The Role of the Intestinal Epithelium in the “Weep and Sweep” Response during Gastro—Intestinal Helminth Infections

Helminths are metazoan parasites infecting around 1.5 billion people all over the world. During coevolution with hosts, worms have developed numerous ways to trick and evade the host immune response, and because of their size, they cannot be internalized and killed by immune cells in the same way as bacteria or viruses. During infection, a substantial Th2 component to the immune response is evoked which helps restrain Th1-mediated tissue damage. Although an enhanced Th2 response is often not enough to kill the parasite and terminate an infection in itself, when tightly coordinated with the nervous, endocrine, and motor systems it can dislodge parasites from tissues and expel them from the gut. A significant role in this “weep and seep” response is attributed to intestinal epithelial cells (IEC). This review highlights the role of various IEC lineages (enterocytes, tuft cells, Paneth cells, microfold cells, goblet cells, and intestine stem cells) during the course of helminth infections and summarizes their roles in regulating gut architecture and permeability, and muscle contractions and interactions with the immune and nervous system.

State-of-the-art preclinical evaluation of COVID-19 vaccine candidates

The coronavirus disease 2019 (COVID-19) results from the infection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and primarily affects the respiratory tissue. Since first reported from Wuhan, China in December 2019, the virus has resulted in an unprecedented pandemic. Vaccination against SARS-CoV-2 can control the further spread of the ongoing pandemic by making people immunised to SARS-CoV-2. Several vaccines have been approved for use in clinics, a lot many are in different stages of development. Diligent interpretations from the preclinical evaluation are crucial to identify the most effective and safest vaccine candidates. Multiple vaccine candidates/variants have been tested in small animal models with relative ease and further in non-human primate models before being taken into clinical development. Here, we review the state-of-the-art strategies employed for a thorough preclinical evaluation of COVID-19 vaccine candidates. We summarise the methods in place to identify indicators which make the vaccine candidate effective in controlling SARS-CoV-2 infection and/or COVID-19 and are safe for administration as inferred by their (1) biophysical/functional attributes (antigen expression, organization, functionality, and stability); (2) immunogenicity in animal models and protective correlates [SARS-CoV-2 specific binding/neutralising immunoglobulin titer, B/T-cell profiling, balanced T-helper type-1 (Th1) or type-2 (Th2) response (Th1:Th2), and anamnestic response]; (3) protective correlates as interpreted by controlled pathology of the respiratory tissue (pulmonary clinical and immunopathology); and finally, (4) strategies to monitor adverse effects of the vaccine candidates.

Schistosome immunomodulators

Schistosomes are long lived, intravascular parasitic platyhelminths that infect >200 million people globally. The molecular mechanisms used by these blood flukes to dampen host immune responses are described in this review. Adult worms express a collection of host-interactive tegumental ectoenzymes that can cleave host signaling molecules such as the “alarmin” ATP (cleaved by SmATPDase1), the platelet activator ADP (SmATPDase1, SmNPP5), and can convert AMP into the anti-inflammatory mediator adenosine (SmAP). SmAP can additionally cleave the lipid immunomodulator sphingosine-1-phosphate and the proinflammatory anionic polymer, polyP. In addition, the worms release a barrage of proteins (e.g., SmCB1, SjHSP70, cyclophilin A) that can impinge on immune cell function. Parasite eggs also release their own immunoregulatory proteins (e.g., IPSE/α1, omega1, SmCKBP) as do invasive cercariae (e.g., Sm16, Sj16). Some schistosome glycans (e.g., LNFPIII, LNnT) and lipids (e.g., Lyso-PS, LPC), produced by several life stages, likewise affect immune cell responses. The parasites not only produce eicosanoids (e.g., PGE2, PGD2—that can be anti-inflammatory) but can also induce host cells to release these metabolites. Finally, the worms release extracellular vesicles (EVs) containing microRNAs, and these too have been shown to skew host cell metabolism. Thus, schistosomes employ an array of biomolecules—protein, lipid, glycan, nucleic acid, and more, to bend host biochemistry to their liking. Many of the listed molecules have been individually shown capable of inducing aspects of the polarized Th2 response seen following infection (with the generation of regulatory T cells (Tregs), regulatory B cells (Bregs) and anti-inflammatory, alternatively activated (M2) macrophages). Precisely how host cells integrate the impact of these myriad parasite products following natural infection is not known. Several of the schistosome immunomodulators described here are in development as novel therapeutics against autoimmune, inflammatory, and other, nonparasitic, diseases.

