Objectives: The objective of this systematic review was to synthetize the published data on the relationships between elevated blood C-reactive protein (CRP) levels and schizophrenia (SZ) onset risk, illness characteristics and treatments, cognition and physical health.Method: The systematic bibliographic searches have been carried out according to the Cochrane methodology. Medline, web of science, Google Scholar with each database being searched from inception to November 2017.Results: 53 studies were included in the present review. While meta-analyses including case-control studies suggest a clear association between CRP and SZ, one other study has suggested that CRP-associated genes were associated with a lower risk of SZ onset. Increased CRP has been significantly associated with positive symptoms in acute phase psychosis, while studies including community-dwelling stabilized subjects did not find such an association. Abnormal CRP has been associated with a wide range of cognitive impairment in SZ stabilized individuals. Body Mass index has been extensively associated with increased CRP in SZ subjects; and increased CRP has been identified as a risk factor for metabolic syndrome and cardiovascular risk in SZ subjects. Increased CRP has also been associated with high nicotine dependence in SZ smokers and one study has suggested that increased CRP was associated with sedentary behavior.Conclusion: In the light of the above-mentioned studies, increased hs-CRP may be reasonably suggested as a marker for SZ onset risk, as well as a risk factor for increased positive symptoms, cognitive impairment, hypovitaminosis D, microbiota disturbances, cardiovascular and metabolic syndrome risk in SZ subjects, and increased nicotine dependence in SZ smokers. In case of increased CRP levels, anti-inflammatory strategies  should be also further evaluated.Limits: Most of the studies were cross-sectional and cohort studies are needed to determine the temporal relationship between increased CRP and the psychiatric outcomes. Inflammation is a complex physiologic response to injury or tissue destruction, and involves recruitment of immune cells. The inflammatory hypothesis of major psychiatric disorders, posits that inflammatory processes are involved in the pathogenesis of psychiatric conditions and may underpin some of their neurobiological correlates . SZ is tC-reactive protein (CRP) is the most commonly used biomarker of systemic inflammation worldwide. CRP is a standard laboratory exam and can be measured in the peripheral blood and analyzed in any clinical laboratory around the globe. CRP is therefore a very attractive potential clinical biomarker in psychiatric disorders . CRP is The objective of the present systematic review was to determine if dosing CRP blood levels was relevant to synthetize cross-sectional studies and to determine (i) SZ onset risk in general population (ii) clinical characteristics and treatments (iii) cognitive deficits and (iv) physical health in SZ in- and outpatients.This meta-analysis is based on the PRISMA criteria . The systematic bibliographic searches have been carried out according to the Cochrane methodology. These were performed to find relevant English and non-English language trials from the following databases: Medline, web of science, Google Scholar with each database being searched from inception to November 2017. Medline is considered as the database of highest quality level, and google scholar as the database with the largest referencing of studies. Most of the international congress posters and abstracts are referenced in web of science. Altogether, these three searches enable a comprehensive exploration and limit the risk of publication bias. The associated articles were also explored, to limit the risk of bias associated with the search terms. The primary search strategy was “C-reactive protein” or “CRP” and “schizophrenia” or “ultra high risk psychosis.” Two reviewers (GF and LB) decided on eligibility and extracted data from included studies. The design of the studies, data extraction and data synthesis are described in Table The study selection process is presented in Figure A recent meta-analysis including 18 studies  has concluded that higher CRP levels were associated with increased risk of SZ, especially for young adult patients < 30 years and independently of body mass index . The studies that found no association between increased CRP and symptomatology have recruited community-dwelling stabilized outpatients , 22, 23.One study has found that history of childhood trauma was associated with increased CRP in SZ adulthood , howeverInconsistent findings have been found in regard of the association between abnormal CRP, current depression and antidepressant consumption in SZ, with one study suggesting that increased CRP levels were associated with depressive symptoms, and one other that it was associated with antidepressant consumption , 24. Thiin vitro anti-inflammatory effects of nicotine  choice of this cut-off. No consensual cut-off values have been proposed in psychiatric studies for the analysis of CRP. A recent meta-analysis has pointed out that most of the included psychiatric studies used a cut-off ≥5 mg/L  while thWhile sleep disorders have been suggested to have a bidirectional relationship with inflammation, no study has explored the relationships between abnormal CRP levels and sleep disorders in SZ to date. Except for antipsychotic effects, no longitudinal data has suggested if increased CRP levels were associated with poor prognosis and outcomes in schizophrenia . Further studies should explore if decreasing CRP blood levels may improve SZ outcomes, especially cardiometabolic events, tobacco smoking behavior, quality of life and cognitive functioning. As some add-on anti-inflammatory strategies have shown effectiveness in SZ symptomatology , each anIn the light of the above-mentioned studies, increased hs-CRP may be reasonably suggested as a marker for SZ onset risk, as well as a risk factor for increased positive symptoms, cognitive impairment, hypovitaminosis D, microbiota disturbances, cardiovascular and metabolic syndrome risk in SZ subjects, and increased nicotine dependence in SZ smokers. In case of increased CRP levels, anti-inflammatory strategies  should be further evaluated.GF and LB selected the studies, analyzed the major outcomes and wrote the manuscript. CL and PA reviewed the manuscript. All authors approved the final version.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.


Diffuse cavernous hemangioma of the rectum (DCHR) is a relatively rare disease. A 40-year-old man presented with long-standing lower abdominal discomfort and hematuria. At the time of hospitalization, his vital signs and hemoglobin level were normal. Colonoscopy showed markedly dilated blood vessels in the sigmoid mucosa, which was confirmed on magnetic resonance imaging and computed tomography as cavernous hemangioma. Without surgery, there have been no signs of progression of DCHR during a 3-year follow-up period. Diffuse cavernous hemangioma of the rectum (DCHR) is a rare benign vascular tumor that is characterized by delayed diagnosis.We report a case of a 40-year-old man who was admitted to our hospital due to long-standing lower abdominal discomfort and hematuria. He had no history of fever, abdominal pain, intermittent rectal bleeding, or weight loss. He also had no notable medical history or family history. His vital signs were normal and laboratory testing revealed a normal complete blood count without anemia.Colonoscopy showed markedly dilated blood vessels in the sigmoid mucosa Fig. . AbdominDCHR is a rare clinical condition, which mainly affects young adults. Since the first case of rectal hemangioma reported by Phillips in 1839, there have been only approximately 350 cases reported worldwide . HoweverAlthough this disease presents with nonspecific symptoms, colonoscopy and abdominal CT and MRI can reveal specific findings that can serve as diagnostic clues; these typical and characteristic findings are irregularly thickened rectal wall and multiple calcified foci of pelvic phleboliths. In DCHR, biopsy is contraindicated because of the high risk for bleeding; the findings on CT or MRI are usually sufficient to make the diagnosis . MultiplGenerally, complete surgical resection of DCHR is the only treatment and most cases are operated to control rectal bleeding . AlternaIn conclusion, colonoscopy, abdominal CT and MRI are useful to make a diagnosis of DCHR, and some case, especially those without a history of bleeding, could be followed-up efficiently without excessive invasive examinations.


