Excess mortality in Europe coincides with peaks of COVID-19, influenza and respiratory syncytial virus (RSV), November 2023 to February 2024.

Since the end of November 2023, the European Mortality Monitoring Network (EuroMOMO) has observed excess mortality in Europe. During weeks 48 2023-6 2024, preliminary results show a substantially increased rate of 95.3 (95% CI:  91.7-98.9) excess all-cause deaths per 100,000 person-years for all ages. This excess mortality is seen in adults aged 45 years and older, and coincides with widespread presence of COVID-19, influenza and respiratory syncytial virus (RSV) observed in many European countries during the 2023/24 winter season.

Current knowledge on multiple sclerosis pathophysiology, disability progression assessment and treatment options, and the role of autologous hematopoietic stem cell transplantation.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that affects nearly 2.8 million people each year. MS distinguishes three main types: relapsing-remitting MS (RRMS), secondary progressive MS (SPMS) and primary progressive MS (PPMS). RRMS is the most common type, with the majority of patients eventually progressing to SPMS, in which neurological development is constant, whereas PPMS is characterized by a progressive course from disease onset. New or additional insights into the role of effector and regulatory cells of the immune and CNS systems, Epstein-Barr virus (EBV) infection, and the microbiome in the pathophysiology of MS have emerged, which may lead to the development of more targeted therapies that can halt or reverse neurodegeneration. Depending on the type and severity of the disease, various disease-modifying therapies (DMTs) are currently used for RRMS/SPMS and PPMS. As a last resort, and especially in highly active RRMS that does not respond to DMTs, autologous hematopoietic stem cell transplantation (AHSCT) is performed and has shown good results in reducing neuroinflammation. Nevertheless, the question of its potential role in preventing disability progression remains open. The aim of this review is to provide a comprehensive update on MS pathophysiology, assessment of MS disability progression and current treatments, and to examine the potential role of AHSCT in preventing disability progression.

Intraluminal assessment of inflammatory factors in patients with intracranial aneurysms.

BACKGROUND: The formation, growth, and rupture of intracranial aneurysms (IA) are due to several pathophysiological mechanisms, including focal hemodynamic injury and inflammation of the arterial wall. We investigated the differences between venous, parent artery, and intra-aneurysmal blood by measuring inflammatory factors and antibodies in patients with ruptured (rIA) or unruptured intracranial aneurysms (uIA). METHOD: A prospective study was performed in patients who presented with IA and required endovascular treatment. Blood was drawn from the lumen of the aneurysm sac, the parent artery, and the peripheral veins, to determine the serum concentrations of complement factors C3, C4, IgG, IgM, IgA antibodies, and C-reactive protein (CRP). RESULTS: Thirty-six patients (15 with uIA and 21 with rIA) were enrolled in the study. In both groups, C3, C4, IgM, IgG, and IgA showed a gradual decrease from venous to intra-aneurysmal samples, but only IgG in the parent artery and intra-aneurysmal samples reached a significant decrease in uIA compared with venous samples. Accordingly, C3 and IgG concentrations in the intra-aneurysmal samples showed a significant decrease in rIA compared with venous samples. A significant increase in CRP concentrations was observed in parent artery and intra-aneurysmal samples from patients with rIA compared with patients with uIA; a significant increase in C3 concentrations was observed in parent artery samples from patients with rIA compared with patients with uIA, and a significant decrease in IgM concentrations was observed in venous, parent artery, and intra-aneurysmal samples from patients with rIA compared with patients with uIA. CONCLUSIONS: A decrease in C3 and IgG in the aneurysm sac indicates activation of the complement system in the arterial wall. CRP in the aneurysm sac and lumen of the parent artery was significantly increased in ruptured compared with unruptured aneurysms, whereas venous, parent artery, and intra-aneurysmal IgM were decreased in ruptured compared with unruptured aneurysms. These results argue for the role of an ongoing inflammatory process in aneurysms leading to their growth and rupture.

Wastewater Levels of Respiratory Syncytial Virus Associated with Influenza-like Illness Rates in Children-A Case Study in Larissa, Greece (October 2022-January 2023).

