Laucaitytė, Goda

The gastrointestinal (GI) tract is a major barrier against pathogens, including viruses. The antiviral responses in the GI epithelium have been broadly investigated, but data on the contribution of the stromal cells remain scarce. Melatonin, widely used to treat insomnia, has recently been proposed as an antiviral agent, yet its effect in the GI tract remains poorly understood. We compared the antiviral responses in Caco-2 monocultures and co-cultures with intestinal fibroblasts (HSIFs) and endothelial cells (HUVECs) after stimulation using Poly I:C. We evaluated the apoptosis, proliferation, key antiviral markers (IRF1, IRF3, IFNs, TBK1, STAT3), and mitochondrial and peroxisomal activation with and without melatonin. The Caco-2 cells cultured with the HSIFs and HUVECs demonstrated enhanced proliferation and reduced Poly I:C-induced apoptosis. The co-culture exhibited a more rapid IRF3-IFNλ1 response, higher TBK1 expression, and enhanced peroxisomal activation compared to these properties in the monoculture. Melatonin further reduced apoptosis and modulated organelle-specific antiviral signaling by suppressing peroxisomal activation and promoting mitochondrial activity. Reduced peroxisomal activation was associated with decreased TBK1, IRF3, and IFNλ1 levels and altered STAT3 signaling. These effects were more pronounced when melatonin was applied post-stimulation compared to that under prophylactic use. Fibroblast–endothelial interactions amplify the antiviral responses in the intestinal epithelial cells by activating the TBK1–IRF3–IFNλ1 axis. Melatonin modulates these responses, highlighting its therapeutic potential in viral GI infections.

Melatonin is a hormone naturally produced by the body that has recently been found to have antiviral properties. However, its antiviral mechanisms are not entirely understood. Using Caco-2 cells, we developed a gastrointestinal organoid model to investigate the impact of melatonin on cellular organoid culture response to Poly I:C-induced viral inflammation in the gastrointestinal tract. Melatonin was found to have different effect when applied as a pretreatment before the induction of viral inflammation or as a treatment after it. Melatonin pretreatment after Poly I:C stimulation did not protect organoids from size reduction but enhanced cell proliferation, especially when lower (1 and 10 µM) melatonin concentrations were used. On the other hand, treatment with melatonin after the induction of viral inflammation helped to maintain the size of the organoids while reducing cell proliferation. In pretreated cells, reduced IFNLR1 expression was found, while melatonin treatment increased IFNLR1 expression and reduced the production of viral cytokines, such as IFNλ1 and STAT1-3, but did not prevent from apoptosis. The findings of this study emphasize the importance of type III IFNs in antiviral defense in epithelial gastrointestinal cells and shed more light on the antiviral properties of melatonin as a potential therapeutic substance.

Background Melatonin is a natural compound produced by the brain. Recently, it has been studied as a treatment option for various infectious diseases due to its possible antiviral properties. Acute viral gastrointestinal infections are a great burden for the healthcare system. Our study aimed to develop a gastrointestinal organoid model and to evaluate melatonin as potential therapeutic option in Poly I:C stimulated Caco-2 cells. Methods The human intestinal epithelial Caco-2 cell line passages 10 to 20 was used for the in vitro infection model. The cells were cultured in DMEM media at 37°C 5% CO2 in an incubator for 21 days until they formed organoids. The monolayer was then pretreated with melatonin (at 1, 10, 50 or 100 μM concentrations) for 24 hours following by stimulation with 1, 10 or 50 ng/ml Poly I:C for another 24 hours. Further, organoids were evaluated by size, and quantity. Proliferation was measured via CCK-8. FACS was used for apoptosis investigation and STAT1-3 measurements. Protein expression (IFNβ, IFNλ) was tested via ELISA according to manufacturer's instructions. Statistical analysis was performed using SPSS 29.0. Results The number of organoids was higher in pretreated cells than controls, but the size of organoids was substantially reduced. Higher proliferation was seen in melatonin-pretreated cells when they were pretreated and stimulated with 1 or 10 ng/ml Poly I:C; however, at higher Poly I:C concentrations (50 ng/ml), the proliferation-promoting effect was not sustained. Interestingly, melatonin did not reduce the cell apoptosis caused by the infection. INFλ expression was higher in melatonin-pretreated Poly I:C-stimulated cells, especially at the lower Poly I:C concentrations (Figure 1). An increase in INFλ amounts correlated with higher concentrations of infection. Meanwhile, a significant decrease in STAT1 and STAT2 was observed with higher melatonin concentrations. The same tendencies were noted with STAT3. Conclusions Our data show that melatonin promotes cell proliferation and induces antiviral effect via type III IFN pathway. This suggests a promising treatment option in acute viral intestinal diseases. However, more studies need to be done.