Bunyavirales, family Hantaviridae), known as important zoonotic human pathogens, possess the capacity to exchange genome segments via genetic reassortment due to their tri-segmented genome. Although not as frequent as in the arthropod-borne bunyaviruses, reports indicating reassortment events in the evolution of hantaviruses have been recently accumulating. The intra- and inter-lineage reassortment between closely related variants has been repeatedly reported for several hantaviruses including the rodent-borne human pathogens such as Sin Nombre virus, Puumala virus, Dobrava-Belgrade virus, or Hantaan virus as well as for the more recently recognized shrew-borne hantaviruses, Imjin and Seewis. Reassortment between more distantly related viruses was rarely found but seems to play a beneficial role in the process of crossing the host species barriers. Besides the findings based on phylogenetic studies of naturally occurring strains, hantavirus reassortants were generated also in in vitro studies. Interestingly, only reassortants with exchanged M segments could be generated suggesting that a high degree of genetic compatibility is required for the S and L segments while the exchange of M segment is better tolerated or is particularly beneficial. Altogether, the numerous reports on hantavirus reassortment, summarized in this review, clearly demonstrate that reassortment events play a significant role in hantavirus evolution and contributed to the currently recognized hantavirus diversity.Hantaviruses (order  Hantaviridae.First reports that genome segment exchange, in other words genetic reassortment events, could have been occurring in the evolution of hantaviruses are rather old and emerged soon after the discovery of hantavirus cardiopulmonary syndrome-causing Sin Nombre virus (SNV) in the United States , 2. UntiBunyavirales, family Hantaviridae) are enveloped, single stranded RNA viruses with segmented genome of negative polarity. The genome is composed of three segments, small (S) segment encoding nucleocapsid protein (N protein) [Hantaviruses (order protein) , medium protein) , and larprotein) .Virus entry into cells is mediated by binding to a cell surface receptor. Integrins are considered to be the main receptors for hantaviruses at least in vitro –8 but otWhen transmitted to humans, hantaviruses can cause severe disease. Transmission occurs usually through inhalation of aerosolized rodent excreta and rarely via biting by infected animals , but theHantaviruses produce chronic and asymptomatic infection in their reservoir hosts, small mammals. Besides with hantaviruses typically associated rodents, other small mammals such as shrews, moles, and bats were identified as hantavirus reservoir hosts during the last decade –22. VeryReassortment is defined as exchange of gene segments between viruses that co-infect the same cell, which can result in the formation of progeny viruses that are genetically distinct from both parental viruses. Therefore, reassortment can create viral progeny conferring important fitness advantages. On the other hand, successful reassortment between two parental strains during co-infection requires a high degree of genetic compatibility including intricate packaging signals and RNA–RNA and/or RNA–protein interactions .Reassortment is particularly well known for influenza A virus where it is associated with the antigenic shift and emergence of new pandemic strains . HoweverBunyavirales) with their tri-segmented genome are obvious candidates for reassortment playing role in their evolution. Indeed, reassortment seems to be rather frequently reported within the order, especially within the family Peribunyaviridae (former genus Orthobunyavirus). Particularly interesting is the fact that there are frequent reassortment events found between distinct viruses . Briese et al. [Bunyaviruses , the principal host of SNV, trapped in Nevada and eastern California. Phylogenetic analyses of all three segments indicated that several segment exchanges were possible but those involving M segment were found most frequently. Conflicting signals in the S and M segment-based phylogenetic trees suggesting reassortment were also found in a more recent study involving SNV sequences obtained from deer mice collected in Colorado, New Mexico, and Montana from 1995 to 2007 [First findings indicating reassortment events in hantaviruses were reported for SNV soon after its discovery. Li et al.  analyzed to 2007 .Myodes glareolus). In a series of systematic studies performed in central and northern Finland, notably high frequency of reassortment, 19.1–32%, could be observed [BMALA, SAMBLB, SBMALB where the S, M, and L capital letters stand for the genomic segments and the subscripted A and B letters indicate origin of the given segment to one of the two hypothetical, phylogenetically distinct parents  associated with bank voles  on Saaremaa island, Estonia seems to be a reassortant containing M segment clustering within another A. agrarius-associated genotype, Kurkino, while its position in the S phylogenetic trees is more ancestral and more closely related with the A. flavicollis- and A. ponticus-associated Dobrava and Sochi genotypes, respectively. Similar conflicts in tree topologies suggesting genetic reassortment during DOBV evolution have been observed also for the Sochi genotype. In S segment trees, Sochi sequences form a well-supported sister group to Dobrava genotype but form an out-group to all other DOBV strains in M and L segment trees [Indications for reassortment events found within enotypes , are assnt trees .A. agrarius mice captured from 2003 to 2014 in the Republic of Korea indicated occurrence of natural reassortment events in the evolution of HTNV. In addition to the observation of conflicting tree topologies, the authors provided additional evidence of reassortment by application of a whole array of recombination detecting algorithms on artificially concatenated complete sequences of all three segments. Interestingly, in this case, only a pattern schematically designated as SAMALB could be found [Analysis of 34 complete genome sequences of HTNV acquired from be found . Differebe found .Crocidura lasiura) in the Republic of Korea and China. The reassortment pattern SAMBLA was recently identified in a study from the Republic of Korea [Sorex araneus shrew-borne Seewis virus [Indications for reassortment events were recently reported also for the shrew-borne hantaviruses. Imjin virus (MJNV) is a shrew-borne hantavirus identified in the Ussuri white-toothed shrews . However, in the S segment analyses, JABV clusters only with AAIV and occupies the most ancestral position within the South American hantaviruses [All the above described reassortment events can be considered as intra- or inter-lineage events between closely related strains of the same virus. In most cases, these events also occur within a single reservoir host. However, there are few reports which describe reassortment events between distinct viruses or phylogenetic incongruences at deep nodes. Two of those reports are originating from south America where numerous rodent species have been identified to harbor unique hantavirus strains. Ape Aime-Itapúa virus (AAIV) was identified in Paraguay ; in the . azarae , 65. HowA. agrarius mice and Rattus norvegicus rats revealed that spill over infections of HTNV from its reservoir host, A. agrarius, to R. norvegicus rats might be quite common. Most unexpectedly, two isolates, originally generated in 1988 from R. norvegicus rats, were shown to contain M and L segments of SEOV but the S segment of HTNV. This finding raises the question that, if such reassortants of HTNV and SEOV are possible, what could be their consequences for the public health and why are they not detected more frequently. It remains to be seen whether such viruses remained unnoticed because sequence analyses of more than one segment were not routinely performed until recently or whether those two isolates represent highly exceptional findings.Very surprising findings were reported by Zou et al. . SequencTalpa europaea). Occurrence of two highly divergent viruses in the same reservoir host shows that at least one of the viruses was involved in host-switching processes. Phylogenetic analyses of all three genomic segments showed tree topology inconsistencies at deep nodes, suggesting that Bruges virus may have emerged from ancient reassortment events. The virus appeared to be most closely related to hantaviruses associated with hosts from the Muridae family, to hantaviruses carried by shrews and moles, or showed ancestral position to both these groups in the S, M, and L segment-specific analyses based on complete coding sequences, respectively [Recently, Bruges virus as second hantavirus in addition to Nova virus was identified to be harbored by the European mole , an HPS-causing virus from Florida, which has the cotton rat (Sigmodon hispidus) as its natural host; the reassortant carried the M segment of SNV and S and L segments of BCCV. Interestingly, in both experiments about 30% of the progeny virus plaques appeared to be transiently diploid, i.e. containing both versions of at least one segment.Rodriguez et al.  showed aSimilar experiments were performed with SNV and ANDV by Rizvanov et al. . Again, The very same reassortment pattern was achieved also in the study of McElroy et al. . In lineReassortant could be later generated also between a pathogenic PUUV and a non-pathogenic Prospect Hill virus (PHV). The reassortant contained the glycoprotein coding M-segment derived from PUUV and the S and L segments from PHV. The reassortant together with parental viruses were characterized also in terms of their ability to modulate in vitro innate immune responses including induction of type I and type III interferon and interferon-stimulated gene MxA. In all experiments, the reassortant revealed the same characteristic innate antiviral response pattern as PHV, which is considered to be a non-pathogenic hantavirus. These data are not only consistent with the previous studies on SNV and ANDV reassortants but also led the authors to the conclusion that such reassortant viruses carrying M segment of the pathogenic virus together with S and L segments of non-pathogenic virus (such as PHV) could be used as attenuated vaccines .AMBLA and SBMALB throughout this review) were found. Analogously to the study of Handke et al. [Inspired by the phylogenetic findings of putative reassortment events in DOBV , Kirsanoe et al. , the reaAlso in this reassortment experiment, significantly high proportion of the analyzed clones  were designated as diploids containing both parental versions of at least one segment . In factAlthough not as frequent as in other arthropod-borne bunyaviruses, reassortment seems to be more common among hantaviruses than initially recognized. The intra- and inter-lineage reassortment between closely related variants seems to occur whenever co-infections of two virus variants are possible due to their sympatric occurrence.On the other hand, heterotypic reassortment between more distantly related viruses occurs less frequently but seems to play a supporting role in the process of crossing the species barriers and host switching when, e.g., the newly acquired M segment encoding the envelope glycoproteins might help the virus to establish persistent infection in the new host. Topologic incongruences in the deep nodes of the segment specific trees suggest that such events occurred in the past and contributed to the currently recognized hantavirus diversity.All the reported in vitro reassortment experiments have in common that only reassortants with exchanged M segments could be generated. This finding suggests that a high degree of genetic compatibility including packaging signals and RNA–RNA and/or RNA–protein interactions is required particularly for the S and L segments while the exchange of M segment is better tolerated or is particularly beneficial.Altogether, the numerous reports on hantavirus reassortment varying from naturally occurring intra-lineage reassortants to in vitro generated inter-species reassortants, as summarized in this review, clearly demonstrate that reassortment events play a significant role in hantavirus evolution. Consequently, it will be highly beneficial to invest in obtaining complete sequences of all three genomic segments in the future studies. It can be assumed that advancement of the next-generation sequencing technologies will generate more high-quality data which are needed to further elaborate the current accumulating evidence of reassortment as a significant driving force in hantavirus evolution.