The emergence of the COVID-19 pandemic has led to significant progress in the field of wastewater-based surveillance (WBS) of respiratory pathogens and highlighted its potential for a wider application in public health surveillance. This study aimed to evaluate whether monitoring of respiratory syncytial virus (RSV) in wastewater can provide a comprehensive picture of disease transmission at the community level. The study was conducted in Larissa (Central Greece) between October 2022 and January 2023. Forty-six wastewater samples were collected from the inlet of the wastewater treatment plant of Larissa and analyzed with a real-time reverse transcription polymerase chain reaction (RT-PCR) based method. RSV and SARS-CoV-2 wastewater viral loads (genome copies/100,000 inhabitants) were analyzed against sentinel surveillance data on influenza-like illness (ILI) to identify potential associations. Univariate linear regression analysis revealed that RSV wastewater viral load (lagged by one week) and ILI notification rates in children up to 14 years old were strongly associated (std. Beta: 0.73 (95% CI: 0.31-1.14), p = 0.002, R2 = 0.308). A weaker association was found between SARS-CoV-2 viral load and ILI rates in the 15+ age group (std. Beta: 0.56 (95% CI: 0.06-1.05), p = 0.032, R2 = 0.527). The results support the incorporation of RSV monitoring into existing wastewater-based surveillance systems.

Time Lag between COVID-19 Diagnosis and Symptoms Onset for Different Population Groups: Evidence That Self-Testing in Schools Was Associated with Timely Diagnosis among Children.

Early identification of COVID-19 cases has been vital for reducing transmission and enabling treatment. In Greece, in autumn 2021 when Delta was the predominant circulating variant, unvaccinated citizens had to be tested before attending activities, and self-testing was required twice a week for students (5−17 years). Here, we describe the time of diagnosis by age group and possible exposure to assess testing strategies (September to November 2021). Information on the presence of symptoms at the time of diagnosis was available for 69,298 cases; 24,855 (36%) were asymptomatic or tested the same day as onset (early diagnosis), 21,310 (31%) reported testing one day after, and 23,133 (33%) did so two or more days after the onset of symptoms. The median lag was 2 days (1−14). Early diagnosis significantly differed among age groups (p-value < 0.001) and was higher among children. For every one-year increase of age, the odds of an early diagnosis were reduced by 1%. Cases exposed during training activities or in settings such as accommodation centers and hospitals were more frequently diagnosed early. The percentage of persons having a positive self-test before a rapid test/PCR diagnosis ranged from 7% in the age group of 60 years and above to 86% in the age group of 5−17 years. The provision of self-tests in schools and increased testing in closed settings led to an earlier diagnosis and probably to a decreased transmission of the virus in the period during which Delta was the predominant variant in Greece. However, more effort is needed for early diagnosis of adults in the community, especially after the onset of symptoms.

Shifting gears: Study of immune system parameters of male habitual marathon runners.

Aim: Marathon is a running event in which athletes must cover a distance of 42.195 km. In addition to participating in marathons, marathoners have incorporated extensive running into their lifestyle. In the present study, we investigated the effect of long-term strenuous exercise in the form of marathon running on the immune system. Methods & Results: We collected peripheral blood samples from 37 male marathoners before/after a race and 37 age/sex/body mass index (BMI)-matched healthy sedentary controls. Hematological and biochemical tests revealed race-induced leukocytosis attributable to neutrophilia and significant increases in plasma lactate dehydrogenase (LDH), creatine phosphokinase (CPK), and cortisol concentrations. Phenotypic analysis of lymphocytes revealed race-induced significant decrease in the number of lymphocytes, memory helper T (Th) cells, naive, memory and activated cytotoxic T (Tc) cells, natural killer (NK), NKT, and B1 cells, and a significant increase in the number of activated Th and regulatory Th cells (Tregs). Compared with controls, marathoners maintained significantly lower levels of memory and activated Th cells and higher levels of activated Tc and B1 cells. Measurement of plasma cytokine levels revealed a pro-inflammatory cytokine polarization that increased after the race. Examination of gene expression of cytokines and Th-cell signature transcription factors in peripheral blood mononuclear cells revealed a significant decrease in tumor necrosis factor α (TNF-α) and interleukin (IL)-17, and a significant increase in IL-6, IL-10 and forkhead box P3 (FoxP3) after the race. Compared with controls, marathoners maintained significantly higher levels of TNF-α. Assessment of the suppressive capacity of Tregs in co-cultures of isolated effector Th cells and Tregs showed significantly increased suppressive capacity of marathoners' Tregs after the race. Conclusions: Compared with controls, marathoners live with permanent changes in certain immune parameters. Marathoners exhibit a stable pro-inflammatory cytokine polarization that increases after the race and is counterbalanced by increased numbers of Tregs overexpressing FoxP3 and having increased suppressive capacity.