D-Pinitol Attenuated Ovalbumin-induced Allergic Rhinitis in Experimental Mice Via Balancing Th1/Th2 Response

Allergic rhinitis (AR) is a complex, chronic immunoinflammatory disorder of the membrane lining of the nasal mucosa. D-Pinitol is considered a cyclic polyol with a potential effect against various allergies. In the present study, we evaluated the anti-allergic effect of pinitol on ovalbumin (OVA)-induced AR model in mice. BALB/c mice were initially sensitized with an intraperitoneal injection of OVA and divided into 5 groups (n=18, in each group) for a treating schedule of distilled water (DW), montelukast (10 mg/kg), and pinitol (5, 10, and 20 mg/kg) through the mouth. Two saline-injected groups were considered as controls by orally administrating DW and pinitol 20. Thereafter, test and control groups were intranasally challenged by OVA and saline, respectively. Our results showed that the OVA challenge caused a marked elevation in AR symptoms like nasal rubbing, sneezing, and discharge which were remarkably diminished using pinitol (10 and 20 mg/kg) and the results were comparable with montelukast. Additionally, increased levels of total and OVA-specific serum Immunoglobulin (Ig) E and IgG1 were significantly attenuated by pinitol as compared to the control group but not the montelukast group. In AR-induced mice, pinitol had significant modulatory effects on representative markers of Th2 (GATA binding protein 3), signal transducer and activator of transcription-6, Interleukins (IL)-4, IL-5, IL-13, suppressors of cytokine signaling 1, Toll-like receptor 4, and myeloid differentiation factor 88), and Type 1 T helper (Th1) immune responses (T-box protein expressed in T cells and Interferon-gamma) as well as the histopathological aberrations induced in the nasal mucosa. In conclusion, Pinitol had potential effects on OVA-induced AR mice through amelioration of nasal symptoms and balancing the Th1/Th2 immune responses during the allergic rhinitis condition.

Assessment of Allergen-Responsive Regulatory T Cells in Experimental Asthma Induced in Different Mouse Strains

Background. Regulatory T cells (Tregs) are important in regulating responses to innocuous antigens, such as allergens, by controlling the Th2 response, a mechanism that appears to be compromised in atopic asthmatic individuals. Different isogenic mouse strains also have distinct immunological responses and susceptibility to the experimental protocols used to develop lung allergic inflammation. In this work, we investigated the differences in the frequency of Treg cell subtypes among A/J, BALB/c, and C57BL/6, under normal conditions and following induction of allergic asthma with ovalbumin (OVA). Methods. Subcutaneous sensitization followed by 4 consecutive intranasal OVA challenges induced asthma characteristic changes such as airway hyperreactivity, inflammation, and production of Th2 cytokines (IL-4, IL-13, IL-5, and IL-33) in the lungs of only A/J and BALB/c but not C57BL/6 strain and evaluated by invasive whole-body plethysmography, flow cytometry, and ELISA, respectively. Results. A/J strain naturally showed a higher frequency of CD4+IL-10+ T cells in the lungs of naïve mice compared to the other strains, accompanied by higher frequencies of CD4+IL-4+ T cells. C57BL/6 mice did not develop lung inflammation and presented higher frequency of CD4+CD25+Foxp3+ Treg cells in the bronchoalveolar lavage fluid (BALF) after the allergen challenge. In in vitro settings, allergen-specific stimulation of mediastinal LN (mLN) cells from OVA-challenged animals induced higher frequency of CD4+IL-10+ Treg cells from A/J strain and CD4+CD25+Foxp3+ from C57BL/6. Conclusions. The observed differences in the frequencies of Treg cell subtypes associated with the susceptibility of the animals to experimental asthma suggest that CD4+CD25+Foxp3+ and IL-10-producing CD4+ Treg cells may play different roles in asthma control. Similar to asthmatic individuals, the lack of an efficient regulatory response and susceptibility to the development of experimental asthma in A/J mice further suggests that this strain could be preferably chosen in experimental models of allergic asthma.