In this review, we discuss the immunomodulatory properties supporting the therapeutic use of MSCs in GVHD and contextualize the main clinical findings of recent trials using these cells. Critical parameters for the clinical translation of MSCs, including consistent production of MSCs according to Good Manufacturing Practices (GMPs) and informative potency assays for product quality control (QC), are addressed.Graft versus host disease (GVHD) is a common condition in patients subjected to allogeneic hematopoietic stem cell transplantation (HSCT). The immune cells derived from the grafted stem cells attack recipient’s tissues, including those from the skin, liver, eyes, mouth, lungs, gastrointestinal tract, neuromuscular system, and genitourinary tract, may lead to severe morbidity and mortality. Acute GVHD can occur within few weeks after the allogeneic cells have engrafted in the recipient while chronic GVHD may occur any time after transplant, typically within months. Although treatable by systemic corticosteroid administration, effective responses are not achieved for a significant proportion of patients, a condition associated with poor prognosis. The use of multipotent mesenchymal stromal cells (MSCs) as an alternative to treat steroid-refractory GVHD had improved last decade, but the results are still controversial. Some studies have shown improvement in the life quality of patients after MSCs treatment, while others have found no significant benefits. In addition to variations in trial design, discrepancies in protocols for MSCs isolation, characterization, and  Allogeneic hematopoietic stem cell transplant (HSCT) is a treatment for high risk hematological and malignant diseases. Conditioning regimen, immunosuppressive strategies, supportive care and prophylaxis for infectious disease are improving, reducing mortality related to transplant . HoweverGraft versus host disease occurs when immunologically competent donor T lymphocytes recognize recipient’s tissue as foreign resulting in damage in many organs systems including skin, liver, gastrointestinal tract, and lung. Clinical manifestations are classified as acute or chronic GVHD. In the past, all clinical manifestations of GVHD occurring before 100 days of transplant where classified as acute GVHD. When clinical manifestations occurred later on, after 100 days of transplant, it was considered chronic GVHD. In 2005, the National Institutes of Health (NIH) Consensus Conference determined new criteria of diagnosis and scoring and abolished the 100 days criterion. NIH Consensus considered acute and chronic GVHD as two distinguished symptoms without restriction of time .Among all patients undergoing allogeneic HSCT 30–50% will have acute GVHD (grade 1–4) and 14% of patients exhibit chronic GVHD. The most important acute GVHD risk factor is human leukocyte antigen (HLA) mismatch . Transplin vitro and in vivo and natural killer cells (NK cells) have been shown to modulate GVHD in a clinical trial reducing incidence of GVHD . Four studies using umbilical cord-derived MSC to prevent GVHD and one study using adipose-derived MSCs to treat GVHD have also been reported. These studies have compared MSCs treatment only with conventional treatment or historical control. Thus far, no studies have compared the impact of different biological sources of MSC in clinical outcome. Variabilities in trial design is one main limitation for carrying out a meta-analysis to determine which biological source is better or if they are all equivalent in terms of adverse effects and therapeutic benefits .de novo malignancies have been reported, although the clinical follow-up in the examined trials were rather short, ranging from 3 to 60 months . Infusion was safe and well tolerated in all studies (doses ranging from 0.1 to 10 million cells per kg) and there was no report of oncogenesis in the follow-up period. Trials using MSCs prophylactically had a median follow-up of 10–60 months. Follow-up of trials using MSCs as treatment varied from 2 to 23 months .As immunosuppressive treatment can reduce graft-versus-leukemia (GVL) effect, two studies reported relapse of malignant disease in short follow up . HoweverSafety regarding the use of MSCs also involves evaluation of possible complications related to embolism in capillary-rich organs. Among the most common routes of MSCs delivery, including topical application, intramuscular or direct injection into organs, intravenous and intra-arterial infusion, the preferred route is the intravenous . DespiteA study by In another experimental model, Altogether these studies indicate that patients in cell therapy protocols should be monitored for possible embolism or thrombus events related to MSCs infusion and, when applicable, antithrombotic therapies could be applied. Preclinical studies like these are of utmost importance to prevent translational failure. To this end, critical parameters such as the therapeutic window, delivery route, type of cells, immunogenicity, comorbidity, and concomitant treatment, should be considered in these studies.Evidence of efficacy is difficult to determine since the clinical studies published so far are highly heterogeneous. Some studies use MSCs as prophylactic scheme, with infusion in pre-determined days. Six studies analyzed incidence of acute GVHD and other six studies evaluated chronic GVHD , 2. PossOnly two controlled trials evaluated efficacy in acute GVHD treatment without difference in clinical manifestation in both groups (MSCs versus no MSCs). In one study with pediatric patients, complete and partial responses were reported in 58 and 17% patients, respectively . One trial evaluated MSC for treatment of chronic GVHD in 40 patients. In this study, complete and partial responses were observed in 15 and one patients, respectively  .As for safety, the cell administration route is a variable potentially impacting therapeutic effects. MSC systemic delivery is one of the most common administration route in cell therapy since it does not require an invasive procedure but, on the other hand, it relies on a transendothelial cell migration process toward the lesion site, which could have a direct influence in the treatment efficacy Mesenchymal stromal cells should be able to exit circulation and migrate to tissues/organs in order to repair local lesions due to GvHD. This process initially involves a contact between the cells and the endothelium and, subsequently, transmigration toward the target tissue/organ. MSCs rolling depends on presence of platelets  and diffEnhancing migration and homing of MSCs to target tissue/organ could be achieved by genetic engineering these cells to increase the expression of chemokines, integrins or selectins . This ap6 cells. Since this pioneer study, many others have been performed over the last decade establishing an excellent safety profile for both, autologous and allogeneic MSC infusions, over a range of cell doses and different schemes  or low dose (2 × 106 MSC/kg). Patients were stratified for dose levels between grades II and grades III/IV of acute GVHD. Standard steroid therapy using glucocorticosteroids and cyclosporine or tacrolimus, was continued at therapeutic dose levels. Seventy seven percent of patients had complete response and 16% partial response  without difference in both arms . In these studies, patients were treated with MSCs doses ranging from 10MSC dose .The MSCs anti-inflammatory properties as well as homing to sites of inflammation, immunomodulatory properties, and trophic influence on tissue repair, have made these cells very popular for clinical studies . Up to Fex vivo for clinical purposes, as an alternative method for regenerative therapy in many diseases. In any case, regulatory issues related to the safety, efficacy and quality of MSC therapies must be considered while preparing a cell- or tissue-based product for clinical and commercial use. Quality assurance (QA) and quality control (QC) must be provided in any cell manipulation under good manufacturing practices (GMP) grade.Currently, distinct strategies are used to produce human MSCs Thus far, there is no consensus on the production of MSCs regarding GMP system. The QC standards may be discussed individually on each project following the local regulatory agency authority. In Brazil, the regulatory agency , establish specific criteria for advanced therapy products to assure the quality, safety and efficacy of cell therapy in the country (Resolutions: RDC 214/2018 and RDC 260/2018). Cell viability, cytogenetics, potency tests and sterility tests to verify contamination by mycoplasma, aerobic and anaerobic bacteria, fungus, and special microorganisms such as filamentous fungus, are routinely used to ensure the quality of the cell product. Main GMP issues addressed by regulatory agencies in different countries are comparable and can be assessed in ex vivo expansion. A