The recent outbreak of COVID-19 in the world is currently a big threat to global health and economy. Convalescent plasma has been confirmed effective against the novel corona virus in preliminary studies. In this paper, we first described the therapeutic schedule, antibody detection method, indications, contraindications of the convalescent plasmas and reported the effectiveness of convalescent plasma therapy by a retrospective cohort study. In December 2019, pneumonia associated with a novel corona virus 2019 (COVID-19) caused an outbreak, which has posed significant threats to global health and economy , while tInclusion Criteria (All six criteria must be met)More than 3 weeks after the onset of symptoms of the COVID-19 and complete resolution of symptoms at least 14 days prior to donation.In accordance with relieved isolation and discharge standards following the latest version of the therapeutic schedule.Age: 18 -55 years old.Weight: male≥50kg, female≥45kg.No history of blood-transmitted diseases.Eligible donors must be assessed by clinicians according to treatment.Exclusion CriteriaWith a history of pregnancy or transfusion whose HNA antibody and HLA antibody are positive.Individual’s physical condition is not eligible assessed by clinicians.Verify the identitiesSign the informed consentHealth inquiry, physical examination, laboratory examination of blood samples .CollectionEquipment and Operations: fully automatic apheresis machine or a fully automatic blood cell separator .Volume: 200-400ml (The exact volume should be assessed by clinicians).The interval between plasma collection should be more than two weeks.StorageFollow the principle of sterility, repackaging the plasma 100-200ml each.Store at 2-6° C for 48 hours. For long-term storage, it should be rapidly frozen to -20° C.Packaging: Labelling Requirements: refer to technical operation procedures of blood station.Method: Elisa (Enzyme-linked immune sorbent assay)*, Colloidal gold label technology*, Chemiluminescence.*Note: We recommend using ELISA to detect the novel coronavirus antibody titer since the colloidal gold method was not suitable for titer detection and the false negative rate was high. The analysis of 17 samples showed that the positive rate and sensitivity of ELISA were significantly better than colloidal gold  positive of novel coronavirus nucleic acid test .Principle of infusionAccording to the principle of cross-matching of blood, ABO homogenous plasma is preferred.A slow infusion is required at the first 15 minutes in order to pay attention to the occurrence of adverse reactions of blood transfusion.Risk disclosure and sign the informed consentThe individual CP therapeutic process and outcomes of the 19 patients treated with CP transfusion were shown in The use of convalescent plasma has a long history. At the end of the 19th century, researchers found that recovery patients’ plasma was effective in diphtheria and tetanus patients. Use of convalescent plasma has been studied in outbreaks of other respiratory infections, including the 2003 SARS-CoV-1, 2009-2010 H1N1 influenza virus pandemic and the 2012 MERS-CoV epidemic . PreviouIn our study, 19 COVID-19 patients were treated with CP and recovered quickly from the disease. The clinical symptoms were significantly improved as the individual critical/severe illness condition turned to moderate after CP treatment. The viral shedding was minimized and the clinical conditions of these patients improved as indicated by the viral nucleic acid test. We also found that the CP treatment was more effective in the patients with a higher neutralizing antibody titer transfusion. More importantly, all the patients in exposure group were survived and discharged, suggesting that the CP treatment was associated with a better outcome and a lower fatality.In the war of fighting against emerging and pandemic COVID-19, the advantages of the convalescent plasma have been validated by the practice and clinical outcome in China. Therefore, it provided an unprecedented opportunity to perform clinical studies and trials of the efficacy of convalescent plasma transfusion during the pandemic of COVID-19. Meanwhile, the convalescent plasma transfusion is worthy of application and promotion in SARS-CoV-2-infected patients globally due to its safety and efficacy.A retrospective cohort study was conducted from January 22, 2020 to February 28, 2020. 19 patients with COVID-19 in Hunan Province confirmed by real-time viral RNA test and clinical manifestation were hospitalized and received transfusions from the CP donors. To compare the effectiveness of treatment defined by fatality rate, a control group comprised of 43 COVID-19 patients who did not receive CP treatment was selected from Hunan (23 cases) and Hubei (20 cases) provinces using a stratified random sampling method by age, gender and severity of the disease. Baseline characteristics  of patients between the exposure group and control group were shown in Supplementary Tables


The biguanide metformin has been used as first-line therapy in type 2 diabetes mellitus (T2DM) treatment for several decades. In addition to its glucose-lowering properties and its prevention of weight gain, the landmark UK Prospective Diabetes Study (UKPDS) demonstrated cardioprotective properties in obese T2DM patients. Coupled with a favorable side effect profile and low cost, metformin has become the cornerstone in the treatment of T2DM worldwide. In addition, metformin is increasingly being investigated for its potential anticancer and neuroprotective properties both in T2DM patients and non-diabetic individuals. In the meantime, new drugs with powerful cardioprotective properties have been introduced and compete with metformin for its place in the treatment of T2DM. In this review we will discuss actual insights in the various working mechanisms of metformin and the evidence for its beneficial effects on (the prevention of) cardiovascular disease, cancer and dementia. In addition to observational evidence, emphasis is placed on randomized trials and recent meta-analyses to obtain an up-to-date overview of the use of metformin in clinical practice. Metformin, a synthetic biguanide, was first synthesized a 100 years ago, in 1922 [It took another 25 years before metformin was allowed on the US market in 1995. This was due to concerns about the safety profile, especially the occurrence of lactic acidosis. To further strengthen the safety profile of metformin, the FDA required a post-marketing safety surveillance study, which was published in 2005. This Comparative Outcomes Study of Metformin Intervention vs. Conventional Approach (COSMIC) showed no difference in serious side effects between metformin and conventional therapy and, in particular, no single case of lactic acidosis [In the meantime, in 1998, the United Kingdom Prospective Diabetes Study (UKPDS) had been published showing that metformin, in addition to lowering the blood glucose, reduced cardiovascular mortality in obese T2DM patients. This finding was further strengthened by the 2008 publication of the 10-year follow of the UKPDS, which showed that significant reductions persisted in the metformin group for any diabetes-related endpoint (risk reduction (RR) 0.79, 95% confidence interval (CI) 0.66–0.95), myocardial infarction (RR 0.67 95% CI 0.51–0.89) and death from any cause (RR 0.73 95% CI 0.59–0.89) [Based on a steadily increasing evidence base of positive effects of metformin coupled with a good safety profile, its prevention of weight gain and low cost, metformin has become the most widely prescribed drug in the treatment of type 2 diabetes mellitus (T2DM) and is recommended in European and American guidelines as first-line therapy in T2DM .However, its leading position is now being debated since two new drug classes with strong cardiovascular protective properties have come on the market. This concerns sodium-glucose cotransporter 2 (SGLT2) inhibitors and glucagon-like peptide 1 (GLP-1) receptor agonists. Given the robust evidence of these new drug classes in large cardiovascular outcome trials, the place of metformin is being reassessed.Apart from cardiovascular benefits, there is growing evidence that metformin may be beneficial for other age-related diseases, such as cancer, cognitive impairment and dementia. Additionally, metformin has been studied in various other diseases, such as polycystic ovary syndrome (PCOS), osteoporosis, periodontitis, inflammatory bowel disease, non-alcoholic fatty liver disease, COVID-19 and as anti-aging drug in general .Thus, the field of application for metformin has broadened instead of diminished in recent decades, and metformin is being repurposed as a geroprotective agent that may promote healthy aging and extend lifespan. This review will give an update of the available evidence of the beneficial health outcomes of metformin regarding cardiovascular disease, cancer, cognitive impairment and dementia, both in the diabetic population and the non-diabetic population.Biguanides are known to have a wide variety of therapeutic indications. Among others they are used as antidiabetic, antimalarial, anticancer, antimicrobial and antiviral agents . From alThe first clinical application of metformin was as anti-flu agent in malaria patients by Garcia in 1950 . The gluThe most relevant side effect of biguanides is mitochondrial toxicity leading to lactic acidosis. Although biguanides inhibit isolated mitochondrial complex 1, only those biguanides that enter mitochondria do so in vivo. Biguanide-associated activation of AMP-activated protein kinase (AMPK), which results in glucose lowering, also is related to access to the mitochondrial compartment . This isMetformin is a hydrophilic cation with metal-binding properties, especially