Thalidomide Exerts Anti-Inflammatory Effects in Cutaneous Lupus by Inhibiting the IRF4/NF-ҡB and AMPK1/mTOR Pathways

Thalidomide is effective in patients with refractory cutaneous lupus erythematosus (CLE). However, the mechanism of action is not completely understood, and its use is limited by its potential, severe side-effects. Immune cell subset analysis in thalidomide’s CLE responder patients showed a reduction of circulating and tissue cytotoxic T-cells with an increase of iNKT cells and a shift towards a Th2 response. We conducted an RNA-sequencing study using CLE skin biopsies performing a Therapeutic Performance Mapping System (TMPS) analysis in order to generate a predictive model of its mechanism of action and to identify new potential therapeutic targets. Integrating RNA-seq data, public databases, and literature, TMPS analysis generated mathematical models which predicted that thalidomide acts via two CRBN-CRL4A- (CRL4CRBN) dependent pathways: IRF4/NF-ҡB and AMPK1/mTOR. Skin biopsies showed a significant reduction of IRF4 and mTOR in post-treatment samples by immunofluorescence. In vitro experiments confirmed the effect of thalidomide downregulating IRF4 in PBMCs and mTOR in keratinocytes, which converged in an NF-ҡB reduction that led to a resolution of the inflammatory lesion. These results emphasize the anti-inflammatory role of thalidomide in CLE treatment, providing novel molecular targets for the development of new therapies that could avoid thalidomide’s side effects while maintaining its efficacy.

221 Effects of energy drinks on inflammatory response: study in vivo in rats

In recent years, the consumption of energy drinks (EDs) has increased constantly among young people because of their capacity to enhance alertness and improve mental and physical performance, reducing fatigue. EDs are beverages containing a high and variable amount of caffeine, which exerts effects on many tissues of the cardiovascular system. In addition to caffeine, they contain several other psychoactive substances including the amino acid taurine, the glucose derivative glucuronolactone, as well as herbal extracts such as guaranà (another source of caffeine, with caffeine-like effects) and ginseng, often present in not well-known concentration. In our project we aim to evaluate whether the consumption of EDs causes alterations in the organs involved in their contact, absorption and metabolism in an animal model, i.e. the rat. We used 28 Sprague Dawley adult male rats, weighing 230–250 g and fed with a standard laboratory diet, randomly divided into four groups (N = 7). Every group received a different treatment (ED, soda-cola, sweetened coffee or water-controls-) for 5 days. All animals were anesthetized and underwent histological analysis and blood sampling at the end of the treatment. We observed eosinophilic infiltrates in gastrointestinal tract, but not in cardiovascular system. We quantified various indicators of tissue damage and cytokines in plasma, including ICAM-1, L-selectin, TIMP-1, VEGF, IL-10, IL-2, IL-4, IL-6, IL-1β, IL-13, IL-33, TNF-α, and IFN-γ. In contrast with the eosinophilic infiltration, we did not detect a systemic increase of Th2 cytokines (i.e. IL-4 and IL-13) after treatment: thus, even if the experiments evaluating the possible presence of an unbalanced Th2 response in the tissues featured by eosinophilic infiltration are still lacking, we exclude further deepening on Th2 immune responses. Interestingly, we observed a decrease of TIMP-1 in plasma from rats orally supplemented with ED, soda-cola or sweetened coffee compared to animals treated with water. TIMP-1 is a major player in preserving tissue integrity and controlling wound healing by balancing the enzymatic activity of matrix metalloproteinases (MMPs) and regulating extracellular matrix turnover. Moreover, elevated levels of the adhesion molecules ICAM-1 and L-selectin were found in plasma from rats receiving ED or soda-cola, together with a high concentration of IL-33 in animals assuming the ED. The circulating form of ICAM-1 is associated with inflammation, particularly due to endothelial damage, and L-selectin and IL-33 play an important role in inflammatory conditions as well. These observations suggest that the consumption of EDs could alter the architecture, and hence function, of the organs involved in their contact, absorption and metabolism. Indeed, the alterations we observed in the periphery could reflect the establishment of inflammation and deregulated mechanisms of damage repair locally in the target tissues.