The Role of Leptin in Childhood Immune Thrombocytopenia (ITP): An Anti-Inflammatory Agent?

To investigate the effect of leptin in childhood ITP, we measured plasma leptin in 39 children with acute ITP, after treatment and in remission, and in 33 healthy age/BMI-matched controls. We also cultured ITP and control peripheral blood mononuclear cells (PBMCs) with recombinant leptin to assess its direct effect on pro/anti-inflammatory cytokine gene expression. A significant increase in leptin was observed in children with active disease compared to controls. A significant inverse correlation of leptin with platelet count was also observed in children with acute ITP. Leptin remained high after treatment with IVIg, whereas steroid treatment lowered leptin below control levels. In remission, leptin was in the control range. Cytokine gene expression was significantly increased in children with acute ITP compared with controls, with highest expression for IFN-γ and IL-10. IVIg/steroid treatment significantly decreased IFN-γ and IL-10 expression. In remission, IFN-γ and IL-10 expression remained low. Addition of leptin to PBMCs isolated from patients in remission resulted in a significant increase in IL-10 gene expression compared to controls. Further experiments with purified T-cells and monocytes identified monocytes as the source of leptin-induced IL-10. We suggest that leptin acts as an active anti-inflammatory agent in childhood ITP by promoting IL-10 secretion by monocytes.

Transcription factor Ets-2 regulates the expression of key lymphotropic factors.

Transcription factor Ets-2 downregulates the expression of cytokine genes and HIV-1 in resting T-cells. Herein, we studied whether Ets-2 regulates the expression of lymphotropic factors (LFs) NFAT2, NF-κΒ/p65, c-Jun, c-Fos, which regulate the activation/differentiation of T-cells, and kinase CDK10, which controls Ets-2 degradation and repression activity. In silico analysis revealed Ets-2 binding sites on the promoters of NFAT2, c-Jun, c-Fos. The T-cell lines Jurkat (models T-cell signaling/activation) and H938 (contains the HIV-1-LTR) were transfected with an Ets-2 overexpressing vector, in the presence/absence of mitogens. mRNA and protein levels were assessed by qPCR and Western immunoblotting, respectively. Ets-2 overexpression in unstimulated Jurkat increased NFAT2 and c-Jun mRNA/protein, c-Fos mRNA and NF-κΒ/p65 protein, and decreased CDK10 protein. In unstimulated H938, Ets-2 upregulated NFAT2, c-Jun and CDK10 mRNA/protein and NF-κΒ/p65 protein. In stimulated Jurkat, Ets-2 increased NFAT2, c-Jun and c-Fos mRNA/protein and decreased CDK10 mRNA/protein. In stimulated H938 Ets-2 increased NFAT2, c-Jun and c-Fos protein and reduced CDK10 protein levels. Furthermore, Ets-2 overexpression modulated the expression of pro- and anti-apoptotic genes in both cell lines. Ets-2 upregulates the expression of key LFs involved in the activation of cytokine genes or HIV-1 in T-cells, either through its physical interaction with gene promoters or through its involvement in signaling pathways that directly impact their expression. The effect of Ets-2 on CDK10 expression in H938 vs Jurkat cells dictates that, additionally to Ets-2 degradation, CDK10 may facilitate Ets-2 repression activity in cells carrying the HIV-1-LTR, contributing thus to the regulation of HIV latency in virus-infected T-cells.

