The Cross-talk Between Intestinal Microbiota and MDSCs Fuels Colitis-associated Cancer Development.

Intestinal chronic inflammation is associated with microbial dysbiosis and accumulation of various immune cells including myeloid-derived suppressor cells (MDSC), which profoundly impact the immune microenvironment, perturb homeostasis and increase the risk to develop colitis-associated colorectal cancer (CAC). However, the specific MDSCs-dysbiotic microbiota interactions and their collective impact on CAC development remain poorly understood. In this study, using a murine model of CAC, we demonstrate that CAC-bearing mice exhibit significantly elevated levels of highly immunosuppressive MDSCs, accompanied by microbiota alterations. Both MDSCs and bacteria that infiltrate the colon tissue and developing tumors can be found in close proximity, suggesting intricate MDSC-microbiota cross-talk within the tumor microenvironment. To investigate this phenomenon, we employed antibiotic treatment to disrupt MDSC-microbiota interactions. This intervention yielded a remarkable reduction in intestinal inflammation, decreased MDSC levels, and alleviated immunosuppression, all of which were associated with a significant reduction in tumor burden. Furthermore, we underscore the causative role of dysbiotic microbiota in the predisposition toward tumor development, highlighting their potential as biomarkers for predicting tumor load. We shed light on the intimate MDSCs-microbiota cross-talk, revealing how bacteria enhance MDSC suppressive features and activities, inhibit their differentiation into mature beneficial myeloid cells, and redirect some toward M2 macrophage phenotype. Collectively, this study uncovers the role of MDSC-bacteria cross-talk in impairing immune responses and promoting tumor growth, providing new insights into potential therapeutic strategies for CAC. SIGNIFICANCE: MDSCs-dysbiotic bacteria interactions in the intestine play a crucial role in intensifying immunosuppression within the CAC microenvironment, ultimately facilitating tumor growth, highlighting potential therapeutic targets for improving the treatment outcomes of CAC.

Functional Assays Evaluating Immunosuppression Mediated by Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are heterogenous populations of immature myeloid cells that can be divided into two main subpopulations, polymorphonuclear (PMN) MDSCs and monocytic (M) MDSCs. These cells accumulate during chronic inflammation, characterizing an array of pathologies such as cancer, inflammatory bowel disease, and infectious and autoimmune diseases, and induce immunosuppression. The suppressive effects of MDSCs on the immune system are studied mainly when focusing on their features, functions, and impact on target cells such as T cells, natural killer cells, and B cells, among others. Herein, we describe methods for the analysis of MDSC immunosuppressive features and functions, measuring different mediators that contribute to their activities and how they impact on T cell function. The protocols described are a continuation to those in a companion Current Protocols article by Reuven et al. (2022), which uses a generated single-cell suspension and isolated cells to test their activity. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Evaluating MDSC suppressive features Alternate Protocol 1: Dichlorofluorescein diacetate-based reactive oxygen species detection Support Protocol 1: Detection of nitric oxide secretion Support Protocol 2: Measurement of arginase activity Basic Protocol 2: Evaluating MDSC suppressive function Alternate Protocol 2: In vitro effects of MDSCs on expression of T cell receptor complex during activation Support Protocol 3: Effect of MDSCs on interferon γ production Basic Protocol 3: Effect of MDSCs on T cell proliferation Basic Protocol 4: Effect of MDSCs on T cell cytotoxic activity Alternate Protocol 3: In vivo cytotoxicity assay Basic Protocol 5: Analysis of MDSC differentiation.

