Lymphoid Formations Research Articles

Methods for Selective Gene Expression Profiling in Single Tertiary Lymphoid Structure Using Laser Capture Microdissection.

Tertiary lymphoid structures (TLS) are de novo lymphoid formations that are induced within tissues during inflammatory episodes. TLS have been reported at various anatomic sites and in many different contexts like cancer, infections, autoimmunity, graft rejection, and idiopathic diseases. These inducible, ectopic, and transient lymphoid structures exhibit the prototypical architecture found within secondary lymphoid organs (SLO) and have been increasingly recognized as a major driver of local adaptive immune reaction. As TLS emerge within tissues, the isolation in situ and the molecular characterization of these structures are challenging to perform. Laser capture microdissection (LCM) is a powerful tool to isolate selective structural components and cells from frozen or paraffin-embedded tissues. We and other groups previously applied LCM to decipher the molecular network within TLS and uncover their intrinsic connection with the local microenvironment. In this chapter, we describe a detailed LCM method for selecting and isolating TLS in situ to perform comprehensive downstream molecular analyses.

Machine Vision–Detected Peritumoral Lymphocytic Aggregates Are Associated With Disease-Free Survival in Patients With Papillary Thyroid Carcinoma

Papillary thyroid carcinoma (PTC) is the most prevalent form of thyroid cancer, with a disease recurrence rate of around 20%. Lymphoid formations, which occur in nonlymphoid tissues during chronic inflammatory, infectious, and immune responses, have been linked with tumor suppression. Lymphoid aggregates potentially enhance the body’s antitumor response, offering an avenue for attracting tumor-infiltrating lymphocytes and fostering their coordination. Increasing evidence highlights the role of lymphoid aggregate density in managing tumor invasion and metastasis, with a favorable impact noted on overall and disease-free survival (DFS) across various cancer types. In this study, we present a machine vision model to predict recurrence in different histologic subtypes of PTC using measurements related to peritumoral lymphoid aggregate density. We demonstrate that quantifying peritumoral lymphocytic presence not only is associated with better prognosis but also, along with tumor-infiltrating lymphocytes within the tumor, adds additional prognostic value in the absence of well-known second mutations including TERT. Annotations of peritumoral lymphoid aggregates on 171 well-differentiated PTCs in the Cancer Genome Atlas Thyroid Carcinoma (TCGA-THCA) data set were used to train a deep-learning model to predict regions of lymphoid aggregates across the entire tissue. The fractional area of the tissue regions covered by these lymphocytes was dichotomized to determine the following 2 risk groups: a significant and low density of peritumoral lymphocytes. DFS prognosticated using these risk groups via the Kaplan-Meier analysis revealed a hazard ratio (HR) of 2.51 (95% CI: 2.36, 2.66), tested on 170 new patients also from the TCGA-THCA data set. The prognostic performance of peritumoral lymphocyte aggregate density was compared against the univariate Kaplan-Meier analysis of DFS using the fractional area of intratumoral lymphocytes within the primary tumor with an HR of 2.04 (95% CI: 1.89, 2.19). Combining the lymphocyte features in and around the tumor yielded a statistically significant improvement in prognostic performance (HR, 3.17 [95% CI: 3.02, 3.32]) on training and were independently evaluated against 62 patients outside TCGA-THCA with an HR of 2.44 (95% CI: 2.19, 2.69). Multivariable Cox regression analysis on the validation set revealed that the density of peritumoral and intratumoral lymphocytes was prognostic independent of histologic subtype with a concordance index of 0.815.

Discovery of an independent poor-prognosis subtype associated with tertiary lymphoid structures in breast cancer.

Tertiary lymphoid structures (TLSs) are ectopic lymphoid formations that arise in non-lymphoid tissues due to chronic inflammation. The pivotal function of TLSs in regulating tumor invasion and metastasis has been established across several cancers, such as lung cancer, liver cancer, and melanoma, with a positive correlation between increased TLS presence and improved prognosis. Nevertheless, the current research about the clinical significance of TLSs in breast cancer remains limited. In our investigation, we discovered TLS-critical genes that may impact the prognosis of breast cancer patients, and categorized breast cancer into three distinct subtypes based on critical gene expression profiles, each exhibiting substantial differences in prognosis (p = 0.0046, log-rank test), with Cluster 1 having the best prognosis, followed by Cluster 2, and Cluster 3 having the worst prognosis. We explored the impact of the heterogeneity of these subtypes on patient prognosis, the differences in the molecular mechanism, and their responses to drug therapy and immunotherapy. In addition, we designed a machine learning-based classification model, unveiling highly consistent prognostic distinctions in several externally independent cohorts. A notable marker gene CXCL13 was identified in Cluster 3, potentially pivotal in enhancing patient prognosis. At the single-cell resolution, we delved into the adverse prognosis of Cluster 3, observing an enhanced interaction between fibroblasts, myeloid cells, and basal cells, influencing patient prognosis. Furthermore, we identified several significantly upregulated genes (CD46, JAG1, IL6, and IL6R) that may positively correlate with cancer cells' survival and invasive capabilities in this subtype. Our study is a robust foundation for precision medicine and personalized therapy, presenting a novel perspective for the contemporary classification of breast cancer.