with copper. Because of its hydrophilic character, it is not able to diffuse through lipid-rich membranes and needs a transporter. Organic cation transporter 1 (OCT1) is an important metformin transporter and the OCT1 expression of cells affects the uptake of metformin. Polymorphisms of this transporter however do not influence the glycemic response to metformin .Oral bioavailability of metformin is between 50 and 60%. Apart from the kidneys and the bladder which dispose unmetabolized metformin, increased concentrations of metformin are primarily found in the intestinal and colonic wall and the liver with tissue-to-plasma ratios of 300 and 3, respectively .The main glucose-lowering property of metformin is a reduction in the fasting blood glucose. Historically this has been ascribed to a reduction in hepatic gluconeogenesis and hence a reduction in hepatic endogenous glucose production. Especially in long-duration T2DM, this is an important mechanism since increased hepatic glucose output is a well-known pathophysiological mechanism .However, a systematic review showed that reduced endogenous glucose production only explained part of the reduction in fasting glucose and that increased glucose disposal is another major part . This haThe major site of action regarding glucose disposal may be the intestinal tract. Metformin restores glucose uptake in the small intestine and colon. A 26-week, randomized, placebo-controlled trial evaluated the intestinal glucose uptake with fluorodeoxyglucose positron-emission tomography (FDG-PET) imaging, showing a 2-fold increase in the small intestine and 3-fold increase in the colon in metformin users . In addiAn important intestinal site of action is also in accordance with studies that showed that metformin was as effective with delayed absorption targeting the ileum, as compared with immediate resorption. The glycemic effects of metformin persisted despite lower plasma metformin levels in the delayed absorption group . AdditioAdditionally, the side effects of metformin are related to the gastrointestinal tract. Up to 30% of metformin users experience nausea, vomiting, diarrhea, bloating or abdominal pain . An impaApart from the liver and the intestines, increased glucose uptake in skeletal muscle has been reported as an additional glucose-lowering mechanism .The pleiotropic effects of metformin, transgressing the boundaries of T2DM, may be explained by several cellular mechanisms which have been discovered during the last decades. Central in the cellular working mechanism of metformin is the mitochondrion. Metformin is able to modulate mitochondrial function and hence the bioenergetic status of the cell. In hypermetabolic states metformin may favorably lower mitochondrial respiration, while in hypometabolic states or resting conditions, mitochondrial respiration may be enhanced .Apart from being the major energy source, mitochondria also play a key role in apoptosis and the production of reactive oxygen species (ROS). By modulating mitochondrial ion channels, metformin can reduce apoptosis in neuronal cells . InhibitIn the following sections we will discuss some main mechanisms by which metformin exerts its effects . HoweverThere are several molecular pathways by which metformin may modulate mitochondrial function . One of This inhibition of respiratory complex 1 may apply in the enterocyte in which high metformin concentrations are present. However, tissue concentrations in the liver are much lower. Because of the negative inner mitochondrial membrane charge, such high metformin concentrations may theoretically be achieved in hepatic mitochondria, but this remains to be proven .Apart from inhibition of complex 1, metformin may influence the mitochondrial glycerophosphate shuttle altering the cytosolic redox state . This coAnother possible mitochondrial target is the voltage-dependent anion channel (VDAC-1). Metformin is able to displace hexokinase from these channels, limiting cytoplasmal access to mitochondrial ATP and hence limiting the energy supply of the cell . DependiAlthough mitochondrial pathways are important, metformin also has effects that are mediated by other pathways. For example, metformin has effects on membrane fluidity and glucose metabolism in erythrocytes (which do not contain mitochondria) .AMPK is a key regulator in modulating the bioenergetic status of the cell. Metformin is an activator of AMPK. AMPK, as a cellular energy sensor, is activated by a decrease in ATP and an increase in AMP. Metformin could activate AMPK by inhibiting mitochondrial respiration and hence lower ATP levels, but other pathways have been reported to be involved.For example, metformin is able to increase AMP levels by slowing down the degradation of AMP . AdditioActivation of AMPK results in an energy conserving state  in which ATP-consuming anabolic pathways such as gluconeogenesis are switched off. Simultaneously a downstream cascade of signaling molecules is activated promoting a catabolic state with glycolysis and oxidation of fatty acids to restore the energy balance.The inhibition of anabolic pathways is not restricted to gluconeogenesis and also applies to lipogenesis, proteins and ribosomal RNA. An important enzyme which is inhibited by metformin is mammalian target of rapamycin (mTOR).This multiprotein complex is a major anabolic regulator that has a role in the metabolism and proliferation of malignant cells which is an important link to potential anticancer effects of metformin. mTOR inhibition is both regulated through AMPK activation but in AMPK knockout mammalian cells also AMPK-independent pathways inhibiting mTOR have been shown to exist .In addition to AMPK-mediated effects, metformin also has AMPK-independent actions. It was shown that metformin is able to inhibit gluconeogenesis in AMPK-knockout mice . To furtIn AMPK-knockout mice, metformin was able to decrease gluconeogenic flux without direct inhibition of gluconeogenic gene expression (as AMKP activation would do). This might be explained by allosteric regulation of gluconeogenic key enzymes such as phospho-fructokinase. Therefore, activation of AMPK may be a secondary mode of regulation that is sufficient but dispensable .This applies also to the anti-inflammatory properties of metformin. Metformin is associated with a reduction in several pro-inflammatory mediators, among which are the master regulator nuclear factor kappa B (NF-kB), interleukin 6 and tumor necrosis factor alpha . This efTumor cells display an increased metabolism in which copper acts as a cofactor. Regarding the metal-binding properties of metformin, the binding of copper may be important. Binding of mitochondrial copper by metformin will slow down the mitochondrial electron transport chain and inhibit the differentiation of tumor cells from the epithelial to the metastatic prone mesenchymal state . AdditioLastly, lactate as an alternative metabolic substrate may underlie some of the beneficial effects of metformin. Already damaged organs especially, such as diabetic heart, kidney and neurons, have reduced capacity for glucose utilization and lactate may fill the gap. Metformin stimulates intestinal lactate production. Provided that toxic metformin concentrations are avoided, the physiological elevation of lactate may be beneficial rather than harmful .Several meta-analyses evaluating the effect of metformin on cardiovascular outcomes have been performed, but, depending on the selection criteria, few adequate randomized trials could be included in those meta-analyses.Since 2006, metformin is considered first-line therapy in the consensus statement of the American Diabetes Association  and the European Association for the Study of Diabetes . This recommendation was mainly based on the UKPDS study awaiting further confirmation. Instead of further confirmation trials comparing metformin with placebo or with an active comparator, most large cardiovascular outcome trials have been performed with new classes of medication , mostly on top of metformin.Recent meta-analyses of randomized controlled trials with metformin mostly concluded an absence of evidence or a neutral effect regarding beneficial effects of metformin on cardiovascular outcomes  57,58,5,60. HoweThe first is the UKPDS trial of 1998 for which the 10-year follow-up results were published in 2008 . The maiCompared with current guidelines, the glycemic treatment goals for the conventional treatment group and the standard of care are outdated and reflect suboptimal treatment. Treatment options apart from diet were insulin or sulfonylurea (SU) for non-obese persons with the addition of metformin for obese persons. Because the intensive treatment goal was hard to achieve with monotherapy, there were many cross-over patients who were prescribed combination therapy. The main analysis was however an intent to treat analysis.n = 342) was randomized vs. diet (n = 411) showed risk reductions of 32% (95% CI 13–47) for any diabetes-related endpoint, 42% for diabetes-related death (95% CI 9–63), and 36% for all-cause mortality (95% CI 9–55) [The pre-specified obese subgroup of 1704 patients in which metformin (CI 9–55) .Compared with other intensive therapies (insulin or SU), metformin performed better for any diabetes-related endpoint, all-cause mortality and stroke. This means that the effects found are not only related to improved glycemic control  but are related to non-glycemic mechanisms either caused by beneficial effects of metformin or by detrimental effects of the comparator therapy.A substudy that was performed within the UKPDS in which addition of metformin to patients who failed on SU monotherapy was compared with staying on SU showed an increased mortality for patients who were randomized to metformin. This was an