Biotherapy of mice’s serum 13cH modifies parasitological and immunological parameters of Trypanosoma cruzi infection.

Background: T. cruzi biotherapies’ alter the infection course by this protozoan [1,2], fact that encourages the evaluation of other highly diluted medicines which modulates host’s immune system. Aim: Evaluate effect of biotherapy produced from mices’s serum in experimental infection by T. cruzi. Methodology: A blind, randomized and controlled study was performed. Animals: 60 male Swiss mice, four weeks old were inoculated intraperitoneally with 1400 trypomastigotes-Y strain and divided: MNI: mice non-infected by T. cruzi; IC: treated with hydroalcoholic solution 7%; BSI13cH: treated with mice’s serum infected by T. cruzi 13cH. Biotherapies: produced from mice’s serum infected by T. cruzi in 13cH dynamization [3]. Treatment: mice were treated 48 hours before and after infection. Subsequently animals were treated 56/56 hours until 9th day of infection (d.i). The medicine was diluted in water (1/100mL) and offered ad libitum, for 16 hours. Parasitological parameters: were evaluated pre-patent and patent period, parasitemia peak, total parasitemia and mortality [4]. Cytokine dosage: IL-4, IL-17, TNF-α and IFN-γ were measured in serum on 0, 8th and 12th d.i., by enzyme immunoassay. Ethics: study was approved by Ethics Committee for Experiments in Animals/UEM. Statistic: data were compared with Mann Whitney or Student t test, significance 5%. Results: BSI13cH showed tendency to increase total parasitemia (p=0.06) and parasitemia peak (p=0.05), with lower patent period (p=0.03) and higher mortality (p=0.03) compared to IC. In dosage of cytokines BSI13cH group showed on 0 d.i. a decrease in IL-17 (p = 0.02) and increased IL-4 (p = 0.01) compared to MNI (baseline value), probably caused by modulation of medication administration 48 hours before infection. IL-17 concentration didn’t vary throughout the infection to BSI13cH, different IC that tended to decrease concentration of cytokine on 8th d.i. IL-4 increased significantly on 0 d.i. to BSI13cH, with subsequent return to baseline values. IC group didn’t change significantly IL-4 value along the infection. IFN-γ concentration on 12th d.i. to BSI13cH was lower (p = 0.00) than IC, which increased this cytokine on 8th and 12th di. TNF-α concentration of BSI13cH followed the same evolution as IC, with an increase on 8th and 12th d.i. (Figure 1). The medicine seems initially promote Th2 response (IL-4), hindering the development of effective Th1 response (INF-γ), causing an increase of parasitemia and animals' death. Conclusions: BSI13cH demonstrated effect in experimental infection by T. cruzi with increased parasitemia, animals’ premature death and modulated immune response differently of IC.

Characterization of allergic inflammation in chronic uterine cervicitis

Female genital tract chronic inflammation is common in clinics; the pathogenesis is not fully understood yet. House dust mite (HDM) involves the pathogenesis of many chronic diseases in human. This study aims to identify HDM-specific allergic response in the cervix of patients with cervical inflammation. Patients (n=80) with chronic cervicitis (CC) and non-CC control (NC) subjects (n=80) were recruited into this study. Vaginal lavage fluids (VLF) were collected from CC patients and NC subjects. Cellular components and fluid part of VLF were separated by centrifugation, and analyzed by flow cytometry and enzyme-linked immunosorbent assay. We found that a portion (52 out of 80) of CC patients responded to HDM, manifesting positive skin prick test, and HDM-specific IgE and IgG was detected in the VLF (designated CCp patients). VLF of CCp patients showed a Th2 dominant profile. HDM-specific Th2 cells were detected in VLF in CCp patients. Exposure to HDM in the culture induced proinflammatory cytokine release from CCp VLF CD4 + T cells. Exposure to CCp VLF CD4 + T cell-conditioned medium induced de novo Th2 response. Direct exposure to HDM induced allergic response in the cervix of CCp patients. In summary, a portion of CC patients respond to HDM challenge in the cervix. Exposure to HDM induces an allergy-like response in the cervix of CCp patients.