
Allison Cabinian ◽  
Daniel Sinsimer ◽  
May Tang ◽  
Youngsoon Jang ◽  
Bongkum Choi ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Immune Cells ◽  
Immune Cell ◽  
Lymphocyte Activation ◽  
Premature Death ◽  
Enteric Pathogens ◽  
Immune Protection ◽  
Intestinal Immunity ◽  
Cell Responses ◽  
And Function

BackgroundInteractions between host immune cells and gut microbiota are crucial for the integrity and function of the intestine. How these interactions regulate immune cell responses in the intestine remains a major gap in the field.AimWe have identified the signalling lymphocyte activation molecule family member 4 (SLAMF4) as an immunomodulator of the intestinal immunity. The aim is to determine how SLAMF4 is acquired in the gut and what its contribution to intestinal immunity is.MethodsExpression of SLAMF4 was assessed in mice and humans. The mechanism of induction was studied using GFPtg bone marrow chimaera mice, lymphotoxin α and TNLG8A-deficient mice, as well as gnotobiotic mice. Role in immune protection was revealed using oral infection with Listeria monocytogenes and Cytobacter rodentium.ResultsSLAMF4 is a selective marker of intestinal immune cells of mice and humans. SLAMF4 induction occurs directly in the intestinal mucosa without the involvement of the gut-associated lymphoid tissue. Gut bacterial products, particularly those of gut anaerobes, and gut-resident antigen-presenting cell (APC)TNLG8A are key contributors of SLAMF4 induction in the intestine. Importantly, lack of SLAMF4 expression leads the increased susceptibility of mice to infection by oral pathogens culminating in their premature death.ConclusionsSLAMF4 is a marker of intestinal immune cells which contributes to the protection against enteric pathogens and whose expression is dependent on the presence of the gut microbiota. This discovery provides a possible mechanism for answering the long-standing question of how the intertwining of the host and gut microbial biology regulates immune cell responses in the gut.

scholarly journals Peutz-Jeghers syndrome: Quantitative study on enterochromaffin cells in hamartomatous intestine polyps

2013 ◽  
Vol 141 (9-10) ◽  
pp. 602-607
Author(s):  
Miljan Krstic ◽  
Vuka Katic ◽  
Slavica Stojnev ◽  
Dragan Mihailovic ◽  
Marijola Mojsilovic ◽  
...  
Keyword(s):  
Clinical Manifestations ◽  
Enterochromaffin Cells ◽  
Ileal Mucosa ◽  
Cell Hyperplasia ◽  
Ki 67 ◽  
Intestinal Polyps ◽  
Serotonin Secretion ◽  
Formalin Fixed Paraffin Embedded ◽  
Ec Cells ◽  
Peutz Jeghers Syndrome

Introduction. Peutz-Jeghers (PJ) syndrome is a rare familial disorder with the autosomal transmission characterized by multiple intestinal polyps, mucocutaneous pigmentation and increased incidence of various malignancies. Some clinical manifestations of PJ syndrome may be associated with the serotonin secretion from the enterochromaffin cells (EC). Objective. Since no data have been reported so far regarding EC cells in PJ polyps, the aim of our study was to quantitatively investigate EC population in hamartomatous intestinal polyps in patients with the PJ syndrome. Methods. The samples of surgically removed PJ polyps from family members with the PJ syndrome were collected during 34-year follow-up period. Formalin-fixed paraffin-embedded specimens of twenty-one PJ polyps were stained with HE, AB-PAS, Van Gieson, Fontana-Masson, FIF and Grimelius. For immuno- histochemical analysis, the following antibodies were used: chromogranin A, serotonin, Ki-67, desmin, vimentin and cytokeratin in order to eliminate differential diagnostic possibilities and to confirm diagnosis of PJ polyps. Results. Strong EC cell hyperplasia was observed within the tissue of the investigated polyps. Statistical analysis demonstrated significantly higher content of EC cells in PJ polyps than in the normal ileal mucosa. Conclusion. Marked hyperplasia of EC cells within the PJ polyps may be the most important contributor to functional disorders in patients with the PJ syndrome.

scholarly journals Therapeutic Effect of Exogenous Treg Cells on Collagen-Induced Arthritis and Rheumatoid Arthritis

2019 ◽  
Author(s):  
Shutong Li ◽  
Hongxing Wang ◽  
Hui Wu ◽  
Guoqing Zhang ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Synovial Fibroblast ◽  
Treg Cells ◽  
Cell Counting ◽  
Fibroblast Cells ◽  
Collagen Induced Arthritis ◽  
Gm Csf