study by Nikitina and colleagues estimated that around 10% of MSC samples contain chromosomal aberrations after expansion, although the clinical consequences of such aberrations are unknown (According to the Foundation for the Accreditation of Cell Therapy (FACT) and Brazil’s ANVISA, cytogenetic testing is mandatory to release a cell product. Most manufacturing units perform karyotyping on the final cell product and the results are an important component of the release criteria. In case of any chromosomal abnormalities in the manipulated cells, the incoming samples may be karyotyped to ensure that the donor does not have any constitutive chromosomal abnormalities and that the alteration has probably originated in a cell clone during  unknown .However, since the potential clinical impact of minor changes in karyotyping is difficult to evaluate, not all services consider the cytogenetic testing as a release criterion .There are many different variations in existing manufacturing protocols for MSC production that may influence the final characteristics of the cells. The type of media supplementation is a typical example. Fetal bovine serum (FBS) is the most common supplementation used. FBS concentration in media ranges from 2 to 20%, with 10% FBS being the most common concentration. This variability on FBS concentration may result in different amounts of growth factors to stimulate cell survival and proliferation . Xeno-frEx vivo MSC expansion may be performed by conventional cell culture techniques or by using bioreactors. Considering autologous use, it is possible to produce lower cell quantity and the scale-out approach can be performed, using planar culture systems with multiple flasks in cell factories. For allogeneic use, MSCs can be expanded to a large number of cells in bioreactor systems  and cryopreserved in cell banks for future use . There are several bioreactors available for cell expansion such as stirred tank bioreactor, rocking bioreactor, hollow fiber bioreactor, and fixed bed bioreactor. The choice of bioreactor will depend on the aim to be achieved, i.e., final amount of cells required for infusion, type of cell (adherent or suspension growing cells), mid/long term use, etc .After MSC expansion, cells are harvested by using an enzyme, usually trypsin. Since this enzyme is normally of porcine origin, alternative GMP recombinant enzymes are available and their use should be prioritized. Mechanical detachment of cells cultured under GMP facilities is also possible by using a cell scraper tool, although detachment must be done gently in order to avoid cell damage and death. While enzymatic MSC detachment can be adapted in expansion protocols using bioreactors, the same adaptation is harder to achieve for mechanical detachment techniques.When high MSC doses and multiple cell infusions are required, which is typically the case of GVHD treatment, cryopreservation of previously expanded cells is an optimal solution to have high amounts of GMP-grade cells readily available for infusion. Nonetheless, cryopreservation is a critical step in MSC manipulation. Although either cryopreservation bags or regular cryotubes could be used for this purpose, the use of the latter is limited by the low volume of cell preparation that can be stored per vial . Cryopreservation media usually contain DMSO as cryoprotectant but it can cause some adverse effects when infused in patients. Typically, cells are stored using a ratio of 10% DMSO and 90% of serum. Thus, after thawing, some laboratories centrifuge and wash cells to remove DMSO. An alternative is to use cryoprotectants containing methylcellulose, sucrose, trehalose, glycerol, hydroxyethyl starch, and polyvinylpyrrolidone. Some companies already developed serum-free and xeno-free cryopreservation media  to circumvent toxicity. Importantly, validation of progressive freezing and thawing cycles is required to avoid significant loss of cell viability.ex vivo expanded MSC need to meet the quality standards required by regulatory agencies.Despite the lack of consensus in critical steps of MSCs production under GMP conditions, different protocols are available to attain high yields of expanded MSCs. However, these Cellular products intended for clinical use must also pass functional evaluation, a key part of a GMP quality control program. Currently, there is no specific release criterion required by the authorities for testing MSC potency. The most used and accepted potency tests for MSCs evaluate their ability to differentiate into three different cell types , to inhibit T-lymphocyte proliferation or to promote endothelial tube formation.in vitro assays that can be easily adapted for routine screening of cell preparations. Nonetheless, adoption of different assays for extensive functional characterization of cells is expensive and time consuming, which may delay the availability of freshly produced MSCs for infusion in patients at critical clinical conditions. Alternatively, a key test could be performed to evaluate a specific MSC property that is correlated with the aimed therapeutic effect. To this end, knowledge of the mechanism of cell action in vivo is of fundamental importance, which reinforces the relevance of basic stem cell biology studies.All these tests involve For instance, exploration of MSC immunosuppressive potential is well established in immunological-based diseases such as diabetes, multiple sclerosis, and GVHD. For this type of application, assaying MSC immunomodulatory activity would better suit a potency test then assaying MSC multipotency. This could be achieved by co-culture assays with T cells to evaluate MSC effects on T cell proliferation and/or by cytokine release by MSC .+ and CD8+ cells. This study suggested that cell-cell contact between MSC and effector cells was not necessary to inhibit T-lymphocyte proliferation (in vitro (feration . Howeverferation . MSC do in vitro .The immuno-suppressive activity of MSCs may also be determined after exposure to IFN-gamma. In this assay, presence of primed MSC could be indicated by expression of MHC class I, MHC II, PDL-1, or other modulatory molecules. Release of certain chemokine receptors such as CXCR3, CXCR4, CXCR5, and CCR7 could also be assayed to indicate immunomodulatory active MSC . As prevlow/D7fib+ve/LNGF+ve cells in samples derived from adults compared with samples derived from children. MSCs obtained from elderly people also showed increased levels of ROS, p21, and p53 proteins. The authors suggested that active MSCs derived from bone marrow decrease in number with age and that these cells are not as potent as the ones isolated from younger patients. Such aging effect on MSC properties should be considered when defining inclusion criteria for MSC donors in cell therapy protocols.Specific MSC properties involved in their expected therapeutic effect may be affected by different factors, including cryopreservation . Thus, pex vivo expansion of cells should help minimize product variability. Also, the choice of potency test is critical in evaluating suitability of the final product, since the desirable therapeutic effect may differ among clinical trials. In the case of GVHD, potency tests addressing MSCs immunomodulatory activity are key factors for obtaining high quality MSCs for therapeutic purposes. Allogeneic MSCs transplantation seem highly appropriate for GVHD treatment, due to the poor clinical condition of patients for tissue donation and lack of necessity of HLA match. In this scenario, the clinical use of cryopreserved third party MSC products offers the additional advantage of faster cell product availability, compared with autologous transplantation. Additional tests to address the cytotoxicity of recipient’s cells toward allogeneic MSCs should help refining the selection of eligible patients. Distribution of MSC products stored in cryobanks to different hospitals is also feasible, allowing potential therapeutic benefits for a greater number of patients in need.Evidences in the literature support safety of MSCs treatment for GVHD, whereas efficacy of this type of cell therapy still needs further clarification. Efficacy of MSC-based treatment is more evident for chronic GVHD patients. Double-blind randomized controlled trials with steroid-refractory GVHD patients should be designed to better address MSCs efficacy. Well-defined inclusion/exclusion criteria for patient accrual and a standardized protocol for GMP production of MSCs should facilitate multicenter studies and acquisition of faster results for clinical outcome assessment. Regarding MSCs production, uniformity in donor age and automated All authors wrote the manuscript and revised the final submitted version.OO was a visiting scholar at Departamento de Hemoterapia e Terapia Celular, Hospital Israelita Albert Einstein.The remaining 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.