Phenotypic Characterization and Isolation of Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are heterogenous populations of immature myeloid cells that can be divided into two main subpopulations, polymorphonuclear (PMN) MDSCs and monocytic (M) MDSCs. These cells accumulate during chronic inflammation and induce immunosuppression evident in an array of pathologies such as cancer, inflammatory bowel disease, and infectious and autoimmune diseases. Herein, we describe methods to isolate and characterize MDSCs from various murine tissue, as well as to phenotype blood-derived MDSCs from patients. The protocols describe methods for isolation of total MDSCs and their subpopulations, for characterization, and for evaluation of their distribution within tissue, as well as for assessing their maturation stage by flow cytometry, immunofluorescence analyses, and Giemsa staining. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Single-cell suspension generation from different tissue Alternate Protocol 1: Single-cell suspension generation from subcutaneous melanoma tumors Basic Protocol 2: Characterization of MDSC phenotype Basic Protocol 3: Cell separation using magnetic beads: Separating pan-MDSCs or PMN-MDSC and M-MDSC subpopulations Alternate Protocol 2: Staining and preparing MDSCs for sorting Support Protocol: PMN-MDSC and M-MDSC gating strategy in mouse Basic Protocol 4: Immunofluorescence analysis of MDSCs Basic Protocol 5: Handling human blood samples and characterizing human MDSCs Alternate Protocol 3: Flow cytometry staining of thawed human whole blood samples.

Specific inflammatory osteoclast precursors induced during chronic inflammation give rise to highly active osteoclasts associated with inflammatory bone loss.

Elevated osteoclast (OC) activity is a major contributor to inflammatory bone loss (IBL) during chronic inflammatory diseases. However, the specific OC precursors (OCPs) responding to inflammatory cues and the underlying mechanisms leading to IBL are poorly understood. We identified two distinct OCP subsets: Ly6ChiCD11bhi inflammatory OCPs (iOCPs) induced during chronic inflammation, and homeostatic Ly6ChiCD11blo OCPs (hOCPs) which remained unchanged. Functional and proteomic characterization revealed that while iOCPs were rare and displayed low osteoclastogenic potential under normal conditions, they expanded during chronic inflammation and generated OCs with enhanced activity. In contrast, hOCPs were abundant and manifested high osteoclastogenic potential under normal conditions but generated OCs with low activity and were unresponsive to the inflammatory environment. Osteoclasts derived from iOCPs expressed higher levels of resorptive and metabolic proteins than those generated from hOCPs, highlighting that different osteoclast populations are formed by distinct precursors. We further identified the TNF-α and S100A8/A9 proteins as key regulators that control the iOCP response during chronic inflammation. Furthermore, we demonstrated that the response of iOCPs but not that of hOCPs was abrogated in tnf-α-/- mice, in correlation with attenuated IBL. Our findings suggest a central role for iOCPs in IBL induction. iOCPs can serve as potential biomarkers for IBL detection and possibly as new therapeutic targets to combat IBL in a wide range of inflammatory conditions.

THE VALUE OF BLINK REFLEX IN EARLY DIAGNOSIS OF MULTIPLE SCLEROSIS.

The aim was to determine differences of blink reflex in clinically definite multiple sclerosis (CDMS) and clinically isolated syndrome (CIS) in patients presented with symptoms and signs of brainstem impairment. The study included 20 patients diagnosed with CDMS, 20 with CIS, and 20 healthy controls. We recorded latencies of early (R1) and late component ipsilaterally (R2) and contralaterally (R2'), and occurrence of irritative component (R3). We analyzed data on sex, age, signs of brainstem impairment and magnetic resonance imaging (MRI) findings for the presence of brainstem demyelinating lesions. There was no statistically significant difference between patient groups according to sex, age, symptoms of brainstem involvement and MRI findings. There was no statistically significant difference in R1 component latencies and R2 latencies on the right side. Latencies of R2 on the left and R2' on the right were statistically longer in CDMS group. There was no difference in the appearance of R3 component. In conclusion, blink reflex was found to be a very sensitive and useful diagnostic tool in the assessment of brainstem structures, especially because abnormalities are seen not only in CDMS but also in CIS. Slowing of the late component as a sign of dysfunction in the efferent part of the reflex arc is not very specific but is a highly sensitive finding.

Strenuous Physical Training, Physical Fitness, Body Composition and Bacteroides to Prevotella Ratio in the Gut of Elderly Athletes.