unexpected finding for which the authors performed an extra analysis in the complete UKPDS study population to evaluate the combination of metformin and SU. No firm conclusions could be drawn regarding possible harm and further study was advised. Later follow-up data showed that the increased mortality in this group subsided over time; in retrospect, it was regarded as a chance finding .In contrast, the beneficial effects of metformin turned out to be robust and lasting as shown in the 2008 follow-up publication. In the metformin group, significant risk reductions persisted for any diabetes-related endpoint, myocardial infarction and death from any cause .The second trial is the 2009 “Hyperinsulinemia: The Outcome of its Metabolic Effects” (HOME) trial . This plDespite randomization, patients in the metformin group were slightly older and had a more extensive cardiovascular history. Therefore, in the statistical analysis, the crude results were adjusted for these baseline differences.The macrovascular aggregate score included both mortality and cardiovascular morbidity. The beneficial effect of metformin was primarily observed in reduced manifestations of cardiac ischemia. Absolute mortality however was greater in the metformin group compared with placebo. Because meta-analyses often use crude mortality or crude cardiovascular events, they use individual components of the secondary endpoint which does not reflect the combined macrovascular aggregate score.There have been several additional analyses of the HOME trial focusing on cardiovascular aspects. It was shown that metformin was associated with improvement in some markers of endothelial function . With reThe third trial is the 2013 “Study on the Prognosis and Effect of Antidiabetic Drugs on T2DM with Coronary Artery Disease” (SPREAD-DIMCAD) . This trThe primary endpoint was time to a macrovascular aggregate score including death from any cause and myocardial infarction. The hazard ratio of the metformin users compared with the SU group for the primary endpoint was 0.54 (95% CI 0.30–0.90).A strength of this study is that the effects of SU treatment and metformin can be assessed separately. In this population of high-risk patients, the benefit of metformin compared with SU was shown, although there was no comparison with placebo. Therefore, the effects could be both explained by beneficial effects of metformin or by deleterious effects of the SU treatment.An example of a large randomized controlled trial (RCT) in which an opposite trend was shown is the 2006 “A Diabetes Outcome Progression Trial” (ADOPT) study . In thisAnother trial which is used in meta-analyses is a trial in which contraindications of metformin were evaluated . In thisA recent summary of the available cardiovascular evidence regarding metformin was provided by the Italian Society of Diabetology and the Italian Association of Clinical Diabetologists . They puWith regard to the all-cause mortality endpoint, over 90% of the weight of their meta-analysis was composed of the UKPDS, the HOME, the SPREAD-DIMCAD and the ADOPT trials. The authors underline the small sample size, the need for a long follow up to assess mortality rates and the paucity of placebo-controlled studies. Active comparator studies induce altered cardiovascular risk in the comparator arm. A decrease in all-cause mortality in metformin users became significant when active comparator studies were excluded.Another recent meta-analysis had a broader scope and included both metformin monotherapy and combination therapy including over 7000 patients . AlthougIn contrast to the paucity of well-designed randomized trials, there is an overwhelming amount of observational data. A recent meta-analysis of cohort studies included over a million patients and showed a strong beneficial effect of metformin with an odds ratio of 0.57 (95% CI 0.48–0.68) for combined mortality and cardiovascular events compared with T2DM patients treated with other drugs .Another observational study concerns a meta-analysis which includes comparison with non-diabetic individuals. This analysis shows a reduced risk for all-cause mortality for metformin users compared with non-diabetic individuals . Compared with diabetes patients receiving non-metformin therapy the risk reduction was stronger  .Additionally, analyses in high-risk patient populations show benefit of metformin therapy. A meta-analysis in diabetes patients with established coronary artery disease showed a reduction in cardiovascular mortality , all-cause mortality  and the incidence of cardiovascular events  .Another meta-analysis in patients with T2DM and chronic kidney disease (stage G1–G3) showed a reduction in all-cause mortality  and the incidence of cardiovascular events  .Since the introduction of new classes of medication for the treatment of T2DM, there have been large cardiovascular outcome trials that showed the benefits of both SGLT2 inhibitors and GLP1 receptor agonists. The cumulative evidence for cardiovascular risk reduction in the new agents is more robust than the available evidence of metformin. However, given the fact that metformin is still first-line therapy, most (60–82%) of these trials have been performed with metformin as baseline therapy . AlthougNotwithstanding the proven cardiovascular benefits of GLP-1 receptor agonists and SGLT-2 inhibitors, the additional benefits of metformin as baseline therapy have not been disproved. Additional head-to-head comparisons between metformin and GLP-1 receptor agonists and SGLT-2 inhibitors in a large, randomized trial are not to be expected and neither are cardiovascular outcome studies with metformin vs. placebo.Therefore, we have to make decisions based on our current evidence base. This evidence still fits with the position of metformin as baseline therapy combined with new drug classes for high-cardiovascular-risk patients, instead of competing with them. To trade in metformin as baseline therapy for new drug classes would deny the patient any additional benefits of metformin therapy. Because metformin does not increase the risk of hypoglycemia, it is very well suited for combination therapy.People with T2DM have a higher risk of cancer and increased mortality rates due to cancer compared with those without diabetes. Although cardiovascular disease used to be the most important cause of diabetes-related death, there has been a transition to cancer as leading cause in high-income countries .A recent epidemiological analysis showed a 30% decline in all-cause death rates over the last 15 years in the UK . AlthougThe same pattern was seen in a Hong Kong study, in which cancer rates also surpassed the cardiovascular death rates in patients with diabetes . In thisIn 2014, an umbrella review was published of meta-analyses from observational data regarding the association between T2DM and the risk of developing or dying from certain forms of cancer . The mosGeneric mechanisms in diabetes, such as hyperglycemia and the resulting hyperinsulinemia with concurrent increased free IGF-1, might be harmful and contribute to the formation and growth of malignant tumors ,35. MetfIn addition to these indirect effects, metformin has multiple drug-specific effects with regard to cancer. These include the effects of metformin as AMPK activator with resulting mTOR inhibition. However, also a decrease in inflammation and oxidative stress, and a reduction in glycolysis, which is used by many rapidly growing tumors as preferred metabolic route . MetformAn observational study in Scotland compared median time to cancer diagnosis in 4000 metformin users compared to 4000 non-metformin users with T2DM. This study showed an unadjusted hazard ratio of 0.46  for the incidence of cancer. After adjustment the hazard ratio was 0.63  . An overA Chinese meta-analysis with more than 10 million T2DM patients showed an overall reduction of 20% in cancer incidence in metformin users compared with other diabetes drugs  with in subgroup analysis a decreased risk in bladder, colorectal, gastric, liver, lung, pancreatic and prostate cancer [In the following section we will discuss the effect of metformin on the three major cancers worldwide: breast, colon and lung cancer .Breast cancer was the most prevalent cancer worldwide in 2020, with over 2 million new cases. The risk of breast cancer is increased in T2DM. As most cases of breast cancer are estrogen-sensitive, the hormone receptor status of the tumor could influence treatment outcomes.Estrogen-receptor-positive (ER+) tumors are the most frequent type of breast tumor (80%). The hormone receptor status of breast tumors differs between T2DM patients and non-diabetic patients with breast cancer. Diabetes patients especially on metformin therapy tend to have less ER+ tumors and instead more ER− and even triple negative tumors . PossiblApart from improving insulin sensitivity, decreasing glucotoxicity and activating AMPK with multiple downstream pathways, metformin decreases estradiol levels with roughly 30% independent of body mass index . This coThe effect of metformin on breast cancer remains controversial. Observational data, while showing mixed results, do support a protective effect of metformin on breast cancer incidence. An example is a Taiwanese retrospective study of almost 500,000 patients showing a reduced incidence rate of breast cancer among metformin using T2DM patients with a rate reduction from 5 per 1000 person-years in non-metformin users compared with 2 per 1000 person-years in metformin user .An observational cohort study in Scotland showed, in the subgroup of breast cancer patients, an unadjusted hazard ratio of 0.44 (95% CI 0.26–0.73) and an adjusted ratio of 0.60 (95% CI 0.32–1.10) for incident cancer in metformin users . A recenWith regard to