Morphology of the colon wall at different stages of chronic constipation

Introduction. Chronic constipation affects 14–16% of the developed countries population, thus remaining an important problem of modern coloproctology. To substantiate an objective treatment, it is elevant to look at not only clinical, laboratory, and instrumental data, but also at morphogenesis of structural colon changes at different stages of chronic constipation. Materials and methods. We studied postoperative material of 30 patients with drug-resistant chronic constipation at subcompensated (SC) and decompensated (DC) stages. The comparison group (CG) included 30 cases of colon resection for non-recurrent colorectal cancer. Morphometry of mucous membrane structural elements and neuromuscular and lymphoid apparatus was carried out. Results. Quantitative analysis of structural colon elements at SC stage indicates reversible atrophic changes and thickening of submucosal and muscular membranes, mainly due to edema and hypertrophy. Simultaneously, atrophic changes at DC stage are irreversible, with sclerotic, dystrophic, and atrophic changes being observed in submucosal and muscular membranes. As chronic constipation progresses, the number of nerve cells in the neuromuscular apparatus of the colon decreases significantly. Morphogenesis of lymphoid formations is limited to the development of a compensatory adaptive reaction as protective hyperplasia at SC stage, without simultaneous increase in the specific proportion of secondary lymphoid follicles. Although at DS stage, the average number and specific proportion of lymphoid formations in the mucosal and submucosal layers of the colon increase; the average area of lymphoid formations decreases markedly; and no secondary lymphoid follicles are observed. Conclusion. The established patterns of the colon wall morphogenesis in chronic constipation provide us objective causes to confirm the irreversible progression of pathological processes in all its functionally significant structures. This, based on the morphometric criteria obtained, serves as a recommendation for finding the most optimal treatment option for such patients. Keywords: chronic constipation, morphometry

Morphological assessment of ovaries during stimulation of ovulation in rab-bits with equine serum gonadotropin in different doses

Ovulatory stimulation of rabbits during artificial insemination requires the use of hormonal drugs to stimulate maturation of follicles, effective fertilization and full manifestation of female sexual function. For this purpose, gonadotropins or analogues of gonadotropin-releasing hormone are common in the practice of rabbit breeding. The latter have a selective effect on folliculogenesis and, under certain conditions, are not capable of inducing ovulation in rabbits of “zero” reproductive cycle, that is, those that have not given birth. Instead, gonadotropins, in particular serum gonadotropin obtained from foal mares (eCG), have a pronounced effect on folliculogenesis in animals. eCG is a glycoprotein hormone secreted by trophoblastic epithelial cells of fetal origin. In other species, except for horses, it is able to induce the release of both luteinizing and follicle-stimulating hormone. That is why eCG is widely used together with human chorionic gonadotropin to induce follicle growth and ovulation. However, there are data on complications and pathological conditions that may occur with long-term use of eCG, especially in genital tract. Therefore, the goal of our research was the morphological evaluation of the functional compartments of ovaries when using eCG to stimulate reproductive function of rabbits. Schemes of hormonal treatment of females in the conditions of a private rabbit farm, an experiment was conducted, which included the introduction of eCG in different doses (40 IU for animals of experimental group 1 and 25 IU for experimental group 2) during five reproductive cycles. Selection of material for histological studies, its fixation, production and analysis of tissue sections stained with hematoxylin and eosin were performed according to commonly used methods. The complex of morphofunctional changes in the ovaries of the rabbits of experimental groups corresponds to the pathomorphological picture of the syndrome of hyperstimulated ovaries. In our experiment, obtained changes were more pronounced at a dose of 40 IU, using which we additionally detected signs of increased immunoreactivity in body of rabbits, as evidenced by the presence of nodular lymphoid formations in outer theca of secondary and tertiary follicles. Thus, as a safe regimen for the use of eCG for ovulatory stimulation in rabbits, we recommend a dosage of 25 IU for 1–2 reproductive cycles.