The most important part of the customized cell is a synthetic genome. Advanced genomic writing technologies are required to build such an artificial genome. Recently, the partially-completed synthetic yeast genome project represents a milestone in this field. In this mini review, we briefly introduce the techniques for de novo genome synthesis and genome editing. Furthermore, we summarize recent research progresses and highlight several applications in the synthetic genome field. Finally, we discuss current challenges and future prospects.The ultimate goal of  In this sense, one of the core questions for the conventional biological studies is how the phenotypic features of a specific organism are controlled by its genome  re-engineering an existing organism, such as engineering metabolic pathways or constructing genetic circuits to achieve pre-designed functions in the target organism. Metabolic engineering can alter intrinsic metabolic pathways of an organism and/or add new metabolic pathways. Implementation of complex genetic circuits enables newly-designed functions within the target organism. In order to achieve these goals, genome writing technologies with different scales and complexities are needed. Genome writing can be achieved by de novo genome synthesis technology and genome editing technology De novo genome synthesis technologies are better suited for re-writing large-scale genomes with high complexity, whereas genome editing technologies are suitable for small-scale writing tasks on the limited genome loci. In this review, we mainly focus on genome writing by detailing relevant techniques and briefly summarize recent progresses and possible industrial applications. Finally, we elaborate on the current challenges and future directions.In general, synthetic biology can be roughly divided into two subareas De novo genome synthesis typically starts with assembly of synthetic single-stranded DNA oligos. These oligos can be annealed followed by PCR amplification to get the lowest dimension of double-stranded DNA fragments, which are usually less than 2 kilobases (Kb) in length. A complete artificial synthetic genome can then be obtained through stepwise DNA assembly. The assembly process is achieved at four scales: Kb-scale assembly, megabase (Mb)-scale assembly, chromosome-scale assembly, and genome-scale assembly.Gibson assembly and Golden Gate assembly can be used for the Kb-scale assembly. The principle of Gibson assembly is similar to the process of homologous recombination, which is suitable for assembly of regular sequences but not highly-repetitive sequences. Gibson assembly is usually capable of piecing together up to five DNA fragments Mb-scale assembly relies on the use of bacteria or yeast cells. Pre-assembled Kb-scale DNA fragments need to be transformed into these cells for a higher level of assembly. After several rounds of assembly, large DNA fragments in the range of a few hundred Kb to several Mb can be obtained. Several methods are available to assemble a Mb-scale DNA fragment, such as the switching auxotrophies progressively for integration (SwAP-In) e.g., Mb-scale DNA donor. Usually, Mb-scale DNA fragments can be maintained as a yeast artificial chromosome within yeast cells. To complete the assembly, the donor yeast cell is typically fused with the host cell Chromosome-scale assembly starts with pre-assembled large DNA fragments, Genome-scale assembly can be accomplished by mating or microcell-mediated chromosome transfer (MMCT) de novo synthesized genome is not needed. Albeit on a much smaller scale, genetic manipulation of existing genomes via genome engineering is often sufficient. Targeted genome editing is typically achieved following two steps In most cases, a i.e., non-homologous end joining (NHEJ) and homology-directed repair (HDR) e.g., epigenetic modification enzymes, these tools can be harnessed for epigenetic modification at target genome sites There are two main mechanisms for DSB repair, de novo genome synthesis and genome engineering may constitute a superior approach for assembling a large artificial genome. For example, existing genome can be edited first and then used as the starting material for de novo genome assembly. Furthermore, available genome editing tools may facilitate the integration of large DNA fragments.Combining Mycoplasma mycoides genome from the JCVI-syn3.0 project, which represents an important milestone in the synthetic biology field. Aimed to build a synthetic minimal genome, they maximally deleted the non-essential genomic DNA from the original genome, and the synthesized genome only has a size of 531 Kb and contains 473 genes Undoubtedly, the most ambitious genome-writing project is the generation of a synthetic cell with a functional synthetic genome. The need to design an artificial genome leads to a fundamental question: what is gene essentiality? Cellular pathways are either essential or non-essential p-acetylphenylalanine in Escherichia coli MG1655 Genome writing does not have to follow the existing rules of genetic encoding and decoding, thereby holding the potential to create new life forms that do not exist in nature De novo design of an artificial genome holds promise for gaining novel insights into cellular processes and metabolic pathways. In order to make an artificial cell with a functional synthetic genome, several rounds of the design-build-test (DBT) processes are needed. The DBT processes may help to discover and understand the mechanisms and processes that are otherwise not accessible with traditional means. Different organisms vary greatly in their composition of genomes and proteomes, as well as their metabolic processes and cellular functions. However, they do share many common features in basic cellular processes etc. However, in different cells or organisms, these processes are likely regulated by different genes and proteins, which often share sequence homologies. De novo synthesis and assembly of different genomes will help gain insights into the conservation and divergence, as well as the crosstalk between genetic elements and proteins from different organisms.In recent years, several genomic DNA synthetic projects have been completed or underway, including viral, prokaryotic, eukaryotic, and mammalian genome  [8], [18Cellular processes such as metabolism are largely controlled by genome. Genome writing technologies can change genome information at a large or small scale, which enables the design and engineer of organism catering to specific applications.in vitro. Finally, the genetically-corrected cells are transplanted back to the patient to cure the disease. In addition to repair defective genes, more sophisticated genome manipulations, e.g., incorporating genetic circuits, has been used for cancer immunotherapy. In 2017, Nissim et al. reported using a genetic AND logic circuit to direct immune cells to specifically target and kill cancer cells Here, we list some examples on how to use genome writing technologies to develop useful products or therapeutics . (1) TheSince the beginning of the 21st century, rapid progresses have been made in the synthetic biology field. However, there still remain many challenges, which require continued investments and efforts.(1) Further understanding of the cellular/biological processes and metabolism pathways is needed. Understanding of the natural biological processes and metabolism pathways is a prerequisite to the development of any new synthetic biology designs and applications. There are more than eight million types of different species living on Earth. Among them, many are complex multicellular organisms that have a multitude of cell types with different compositions and functions. These cells share common fundamental metabolisms, but also differ in the specific metabolic pathway(s). What are the factors involved? What are the relations between these factors? Lots of work needs to be done to answer these questions.(2) Technically, the cost of genome synthesis needs to be reduced and the efficiency increased. At present, implementing a large-scale synthetic genomic project is still costly and time-consuming. For small-scale genome editing, the current techniques are still inefficient in HDR-based precision editing. Genome editing based on the customizable nucleases relies on the intrinsic cellular DNA DSB repair mechanisms. Due to the difficulties of precisely controlling the DNA repair processes, the efficiency for error-free repair is inherently low. In addition, the off-target effects are also an important concern that needs to be further addressed.vice versa. As time goes by, it will become increasingly difficult to define whether such kinds of chimeras are human or animal. In addition, it has been reported that synthetic viruses have the same infectivity compared to their natural counterparts (3) There are bioethics and biosafety concerns etc., we might be able to create new species or resuscitate extinct species according to human needs.In general, synthetic biology marks a new era in the biological field. Different from physics and chemistry, there do not exist simple and clear disciplines to follow in biology area when developing applications. Basically, all species are black boxes with extremely complex structures and functions. Fully understanding the laws is the premise of all applications for every discipline. In the past century, the biological studies have undergone a rapid development, which tremendously promotes our understanding of organismal heredity and development. All these have laid an important foundation for the birth of synthetic biology. In the future, with a combination of genome writing technologies, genetic circuit technologies, new genetic encoding and decoding technologies, The authors declare no competing interests.