randomized trials there are conflicting results. A recent meta-analysis of randomized trials evaluated a mixed outcome of tumor growth, apoptosis and biomarkers in 1600 metformin-treated non-diabetic female breast cancer patients . It showHowever, randomized trials in phase 2 trials fail to show benefit of metformin. In non-diabetic patients, no survival effect of metformin in metastatic/recurrent breast cancer was seen when used as adjunctive therapy ,89,90.In summary, there is strong observational evidence that metformin may reduce the incidence of breast cancer, in particular estrogen-positive tumors ,81,86. AColorectal cancer (CRC) was the third most incident cancer worldwide and the second most common cause of cancer-related death in 2020. Patients with T2DM have an increased risk of CRC and related death.Three recent meta-analyses of observational data confirm a beneficial effect of metformin on the incidence of CRC, all-cause mortality of CRC and lower CRC-specific mortality. An 2021 meta-analysis of 28 studies including over 2 million people showed that the use of metformin reduced the risk of CRC by 29% compared with nonuse  . This fiAlthough most of these meta-analyses were based on observational data, there has been a randomized trial in patients without diabetes on the recurrence of colon adenoma or polyps after resection of an adenoma . A totalWith regard to prognosis of CRC patients, an all-cause mortality benefit was reported of 28%  and CRC-specific mortality of 20%  . AnotherMost of the data are again observational but there have been phase 2 studies. In treatment refractory CRC, 41 patients were treated with the combination of metformin and irinotecan. From other placebo-controlled studies the placebo response rate in this setting was 13% after 12 weeks. In this study 41% reached disease control after 12 weeks, consistent with a beneficial effect of metformin .Another phase 2 study, including 50 patients with refractory metastatic CRC, studied the combination of 5-fluorouracil and metformin. Although the predefined response rate of 20% after 8 weeks was met in the metformin group (22%), it was only a modest response .Apart from potential synergistic effects with chemotherapy, metformin is also used as radiosensitizer. Two clinical trials demonstrated an improved tumor and nodal downstaging and pathological complete response rates for rectal cancer using metformin ,97.Hence, there is strong observational data supported by one randomized trial that metformin is able to reduce the incidence of CRC. Concerning benefits for the treatment of CRC observational data also suggest survival benefits; however, randomized trials are lacking, and phase 2 trials may only indicate a small benefit.Lung cancer was the leading cause of cancer-related death in 2020 and the second most common cancer. Other than breast and colon cancer, its incidence is not increased in diabetes so beneficial effects of metformin cannot be ascribed to generic diabetes treatment effects.A 2020 meta-analysis evaluated the incidence of lung cancer and the use of metformin in 8 observational studies with over 200,000 patients with T2DM. This study found a decreased risk of lung cancer in metformin users  .Two recent meta-analyses combined the observational data and the available randomized trials on the role of metformin in lung cancer survival. In one meta-analysis, the observed overall survival improved both in studies without a time-dependent approach , as well as in studies using a time-dependent approach  . In the Three randomized trials of metformin use in non-small cell lung cancer (NSCLC) non-diabetic patients taken together did not reach statistical significance but showed a trend towards improved survival in the two meta-analyses .One of the included trials randomized 139 non-diabetic lung cancer patients between epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitors and the addition of metformin . Both thA post hoc analysis of this study was published this year in which the effect of body mass index (BMI) on the protective effect of metformin was evaluated. Only patients with BMI > 24 showed beneficial effects of metformin. This could be an explanation for the heterogeneity of the results of the randomized trials, as the overweight rate in Asian populations is markedly lower than that in Hispanic populations .In summary the evidence for a beneficial effect of metformin on both the incidence and the treatment of lung cancer is convincing although data from randomized trials are still lacking behind observational data.In contrast to cardiovascular disease and cancer, the incidence of dementia is rising, and in some countries, it is the most common cause of death (depending on the classification scheme used) .The most invalidating and common form of dementia is Alzheimer’s disease. This is an age-related disease which usually starts in individuals of 65 years and older.With regard to the pathogenesis of Alzheimer’s disease, the most accepted model is the amyloid hypothesis. This model is based on the typical pathological findings of β-amyloid plaques and neurofibrillary tangles composed of abnormal tau protein. However, the metabolic processes leading to the formation of these plaques and tangles remain poorly understood .Both microvascular disease and insulin resistance are linked to cognitive dysfunction either initiating, complicating or accelerating the disease process. Although obesity and insulin resistance worsen cognitive function, they do not lead to the typical pathological hallmarks of Alzheimer’s dementia. Instead, an independent form of insulin resistance confined to the brain is proposed as an additional phenotype of diabetes (type 3 diabetes) ,103.Because of this metabolic background, diabetes treatment may slow down and reduce the formation of amyloid and neurofibrillary tangles. In animal research, metformin has been shown to reduce amyloid formation . In addiIn addition, hypoglycemia is an independent risk factor for dementia ,105. TreSeveral meta-analyses have been performed the last years evaluating the role of metformin in modulating neurodegenerative disease. A 2018 meta-analysis reported a decreased risk for cognitive impairment in T2DM patients using metformin compared with non-metformin users (OR 0.55 95% CI 0.38–0.78) and a decreased risk for dementia (OR 0.76 95% CI 0.60–0.97) .However, an updated meta-analysis in 2020 comprising more trials concluded a neutral effect (OR 1.04 95% CI 0.92–1.17) for overall neurodegenerative disease and in a subgroup analysis also a neutral effect for dementia (OR 0.96 95% CI 0.85–1.08) . In addiA combined network meta-analysis and meta-analysis, also published in 2020, evaluated over 1 million patients and assessed the effect of various antidiabetic agents on the risk of dementia. In comparison with no treatment for diabetes, the risk for dementia in the metformin users was decreased in the network meta-analysis (HR 0.75 95% CI 0.63–0.86) and in the separate meta-analysis (HR 0.86 95% CI 0.74–1.00) .The authors concluded that patients treated with dipeptidyl peptidase-4 (DPP-4) inhibitors presented with the lowest risk of dementia, followed by those treated with metformin and thiazolidinedione, while treatment with insulin was associated with the highest risk. This might be related to the risk of hypoglycemia in insulin users, while this risk is very low in DDP-4 and metformin treatment.One of the recent studies is the “Sydney Memory and Ageing Study” . This prApart from prevention, metformin might also be beneficial in the treatment of established cognitive disease. Two small, randomized trials were performed with metformin in which verbal memory, executive function and cerebral blood flow were improved vs. placebo ,113.Taken together the evidence for a beneficial effect of metformin on cognitive function is not univocal. However, most studies do point to a protective effect of metformin on the risk of cognitive function and the risk of dementia in T2DM patients . CompareApart from a possible beneficial effect, there seems also a risk for a harmful effect of metformin regarding the increased rate of Parkinson’s disease. This might be confounded by an underlying vitamin B12 deficiency. It is known that long term metformin use can lead to vitamin B12 deficiencies . B12 defMetformin is still the most commonly prescribed drug for the treatment of T2DM although new drug classes are competing for its leading position. Although the evidence for cardiovascular protection is stronger for the new drug classes than for metformin, it would be premature to discard metformin in this respect.Combined with SGLT-2 inhibitors or GLP-1 receptor agonists, metformin has a favorable safety profile without increasing the risk of hypoglycemia. It might offer additional benefits, and therefore deserves to keep its foundational position, either in combination therapy in high cardiovascular risk T2DM patients or as first-line monotherapy in lower-risk patients.The unique properties of metformin arising from its effects on cellular energy metabolism and oxidative stress extends its effects from cardiovascular protection to age-related diseases, such as cancer and dementia. Over and above an insulin-sparing effect, metformin distinguishes itself from other antidiabetic drugs with added value. Recent meta-analyses show a decrease in cancer incidence, an increase in cancer specific survival and a decrease in the incidence of dementia in T2DM patients.Although most evidence in this field is observational, there is a growing number of randomized trials. A century after its chemical invention, metformin still deserves an important place, first and foremost in the treatment of T2DM, but possibly even in the larger non-diabetic population.