Abstract B047: Tertiary lymphoid structures and B cells signatures determine clinically relevant T cell phenotype in ovarian cancer

Abstract Tertiary lymphoid structures (TLSs) are ectopic lymphoid formations that arise at sites with persistent inflammatory conditions, including tumors. TLSs are composed predominantly of B cells, T cells, and CD21+ CD23+ follicular dendritic cells and exhibit different levels of the organization, ranging from locally concentrated aggregates of immune cells to well-defined B cell follicles to mature follicles with germinal center formation. Supporting key relevance for TLSs in natural and immunotherapy-driven immunosurveillance, intratumoral TLS density has been associated with improved disease outcome and response to cancer immunotherapy, mainly check-point inhibitors (ICIs) in solid carcinomas. Here, we demonstrate that compared to the immunologically hot tumors like non-small cell lung carcinoma (NSCLC), human high-grade serous ovarian carcinoma (HGSOC) contains only a limited number of TLS with germinal center formation. In parallel, TLS frequency and maturation positively correlate with progenitor stem-like CD8+ T cell phenotype. In line with this notion, B cell depletion in HGSOC biopsies promoted terminally exhausted, poorly ICI-sensitive CD8+ T cells, suggesting a critical role of intratumoral B cells in maintaining T cell effector functions. These findings point to key numerical and functional differences between mature TLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative ICI-based immunotherapies for patients with HGSOC. Citation Format: Irena Kusova Moserova, Lenka Kasikova, Jana Rakova, Jana Tomankova, Michal Henser, Tereza Lanickova, Katerina Mojzisova, Jana Drozenova, Jan Laco, Lukas Rob, Michael Halaska, Ales Ryska, Robert Lischke, Jiri Vachtenheim, David Cibula, Lorenzo Galluzzi, Radek Spisek, Jitka Fucikova. Tertiary lymphoid structures and B cells signatures determine clinically relevant T cell phenotype in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B047.

Detection and quantitative analysis of tumor-associated tertiary lymphoid structures.

Tumor-associated tertiary lymphoid structures (TLSs) are ectopic lymphoid formations within tumor tissue, with mainly B and T cell populations forming the organic aggregates. The presence of TLSs in tumors has been strongly associated with patient responsiveness to immunotherapy regimens and improving tumor prognosis. Researchers have been motivated to actively explore TLSs due to their bright clinical application prospects. Various studies have attempted to decipher TLSs regarding their formation mechanism, structural composition, induction generation, predictive markers, and clinical utilization. Meanwhile, the scientific approaches to qualitative and quantitative descriptions are crucial for TLS studies. In terms of detection, hematoxylin and eosin (H&E), multiplex immunohistochemistry (mIHC), multiplex immunofluorescence (mIF), and 12-chemokine gene signature have been the top approved methods. However, no standard methods exist for the quantitative analysis of TLSs, such as absolute TLS count, analysis of TLS constituent cells, structural features, TLS spatial location, density, and maturity. This study reviews the latest research progress on TLS detection and quantification, proposes new directions for TLS assessment, and addresses issues for the quantitative application of TLSs in the clinic.

CELLULAR COMPOSITION OF THE LYMPHOID STRUCTURES OF THE HUMAN INTRAHEPATIC BILE DUCTS

he aim of our study was to investigate the cellular composition of the lymphoid structures of the human intrahepatic bile ducts. Materials and Methods: The object of the study was the lymphoid structures of the walls of the intrahepatic bile ducts obtained from 45 cadavers of different ages. Lymphoid nodules in the walls of the intrahepatic bile ducts were investigated by T. Hellman’s method. The microanatomy of lymphoid structures was studied on microscopic preparations. Microscopic preparations were stained according to Van Gieson, with hematoxylin-eosin, methylene blue. The digital data obtained during the study was subjected to statistical processing. Results: A macromicroscopic study of lymphoid formations in the intrahepatic bile ducts showed that lymphoid formations in the walls of these organs are represented by lymphoid nodules and diffuse lymphoid tissue. The peripheral contours of the lymphoid nodules are clearly defined. Histologically, the cellular composition of lymphoid nodules and diffuse lymphoid tissue was studied. In the mucous membrane and in the submucosa, small, medium sized lymphocytes, and large lymphocytes, macrophages, plasma, reticular, mast cells, cells in a stage of mitosis, degeneratively altered (destructive), and other forms of lymphoid cells are determined. Conclusions: According to our data, newborns have not only lymphoid nodules, but also a typical cellular composition in the lymphoid tissue, which has a completely “mature character”. The results of morphometric studies showed that in early childhood there is a high percentage of small, medium, large lymphocytes, macrophages, cells with signs of mitosis.