Clinical and immunological parameters of Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic autoimmune disease that primarily affects the exocrine glands, resulting in their functional impairment. In SS, lymphocytic infiltration of salivary and lacrimal glands, and deposition of several types of autoantibodies, mainly anti-SS-A (anti-Ro) and anti-SS-B (anti-La), lead to chronic inflammation, with xerostomia and keratoconjunctivitis sicca. In its primary form (pSS), SS does not involve additional connective tissue diseases, whereas in its secondary and more common form (sSS), SS presents in association with other rheumatic autoimmune diseases, mainly rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). As in most autoimmune diseases, environmental, hormonal and genetic factors are implicated in SS pathogenesis. In SS T cells predominate in mild lesions, whereas B cells predominate in advanced lesions. Th1, Th2, Th17, follicular helper T (Tfh) cells and regulatory cells (Tregs/Bregs), with their characteristic cytokine profiles, have been implicated in the pathogenesis of SS. It has been suggested that Th1 and Th17 cells initiate SS and, as the disease progresses, Th2 and Tfh cells predominate. It is assumed that, as in all autoimmune and inflammatory conditions, tolerance defects contribute to SS pathogenesis. It is intriguing that in SS it remains unclear which types of regulatory cells are functional and whether they ameliorate or worsen the disease. In this review we present a comprehensive update on SS with emphasis on immune system involvement, and suggest new insights into SS immunopathogenesis.

Construction of an M1GS Ribozyme for Targeted and Rapid mRNA Cleavage; Application on the Ets-2 Oncogene.

BACKGROUND: RNase P-mediated cleavage of target RNAs has been proposed as a promising tool for gene silencing. Ets-2 proto-oncogene controls the expression of a wide variety of genes involved in cancer and immunity. OBJECTIVE: Construction of a functional RNase P-based ribozyme (M1GS303) that targets Ets-2 mRNA. METHODS: The accessible sites for targeting of Ets-2 mRNA were identified by footprinting analysis. M1GS303 ribozyme was constructed by cloning. The activity of the ribozyme in the presence or absence of spiramysin in E. coli cells and human cell lines was quantified by RT-PCR. The efficiency of the ribozyme in silencing the endogenous expression of Ets-2 in human cell lines was examined by RT-PCR, western blot and immunofluorescence analysis. RESULTS: In E. coli cells co-transformed with plasmids bearing M1GS303 and the ets-2 target gene, Ets-2 mRNA was decreased by 93% 12h after IPTG induction in the absence, and after 4h in the presence of spiramycin. Ets-2 was rapidly downregulated in the human embryonic kidney cell line HEK293 and the T-cell line Jurkat transfected with an M1GS303 plasmid; the silencing effect of M1GS303 was considerably faster when the cells were cultured with spiramycin. In Jurkat cells, Ets-2-downregulation resulted in upregulation of the expression of IL-2, IL-4 and IFN-α cytokine genes that have Ets-2 binding sites on their promoters, whereas it had no effect on the expression of the IL-10 gene that lacks Ets-2 binding sites on its promoter. CONCLUSIONS: M1GS303 ribozyme cleaves effectively Ets-2 mRNA in bacteria and mammalian cells, and its activity is enhanced by spiramycin. Downregulation of ets-2 gene in the T-cell line Jurkat upregulates IL-2, IL-4 and IFN-α cytokine genes. M1GS technology may be a better alternative to conventional gene-interference therapies and the delineation of the effects of gene silencing in various pathologies.

Ets-2 Acts As a Transcriptional Repressor of the Human Immunodeficiency Virus Type 1 through Binding to a Repressor-Activator Target Sequence of 5'-LTR.