Dental calculus forms on teeth during the life of an individual and its investigation can yield information about diet, health status, and environmental pollution. Currently, the analytical techniques used to visualize the internal structure of human dental calculus and entrapped inclusions are limited and require destructive sampling, which cannot always be justified.We used propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SR-The virtual histological exploration of the samples shows that PPC-SR-PPC-SR- First, though it is widely understood how dental calculus formation occurs and how salivary calcium phosphates inhibit demineralization of enamel and dental decay,,,––Several researchers have explored non-destructive methods to visualize the microscopic structures and internal features of archeological and fossil materials. Scanning electron microscopy has been used to examine the internal structures and inclusions of dental calculus, but only on already-broken surfaces or histological thin sections.in situ samples (on teeth); (2) compare the visibility of the fine calculus inner structure between PPC-SR-In this study, we report preliminary results of the analyses of two dental calculus samples scanned using PPC-SR-2,We examined two samples of dental calculus: one contemporary (CM03) and one archeological (FK01) . CM03 waFK01 formed on the upper right second molar of a roughly 25-year-old female individual recovered from a Chalcolithic burial from Fourknocks II in Ireland.33.1The dental calculus chunk (CM03) and the tooth (FK01) were individually mounted onto the sample holder (plastic pin) using small slivers of beeswax . The sam3.1.1Experiments were performed at the P07 High Energy Material Science Beamline (HEMS),,,The PPC-SR-3.1.2Both specimens were scanned at 3.2The tiff stack of PPC-SR-44.1Both the isolated chunk of dental calculus and the calculus remaining on the tooth were successfully imaged with no visible mechanical damage or color change. Mounting these samples using beeswax proved a successful means to stabilize the samples while retaining the ability to remove the calculus or tooth after scanning to perform further analyses or to return the sample to the museum. The scans revealed a variety of information about the formation of calculus and inclusions. We note that the image quality is better in CM03 than in FK01: the spatial resolution and the signal-to-noise ratio are much improved since CM03 was scanned with a smaller aperture (0.67 versus 1.00) and a higher exposure time , it was straightforward to identify the surface of the dental calculus that once adhered to the tooth surface by its smooth profile. This interior surface also retained negative impressions of regular and periodic incremental markers , much lin enamel . These wUsing virtual histology techniques, and especially 2D thick-slabs , the scans also allow us to visualize the complex interior structures of the calculus that are related to how it formed on the tooth. Both samples show multiple levels of layering, which exhibit as alternating heterogeneous thick layers with increments of varying density. Unexpectedly, many of these layers are not parallel to the calculus surface, the tooth surface, or other layers. Instead, they occur as highly convoluted, marbled forms –5. Figur blurred .The modern calculus sample (CM03) also retains voids that seemingly follow intra-layer spaces and are of highly variable sizes, from a few tens to several hundreds of microns in diameter. The smaller voids often have a very elongated shape and scalloped outline, and lie roughly parallel to the layers of calculus, while the larger ones have irregular outlines, and possibly are the result of the decay or dissolution of organic inclusions. Voids also occur in the archeological sample FK01, but these are much larger  to the exterior also occur. Although FK01 appears as an extremely well-preserved sample of calculus on its exterior , it disp4.3Papaver cf. somniferum). The seed coat (SC) is well preserved, and easily identified in 2D and 3D due to its characteristic textured surface. The endosperm (E) is preserved and visible as well  are Synchrotron radiation micro-tomography allows unparalleled insights into the inner structure of human dental calculus. This non-destructive imaging technique enables researchers to visualize inclusions We show that conventional In contrast to histological and hard tissue sections, which provide layers of 3 to hickness . FurtherSynchrotron radiation micro-tomography also enables us to easily distinguish, in an isolated calculus fragment, the surface of the specimen that was in contact with the oral environment from that which was in contact with the crown enamel or root surface. The clear imprint of perikymata, periradicular bands and the gingival line on the interior surface suggests that it may be possible to use well-preserved calculus as an insight into the record of dental development  in the absence of the tooth itself.Shifts in diet, health, or oral pollution could potentially be captured in dental calculus and identified by a change in the nature of the inclusions, in the layers’ thickness or structure (or even in chemical composition, although this is not possible to assess using PPC-SR-Finally, the comparison of the modern and archeological samples provides some insight into the potential taphonomic processes affecting ancient calculus. While some of the differences between these samples are due to changes in the scanning parameters see , others


The burden of heart failure (HF), developing after myocardial infarction MI, still represents a major issue in clinical practice. Failure of appropriate resolution of inflammation during post-myocardial injury is associated with unsuccessful left ventricular remodeling and underlies HF pathogenesis. Cells of the immune system have been shown to mediate both protective and damaging effects in heart remodeling. This ambiguity of the role of the immune system and inconsistent results of the recent clinical trials question the benefits of anti-inflammatory therapies during acute MI. The present review will summarize knowledge of the roles that different cells of the immune system play in the process of post-infarct cardiac healing. Data on the phenotype, active molecules and functions of the immune cells, based on the results of both experimental and clinical studies, will be provided. For some cellular subsets, such as macrophages, neutrophils, dendritic cells and lymphocytes, an anti-inflammatory activity has been attributed to the specific subpopulations. Activity of other cells, such as eosinophils, mast cells, natural killer (NK) cells and NKT cells has been shown to be highly dependent of the signals created by micro-environment. Also, new approaches for classification of cellular phenotypes based on the single-cell RNA sequencing allow better understanding of the phenotype of the cells involved in resolution of inflammation. Possible perspectives of immune-mediated therapy for AMI patients are discussed in the conclusion. We also outline unresolved questions that need to be solved in order to implement the current knowledge on the role of the immune cells in post-MI tissue repair into practice. Study of treatment and prevention of atherosclerosis complications represents one of the most cutting-edge problems in the modern world. Despite the fact that modern interventional and pharmacological approaches allowed to greatly reduce mortality and morbidity after myocardial infarction (MI), patients remain at the high risk for the recurrent events in both short- and long-term . Even moLeft ventricular remodeling is the underlying basis of HF pathogenesis in patients after acute MI (AMI). The term “remodeling” was coined by N. Sharp in the end of 70s of the 20th century to describe structural and geometrical changes after AMI. Later on, it became more general term. Nowadays, heart remodeling means the complex process of disturbances in myocardial structure and function in response to the damaging myocardial overload and loss of the functioning myocardium .Restoration of the myocardial blood flow with thrombolytic therapy and primary percutaneous coronary intervention, being an obligatory step in myocardial treatment, increases tissue deterioration even further and leads to ischemia/reperfusion myocardial injury . Deeper Nowadays approaches to influence cardiac healing in the clinical practice are absent. Meanwhile state-of-the-art data emphasize an important role of immune system in orchestration of reparation and post-infarction tissue remodeling after AMI , 7. It dUnderstanding of the key processes in the immune system during AMI may allow to create new diagnostic and therapeutic approaches to predict and prevent further HF development. Unfortunately, knowledge of the immune mechanisms during heart remodeling is incomplete and even clinical trials sometimes show contradictory results. Partially this may be explained by the fact, that many cells, which originally were considered to be proinflammatory and harmful by default, also may display powerful healing properties. The current therapeutic approaches are missing techniques which would have not only suppressed inflammation, but