Follicular cholangitis mimicking a common bile duct cancer: a case report

BackgroundFollicular cholangitis (FC) is a benign bile duct disease that was first reported 2003. Pathologically, it is characterized by lymphoplasmacytic infiltration with multiple lymphoid follicle formations under the mucosal layer of the biliary tract. However, as this disease is extremely rare, little is known about its etiology and pathogenesis.Case presentationA 77-year-old woman was diagnosed with middle bile duct stenosis and potential increases in alkaline phosphatase (ALP) and γ-glutamyl transpeptidase levels (γ-GTP). Carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9) and IgG4 levels were all within the normal limits. Contrast-enhanced computed tomography (CE-CT) and magnetic resonance imaging (MRI) revealed bile duct dilation from intrahepatic to upper common bile duct and an irregular mass lesion in distal bile duct. Additionally, multiple overlapping leaf-like folds were detected. 18F-fluorodeoxyglucose positron emission tomography–computed tomography (18F-FDG-PET/CT) did not demonstrate fluorodeoxyglucose uptake. Subtotal stomach-preserving pancreaticoduodenectomy with regional lymph node dissection was performed because common bile duct cancer could not be ruled out. The resected specimen showed diffuse homogeneous middle bile duct wall thickening. Microscopically, the lesion exhibited thick fibrosis with several invaded lymphoplasmacytic cells, and lymphoid follicle formations were detected under the mucosal layer. Immunohistochemical staining (IHC) revealed positive for CD3, CD4, CD20 and CD79a, and these findings led to a final diagnosis of FC. The patient has not experienced recurrence to date (42 months postoperatively).ConclusionsCurrently, accurate preoperative diagnosis of FC is difficult. More cases must be accumulated to generate additional knowledge on its precise diagnosis and proper treatment.

Effect of chicken infectious bronchitis vaccine on morphogenesis and differentiation of cells in caecal tonsils

The study of the chickens’ immune system morphofunctional state allows assessing critical periods of their development and the body as a whole, as well as the effectiveness of vaccine prevention methods. The purpose of this study was to identify morphological and immunohistochemical changes in the caecal tonsils of chickens aged 8, 20, 40, 90, 110 days for vaccine prevention of infectious bronchitis. During the study, the following research methods were used: cytological, histological, immunohistochemical, morphometric, light-optical, statistical. Histological preparations of caecal tonsils of poultry aged 8, 20, 40, 90, 110 days of vaccinated and unvaccinated groups were analysed and studied. Up to 20 days of age, no lymphoid nodules were detected in the caecal tonsils of chickens, both vaccinated and non-vaccinated groups. Histological and morphometric studies were conducted, which showed that vaccination of poultry accelerates the development of lymphoid formations in the early stages of the postnatal period of ontogenesis, especially in chickens aged 20 days, which is manifested by an increase in the number and size of lymphoid nodules of caecal tonsils relative to the control group. Immunohistochemical studies have established that the placement and accumulation of T-lymphocytes with markers CD4+ , CD8+ , as well as B-lymphocytes (CD20+ ) and CD45RA+ cells in caecal tonsils, namely in lymphoid nodules and diffuse accumulation of lymphoid cells, depend on the age of chickens and multiplicity of vaccinations. It was proved that in chickens aged 8 days, the number of T-lymphocyte subpopulations with surface markers CD4+ , CD8+ prevailed over CD45RA+ and CD20+ . With the increase in the number of immunizations (after three-fold vaccination against infectious bronchitis of chickens), immunocytometric studies indicated that in vaccinated chickens aged 40 and 90 days, there was a clear increase in the number of mature B-lymphocytes by 1.58 and 1.37 times, respectively. Considering the fact that the number of CD8+ lymphocytes in vaccinated chickens aged 40 days was 1.49 times greater than the number of CD4+ cells, this led to a sharp decrease in the immunoregulatory index of the caecal tonsils, which must be factored in when carrying out preventive vaccinations

Gene co-expression regulatory analysis associated with tertiary lymphoid structures and endometrial cancer progression