Is top-down control necessarily scarce, slow, and hence unimportant in visual selection? Here we outline the risks of downplaying top-down control. Contrary to Theeuwes’ review, we suggest that not all sources of attention map onto a unitary attentional priority map. Goals and search habits may influence where and how people deploy attention, respectively. Because goals have modulatory effects on sensory processing, their impact on attention is broad and not always deliberate. In addition, when multiple sources influence attention, top-down control often dominates over less deliberate forms of attention. We agree with Theeuwes that selection history can drive attention independent of explicit goals. Nonetheless, top-down control remains a cornerstone of visual selection. In the target article, Theeuwes  builds oFirst, the assumption that attentional control is a product of activation within an integrated priority map obscures the nuanced mechanisms by which selection history drives attention. Holding the priority of each location on a spatial map equivalent, attentional biases can occur via procedural tuning. Recently, we showed that whereas top-down goals likely affect where people direct attention, perhaps in a manner akin to that described by the priority map, implicit location probability learning biases search by changing the preferred vector of attentional shift . The ideSecond, as Theeuwes discusses, the idea that behavioral goals cannot modulate attentional capture by salience directly contradicts contingent attentional capture  and searFinally, we must address the assumption that due to its scarcity, top-down control is less important in driving attention than fast, automatic influences. Even were it less frequent than experience- or salience-driven attention, the idea that top-down control is therefore less important does not directly follow. This logical jump fails to consider the relative magnitudes of the effects of types of control. In Cherry’s work on dichotic listening, for example, only 33% of participants attended to the ignored channel when their name was mentioned, whereas 80% did when told that new instructions may appear on the ignored channel . FurtherIn summary, Theeuwes offers a new dichotomy of fast versus slow control of attention, grouping selection history and bottom-up attention into the category of fast, automatic control. We believe that compelling evidence supports the idea that selection history may influence attention quickly. However, the relegation of top-down control to a slow, volitional system incapable of modulating bottom-up effects and unimportant to daily life is at odds with existing data. Theeuwes provides a well-articulated framework that reduces ambiguity in terminology. Such clarity will steer attention research toward an understanding of how these diverse sources interact to optimize visual selection.The authors have no data accessibility statement to declare.


Autoimmune rheumatic diseases (ARDs) form a heterogeneous group of disorders that include systemic lupus erythematosus (SLE), systemic sclerosis (SSc), rheumatoid arthritis (RA), idiopathic inflammatory myopathies (IIMs), and systemic vasculitis. Coronary microvascular dysfunction (CMD) is quite common in patients with ARDs and is linked to increased cardiovascular morbidity and mortality. Inflammation plays a crucial role in the pathogenesis of both accelerated atherosclerosis and CMD in ARDs, especially in patients affected by SLE and RA. In this regard, some studies have highlighted the efficacy of immunosuppressants and/or biologics in restoring CMD in these patients. By contrast, the role of inflammation in the pathogenesis of CMD-SSc appears to be much less relevant compared to endothelial dysfunction and microvascular ischemia, with calcium-channel blockers providing some benefits. Few studies have endeavored to assess the occurrence of CMD in IIMs and systemic vasculitis, thus warranting further investigations. The present review summarizes the current evidence on the occurrence of CMD in ARDs, focusing on the role of inflammation and possible therapeutic approaches. Autoimmune rheumatic diseases (ARDs) form a heterogeneous group of disorders characterized by multiple organ involvement stemming from systemic inflammation and a dysregulation of the immune system with subsequent tissue damage. ARDs include systemic lupus erythematosus (SLE), systemic sclerosis (SSc), rheumatoid arthritis (RA), inflammatory idiopathic myopathies (IIMs), and systemic vasculitis. Accelerated atherosclerosis and coronary microvascular dysfunction (CMD) are the main causes of cardiovascular (CV) involvement in these patients, leading to increased cardiovascular morbidity and mortality .In the literature pertaining to the CV implications in ARDS, much attention was initially given to the role of inflammation in inducing accelerated atherosclerosis—especially in SLE and RA—whereas the occurrence of CMD has only been more recently investigated. Endothelial dysfunction is thought to be an early event in the pathogenesis of both macro- and microvascular cardiac involvement in ARDs . DecreasThe coronary microcirculation comprises pre-arterioles (diameter 100–500 μm) and arterioles (diameter < 100 μm) and is of the utmost importance in determining myocardial perfusion as it accounts for 80% of the global blood flow resistance in the heart . ConsideKnowing the strong link between CMD and cardiovascular mortality in ARDs, it is crucial to diagnose cardiac involvement as early as possible and identify effective therapeutic strategies.CMD can be detected by both non-invasive and invasive techniques. The coronary flow reserve (CFR) is the ratio between maximal  and baseline coronary blood flow, and a CFR ≤ 2.5 indicates CMD . The assSingle-photon emission computed tomography (SPECT) is another validated imaging modality to detect microvascular impairment. SPECT is a nuclear imaging technique performed at baseline or after stress, which reveals the differences in the distribution of the radionuclide  and, therefore, the relative blood flow to the different regions of the myocardium . HoweverMicrovascular impairment can also be quantified invasively by coronary angiography with the determination of the index of myocardial resistance using an intracoronary pressure wire at baseline conditions and during hyperemia .Systemic lupus erythematosus is a heterogeneous ARD that occurs predominantly in females and causes chronic inflammation in multiple organs. Cardiac manifestations  and myocarditis) are the leading cause of morbidity and mortality. SLE is associated with an accelerated atherosclerotic process, and it has been reported that women affected by SLE carry a 7.5-fold increased risk of developing CAD versus healthy controls ,20. AlthWhile the link between symptomatic patients  and CMD is well established, the data among non-symptomatic patients are still scarce. Recent studies on SLE patients without known heart involvement or CV symptoms reported an interesting association between troponin elevation, strain rate diminution (TTE), native T1 and T2 augmentation (MRI), aortic stiffness augmentation (pulse wave velocity), and myocardial inflammation/interstitial and perivascular fibrosis on histology ,27. ThesAll these findings show that CMD is quite common in SLE and represents a major cause of persistent chest pain in the absence of obstructive CAD in these patients. CMD and accelerated atherosclerosis alike appear to stem the inflammatory burden. Thus, the efficacy of corticosteroids and immunosuppressants in CMD-SLE warrants further investigation .Systemic sclerosis is a complex autoimmune connective tissue disease (CTD) characterized by immune dysregulation, microvascular damage, and fibrosis ,29. FuncSeveral studies with CFR measurement by TTE with adenosine infusion have found that CMD is detectable in about 50–60% of clinically asymptomatic scleroderma patients ,33,34. AAs in other microvascular manifestations of SSc , the main pathogenetic mechanisms underlying primary myocardial involvement seem to be an imbalance between mediators of vasoconstriction and vasodilation, resulting in ischemic damage. In these settings, calcium channel blockers (CCBs), such as nifedipine, have been shown to improve myocardial perfusion assessed by SPECT and prevent the future onset of LV diastolic dysfunction in scleroderma ,35. MoreOn the other hand, a possible additional role of inflammation in CMD-SSc has been hypothesized recently. Conventional cardiac MRI has detected the presence of focal and diffuse myocardial fibrosis and, to