rather targeted its resolution. In the present review we will summarize knowledge of the roles that different cells of the immune system play in the process of post-MI inflammation development and resolution and the subsequent cardiac remodeling. In the end we will outline possible perspectives of immune-mediated therapy for AMI patients and outline pending questions concerning implementation of the current knowledge into practice.Cardiac repair after AMI consists of several phases: early inflammatory phase (first 72 hours after AMI), late reparative and proliferative phase days 7-10), which are followed by maturation phase , 10., which aThe initial processes activated in the early phase are related to the maintenance of the cardiac output and normalization of the left ventricular wall strain. Left ventricular radius of curvature changes, which determinates different wall stiffness and distribution of the intraventricular volume. “Stretching” and thinning of the myocardium in the injured zone takes place, followed by inflammation and resorption of necrotic tissue. This ultimately leads to the so-called “myocardial infarct expansion”. Release of the intracellular content and destruction of extracellular matrix during inflammatory phase are associated with generation of danger-associated molecular patterns (DAMPs), non-specific stereotypic signals, which when recognized by pattern-recognition receptors (PRRs), lead to activation of the immune cells . The ultInflammatory phase should be followed by the reparative and proliferative phases to provide a wound healing and a proper scar formation. Intact myocardium is actively involved in the processes of the late remodeling. The aim of scar is prevention of myocardial rupture and partial limitation of the functional deterioration. Anti-inflammatory signaling, depletion of inflammatory neutrophils, fibroblasts proliferation and deposition of granulation tissue should be switched on to achieve this .The final phase is maturation, associated with remodeling of extracellular matrix and lasting several months. This phase depends on the events that had occurred in the previous phases and is critical for the restoration of the heart function. Unsuccessful maturation may lead to the increase of myocardial stiffness, diastolic dysfunction and development of HF .Cells of the immune system are present in myocardium at all the stages of heart repair. But the original dogma of their exclusively deteriorating role has been significantly shattered in the last decades. Many cellular populations have been shown to encompass both inflammatory and anti-inflammatory subsets, while properties of others have been shown to depend on the signals received from microenvironment. Timely switching from inflammatory to anti-inflammatory activity of the immune systems is crucial during post-myocardial healing. Meer inhibition of inflammation appeared to be harmful to the myocardial function and may even lead to the rupture of myocardium. On the contrary, adequate resolution of inflammation, defined as degradation of the inflammatory products, leading to cessation of inflammation and restoration of homeostasis , is esseCellular traffic in the development of any inflammation is governed by the chemical mediators. Sterile inflammation developing in the course of acute MI is not an exception. The most potent mediators of inflammation are produced from the essential polyunsaturated fatty acids (PUFAs). Splenic leukocytes recruited to the myocardium represent an important source of these mediators, with macrophages being the major cellular subtype being involved . While ain vitro significantly increased their viability and limited the rate of apoptosis  represent the most abundant subpopulation of polymorphonuclear cells in the blood and are the first cells of the immune system arriving to the site of infraction . In humaNeutrophils are recruited to the infarcted myocardium during the first few hours, initiating local inflammation and tissue destruction . Of inteElevated neutrophils are associated with bad prognosis in MI patients. The major detrimental effects are dependent upon the function of myeloperoxidase (MPO) secreted by neutrophils into the extracellular space . NETs reHowever, neutrophils’ recruitment has been shown to be an obligatory requirement for the successful switching from inflammatory response to its resolution. This dual function of neutrophils may explain unsuccessful attempts of translation of anti-neutrophilic therapies into clinics . ApoptosSwitching between the production of inflammatory and pro-resolving lipid mediators also explains the dual role of neutrophils in the MI. Neutrophils are the main cells, expressing 5-lipoxigenase (LOX), which is responsible both for the synthesis of leukotriens and production of the various SPM initiated in the course of intercellular interactions in tissues , 18. As It has been shown that neutrophils undergo the continual change of the proteome in the first week after AMI, with Day 1 cells predominantly producing metalloproteinases, switching to upregulation of apoptosis and ECM reorganization phenotype at Day 3 and predominant expression of the reparative substances, including fibronectin, galectin-3, and fibrinogen at Day 7 .Even more so, according to the recent data neutrophils may be subcategorized into N1- and N2-subpopulations, with CD206-N1-neutrophils being inflammatory and CD206+ N2-neutrophils – ant-inflammatory cells . In miceCurrent studies provided information that neutrophils are characterized by the even more significant heterogeneity than it was originally thought. In humans there were recognized CD177+, olfactomedin 4 (OLFM4)+, T cell receptor αβ+, CD49d+CXCR4+vascular endothelial growth factor (VEGFR)+ neutrophilic subsets. Neutrophils expressing programmed death-ligand (PD-L)1, arginase 1(ARG1), CD10 are reported to have potent immunosuppressive activity . HoweverAnother subpopulation of granulocytes which role in myocardial infarction is usually overlooked is represented by eosinophils. In steady state they constitute 1-3% of leukocytes in peripheral blood and contain primary and secondary granules, as well as lipid bodies . Main acMeanwhile, patients with low eosinophilic cell count were characterized by the bigger infarct size and higher rate of cardiac events during long-term follow-up . Thus, eEosinophils also secrete IL-4, cytokine playing an essential role in differentiation of macrophages towards M2 phenotype, and regulating myocardial tissue regeneration , 54. EosintCD62L+CD101lo eosinophils  with homeostatic functions has recently been described in lungs , arising either from the yolk sac or fetal liver, and providing immune surveillance and regulation of cardiac functions , 56. Evereceptor . These ireceptor .neg; in mice – Ly-6Chi. They constitute about 90% of all circulating monocytes and are characterized by high expression of CD62 and CCR2, which enables their recruitment to the site of injury towards the gradient of MCP-1 and chemokines CCL2 and CCL7. Classical monocytes produce inflammatory cytokines and nitric oxide and are able to differentiate into macrophages and dendritic cells. They peak at Day 3 after MI, and are involved in breakdown of necrotic myocardium  and CD16 (low-affinity Fcγ-receptor). Classical monocytes in humans are described as CD14++CD16ocardium . Even thocardium . Blockagocardium . Increasocardium . Meanwhihi and murine non-classical monocytes are Ly-6Clo. High expression of chemokine receptor CX3CR1 is typical for non-classical monocytes, allowing their homing to the infarction site by gradient of chemokine fractalkine. Their function and origin are less clear than classical monocytes. Mainly they represent a patrolling monocyte population, being found close to the vessel wall in the circulation and scavenging oxidized lipids, cellular debris and pathogens , absent in mice. Most likely these cells represent a transition step between classical and non-classical monocytes. Their function is described primarily as inflammatory  also known as colony stimulating factor 1 (CSF-1). The following post-differentiation stimulation with either LPS/IFN-gamma/TNF-alpha or IL-4/IL-13 will respectively initiate transition of M1/M2 macrophages into the activated state  and did not decrease further on, predominating in the infarct area . Specifihi inflammatory macrophages in the post-infarct myocardial tissue. Yet, animals performed improved LV function in 2 months of observation compared to intact animals, thus, questioning the detrimental role of Ly-6Chi macrophages in cardiac remodeling. It is worth noting that the decreased accumulation of neutrophils was also observed in animals after GATA3-knock out, thus representing the confounding issue of the study  and between M_IL‐4 and M_IL‐10 (previously reported together as M2 macrophages) . The useMast cells represent cells of immune system of myeloid origin, residing in mucosa and connective tissue and mainly being involved in development of allergic conditions. They are abundant in human heart and coronary lesions and were shown to participate both in plaque rupture and processes of myocardial remodeling after infarction –87. MastT (with granules containing only tryptase) and MCCT . Heart mast cells have been described to have MCct phenotype -2 on cardiomyocytes by tryptase. As a result of this activation anti-inflammatory 15-deoxy 12,14 prostaglandin J2 (15-d-PGJ2) is produced from Cyclooxygenase-2 (COX-2) . 