Abstract Background Regulatory T (Treg) cells have anti-inflammatory and anti-autoimmune functions. The proportion and functions of Treg cells are perturbed in rheumatoid arthritis (RA) patients. Methods Human Treg cells were induced to amplify in vitro and cocultured with RA synovial fibroblast cells (RASFs). The proliferation and apoptosis of RASFs were determined by the cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Human Treg cells were also injected to collagen-induced arthritis (CIA) rats via the tail vein. Changes in lymphocyte subtypes and cytokines in the peripheral blood and spleen were observed by flow cytometry. Results After coculture with the Treg cells, the proliferation of RA synovial fibroblast cells decreased (p<0.01), and the rate of apoptosis increased (p=0.037). The human Treg cells were injected into the tail veins of collagen-induced arthritis (CIA) rats. The severity of the CIA was reduced (p<0.01) following the injection, the percentages of rat endogenous Treg cells in the peripheral blood and spleen increased significantly (p=0.007 and p<0.01, respectively), and the proportion of B cells decreased (p=0.031). The levels of interleukin IL-5 and IL-6 and the Th1/Th2 ratio in the peripheral blood were significantly decreased (p=0.013, 0.009 and 0.012, respectively). The number of NK cells and the levels of IL-4, IL-13, TNF-α, IFN-γ and GM-CSF in the peripheral blood and spleen did not change significantly. Conclusion These results suggest that exogenous Treg cells play a therapeutic role in RA and CIA. Treg cell treatment could serve as a therapy for RA.

scholarly journals Correlation Between 18F-FDG Uptake and Immune Cell Infiltration in Metastatic Brain Lesions

2021 ◽  
Vol 11 ◽  
Author(s):  
Young-Sil An ◽  
Se-Hyuk Kim ◽  
Tae Hoon Roh ◽  
So Hyun Park ◽  
Tae-Gyu Kim ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Immune Cells ◽  
Immune Cell ◽  
Standardized Uptake Value ◽  
Brain Lesions ◽  
Cell Infiltration ◽  
Immune Cell Infiltration ◽  
Ki 67 ◽  
Glucose Transporter 1 ◽  
Fdg Uptake

BackgroundThe purpose of this study was to investigate the correlation between 18F-fluorodeoxyglucose (FDG) uptake and infiltrating immune cells in metastatic brain lesions.MethodsThis retrospective study included 34 patients with metastatic brain lesions who underwent brain 18F-FDG positron emission tomography (PET)/computed tomography (CT) followed by surgery. 18F-FDG uptake ratio was calculated by dividing the standardized uptake value (SUV) of the metastatic brain lesion by the contralateral normal white matter uptake value. We investigated the clinicopathological characteristics of the patients and analyzed the correlation between 18F-FDG uptake and infiltration of various immune cells. In addition, we evaluated immune-expression levels of glucose transporter 1 (GLUT1), hexokinase 2 (HK2), and Ki-67 in metastatic brain lesions.ResultsThe degree of 18F-FDG uptake of metastatic brain lesions was not significantly correlated with clinical parameters. There was no significant relationship between the 18F-FDG uptake and degree of immune cell infiltration in brain metastasis. Furthermore, other markers, such as GLUT1, HK2, and Ki-67, were not correlated with degree of 18F-FDG uptake. In metastatic brain lesions that originated from breast cancer, a higher degree of 18F-FDG uptake was observed in those with high expression of CD68.ConclusionsIn metastatic brain lesions, the degree of 18F-FDG uptake was not significantly associated with infiltration of immune cells. The 18F-FDG uptake of metastatic brain lesions from breast cancer, however, might be associated with macrophage activity.

scholarly journals Pulmonary Peripheral Carcinoids after Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia and Tumorlets: Report of 3 Cases

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Carlos Abrantes ◽  
Rui Caetano Oliveira ◽  
Joana Saraiva ◽  
João Bernardo ◽  
Lina Carvalho
Keyword(s):  
Lung Disease ◽  
Neuroendocrine Differentiation ◽  
Neuroendocrine Cells ◽  
Neuroendocrine Cell ◽  
Peripheral Spectrum ◽  
Surgical Biopsy ◽  
Cell Hyperplasia ◽  
Ki 67 ◽  
Typical Carcinoid ◽  
Pulmonary Neuroendocrine Cell

Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) and tumorlets are neuroendocrine cells proliferations smaller than 5 mm. The former confines to bronchial/bronchiolar wall, while the latter broke through epithelial basement membrane. The authors present 3 cases of DIPNECH and tumorlets associated with a typical peripheral carcinoid tumor without underlying lung disease. The patients presented with nonspecific pulmonary symptoms: 3 females, 60, 72, and 84 years old, whose CT-scans showed well-defined pulmonary nodules, 2.2, 1.6, and 1.4 cm, respectively; first patient was submitted to lobectomy and the others underwent surgical biopsy. Whitish/brownish lobulated tumors corresponded to typical carcinoids (less than 2 mitoses/2 mm2and without necrosis); polygonal/elongated cells under lobular pattern expressed CD56, chromogranin A, synaptophysin, and CK7; Ki-67 positivity was between 1 and 3%. Bronchial/bronchiolar wall neuroendocrine cell hyperplasia and several neuroendocrine nodules under 5 mm, with identical morphologic and immunoexpression, were observed, without lung disease. Typical carcinoid associated with DIPNECH and tumorlets without other pulmonary diseases is rare. Sporadic cases may recall embryonal neuroendocrine differentiation potentiality to develop peripheral hyperplasia, most commonly in underoxygenated parenchyma. The described cases are elucidative of peripheral spectrum of neuroendocrine cell tumour evolution, reinforcing higher female incidence as in central carcinoids, still without a clear preneoplastic lesion.