a lesser extent, myocardial inflammation even in SSc with no cardiac symptoms . MavrogeMoreover, SSc patients showed significantly expanded the extracellular volume and larger areas of T1 abnormality than those identified at conventional CMR, which likely reflects a combination of low-grade inflammation and diffuse myocardial fibrosis . As a coThese data point to a continuum between disease onset with microcirculation remodeling and low-grade ischemia, to diffuse myocardial fibrosis and inflammation induced by microvascular rarefaction and immune activation. Early subclinical microvascular heart impairment seems detectable, but aggressive vasodilator and immunosuppressive regimens warrant further investigation in these asymptomatic patients.Rheumatoid arthritis is linked to an increased risk of CV events. Large epidemiological studies have suggested that while traditional CV risk factors may be crucial, they alone cannot account for the added risk in RA patients . The infAlthough the excess CV risk in RA is not fully understood, several mechanisms are believed to be responsible for endothelial dysfunction in these patients: genetic factors, immune dysregulation, inflammation, metabolic disturbances, use of steroid and anti-inflammatory drugs.Toutouzas et al. reported that RA and diabetic patients presented similar rates of ischemic alterations at dobutamine stress TTE, significantly higher vs. controls . NeverthSeveral studies have investigated CMD in patients with RA. Recently, Amigues et al. found that 30% of RA patients showing no clinical CV disease had a pathological  myocardial flow reserve (MFR) assessed by PET-CT . MoreoveAltogether, these studies suggest that CMD and accelerated atherosclerosis share similar pathogenetic mechanisms in RA. Moreover, currently available data suggest that achieving remission in RA does not necessarily ensure the reversal of microvascular endothelial dysfunction. Since the treat-to-target is a cornerstone of the management of patients with RA, these findings suggest that restoring CMD may be considered as an adjunctive potential therapeutic target in RA. The demonstrated effect of some biologic therapies on CMD-RA could represent a key point in choosing biological drugs in RA patients, considering their high CV risk ,47.Systemic vasculitis refers to a heterogeneous group of autoimmune disorders characterized by vessel inflammation. Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAVs) mainly include granulomatosis with polyangiitis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA), and microscopic polyangiitis. AAVs affect small vessels, and, therefore, the cardiac impairment is intrinsically linked to a dysfunction of the coronary microcirculation. Perhaps because the microvascular origin is implicit in the classification of AAVs , there aAs it pertains to large vessels vasculitis , the involvement of small vessels is very rare, and hence, CMD is very uncommon. Behar et al. reported a case of an Asian female affected by Takayasu’s vasculitis and refractory ischemic chest pain caused by CMD .Microvascular dysfunction studied by CFR highlighted abnormalities in coronary microcirculation in Kawasaki disease. A reduced CFR was reported in medium and giant coronary aneurysms, and in stenotic lesions even of mild to intermediate severity. Therefore, abnormalities in the coronary microcirculation and epicardial lesions may occur concomitantly in these patients .Finally, myocardial perfusion defects caused by CMD have been reported in patients with Behçet’s disease ,56.Idiopathic inflammatory myopathies (IIMs) are a group of chronic, autoimmune conditions characterized by the infiltration of inflammatory cells into skeletal muscles . The mosCoronary microvascular disease is common in patients with ARDs, leading to increased CV risk. Inflammation is relevant in the pathogenesis of CMD-ARDs, especially in patients affected by SLE and RA, and, therefore, a more aggressive therapeutic approach with immunosuppressants and biologics might prove useful in these patients to prevent diastolic and systolic dysfunction. By contrast, autoimmunity and inflammation seem much less involved in CMD-SSc, whose treatment to date consists of vasodilators and low-dose aspirin. The occurrence of CMD has been less investigated in systemic vasculitis and IIMs, thus warranting further studies. The early identification of CMD and a more precise clarification of the pathogenetic mechanisms involved in CMD-ARDs is paramount in these patients to define the best therapeutic strategy and avoid major CV events.


Plants and microbes utilize glycoconjugates as structural entities, energy reserves for cellular processes, and components of cellular recognition or binding events. The structural heterogeneity of carbohydrates in such systems is a result of the ability of the carbohydrate biosynthetic enzymes to reorient sugar monomers in a variety of forms, generating highly complex, linear, branched, or hierarchical structures. During the interaction between plants and their microbial pathogens, the microbial cell surface glycans, cell wall derived glycans, and glycoproteins stimulate the signaling cascades of plant immune responses, through a series of specific or broad spectrum recognition events. The microbial glycan-induced plant immune responses and the downstream modifications observed in host-plant glycan structures that combat the microbial attack have garnered immense interest among scientists in recent times. This has been enabled by technological advancements in the field of glycobiology, making it possible to study the ongoing co-evolution of the microbial and the corresponding host glycan structures, in greater detail. The new glycan analogs emerging in this evolutionary arms race brings about a fresh perspective to our understanding of plant–pathogen interactions. This review discusses the role of diverse classes of glycans and their derivatives including simple sugars, oligosaccharides, glycoproteins, and glycolipids in relation to the activation of classical Pattern-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI) defense responses in plants. While primarily encompassing the biological roles of glycans in modulating plant defense responses, this review categorizes glycans based on their structure, thereby enabling parallels to be drawn to other areas of glycobiology. Further, we examine how these molecules are currently being used to develop new bio-active molecules, potent as priming agents to stimulate plant defense response and as templates for designing environmentally friendly foliar sprays for plant protection. Plant cell walls are complex configurations of highly recalcitrant interlocking polysaccharides which insulate against microbial invasion and abiotic stress. Filamentous plant pathogens, which mainly comprise fungi and oomycetes, breach the plant cell wall by releasing enzymes that deconstruct polysaccharides, proteins, and lignin based polymers . The resFigures 2 and Table 1; The activation of the complex array of plant innate defense mechanisms relies on the recognition of pathogen signatures by the host transmembrane pattern recognition receptors (PRRs) . The sigAlthough PAMPs represent a broad range of molecules, which includes carbohydrates, lipids, proteins, peptides, lipopolysaccharides, glycolipids, and glycoproteins , it is oIn the context of glycan-mediated plant immunity, identification of microbial cell-surface glycans, building blocks of fungal/oomycetes cell walls  and bacterial glycoconjugates  that act as regulators of plant defense signaling, presents new perspectives to analyze and understand host–microbe interactions . Within Simple sugars including mono and di-saccharides are central to plant–microbe interactions, serving both as energy sources to drive the PTI and ETI responses as well as themselves acting as signaling molecules that drive signal fluxes leading to localized or systemic defense responses, when challenged with filamentous pathogens .PR) genes, during HR mediated cell death , which is responsible for the conversion of glucose to glucose 6-phosphate, has been most investigated. Among the several isoforms of HXK1, the mitochondria-associated HXK1 is central to the control of programmed cell death (PCD) during microbial pathogenesis and is also responsible for regulation of several pathogenesis related . Trehalose induces the activation of the defense genes Phenylalanine Ammonia-Lyase (PAL) and Peroxidase (POX) during wheat challenge with Blumeria graminis, the causal agent of powdery mildew disease  are considered important