Abstract Endometrial cancer (EC) is the most common cancer diagnosis of the reproductive organs among women in the United States. Previous studies have shown the chemokine CXCL13 was correlated with forming B-cell and other immune cell aggregates in the presence of high endothelial venules (HEV). These ectopic lymphoid formations are referred to as tertiary lymphoid structures (TLS). Some studies have shown that TLS are associated with a reduced risk of recurrence and improved immune response in cancers, while others have described negative cancer outcomes associated with TLS. Few studies have focused on the role of TLS in EC and the pathways involved in tumorigenesis. In this project, we identify chemokine-mediated gene networks associated with TLS formation and EC pathogenicity. We obtained RNA-Seq EC samples (n=643), which included Type I Uterine Corpus Endometrial Cancer (UCEC, n=587) and Type II Uterine Carcinosarcoma (UCS, n= 56) from the Cancer Genome Atlas (TCGA). Hierarchically clustered genes were correlated, functionally annotated for mRNA, and evaluated using a weighted gene network co-expression analysis (WGCNA). Functional enrichment analysis, such as Gene Ontology (GO) and the database for annotation, visualization, and integrated discovery (DAVID), were used to identify and visualize biological functions associated with the hub genes overrepresented in significant modules. Modules associated with metastasis and survival are representative of novel and known pathways. The analysis reveals that CXCL13 may be involved in cell cycle progression and apoptosis pathways. This study provides novel insights into the biological pathways associated with TLS, EC tumorigenesis, and patient survival risks. Supported by Frederick National Laboratory for Cancer Research subcontract IDIQ NCI (75N91019D00024) and MSM/TU/UABCCC Partnership NCI (U54CA118638)

Preventive effect of tertiary lymphoid structures on lymph node metastasis of lung adenocarcinoma.

Ectopic lymphoid formations are called tertiary lymphoid structures (TLSs). TLSs in cancer have been reported to be associated with good prognosis and immunotherapy response. However, the relationship between TLSs and lymph node (LN) metastasis is unclear. We analyzed 218 patients with radically resected lung adenocarcinoma. TLSs were defined as the overlap of T cell zone and B cell zone. Granzyme B + cells were defined as cytotoxic lymphocytes. We evaluated phenotypes of lymphocytes in TLSs, tumor-infiltrating lymphocytes (TILs) and LNs by immunohistochemistry. We divided the patients into mature TLS (DC-Lamp high) and immature TLS (DC-Lamp low) groups. The relationship between TLS maturation and clinicopathological factors was analyzed. The mature TLS group was associated with significantly lower frequency of LN metastasis (P < 0.0001) and early cancer stage (P = 0.0049). The mature TLS group had significantly more CD8 + (P = 0.0203) and Foxp3 + (P = 0.0141) cells in TILs than the immature TLS group had. Mature TLSs were independently associated with a favorable overall survival (hazard ratio [HR] = 0.17, P = 0.0220) and disease-free survival (HR = 0.54, P = 0.0436). Multivariate analysis showed that mature TLS was an independent low-risk factor for LN metastasis (odds ratio = 0.06, P = 0.0003). The number of cytotoxic lymphocytes in LNs was higher in the mature TLS group than in the immature group (20.0 vs. 15.1, P = 0.017). Mature TLSs were associated with an increased number of cytotoxic lymphocytes in draining LNs, a lower frequency of LN metastasis, and favorable outcomes. Mature TLSs may support antitumor immunity by lymphocyte activation.

Features of preoperative diagnosis of chronic lymphoproliferative syndrome of ENT organs in young children