HIV-1 is transcriptionally active in activated T helper (Th)-cells and inactive in naive or resting memory Th-cells. Ets-2 is a preinduction transcriptional repressor of the IL-2 gene in naive Th-cells and a candidate transcriptional repressor of HIV-1 in the same cells, because the -279 to -250 upstream region of HIV-1-LTR [repressor-activator target sequence (RATS)], that participates in HIV-1-LTR transcriptional silencing, encompasses the AAGGAG Ets-2 binding site. In this proof of concept study, we investigated whether Ets-2 represses the expression of HIV-1. To assess whether Ets-2 can repress HIV-1 transcriptional activation acting through RATS, we transfected Jurkat cells with an Ets-2 overexpression plasmid (pCDNA3-ets-2) or Ets-2 silencing plasmids (ets-2-shRNA) and, as target genes, plasmids carrying the whole HIV-1-LTR sequence (HIV-1-LTR-CAT) or two copies of the RATS sequence (2× RATS-CAT) or a point mutation in the Ets-2 binding site (2× mutantRATS-CAT) or CMV-CAT (control). Ets-2 overexpression resulted in a significant reduction of HIV-1-LTR-CAT and 2× RATS-CAT activities in stimulated cells, but not of the 2× mutantRATS-CAT or CMV-CAT. Ets-2 silencing led to increased activities of HIV-1-LTR-CAT and 2× RATS-CAT in unstimulated cells, but had no effect on the activities of 2× mutantRATS-CAT and CMV-CAT. To assess Ets-2 binding to HIV-1-LTR-RATS in naive Th-cells, we isolated naive Th-cell nuclear proteins and passed them through an Ets-2 antibody column; electrophoretic mobility shift assays were performed using an RATS probe mixed with consecutive protein eluates. Ets-2 bound to the HIV-1-LTR-RATS in a dose-dependent manner. To assess Ets-2 binding to RATS in vivo, Jurkat cells were transfected with 2× RATS-CAT and stained for the Ets-2 protein and the RATS sequence by combining immunofluorescence and fluorescence in situ hybridization techniques. In unstimulated cells, Ets-2 bound to RATS, whereas no binding was observed in stimulated cells. To test for RATS specificity, the same experiments were performed with 2× mutantRATS-CAT, and no binding of Ets-2 was observed. The results were corroborated by chromatin immunoprecipitation assays performed with the same cells. Our results show that Ets-2 is a transcriptional repressor of HIV-1. Repression of HIV-LTR-RATS mediated by Ets-2 may account for the low-level transcription and replication of HIV-1 in naive Th-cells, and contribute to the viral latency and maintenance of viral reservoirs in patients, despite long-term therapy.

Transcription Factor Ets-2 Acts as a Preinduction Repressor of Interleukin-2 (IL-2) Transcription in Naive T Helper Lymphocytes.

IL-2 is the first cytokine produced when naive T helper (Th) cells are activated and differentiate into dividing pre-Th0 proliferating precursors. IL-2 expression is blocked in naive, but not activated or memory, Th cells by the transcription factor Ets-2 that binds to the antigen receptor response element (ARRE)-2 of the proximal IL-2 promoter. Ets-2 acts as an independent preinduction repressor in naive Th cells and does not interact physically with the transcription factor NFAT (nuclear factor of activated T-cells) that binds to the ARRE-2 in activated Th cells. In naive Th cells, Ets-2 mRNA expression, Ets-2 protein levels, and Ets-2 binding to ARRE-2 decrease upon cell activation followed by the concomitant expression of IL-2. Cyclosporine A stabilizes Ets-2 mRNA and protein when the cells are activated. Ets-2 silences directly constitutive or induced IL-2 expression through the ARRE-2. Conversely, Ets-2 silencing allows for constitutive IL-2 expression in unstimulated cells. Ets-2 binding to ARRE-2 in chromatin is stronger in naive compared with activated or memory Th cells; in the latter, Ets-2 participates in a change of the IL-2 promoter architecture, possibly to facilitate a quick response when the cells re-encounter antigen. We propose that Ets-2 expression and protein binding to the ARRE-2 of the IL-2 promoter are part of a strictly regulated process that results in a physiological transition of naive Th cells to Th0 cells upon antigenic stimulation. Malfunction of such a repression mechanism at the molecular level could lead to a disturbance of later events in Th cell plasticity, leading to autoimmune diseases or other pathological conditions.

Conjugation with polyamines enhances the antibacterial and anticancer activity of chloramphenicol.