Regular physical activity seems to have a positive effect on the microbiota composition of the elderly, but little is known about the added possible benefits of strenuous endurance training. To gain insight into the physiology of the elderly and to identify biomarkers associated with endurance training, we combined different omics approaches. We aimed to investigate the gut microbiome, plasma composition, body composition, cardiorespiratory fitness, and muscle strength of lifetime elderly endurance athletes (LA) age 63.5 (95% CI 61.4, 65.7), height 177.2 (95% CI 174.4, 180.1) cm, weight 77.8 (95% CI 75.1, 80.5) kg, VO2max 42.4 (95% CI 39.8, 45.0) ml.kg-1.min-1 (n = 13) and healthy controls age 64.9 (95% CI 62.1, 67.7), height 174.9 (95% CI 171.2, 178.6) cm, weight 83.4 (95% CI 77.1, 89.7) kg, VO2max 28.9 (95% CI 23.9, 33.9), ml.kg-1.min-1 (n = 9). Microbiome analysis was performed on collected stool samples further subjected to 16S rRNA gene analysis. NMR-spectroscopic analysis was applied to determine and compare selected blood plasma metabolites mostly linked to energy metabolism. The machine learning (ML) analysis discriminated subjects from the LA and CTRL groups using the joint predictors Bacteroides 1.8E + 00 (95% CI 1.1, 2.5)%, 3.8E + 00 (95% CI 2.7, 4.8)% (p = 0.002); Prevotella 1.3 (95% CI 0.28, 2.4)%, 0.1 (95% CI 0.07, 0.3)% (p = 0.02); Intestinimonas 1.3E-02 (95% CI 9.3E-03, 1.7E-02)%, 5.9E-03 (95% CI 3.9E-03, 7.9E-03)% (p = 0.002), Subdoligranulum 7.9E-02 (95% CI 2.5E-02, 1.3E-02)%, 3.2E-02 (95% CI 1.8E-02, 4.6E-02)% (p = 0.02); and the ratio of Bacteroides to Prevotella 133 (95% CI -86.2, 352), 732 (95% CI 385, 1079.3) (p = 0.03), leading to an ROC curve with AUC of 0.94. Further, random forest ML analysis identified VO2max, BMI, and the Bacteroides to Prevotella ratio as appropriate, joint predictors for discriminating between subjects from the LA and CTRL groups. Although lifelong endurance training does not bring any significant benefit regarding overall gut microbiota diversity, strenuous athletic training is associated with higher cardiorespiratory fitness, lower body fat, and some favorable gut microbiota composition, all factors associated with slowing the rate of biological aging.

The diverse roles of myeloid derived suppressor cells in mucosal immunity.

The mucosal immune system plays a vital role in protecting the host from the external environment. Its major challenge is to balance immune responses against harmful and harmless agents and serve as a 'homeostatic gate keeper'. Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of undifferentiated cells that are characterized by an immunoregulatory and immunosuppressive phenotype. Herein we postulate that MDSCs may be involved in shaping immune responses related to mucosal immunity, due to their immunomodulatory and tissue remodeling functions. Until recently, MDSCs were investigated mainly in cancerous diseases, where they induce and contribute to an immunosuppressive and inflammatory environment that favors tumor development. However, it is now becoming clear that MDSCs participate in non-cancerous conditions such as chronic infections, autoimmune diseases, pregnancy, aging processes and immune tolerance to commensal microbiota at mucosal sites. Since MDSCs are found in the periphery only in small numbers under normal conditions, their role is highlighted during pathologies characterized by acute or chronic inflammation, when they accumulate and become activated. In this review, we describe several aspects of the current knowledge characterizing MDSCs and their involvement in the regulation of the mucosal epithelial barrier, their crosstalk with commensal microbiota and pathogenic microorganisms, and their complex interactions with a variety of surrounding regulatory and effector immune cells. Finally, we discuss the beneficial and harmful outcomes of the MDSC regulatory functions in diseases affecting mucosal tissues. We wish to illuminate the pivotal role of MDSCs in mucosal immunity, the limitations in our understanding of all the players and the intricate challenges stemming from the complex interactions of MDSCs with their environment.