Introduction. To determine the tactics of management of chronic lymphoproliferative syndrome in young children, a comprehensive examination is necessary. Herpesvirus infection (HVI) plays an important role in the etiology of hypertrophy of lymphoid formations of the pharynx.Purpose. To evaluate the results of preoperative diagnosis of chronic lymphoproliferative syndrome in children in early childhood.Materials and methods. In 96 patients aged 1 to 3 years 11 months with lymphoproliferative syndrome more than 3–6 months, endoscopy, otomicroscopy, impedance, ultrasound examination of abdominal organs, cervical and submandibular lymph nodes, serological and molecular genetic analyses of markers of EBV, CMV, HCV-6 in the blood; PCR in pharyngeal tonsil scrapings were performed.Results and discussion. The clinical picture in children with chronic lymphoproliferative syndrome of younger age was dominated by complaints of difficulty in nasal breathing, snoring in 42% of children, recurrent otitis in 58%, manifestations of asthenovegetative and intoxication syndromes and frequent acute respiratory viral infections in 70% of patients. A high degree of infection of children of the younger age group was revealed – HCV-6 in 87%, CMV in 63% of children, 46% – EBV. And high activity of the infectious process in 31% of patients for all three GVI, more often for EBV in 27%. A combination of two or three GVI was detected in 83% of patients. During instrumental examination, a high degree of hypertrophy of the nasopharyngeal tonsils (adenoid vegetations of 2–3 degrees – 67% and 3 degrees – 18%, combined with hypertrophy of the palatine tonsils in 27% of cases), an increase in neck lymph nodes of more than 16mm, including lymph node packs in 28% of younger children and reactive hepatosplenomegaly in 17.7% of patients.Conclusion. To determine the tactics of management of chronic lymphoproliferative syndrome in young children, it is important to assess the severity of lymphoproliferative syndrome according to the clinical picture and instrumental research methods (nasopharyngeal endoscopy, ultrasound examination of neck and abdominal lymph nodes) in combination with laboratory data and characteristics of the stage of activity of the infectious process of herpesvirus etiology.

HISTOLOGICAL AND MORPHOLOGICAL CHANGES IN THE LYMPHOID STRUCTURES OF THE GASTRIC MUCOUS MEMBRANE IN WHITE RATS WITH THE ADMINISTRATION OF SODIUM GLUTAMATE.

The aim: To determine the histological and morphological changes of the lymphoid structures of the stomach in male rats under the influence of oral sodium glutamate at the rate of 15 mg/kg of body weight. Materials and methods: The scientific experiment was performed on 20 white non-linear male rats of reproductive age (4-5 months). The experimental animals were divided into two groups (10 rats in each group), which were orally received monosodium glutamate at a dose of 15 mg/kg body weight every day. We studied the effect of 2 and 4 weekly administration of monosodium glutamate at a dose of 15 mg/kg body weight, respectively, in the I and II groups of experimental animals (depending on the week of their decapitation). Rats of the control groups (n=10) were injected with a placebo for 2 and 4 weeks, namely 0.5 ml of dechlorinated tap water at room temperature. Intact control animals were also divided into two groups, 5 rats each, depending on the week of decapitation: respectively, III group - decapitation on the 2nd week of the experiment; IV group - decapitation on the 4th week of the experiment. After the experiments were completed, animals were decapitated under light ether anesthesia. According to the purpose of the study, pieces of rat stomach measuring 1.0 x 1.0 cm were taken from the front wall of the bottom of the stomach near the great curvature, cardiac and portal parts of the organ. Histological preparations were examined using a MICROmed SEO SСAN light microscope and a Vision CCD Camera. Morphometric studies were carried out according to the method of S. B. Stefanov, using grids No. 3/16. For electron microscopic examination, pieces of the stomach wall of rats were fixed in a 2.5% solution of glutaraldehyde in a 0.1 M phosphate buffer (pH 7.2-7.4) with subsequent fixation in a 2.0% solution of osmium tetroxide. After dehydration in alcohols and acetone, the material was embedded in eponaraldite. Sections were made on an LKB-8800-III ultramicrotome and studied using a JEM - 100-V microscope. To study the structural components of the lymphoid formations of the mucous membrane of different parts of the stomach of rats, semi-thin sections were made for the purpose of sharpening the blocks, which were stained with methylene blue. Results: The analysis of the obtained data of the conducted experiment indicates that the administration of monosodium glutamate in a dose of 15 mg/kg of body weight to rats already after 14 days leads to an increase in the density and size of the lymphoid structures of the GMM. The number of immunocompetent cells between the fundus of the gastric glands and the muscle plate increases in the diffuse lymphoid tissue of the gastrointestinal tract of rats in all its parts, both in the I and II groups of experimental animals. These changes are most pronounced in the cardiac and portal parts of the stomach. In both groups of experimental animals, the migration of interepithelial lymphocytes, macrophages, plasma cells, and tissue basophils to the surface epithelium increases. In both groups of experimental animals (and the II group of rats), lymphoid nodules and lymphoid pre-nodules of the gastric mucous membrane (GMM) are located between the bottom of the gastric glands and the muscular plate of the GMM. A gradual increase of medium lymphocytes in the GMM was established both in animals of I and II groups, while large lymphocytes increased in almost the same amount in experimental animals of both groups. Similar changes occur in the characteristics of the number of plasma cells, macrophages and tissue basophils in the lymphoid pre-nodules of GMM. Conclusions: Administering monosodium glutamate to rats at a dose of 15 mg/kg of body weight for 2 weeks leads to an increase in the density and size of lymphoid structures of the mucous membrane in all parts of the stomach with a predominant increase in the number of immunocompetent cells between the bottom of the gastric glands and the muscle plate. At the same time, more pronounced changes were found in the number of small lymphocytes, which tend to decrease by the 2nd week of the experiment, and vice versa - their density increases by the 4th week of monosodium glutamate administration.