scholarly journals miR-182-5p Promotes Growth in Oral Squamous Cell Carcinoma by Inhibiting CAMK2N1

2018 ◽  
Vol 49 (4) ◽  
pp. 1329-1341 ◽  
Author(s):  
Nan Li ◽  
Chuan-Chuan Nan ◽  
Xue-Yun Zhong ◽  
Jun-Quan Weng ◽  
Hai-Dong Fan ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cell Lines ◽  
Squamous Cell ◽  
Colony Formation ◽  
Cell Counting ◽  
Control Group ◽  
Rt Pcr ◽  
Ki 67

Background/Aims: Emerging evidence suggests that the propagation of oral squamous cell carcinoma (OSCC) is influenced by the abnormal expression of microRNAs (miRNAs). This study aimed to characterize the involvement of miR-182-5p in OSCC by targeting the calcium/ calmodulin-dependent protein kinase II inhibitor CAMK2N1. Methods: miR-182-5p expression was quantified in OSCC tissues and cell lines with reverse transcription polymerase chain reaction (RT-PCR). Cell colony formation, Cell Counting Kit-8 (CCK-8), Ki-67, and nude mouse xenograft assays were used to characterize the role of miR-182-5p in the proliferation of OSCC. A miR-182-5p target gene was identified with western blotting, RT-PCR, and luciferase activity assays. OSCC patient survival based on CAMK2N1 expression was also analyzed. Results: miR-182-5p was up-regulated in in vitro cell lines and in vivo clinical OSCC samples. CCK-8, colony formation, and Ki-67 assays revealed that miR-182-5p promoted the growth and proliferation of OSCC cells. miR-182-5p directly targeted CAMK2N1, as evidenced by luciferase assays and target prediction algorithms. CAMK2N1 operated as a tumor suppressor gene in patients with OSCC. Down-regulating miR-182-5p expression in the CAL-27 cell line restored CAMK2N1-mediated OSCC cell proliferation. miR-182-5p expression inhibited the activation of AKT, ERK1/2, and NF-κB. Mice injected with CAL-27 cells transfected with miR-182-5p-inhibitor demonstrated a significant increase in tumor size and weight and increased CAMK2N1 mRNA and protein expression compared with the miR-negative control group. Conclusion: The miR-182-5p-CAMK2N1 pathway can be potentially targeted to regulate the proliferation of OSCC cells.

Immunohistochemical observation of local inflammatory cell infiltration in the host-tissue reaction site of human hydatid cysts

2018 ◽  
Vol 93 (3) ◽  
pp. 277-285 ◽  
Author(s):  
R. Jafari ◽  
B. Sanei ◽  
A. Baradaran ◽  
M. Kolahdouzan ◽  
B. Bagherpour ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Cells ◽  
Immune Cell ◽  
Tissue Reaction ◽  
Distinct Pattern ◽  
Host Tissue ◽  
Cell Infiltration ◽  
Reaction Site ◽  
Hydatid Cysts ◽  
Ki 67

AbstractThe aim of this study was to evaluate the pattern of local immune cell infiltration in human cystic echinococcosis (CE) by identifying the subtypes of immune cells using immunohistochemistry (IHC). Fifty surgically removed hydatid cyst samples and surrounding tissues were collected from patients referred to Al-Zahra Hospital, Isfahan, Iran. IHC was performed on the surrounding host tissue of hydatid cysts using anti-human CD3, CD19, CD8, CD4, CD68, CD56, Ki-67 and Foxp3 (forkhead box P3) antibodies. The results were then compared to hepatocellular carcinoma and chronic hepatitis. In the host-tissue reaction site of liver hydatid cysts, a distinct pattern of local immune cell response, which outwardly consisted of a pack of the fibrous elements, a layer of palisading macrophages, an eosinophil-containing layer and a layer of accumulated lymphocytes, was observed. However, in some cases there were no positive cells for CD56+ natural killer cells and Foxp3+ regulatory T cells. The CD3+ T cells were the predominant inflammatory cells in all groups, followed by CD19+ B cells. It can be concluded that different immune cells are involved in the local response to human hydatid cysts.