Background: Melatonin is an endogenous hormone recently found to have antiviral properties and investigated as a therapeutic option for various viral diseases. However, the results of the studies are conflicting. We aimed to evaluate the antiviral effect of melatonin in Poly(I:C) stimulated gastrointestinal cells. Method: The human intestinal epithelial Caco-2 cell line (passages 10-20) was used for the in vitro infection model. The cells were cultured in DMEM media at 37°C 5% CO2 in an incubator for 21 days until organoid formation. The monolayer was then either pretreated with melatonin (at 1, 10, 50 or 100 μM concentrations) for 24 hours following by stimulation with different concentrations of Poly(I:C) for another 24 hours or treated with melatonin for 24 hours at above concentrations after stimulation with Poly(I:C) for 24 hours. IFNλ expression was tested using ELISA. Interferon lambda receptor 1 (IFNLR1) expression was tested using flow cytometry. Statistical analysis was performed using SPSS 29.0. Results: Caco-2 cells were found to produce IFNλ in response to Poly(I:C) stimulation and the amounts of INFλ directly correlated with higher concentrations of infection. IFNλ production was higher in melatonin pretreated cells compared to those treated with melatonin after Poly(I:C) stimulation. Melatonin reduced IFNLR1 expression in infected cells. In all of the studied conditions pretreatment with melatonin induced significantly higher IFNLR1 expression in Poly(I:C)-stimulated melatonin-pretreated cells compared to cells treated with melatonin after Poly(I:C) stimulation. In noninfected cells melatonin induced IFNLR1 expression but did not induce IFNλ production. Conclusions/Learning Points: Our data show that melatonin induces IFNλ expression via increased IFNLR1 expression in infected cells. Melatonin as a pretreatment may be a potential therapeutic option priming cells for higher antiviral effect. However, further studies need to be done.

OBJECTIVES:Our study aimed to evaluate possible antiviral properties of melatonin when used as treatment vs pretreatment in Poly(I:C)-stimulated gastrointestinal organoids. METHODS:The human intestinal epithelial Caco-2 cells (passages 10-20) were used for an in vitro infection model. Cultured in DMEM at 37°C 5% CO2 for 21 days, they formed organoids. The monolayer was pretreated with melatonin (1, 10, 50, or 100 µM) for 24 hours, followed by TLR3 stimulation with Poly(I:C) for another 24 hours. Alternatively, cells were treated with melatonin after stimulation. Proliferation was measured using CCK-8. IFNλ1, IFNβ were tested using ELISA. Apoptosis, interferon lambda receptor 1 (IFNLR1) and STAT1-3 expression were analyzed using flow cytometry. Statistical analysis was performed with SPSS 29.0. RESULTS:Caco-2 cells displayed direct correlation between IFNλ1 expression and higher Poly(I:C) concentrations, while IFNβ expression was absent. IFNλ1 levels were highest in melatonin-pretreated cells infected with high Poly(I:C) concentrations, while melatonin treatment reduced IFNλ1 expression. Melatonin reduced IFNLR1 expression in both treated and pretreated cells compared to stimulated controls. Interestingly, it induced IFNLR1 expression in non-infected cells but did not stimulate IFNλ1 production. In pretreated cells, IFNλ1 levels moderately correlated with STAT1-3 expression (highest with STAT2, r=0.509), but did not correlate with IFNLR1. In treated cells, there was a reverse weak or no correlation between STAT1-3 and IFNλ1, while STAT2 showed the strongest correlation with IFNLR1 (r=0.318). Melatonin pretreatment reduced apoptosis, while melatonin treatment on infected cells did not protect them from apoptosis despite decreasing levels of IFNλ1, IFNLR1 and STAT1-3. CONCLUSIONS:Melatonin demonstrated a potential antiviral effect via III IFN pathway by reduction of IFNλ1 expression in treated cells and induction of IFNLR1 expression in noninfected cells. Interestingly, its impact on STAT1-3 and IFNλ1 correlation varied between pretreated and treated cells, highlighting the intricate interplay in the modulation of antiviral response.