10P Deep learning-based prediction of patient’s TLS status from HE images in pan-cancer cohort

Tertiary lymphoid structures (TLS) are ectopic lymphoid formations which are composed predominantly of B cells, T cells and dendritic cells. The presence in the tumor compartment of mature TLS - characterized by mature follicles containing germinal centers - has been strongly associated with improved survival upon cancer immunotherapies for patients with solid tumors. For this reason, TLS could be used as a predictive factor biomarker to identify the patients more likely to benefit from immune checkpoint inhibitors.

Tertiary lymphoid structures accompanied by fibrillary matrix morphology impact anti-tumor immunity in basal cell carcinomas.

Tertiary lymphoid structures (TLS) are specialized lymphoid formations that serve as local repertoire of T- and B-cells at sites of chronic inflammation, autoimmunity, and cancer. While presence of TLS has been associated with improved response to immune checkpoint blockade therapies and overall outcomes in several cancers, its prognostic value in basal cell carcinoma (BCC) has not been investigated. Herein, we determined the prognostic impact of TLS by relating its prevalence and maturation with outcome measures of anti-tumor immunity, namely tumor infiltrating lymphocytes (TILs) and tumor killing. In 30 distinct BCCs, we show the presence of TLS was significantly enriched in tumors harboring a nodular component and more mature primary TLS was associated with TIL counts. Moreover, assessment of the fibrillary matrix surrounding tumors showed discrete morphologies significantly associated with higher TIL counts, critically accounting for heterogeneity in TIL count distribution within TLS maturation stages. Specifically, increased length of fibers and lacunarity of the matrix with concomitant reduction in density and alignment of fibers were present surrounding tumors displaying high TIL counts. Given the interest in inducing TLS formation as a therapeutic intervention as well as its documented prognostic value, elucidating potential impediments to the ability of TLS in driving anti-tumor immunity within the tumor microenvironment warrants further investigation. These results begin to address and highlight the need to integrate stromal features which may present a hindrance to TLS formation and/or effective function as a mediator of immunotherapy response.

Assessing the Relationship between Residual Cancer Burden and the Tumor Immune-Microenvironment in Early-Stage, Hormone Receptor-Positive Breast Cancer Following Preoperative Radiation Therapy in MC1732 Clinical Trial

<h3>Purpose/Objective(s)</h3> Tumor-infiltrating lymphocytes (TILs) and ectopic lymphoid formations known as tertiary lymphoid structures (TLS) are prognostically significant in triple-negative breast cancer (TNBC). However, there is a paucity of data on this matter in hormone receptor-positive (HR+) breast cancer and whether these immunological parameters are impacted by preoperative radiation therapy (RT). The purpose of this work is to assess the relationship between residual cancer burden (RCB) and TILs/TLS in HR+ early-stage breast cancer following preoperative radiation therapy. <h3>Materials/Methods</h3> During 2019-2020, patients with cT0-T2, N0, M0 breast cancer were enrolled in a clinical trial (MC1732) in which they received hypofractionated whole-breast RT in 25 Gy in 5 fractions 4-8 weeks prior to breast-conserving surgery (BCS). RCB (determined per MD Anderson calculator), estimated % cellularity, TILs, and TLS were assessed in the surgical tissues. Low TIL was defined as < 10%, intermediate TIL as 11-49%, and high TIL as > 50%. The relationships between RCB class, numerical RCB values, and immunological parameters were explored using Kruskal-Wallace/Wilcoxon Rank Sum Test, linear regression, and Kendall's tau correlations. TILs and TLS were log-transformed to overcome extreme distributions. Lastly, a predictive model for pathologic outcomes was developed using backward elimination. <h3>Results</h3> Among the 22 patients analyzed for RCB, all were ER/PR positive. One patient was HER2 positive, and 13/22 had Grade 1 disease. Pathologic complete response (pCR) was noted in 18%, RCB-I in 60%, and RCB-II in 22%. In post-treatment samples, low, intermediate, and high TIL were found in 77%, 18%, and 5% of patients, respectively. Approximately 36% of patients had at least one TLS present after radiation. Kruskal-Wallace/Wilcoxon Rank Sum Test indicated a significant association between RCB class and % TILs and TLS (p = 0.010 and 0.003, respectively.) Kendall's tau correlation indicated a statistically significant positive correlation between RCB values and % TILs (p = 0.02) and TLS (p = 0 .001). Linear regression revealed that for every one-unit increase in RCB value, there was a statistically significant 4.3 unit increase in the % TILs (p <0.001), and a 2.48 unit increase in TLS (p <0.001). Finally, per the backward selection model, the number of TLS was associated with increased % cellularity at the time of surgery. <h3>Conclusion</h3> Our data suggest that increases in TILs and TLS following preoperative radiation are associated with increased residual disease in HR+ early-stage breast cancer. Given the fact that there are multiple types of potential immune infiltrates, further work is needed to elucidate the mechanisms behind these findings and specific types of immune infiltration.