Pharmacogenomics at the center of precision medicine: challenges and perspective in an era of Big Data.

Pharmacogenomics (PGx) is one of the core elements of personalized medicine. PGx information reduces the likelihood of adverse drug reactions and optimizes therapeutic efficacy. St Catherine Specialty Hospital in Zagreb/Zabok, Croatia has implemented a personalized patient approach using the RightMed® Comprehensive PGx panel of 25 pharmacogenes plus Facor V Leiden, Factor II and MTHFR genes, which is interpreted by a special counseling team to offer the best quality of care. With the advent of significant technological advances comes another challenge: how can we harness the data to inform clinically actionable measures and how can we use it to develop better predictive risk models? We propose to apply the principles artificial intelligence to develop a medication optimization platform to prevent, manage and treat different diseases.

Strategy for improved therapeutic efficiency of curcumin in the treatment of gastric cancer.

Gastric cancer is a common oncological disease. Although enormous efforts have been expended, possible therapeutic modalities are still limited. For this reason, new therapeutic approaches and agents are highly requested and intensively developed. One strategy is the application of natural agents, such as curcumin, with proven anticancer effects and low toxicity for patients. Therefore, this review discusses the potential application of curcumin in the therapy of gastric cancer and its potential incorporation in therapeutic regimens. Because one of the largest impediments for widespread curcumin application is its limited bioavailability (caused mainly by its very low water solubility), studied strategies (drug delivery systems and curcumin derivatization) aimed to solve this obstacle are discussed in more detail.

Pentamethinium salts as ligands for cancer: Sulfated polysaccharide co-receptors as possible therapeutic target.

A series of pentamethinium salts with benzothiazolium and indolium side units comprising one or two positive charges were designed and synthesized to determine the relationships among the molecular structure, charge density, affinity to sulfated polysaccharides, and biological activity. Firstly, it was found that the affinity of the pentamethinium salts to sulfated polysaccharides correlated with their biological activity. Secondly, the side heteroaromates displayed a strong effect on the cytotoxicity and selectivity towards cancer cells. Finally, doubly charged pentamethinium salts possessing benzothiazolium side units exhibited remarkably high efficacy against a taxol-resistant cancer cell line.

Continuing war on pain: a personalized approach to the therapy with nonsteroidal anti-inflammatory drugs and opioids.

Successful pain management requires the delivery of analgesia with minimal risk of adverse drug reactions. Nonsteroidal anti-inflammatory drugs and opioids remain the mainstay of treatment for the majority of patients. Unfortunately, almost 50% of all patients experience inadequate pain relief and serious side effects. Allelic variants in genes coding for target proteins, transporters and enzymes, which govern analgesic drugs action and their fate in the organism, might explain inter-individual variability in pain severity and in drug-induced pain relief and toxicities. Additionally, it seems that epigenetic changes contribute to the highly variable response to pain treatment. Therefore, pharmacogenomic testing might be a valuable tool for personalization of pain treatment, with a multidisciplinary team approach involved.

Epigenetic agents in combined anticancer therapy.

In the last decade, epigenetic drugs (such as inhibitors of DNA methyltransferases and histone deacetylases) have been intensively used for cancer treatment. Their applications have shown high anticancer effectivity and tolerable side effects. However, they are unfortunately not effective in the treatment of some types and phenotypes of cancers. Nevertheless, several studies have demonstrated that problems of drug efficacy can be overcome through the combined application of therapeutic modulates. Therefore, combined applications of epigenetic agents with chemotherapy, radiation therapy, immunotherapy, oncolytic virotherapy and hyperthermia have been presented. This review summarizes and discusses the general principles of this approach, as introduced and supported by numerous examples. In addition, predictions of the future potential applications of this methodology are included.

Optical probes and sensors as perspective tools in epigenetics.