15-d-PGDendritic cells (DCs) represent an extremely heterogeneous population of immune cells, both of myeloid and lymphoid origin. Their main function is presentation of antigens to T-lymphocytes. DCs express MHC II, similarly to monocytes and macrophages, but belong to a distinct cellular population . They alTwo major DC subpopulations were identified in men and mice, such as plasmacytoid DCs (pDCs) and conventional DCs (cDCs), further classified into CD141+/CD103+ cDCs1 and CD1c/CD11b+ cDCs2 lineages . Both pDDCs may modify the process of post-MI healing through activation of T-lymphocytes in draining lymph nodes, production of inflammatory cytokines and direct activation of fibroblasts . BesidesExperimental works using depletion of DCs to study their function show inconsistent results. Depletion of conventional DCs in chimeric mice was associated with decrease of macrophages at day 7 post-MI, followed by reduction of infarct size, prevention of ventricular remodeling and improvement of cardiac function . HoweverThus, the ultimate result of the DCs activity in the course of AMI depends on the dendritic cells’ subpopulation being involved and subsequent type of the recruited effector cells. Ideally, activation of the conventional DCs should be followed by the increase of the tolerogenic DCs’ activity to provide timely resolution of inflammation.T-lymphocytes are the cells of the adaptive immunity. Presentation of the specific antigen by antigen-presenting cells in secondary lymphoid organs is an obligatory stage in their activation and clonal expansion . PopulatCells of the adaptive immunity attracted attention of the researchers not so long ago, and their function during MI remains elusive and stem from experimental works. It has been shown that RAG1 knock-out (KO) mice, which lacked T-lymphocytes, developed infarction of smaller sizes, compared to the wild-type animals. The primary role for modulation of the injury size has been ascribed to CD4+ T-lymphocytes, with interferon-γ (IFN-γ) and IL-17 being tightly involved in the process , 105. Thvia cleavage of CD31, which normally inhibits signaling through T-cell receptor (TCR)  .At the same time, pro-inflammatory milieu, formed during MI, creates conditions favorable for activation of autoreactive clones of T-lymphocytes, which may trigger autoimmune destruction of the myocardial tissue . Heart-sT-lymphocytes are attracted to injured myocardium and are detained there. One of the possible chemokines, involved in this process, is fractalkine, recognized by T-lymphocytes’ receptor CX3CR1 . T-cellsCytotoxic CD8+ T-lymphocytes were also shown to be involved in post-infarction inflammation . CD8+ T-Protective effects mediated by T-lymphocytes may be mediated by the distinct CD4+ and CD8+ subsets positive for expression of angiotensin receptor AT2 , 115. Buin situ conversion to Tregs, which were characterized by enhanced expression of inhibitory check-point molecules, such as CTLA-4, TIGIT (T cell immunoreceptor with Ig and ITIM domains), PD-1, BTLA (B- and T-lymphocyte attenuator)  .Ectoenzymes CD39 and CD73 are expressed on the membrane of Tregs. Ectonucleoidase activity of these molecules input to the increase of the local adenosine concentration, which has been shown to suppress inflammatory activity of conventional CD4+ T-lymphocytes in myocardial injury , 118. Trvia agonistic antibodies led to the reduction of the recruitment of neutrophils, monocytes and lymphocytes to the site of myocardial injury and suppressed activation of CD8+ lymphocytes  represent a recently described subpopulation of cells that originate from myeloid precursors in bone marrow, blocked at different steps of their development. This subpopulation is highly heterogeneous and at present includes polymorphonuclear MDSC (PMN-MDSC), monocytic (M-MDSC) and early-derived MDSC (eMDSC) . In steaAdministration of rapamycin suppressed activation of the mammalian target of rapamycin (mTOR) and activated MDSC during acute kidney injury . This acMDSCs were elevated in patients with HF, and their depletion aggravated heart function . The kinB-lymphocytes represent cells of the adaptive immunity, characterized by the production of antibodies and mediating humoral immune response. Two major types of B-lymphocytes have been described: B1-lymphocytes  and B2-lymphocytes  . B2 lympThe main effector molecules produced by B-lymphocytes are antibodies. Antibody production in performed by plasma cells – the terminal stage of B-lymphocyte differentiation. Antibodies increased destruction of myocardial tissue, as blocking of IgM led to the significant reduction in ischemia-reperfusion injury . At the high monocytes to myocardium, has been shown to be dependent upon B-lymphocytes. Depletion of B2-lymphocytes from circulation by anti-BAFF of anti-CD20 antibodies disrupted monocytes recruitment and was associated with the reduction of infarct size and favorable heart remodeling  cells constitute 2-5% of all peripheral blood leukocytes and belong to the heterogeneous group of innate lymphoid cells (ILC), type I ILC in particular. They exhibit lymphoid cellular morphology, but do not have antigen specificity. Instead, they express a great variability of invariant receptors, recognizing both self and non-self antigens. NK activation depends on the balance between activation and inhibitory signals they receive during interaction with their target cells. NK cells express transcriptional factor T-bet and produce IFN-γ, perforin and granzyme B .NKs’ role during MI still remains poorly characterized. NK cells appeared to be the major source of IFN-γ during angiotensin (AT) II induced vascular dysfunction . NK cellAt the same time there are data pointing to the possibility of insufficient NK function during MI. Patients with acute coronary syndrome were characterized by the lower numbers of NK cells in the peripheral blood compared to patients with stable angina . One of A distinct minor subpopulation of invariant natural killer T (iNKT) cells  simultaneously expresses NK activating and inhibitory receptors and an invariant αβ TCR which can recognize lipid and glycolipid antigens (mainly DAMPs) . Infiltrvia production of IL-4 and switching of macrophages to patrolling phenotype  is one of the most potent stimulators of both NK and iNKT activity. Administration of αGC to mice with ischemia/reperfusion reduced the infarction size, but whether this effect was mediated by iNKT cells or represents epiphenomenon remains disputable .via targeting of the single signaling pathways or definite cellular populations  may activate either inflammatory or anti-inflammatory genes depending on the nature of the transcription factor being recruited .The issue of targeted delivery of immune-modulating medications during MI also needs to be elaborated to avoid undesirable systemic effects. Current approaches propose to use hydrogels, patches, inhaled nano-particles, extracellular vesicles including exosomes and are reviewed in detail elsewhere . All theNew diagnostic approaches, such as fluorescence-based techniques, single-cell RNA sequencing and mass spectrometry imaging and profiling, which become more and more wide-spread and affordable, allow to evaluate single cell metabolomics and transcriptomics and propose personalized targeted ways of metabolic and epigenetic reprogramming depending on the initial state of immunometabolism , 158.In the perspective it might be possible to find a powerful signaling mechanism to manipulate cellular heterogeneity in the course of the immune response during AMI and influence not only a single subpopulation, but redirect functional activity of the whole cellular diversity in a desired way. Identification of molecules that may be used during AMI to obtain time-dependent equilibrium in immune regulation through metabolic and epigenetic rearrangement represents a promising area of future research.via total depletion of a definite cellular subset has not been associated with a more favorable outcome, or has led to inconsistent results. Rather, keeping a time-dependent balance in the work of immune cells, when inflammatory activity is followed by timely switching on of the resolution and reparative mechanisms, is what matters in the successful heart healing and remodeling following infarction.Increase of patients’ survival after AMI is associated with spread of HF frequency in population. Hence study of consequences of cellular response to myocardial injury will remain an important research area withholding both fundamental and practical significance. Summarizing our review, we can state that there are no “good” or “bad” players in the realm of the immune cells involved in myocardial remodeling after infarction. Each subset of immune cells present in the heart may either increase injury and inflammation or initiate healing of the cardiac tissue. Suppression of inflammation The substantial question remains, which is the nature of the factors that orchestrate the