Dynamics of the cellular composition of lymphoid nodules in the lungs of guinea pigs sensitized with ovalbumin

The article discusses the morphological aspects of the dynamics of the cellular composition of lymphoid nodules in the lungs of guinea pigs as a result of an experimental ovalbumin-induced allergic process. We studied the reactivity of immunocompetent cells of lymphoid formations of the lungs after three times subcutaneous sensitization and subsequent 8-day intranasal aeroallergization with ovalbumin in the early and late stage period of the allergic inflammatory process by microscopic, morphometric and statistical methods. By help of morphometric analysis we demonstrate the general regularity of reactivity of a local specific link of the pulmonary immune system to the action of an allergen, which consists in the elevation of the average amount of immune cells of lymphoid nodules of the lungs, starting from the 30th to the 44th day after the start of the experiment. The maximal coefficient of increase by 5.8 times was observed in counting plasma cells among all types of immunocompetent cells of lymphoid nodules in the lungs during the experiment. It has been statistically proven that the implementation of the ovalbumin-induced allergic inflammatory process in the lungs proceeds according to the humoral type and the duration of its course is not limited by the direct influence of the allergen, it also continues after the end of its action, which is a manifestation of changes in compensatory-adaptive processes in the pulmonary immune system with ovalbumin-induced allergic inflammation.

Macromicroscopic Anatomy of the Lymphoid Formations of the Vaginal Vestibule and Intrahepatic Bile Ducts and Human

Macromicroscopic Anatomy of the Lymphoid Formations of the Vaginal Vestibule and Intrahepatic Bile Ducts and Human

Морфогистохимическая оценка влияния кормовой минеральной добавки на органы пищеварения при экспериментальной иммуносупрессии у животных

Abstract. The conditions of keeping animals and birds are increasingly provoking stressful situations for them. The occurrence of stress helps to reduce the body's resistance and creates conditions for the development of diseases of various etiologies. With a decrease in resistance, the quantity of products and their quality decreases. It is possible to prevent stress in animals and poultry by using adaptogens of various origins. The search for optimal means that meet all the requirements of prevention remains to this day an urgent problem of practical veterinary medicine. The purpose of this study was to give a morphohistochemical assessment of the effect of a feed mineral supplement on the digestive organs during experimental immunosuppression in laboratory animals. In the conditions of experimental immunosuppression, to identify the effect of mineral feed additives on the structure of organ cells and their metabolism. Methods. To confirm the effectiveness of the mineral adaptogen, histological, morphometric and histochemical studies of the digestive organs were performed. As a result of the conducted research on preventive feeding of a feed mineral supplement of domestic production, its immune- and organoprotective role was revealed. The most sensitive organs to the action of adverse factors (lymphoid formations in the intestinal wall) have been identified. Morphometrically and histochemically determined the reliability of morphological changes in them. During prophylactic feeding of a feed mineral supplement of domestic production in the digestive organs, the revealed changes did not have total destruction, approaching the structure and metabolic processes in cells to the structure of these organs in intact animals. Scientific novelty. For the first time, a comparative microstructural, histochemical and morphometric assessment of the digestive organs in the body of laboratory animals under conditions of artificially induced immunosuppression was carried out, confirming the immune- and organoprotective effect of a feed mineral supplement of domestic production, which can be considered as a mineral adaptogen.