Modifications of DNA cytosine bases and histone posttranslational modifications play key roles in the control of gene expression and specification of cell states. Such modifications affect many important biological processes and changes to these important regulation mechanisms can initiate or significantly contribute to the development of many serious pathological states. Therefore, recognition and determination of chromatin modifications is an important goal in basic and clinical research. Two of the most promising tools for this purpose are optical probes and sensors, especially colourimetric and fluorescence devices. The use of optical probes and sensors is simple, without highly expensive instrumentation, and with excellent sensitivity and specificity for target structural motifs. Accordingly, the application of various probes and sensors in the recognition and determination of cytosine modifications and structure of histones and histone posttranslational modifications, are discussed in detail in this review.

The Croatian Model of Integrative Prospective Management of Epilepsy and Pregnancy

Epilepsy is the most common neurological complication in pregnancy. Women with epilepsy have a higher risk of complications in pregnancy. In Croatia, women with epilepsy are treated by neurologists at tertiary centers according to the place of residence. We prospectively followed-up pregnancies in women with epilepsy and healthy controls, and analyzed the factors responsible for their delivery outcomes and development of their babies. Healthy pregnant women had a higher level of education and economic status, but pregnant women with epilepsy took folic acid in a higher proportion than controls, possibly due to timely preconception counseling. Complications during pregnancy depended on the number of antiepileptic drugs and epilepsy control. We noticed some behavioral and cognitive aspects in children exposed in utero to valproic acid, which required follow up. The rate of congenital malformations was not increased. In conclusion, women with epilepsy should receive preconception counseling about the risk for pregnancy, but also about the possibilities to minimize that risk. We have introduced a model of integrative management of pregnancy and epilepsy based on close collaboration among different clinical experts in Croatia, in order to provide prompt counseling and timely intervention.

New method for recognition of sterol signalling molecules: methinium salts as receptors for sulphated steroids.

In this work, we studied indolium and benzothiazolium pentamethine salts 1-3 as novel type of receptors for the recognition of sulphated signalling molecules (sulphated steroids: oestrone, pregnenolone and cholesterol sulphate). A recognition study was performed in an aqueous medium (1mM phosphate buffer (H2O:MeOH; 99:1 (v/v))) at pH 7.34. The tested salts displayed a high affinity for these sulphated analytes, mainly for cholesterol sulphate. However, no interaction between the salts and control, non-sulphated sterol analytes (cholesterol and bile acid) was observed. The highest affinity for the sulphated steroids was observed for benzothiazole salt 1. This salt also displayed different spectral behaviour from that observed for carbocyanine salts 2 and 3. In this presence of cholesterol sulphate, benzothiazole salt 1 displayed significant spectral changes depending on the medium used: a blue shift in the aqueous medium and a red shift in the methanolic one (H2O:MeOH; 2:1 (v/v)). Subsequently preliminary in vivo study showed that, salt 1 significantly inhibits a growth of breast carcinoma on Nu/nu mice model.

Effects of high tidal volume mechanical ventilation on production of cytokines, iNOS, and MIP-1ß proteins in pigs.

The aim of this study was to investigate longitudinal changes of the pulmonary inflammatory process as a result of mechanical stress due to mechanical ventilation. The concentrations of IL-8, TNF-α, MIP-1ß, nitrites/nitrates, and inducible nitric oxide synthases (iNOS) were investigated indicate in bronchoalveolar lavage (BAL). Twenty-three piglets were divided into three groups. Group I: animals breathing spontaneously; group II: mechanical ventilation (tidal volume (TV) = 7 mL/kg, PEEP = 5 cmH(2)O); group III: mechanical ventilation (TV = 15 mL/kg, PEEP = 0 cmH(2)0). Concentrations of BAL nitrites/nitrates from groups II and III increased during the first hour of mechanical ventilation (P = .03 and .02, respectively). The highest expression of iNOS was observed during the first hour in groups II and III. IL-8 concentration increased significantly in groups II and III. Production of TNF-α increased significantly in group III during the second and third hour (P = .01). Concentration of MIP-1ß was significantly increased in groups II and III after the first hour (P = .012 and P = .008, respectively).