preservation of the balanced immune response during myocardial infarction, and which are the factors that lead to the impairments in this process? We need to keep in mind, that patients face myocardial infarction already having a dramatic background of various health problems, including but not limited to atherosclerosis, other chronic inflammatory disorders, metabolic disturbances, increased blood pressure, aging, history of smoking and chronic stress, etc., all of which being associated with immune dysregulation of various kind and different scale. The amount of fundamental and clinical data obtained in the field up to date is enormous, but does not lead to the significant improvements. We need to admit that further breakthrough in management of post-infarction remodeling requires a novel instrument to systematize an exciting data. Possible solution may be the use of high-throughput methods and artificial intelligence to develop “digital counterparts” of inflammation which will allow creation of “inflammatory portrait” for each patient and identification of an existing gap in the work of immune cells in each single case. As a matter of fact, this foreshadows the end of the population-based medicine and beginning of the personalized medicine era.VR and TS conceived the concept and designed the manuscript outline. IV and MS wrote the manuscript. IV prepared figures. TS and VR performed critical evaluation of the manuscript. IV prepared manuscript for publication. All authors contributed to the article and approved the submitted version.This work was supported by the Ministry of Science and Higher Education of the Russian Federation [theme of fundamental research AAAA-A15-115123110026-3].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CBX7, JADE2, IGF2BP3, OR2W6P, PRAME, TICRR, KIF4A, and PIMREG) could accurately identify patients' prognosis and the signature had close interactions with the immunodominant tumor environment, which may provide insight into personalized prognosis prediction and new therapies for PBT patients.Genomic studies have provided insights into molecular subgroups and oncogenic drivers of pediatric brain tumors (PBT) that may lead to novel therapeutic strategies. Participants of the cohort Pediatric Brain Tumor Atlas: CBTTC (CBTTC cohort), were randomly divided into training and validation cohorts. In the training cohort, Kaplan-Meier analysis and univariate Cox regression model were applied to preliminary screening of prognostic genes. The LASSO Cox regression model was implemented to build a multi-gene signature, which was then validated in the validation and CBTTC cohorts through Kaplan-Meier, Cox, and receiver operating characteristic curve (ROC) analyses. Also, gene set enrichment analysis (GSEA) and immune infiltrating analyses were conducted to understand function annotation and the role of the signature in the tumor microenvironment. An eight-gene signature was built, which was examined by Kaplan-Meier analysis, revealing that a significant overall survival difference was seen, either in the training or validation cohorts. The eight-gene signature was further proven to be independent of other clinic-pathologic parameters via the Cox regression analyses. Moreover, ROC analysis demonstrated that this signature owned a better predictive power of PBT prognosis. Furthermore, GSEA and immune infiltrating analyses showed that the signature had close interactions with immune-related pathways and was closely related to CD8 T cells and monocytes in the tumor environment. Identifying the eight-gene signature ( Brain tumors are the most common solid tumor in pediatrics, accounting for 23.7% of new cancer diagnoses in children , and theAlthough the cure rate of PBT has increased in the past two decades of the 20th century, which was primarily due to advances in imaging, neurosurgery, and radiation oncology technologies and the introduction of combined chemotherapy, unfortunately, the overall survival has remained static . The lachttps://kidsfirstdrc.org>) is a new, collaborative, pediatric research effort to understand the genetic causes and links between childhood cancer and structural congenital disabilities. This is a brand-new public database that has only been launched in recent years. It is vital that this data is expressly set up for children's tumor research, and few researchers have begun to mine this database.The Gabriella Miller Kids First Data Resource Center  with the cohort name: Pediatric Brain Tumor Atlas: CBTTC (CBTTC cohort). The study was conducted following the Declaration of Helsinki, and the Ethical Committee of Zhumadian Central Hospital approved the study.The gene expression profiles of PBT from 973 patients and their clinical and survival data were downloaded from Kids First Xena Hub (<http://www.r-project.org>) and the “glmnet” package (<http://cran.r-project.org>). Ten-time cross-validations were applied to detect the best penalty parameter lambda  and validation cohort . In the training cohort, Kaplan-Meier analysis was used to screen the prognostic genes with a cutoff of P value <10.0E-15 in the log-rank test. Furthermore, univariate Cox regression analysis was performed on the training cohort to find prognostic genes with P values <10.0E-15. The intersected genes identified in Kaplan-Meier and univariate Cox analyses were then entered into the LASSO Cox regression model analysis, which was implemented in the training cohort utilizing R software . GSEA was performed based on the downloaded gene set collections using GSEA software . The entire CBTTC cohort was taken to GSEA to reveal the functions and pathways in the differentially expressed genes between high-risk and low-risk groups. Only gene sets with family-wise error rate (FWER) P values <0.05 were considered significant.The Hallmark (v7.1) and C7 (v7.1) gene set collections were downloaded from the Molecular Signatures Database v7.1 download page  served as a pointer of prognostic accuracy. The R package “pROC” was used for ROC analysis, and the “Delong” method was used to study the significant differences among ROC curves. In all analyses, a P value <0.05 was considered statistically significant.The flowchart of the present research is shown in Ninety-two genes were extracted from the Kaplan-Meier analysis (Table S1), while 136 genes were identified as significant in the Cox regression analysis (Table S2). Taken together, 72 genes in the intersection of the two results are defined as prognostic genes for subsequent analyses (Table S3). These prognostic genes were then subjected to LASSO Cox regression analysis, and regression coefficients were calculated. The coefficient of each gene is illustrated in CBX7 and JADE2 were under-expressed in high-risk patients, while IGF2BP3, OR2W6P, PRAME, TICRR, KIF4A, and PIMREG were highly expressed in high-risk patients. We also verified the performance of this eight-gene signature in the validation cohort. As shown in The distribution of risk scores and survival status and the expression profiles of the eight-gene signature of the patients in the training cohort were plotted and are shown in Kaplan-Meier survival analysis showed that patients in the high-risk group were associated with a poor prognosis trend in the training cohort  were correlated with the eight-gene signature risk score in PBT patients, further supporting that the signature interacted closely with the tumor environment. Combining the CIBERSORT and MCP-counter methods, we found CD8 T cells and monocytes were in a close relationship with the eight-gene signature in the tumor environment of PBT. Strategies targeting the tumor microenvironment of pediatric brain cancers have the potential to improve the efficacy of standard and genome-based molecular therapeutics and to help resolve many of the challenges associated with developing new drugs and running clinical trials for a relatively small PBT population . The speOur research also had some limitations. For the study of PBT, currently available public databases are very limited. The datasets in GEO and TCGA are not eligible for validation purposes because of the population's age distribution. Our eight-gene signature came from retrospective data, and more prospective data is needed for proving the clinical utility of it. In addition, due to the very limited clinical characteristics of patients included in the CBTTC cohort, we could not perform certain clinical subgroup analyses. Furthermore, there are currently no wet experimental data explaining the relationship between these eight genes and their mechanism in PBT samples. Therefore, more research is needed to clarify the potential relationship.In conclusion, our research defined a robust eight-gene signature in PBT. It was a comprehensive analysis of the new Kids First database. This signature was related to PBT's overall survival and accurately identified the prognostic risk of patients. Notably, we assessed the reliability and accuracy of the signature in a validation cohort. In addition, the functions and immune infiltrating analyses showed that the signature had close interactions with CD8 T cells and monocytes in the tumor environment, which may advance the development of new therapies for PBT treatment.