Chloramphenicol (CAM) is a broad-spectrum antibiotic, limited to occasional only use in developed countries because of its potential toxicity. To explore the influence of polyamines on the uptake and activity of CAM into cells, a series of polyamine-CAM conjugates were synthesized. Both polyamine architecture and the position of CAM-scaffold substitution were crucial in augmenting the antibacterial and anticancer potency of the synthesized conjugates. Compounds 4 and 5, prepared by replacement of dichloro-acetyl group of CAM with succinic acid attached to N4 and N1 positions of N(8),N(8)-dibenzylspermidine, respectively, exhibited higher activity than CAM in inhibiting the puromycin reaction in a bacterial cell-free system. Kinetic and footprinting analysis revealed that whereas the CAM-scaffold preserved its role in competing with the binding of aminoacyl-tRNA 3'-terminus to ribosomal A-site, the polyamine-tail could interfere with the rotatory motion of aminoacyl-tRNA 3'-terminus toward the P-site. Compared to CAM, compounds 4 and 5 exhibited comparable or improved antibacterial activity, particularly against CAM-resistant strains. Compound 4 also possessed enhanced toxicity against human cancer cells, and lower toxicity against healthy human cells. Thus, the designed conjugates proved to be suitable tools in investigating the ribosomal catalytic center plasticity and some of them exhibited greater efficacy than CAM itself.

Cord blood leptin levels of healthy neonates are associated with IFN-γ production by cord blood T-cells.

Leptin is a hormone synthesized by adipocytes and other tissues, including the placenta, and it regulates food intake and energy expenditure, reproductive and immune functions. To investigate the role of leptin in neonatal immunity, we measured serum leptin and cytokine (IFN-γ, TNF-α, IL-2, IL-4, IL-10, IL-12) levels in the cord blood (cb) of 510 healthy neonates, 14 small for gestational age (SGA), 312 appropriately grown for gestational age (AGA) and 184 large for gestational age (LGA). Median serum leptin concentration in the whole sample was 11 ng/ml. In 11.2% neonates (1 SGA, 32 AGA, 24 LGA), leptin levels were >90th percentile (median 39 ng/ml). In 33.3% of those (3.72% of total sample) with the highest leptin levels (median 46 ng/ml), significantly elevated levels of serum IFN-γ were also found (mean 27.11 pg/ml, range 17.5-38.5 pg/ml). In neonates with leptin levels ∼50th percentile (median 12 ng/ml) or <10th percentile (median 1 ng/ml), serum IFN-γ levels were negligible. All other cytokines measured, were < the assays' detection limits. To investigate whether leptin can independently influence cytokine gene expression by cb T-cells and monocytes (Mc), we cultured cb T-cells or Mc, isolated from randomly selected AGA neonates or adult peripheral blood, with leptin. This resulted in upregulation of IL-2, IFN-γ and IL-4 gene expression in cb and adult T-cells and IL-10 expression mainly in cb-Mc. Significantly higher expression of IFN-γ occurred in female cb-T-cells cultured with leptin, compared with male cb-T-cells. In conclusion, the concurrent presence of high concentrations in both leptin and IFN-γ in cb of healthy infants, and leptin's ability to directly upregulate cytokine gene expression in cb T and Mc cells, indicate that abnormally high leptin levels can independently influence the immune system of healthy newborns, and may mediate gender differences in the development of a Th1 polarized immune response.

Expression patterns of leptin receptor (OB-R) isoforms and direct in vitro effects of recombinant leptin on OB-R, leptin expression and cytokine secretion by human hematopoietic malignant cells.

Several studies have implicated leptin in the pathophysiology of neoplasias. We investigated the direct effect of leptin on malignant hematopoietic tissue that included: primary acute myeloid leukemia (AML) cells, leukemic cell lines and bone marrow biopsies from multiple myeloma (MM) patients. PBMC, T-cells, B-cells and monocytes from healthy subjects served as controls. We defined the patterns of OB-R isoform expression in AML cells and leukemic cell lines in comparison to control cells by RT-PCR. rLeptin upregulated the expression of OB-R and endogenous leptin in AML blasts and certain cell lines but not in control cells. Cytometric Bead Array analysis of pro- and anti-inflammatory cytokines showed that rleptin upregulates IL-6 secretion by AML cells, various cytokines by the leukemic cell lines tested and IL-10 secretion by control PBMC, contributed by monocytes. Western immunoblotting revealed that the effect of rleptin was independent of JAK-2/phospho-JAK-2 protein levels. Finally, MM biopsies stained positive for leptin and, to a lesser extend, OB-R. Immunoreactivity was confined mostly to the nucleus of the myeloma cells. Normal myelocytes, promyelocytes and megakaryocytes stained weakly positive, and erythroid cells were constantly negative. We propose that the leptin/OB-R system is strongly and directly involved in supporting the growth of hematopoietic malignancies.