Correlation between MDSC and Immune Tolerance in Transplantation: Cytokines, Pathways and Cell-cell Interaction

2019 ◽  
Vol 19 (2) ◽  
pp. 81-92 ◽  
Author(s):  
Tianying Yang ◽  
Jiawei Li ◽  
Ruimin Li ◽  
Chunchen Yang ◽  
Weitao Zhang ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Graft Survival ◽  
Immune Tolerance ◽  
Immune Cells ◽  
Cell Interaction ◽  
Erk Signaling ◽  
Gm Csf ◽  
Cytokines And Chemokines ◽  
Direct Suppression ◽  
Cell Cell

MDSCs play an important role in the induction of immune tolerance. Cytokines and chemokines (GM-CSF, IL-6) contributed to the expansion, accumulation of MDSCs, and MDSCs function through iNOS, arginase and PD-L1. MDSCs are recruited and regulated through JAK/STAT, mTOR and Raf/MEK/ERK signaling pathways. MDSCs’ immunosuppressive functions were realized through Tregs-mediated pathways and their direct suppression of immune cells. All of the above contribute to the MDSC-related immune tolerance in transplantation. MDSCs have huge potential in prolonging graft survival and reducing rejection through different ways and many other factors worthy to be further investigated are also introduced.

scholarly journals P01.10 IFNy secretion of adaptive and innate immune cells as a parameter to display leukaemia derived dendritic cell (DCleu) mediated immune responses in AML

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A13.1-A13
Author(s):  
LK Klauer ◽  
O Schutti ◽  
S Ugur ◽  
F Doraneh-Gard ◽  
N Rogers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Immune Cells ◽  
Gm Csf ◽  
Adaptive Immune ◽  
Cik Cells ◽  
Adaptive Immune Cells ◽  
Suitable Parameter

BackgroundMyeloid leukaemic blasts can be converted into leukaemia derived dendritic cells (DCleu) with blastmodulatory Kit-I and Kit-M, which have the competence to regularly activate T and immunoreactive cells to gain anti-leukaemic activity or rather cytotoxicity. As innate and adaptive immune responses are notably promoted by the cytokine interferon gamma (IFNy), we hypothesised that the IFNy secretion could be a suitable parameter to display DC/DCleu mediated immunologic activity and even anti-leukaemic cytotoxicity.Materials and MethodsDC/DCleu were generated from leukaemic WB with Kit-I (GM-CSF + OK-432) and Kit-M (GM-CSF + PGE1) and used to stimulate T cell enriched immunoreactive cells. Initiated anti-leukaemic cytotoxicity was investigated with a cytotoxicity fluorolysis assay (CTX). Initiated IFNy secretion of innate and adaptive immune cells (T cells, TCD4+ cells, TCD8+ cells, NKCD56+ cells, NKCD161+ cells, CIKCD56+ cells, CIKCD161+ cells and iNKT) was investigated with a cytokine secretion assay (CSA). In some cases IFNy production was additionally evaluated with an intracellular cytokine assay (ICA). Conclusively, the IFNy secretion of immunoreactive cells was correlated with the anti-leukaemic cytotoxicity.ResultsSignificant amounts of DC and DCleu as well as migratory DC and DCleu could be generated with Kit-I and Kit-M without induction of blast proliferation. T cell enriched immunoreactive cells stimulated with DC/DCleu showed an increased anti-leukaemic cytotoxicity and an increased IFNy secretion of T, NK and CIK cells compared to control. Both the CSA and ICA yielded comparable amounts of IFNy positive innate and adaptive immune cells. The correlation between the IFNy secretion of immunoreactive cells and the anti-leukaemic cytotoxicity showed a positive relationship in T cells, TCD4+ cells, TCD8+ cells and NKCD56+ cells.ConclusionsWe found blastmodulatory Kit-I and Kit-M competent to generate DC/DCleu from leukaemic WB. Stimulation of T cell enriched immunoreactive cells with DC/DCleu regularly resulted in an increased anti-leukaemic cytotoxicity and an increased IFNy dependent immunological activity of T, NK and CIK cells compared to control. Moreover the anti-leukaemic cytotoxicity positively correlated with the IFNy secretion in T cells, TCD4+ cells, TCD8+ cells, NKCD56+ cells. We therefore consider the IFNy secretion of innate and adaptive immune cells to be a suitable parameter to assess the efficacy of in vitro and potentially in vivo AML immunotherapy. The CSA in this regard proved to be a convenient and reproducible technique to detect and phenotypically characterise IFNy secreting cells of the innate and adaptive immune system.Disclosure InformationL.K. Klauer: None. O. Schutti: None. S. Ugur: None. F. Doraneh-Gard: None. N. Rogers: None. M. Weinmann: None. D. Krämer: None. A. Rank: None. C. Schmid: None. B. Eiz-Vesper: None. H.M. Schmetzer: None.

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