Background. Melatonin, a natural compound, has been discovered to play a role in antiviral processes and is being actively studied as a potential therapeutic agent for various infectious diseases. Signal transducer and activator of transcription (STAT) proteins serve as key factors mediating antiviral and proinflammatory responses in case of viral invasion. Aim. The aim of the study was to evaluate the effect of melatonin on STAT expression in Poly(I:C)- stimulated gastrointestinal organoids. Methods. The human intestinal epithelial Caco-2 cell line (passages 10–20) was used for the in vitro infection model. The cells were cultured in DMEM media at 37°C 5% CO2 for 21 days until they formed organoids. The monolayer was then either pretreated with melatonin (at concentrations of 1, 10, 50 or 100 μM) for 24 hours, followed by stimulation with different concentrations of Poly(I:C) for another 24 hours, or treated with melatonin for 24 hours at aforementioned concentrations after stimulation with Poly(I:C) for 24 hours. STAT1-3 expression was analysed using flow cytometry. Statistical analysis was performed using SPSS 29.0. Results. Poly(I:C) induced higher levels of STAT1-3 in Caco-2 cells compared to control cells. Pretreatment with melatonin slightly reduced STAT1-3 production in infected Caco-2 cells. Noninfected Caco-2 cells pretreated with melatonin exhibit higher levels of STAT1-3 than control cells. In cells treated with melatonin after Poly(I:C) stimulation, STAT1-3 production was lower than in pretreated cells. There was a moderate correlation between STAT1-3 production, with the strongest correlation observed between STAT1 and STAT3 (r = 0.683, p < 0.001). However, neither a higher concentration of melatonin nor a higher Poly(I:C) concentration demonstrated a stable effect on STAT1-3 production. The levels of STAT1- 3 did not show a significant correlation with apoptosis neither in pretreated, or treated cells. Conclusion. In our study, melatonin, when used as a pretreatment, revealed its ability to elicit an antiviral effect even in noninfected cells, while its application as treatment demonstrated a decrease in STAT1-3 levels. Our initial findings underscore the significance of melatonin as a potential therapeutic approach for virus-induced infections, highlighting the crucial need for judicious and timely consideration in its administration. Further studies are needed to emphasize melatonin’s importance in antiviral effects.

Backgrounds: Children who have contracted COVID are at risk of experiencing a variety of long-lasting symptoms collectively known as prolonged COVID syndrome. According to world data, about 30% of children infected with SARS-CoV-2 will experience certain symptoms. In this study, we aimed to create a continuous follow-up program for children after COVID infection. Moreover, our aim was to improve longterm periodic communication with the patient's parents to discuss her/his condition and timely recognize the signs of prolonged COVID. In addition, provide individualized assistance, monitoring of the patient and symptoms, assessment of the persistence of symptoms, and provide specific treatment. Methods: Continuous follow-up program in the frame of a prospective observational study was created and is conducted in our hospital. The program started in March 2022. Criteria for the inclusion: 1 month up to 18 years of age, diagnosed with COVID-19, or have been diagnosed with MIS-C syndrome according to the clinical criteria. After the inclusion, in a period of one year, 5 medical assessments of the state of health are planned to be carried out. In case of specific symptoms, a patient is referred to a specialist. Results: Currently, ~40 children with their caregivers are included into the program. The median age for inclusion – 9y, 42%-female. Nine children required additional consultation with a pediatric neurologist with the most frequent complaint of headaches. 4 children have ongoing consultations with a psychiatrist and continuous visits are planned. Six children were referred to a pediatric rheumatologist complaining of prolonged joint aches and the assessment regarding autoimmune diseases is ongoing. Conclusions/Learning Points: Our further plans are to collect more data and analyze the symptoms and signs according to age and gender.