Visual evoked potentials in patients with pineal gland cyst.

AIMS: The functional effect of the pineal gland cyst is difficult to evaluate with visual field examination. The aim of this study is to investigate the usefulness of visual evoked potentials (VEP) in patients with pineal gland cyst due to the possible compression on the visual pathway. SUBJECTS AND METHODS: Black-and-white pattern-reversal checkerboard VEP were recorded in 75 patients (50 females and 25 males, mean age 26.3 ± 15.7 and 25.6 ± 17.6 years, respectively) with pineal gland cyst detected on magnetic resonance of the brain (subject group) and 75 age and sex-matched control subjects (control group). Amplitudes and P100 latencies were collected and later grouped as: (1) normal finding; (2) prechiasmal; (3) prechiasmal and postchiasmal; and (4) postchiasmal dysfunction. RESULTS: P100 latencies differed significantly between subject (110.26 ± 13.23 ms) and control group (101.01 ± 5.36 ms) (p < 0.01). Findings of the VEP differed significantly (p < 0.01) between subject and control group, mainly due to the postchiasmal dysfunction frequency in subject group. Findings of the VEP differed significantly according to the pineal gland cyst volume (p = 0.006) with more frequent postchiasmal dysfunctions among subjects with larger cysts. Postchiasmal changes were significantly more frequent in patients with described compression of the cyst on surrounding brain structures (p = 0.016). CONCLUSIONS: Postchiasmal dysfunction on VEP can be seen in patients with pineal gland cyst, mostly with larger cysts and with compression of the cyst on surrounding brain structures. VEP serve as a useful method to determine functional impairment of the visual pathway in patients with pineal gland cyst.

Diagnostic value of some less frequently considered blink reflex parameters in idiopathic trigeminal neuralgia.

AIM OF THE STUDY: We have done a study investigating the value of some less frequently considered Blink reflex parameters for establishing the diagnosis of idiopathic trigeminal neuralgia. PATIENTS: The study was done on 50 patients suffering from idiopathic trigeminal neuralgia, diagnosed according to the guidelines of the International Classification of Headache Disorders, with no other apparent illness. METHODS: We have stimulated the supraorbital nerve at the forehead (foramen n. supraorbitalis) and recorded the reflex response on both mm. orbiculares oculi. Incidence of following findings was determined: (1) occurrence of ipsilateral R3 component, (2) prolonged duration (>25 ms) of R2 when stimulating the affected side and (3) occurrence of R1 component during the stimulation of contralateral supraorbital nerve. We have compared these findings to those of 50 healthy subjects from the control group (Chi-square, p < 0.05). Sensitivity, specificity and diagnostic value for individual parameters were determined. RESULTS AND CONCLUSION: All three parameters tested proved to have a significantly higher incidence in the group of subjects. The occurrence of R3 component on the affected side showed the highest diagnostic value. SIGNIFICANCE: We believe these findings could electrophysiologically reinforce the clinically established diagnosis of idiopathic trigeminal neuralgia.

Characterization of ovine Toll-like receptor 9 protein coding region, comparative analysis, detection of mutations and maedi visna infection.

One of the major roles of innate immunity system is the recognition and the determination of the nature of the antigen. This ability is encompassed by specific receptors as Toll-like receptors (TLRs). TLR9 recognizes bacterial and viral CpG motifs, while their potent immunostimulation effect seems to be promising for lentiviral therapies. Recent studies, however, show the presence of a big polymorphism within the TLR genes and the linkage between substitutions and susceptibility to various infections. Moreover, different recognition ability seems to be utilized by different species and possibly breeds. In this study, we characterized the protein coding region of ovine TLR9 gene. By using comparative analysis of two closely related species and humans, we suggest, which characteristics of protein could be responsible for altered recognition. Furthermore, analyzing the presence of the substitutions, we show the intraspecies polymorphism and its possible implications, while attempting to define the association of discovered substitutions with the maedi visna infection.