Introduction. Multi systemic Smooth Muscle Dysfunction Syndrome (MSMDS) is a rare disease with fewer than 50 cases known worldwide. MSMDS is caused by a genetic mutation in the ACTA-2 gene. Consequently, smooth muscle contraction is less effective, causing symptoms to appear in those organs where smooth muscle cells are abundant. Due to smooth muscle insufficiency, bronchomalacia can be one of the disease manifestations. Heart, lungs, brain, and bladder can be affected at the same time leading to challenging management. Case report. We present a case of a 3-month-old girl with MSMDS. She was hospitalized because of recurrent respiratory tract infections and respiratory failure. Her saturation without supplemental oxygen was 80%, therefore high flow nasal cannulas were applied. Bronchoscopy revealed massive left mainstem bronchomalacia. To maintain left lung function, self-expanding stent was inserted into the left mainstem bronchus. However, after 3 months desaturation episodes reoccurred. There was a need to change to a longer stent because previous one reappeared to be too small. Longer silicone stent was placed into the left bronchus. Her condition has improved significantly, and she was discharged with the continuous positive airway pressure(CPAP) therapy at home. It has been more than a year since the stent was inserted, and the patient demonstrates normal neurological development according to her age expectations. The patient had not sought any medical treatment according to her respiratory system since her last hospitalization. Conclusions. MSMDS is a rare congenital disease requiring multidisciplinary teamwork. These patients usually have breathing insufficiency due to bronchomalacia and need very careful, less invasive treatment. It is important that all members of the child’s healthcare team would be aware of current recommendations and identify the importance of collaboration when it comes to clinical decision-making.

Fever is the most common complaint of children who are attending a pediatric emergency department (PED). Most of the fever cases are of viral origin; however, the most common markers, such as leucocyte, neutrophil count, or C-reactive protein, are not sensitive or specific enough to distinguish the etiology of fever, especially if children present at the early phase of infection. Currently, platelets have been attributed a role as important sentinels in viral and bacterial infection pathogenesis. Thus, our aim was to analyze different platelet indices, such as PNLR (platelet-to-neutrophil/lymphocyte ratio), PNR (platelet-to-neutrophil ratio) as well as specific secreted proteins, such as sP-selectin, CXCL4, CXCL7, and serotonin. We included 68 children who were referred to PED with the early onset of fever (<12 h). All children with comorbidities, older than five years, and psychiatric diseases, who refused to participate were excluded. All the participants were divided into viral, bacterial, or serious bacterial infection (SBI) groups. All the children underwent blood sampling, and an additional sample was collected for protein analysis. Our analysis revealed statistically significant differences between leucocyte, neutrophil, and CRP levels between SBI and other groups. However, leucocyte and neutrophil counts were within the age norms. A higher PNLR value was observed in a bacterial group, PNR-in viral. As we tested CXCL7 and sP-selectin, alone and together those markers were statistically significant to discriminate SBI and sepsis from other causes of infection. Together with tachypnoe and SpO2 < 94%, it improved the prediction value of sepsis as well as SBI. CXCL4 and serotonin did not differ between the groups. Concluding, CXCL7 and sP-selectin showed promising results in early SBI and sepsis diagnosis.

Background and aim. Since COVID pandemics, ~50% of children can have asymptomatic infection. However, they tend to have post-covid symptoms. Due to unexplained symptom origin (without confirmed COVID), they are referred to different paediatric specialists. Aims:to analyse SARS-CoV-2 antibody levels in children referred to our University Hospital ambulatory care with unexplained symptoms, to clarify percentage of asymptomatic covid cases and related post-covid symptoms. Methods:prospective study was conducted including children up to age of 18y without previous SARS-CoV-2 infection, were not breastfed during the last months, nor vaccinated against SARS-CoV-2. Parents were asked to fill-in the questionnaire about symptoms within one year before referral. Results:in total, 124 children were included into the study, 47.5% were female. Median age was 8.6y. 63.7% had different chronic diseases, e.g., asthma or arthritis and 56.4% were allergic. 41% noted that they have had symptoms of acute infection within one year, 24% had family member who tested positive for SARS-COV-2. 24.2% of children had antibodies against SARS-CoV-2 (Ab+). No correlation between positive test of family member and Ab+ child was found. No significant difference was observed between Ab+ versus Ab- within age groups, with or without chronic diseases. There were 57 children with unexplained symptoms and 26% of those children had Ab+. Most of those symptoms were respiratory (dyspnoea, cough), followed by neurological symptoms (insomnia, tiredness). Conclusions:we did find 24% of the Ab+ children who were defined as never ill with COVID. Majority of unexplained symptoms within the Ab+ group were respiratory and/or neurological.