sugar signals, modulating defense responses through complex sugar sensing pathways  leading to protection against pathogens via PCD. The response of the transcription factors to sugar levels is, however, variable as seen for Figure 4), which serve as an osmoprotectant of plants, as a transporter of sugar in phloem sap, and as storage sugars  , which in turn enhanced resistance against Botrytis cinerea and Erwinia carotovora in pathogen challenged cucumber and tobacco plants (Camellia sinensis GolS gene (CSGolS) have shown that CsGolS1 regulation was mainly related to abiotic stresses such as water deficiency, low temperature, and ABA treatment. On the other hand, significant regulation of CsGolS2 and CsGolS3 was seen in relation to biotic stresses such as E. oblique (moth) attack, SA, and MeJA treatment  needs to be further investigated to understand the underlying mechanisms. Furthermore, the roles played by galactinol derivatives in response to stresses including pathogen attack remain to be elucidated  . This ene (GolS) . The indo plants . Prelimireatment . Althougucidated .in vitro quencher of reactive oxygen species that limits cell damage   regarding the interplay between mannitol and mannitol dehydrogenase during pathogenesis of the mannitol-secreting pathogens and how it may be balanced between detection of pathogens by plants and evasion of defense response by the pathogen.Polyols, often referred to as sugar alcohols, are the reduced form of aldose and ketose sugars . Mannitol damage . Interesl damage . The funextracts . Mannitont stain . The rol/Δm1pdh) . In ligh/Δm1pdh) . The cur/Δm1pdh) . This asMost oligosaccharides implicated in plant–pathogen interactions are generated by the enzymatic degradation of polysaccharides from the structural constituents of fungal cell wall or pathogen virulence factors . The actPhytophthora sojae mycelial cell wall hydrolysate, which mediated plant defense by the induction of PAL activity in plants . This strategy was effective in reduction of B. cinerea infection of grapevine leaves upon treatment with these acetylated oligoglucuronans , bean (Vicia faba), lupin , pea, Medicago truncatula, and Lotus japonicus  and basic (Chit1b) chitinases and β-1,3-glucanase which are responsible for hydrolysis of chitin and β-1,3-glucans in fungal cell walls . Exogenoactivity . In grapdextrins . Also, infection . HoweverN-acetylglucosamine, is a major constituent of fungal cell walls and is the most well characterized filamentous PAMP , was identified as a plasma membrane localized receptor in rice cells, which was responsible for defense signal transduction  contains additional extracellular carbohydrate binding lysine motifs (LysMs), as well as an intracellular kinase domain essential for chitin based defense signaling 6, a hexasaccharide from the necrotrophic fungus Plectosphaerella cucumerina which induces innate defense responses such as increased levels of cytosolic Ca2+, activation of MAPKs cascades and elevated expression of PR proteins  motif in subdomain VII, essential for signaling. Also, several LysM receptors have been found to recognize and distinguish pathogenic/mutualistic carbohydrate signatures emerging from GlcNAc derived and microbial exopolysaccharide sources. It is therefore plausible that LYS12 is involved in monitoring carbohydrate PAMPs produced by P. palmivora as well as the damage associated signatures emerging from the host plant, thereby regulating disease progression  and root-nodule symbiosis (Nod factors) . On the and rice . Thus chL-glucuronic and D-mannuronic residues , which is an ET-mediated shared defense signal for protection against necrotrophic fungi and herbivorous insects.  are reported to act against the oomycete pathogen Phytophthora brassicae and the ascomycete M. oryzae, respectively (Arabidopsis interacts with oligosaccharides via two tripeptide Arg-Gly-Asp (RGD) in the lectin domain  is a vital structural component of the outer layer of Gram negative bacteria with three distinct functional domains which include: the lipophilic lipid A (LA) the di-glucosamine moiety which carries 4–7 fatty acids, an oligosaccharide core region, and the  repeats . The oli repeats . The LPSesponses . The sigFigure 7; LPS mediated defense signaling is well characterized in the mammalian system where it binds to the LPS receptor, TLR4/MD-2 (toll-like receptor 4/myeloid differentiation factor-2), to trigger activation of phagocytosis, production of pro-inflammatory cytokines, and interferons and antimicrobial peptides  and digalactosyldiacylglycerol (DGDG), are important plant glycerolipids, known for their role in mediating in systemic acquired resistance (SAR) based plant defenses. It was seen that . Tomato . This prPhosphoinositol sphingolipids and glucosylceramides (GlcCer) are another group of glycosylated lipids found in fungi that feature a characteristic C-9 methyl group present on the long chain fatty acid base . These fPseudomonas aeruginosa have been shown to induce defense responses in grapevines, tobacco, and Arabidopsis cells via oxidative burst, ROS production, and inducing systemic acquired resistance (Natural rhamnolipids produced by sistance . In thissistance .Our current understanding of the roles that carbohydrates play in PAMP based defense priming in plants remains fragmented. Although the potential role of glycans as elicitors of plant defense signaling cascades has been identified as an important aspect, there remains a huge information gap when compared to what we currently understand about proteinaceous receptors of these glycan elicitors. The likely reason for this is the complexity of carbohydrate structures and the fact that the techniques used to analyze glycans lag behind those for protein and gene analysis. Improvements in oligosaccharide extraction, purification and identification techniques are beginning to enable studies using more specific glycan species rather than crude extracts containing polydisperse carbohydrate species. The understanding of the roles played by carbohydrates in plant–pathogen interactions falls behind knowledge in human–pathogen interactions, which has attracted attention due to the implications in medicine development and disease treatment. It is only recently that we have begun to realize the importance carbohydrates have in plant protection strategies and appreciate the potential that carbohydrates have as specific intervention technologies.In their natural habitat, plants are exposed to a wide variety of symbiotic and pathogenic microflora at any given time, which paves the way for a novel and extensive area of research to investigate the controlled and diversified signaling cascades generated within plants in response to simultaneous attack from different microbes. Plant genomes encode a large number of RLKs, RLPs, and lectins; biochemical and structural studies on these receptors will lead to better knowledge of their workings and allow identification of the most suitable oligosaccharide candidates.Coming to the concept of sustainable agriculture, there is potential to use oligosaccharides for crop protection, to induce pathogen resistance and prime plants for microbial attack. The use of biodegradable, environmentally friendly glycans and glycan mimics will help to replace the conventionally used chemicals with safer alternatives. This will enable more sustainable agriculture and help avoid major crop losses, allowing better food security, given mankind’s growing demand for food quality and quantity.CC, EK, and MR conceived the idea and prepared the draft of the manuscript. RF supervised the development of topics for discussion and edited the draft manuscript.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.


Saccharomyces cerevisiae. These ALS-associated proteins are not orthologs of yeast prion proteins, but are similar in having long, intrinsically disordered domains that are rich in hydrophilic amino acids. These so-called prion-like domains are particularly aggregation-prone and are hypothesized to participate in the mislocalization and misfolding processes that occur in the motor neurons of ALS patients. Methods developed for characterizing yeast prions have been adapted to studying ALS-linked proteins containing prion-like domains. These yeast models have yielded major discoveries, including identification of new ALS genetic risk factors, new ALS-causing gene mutations and insights into how disease mutations enhance protein aggregation.Many proteins involved in the pathogenic mechanisms of amyotrophic lateral sclerosis (ALS) are remarkably similar to proteins that form prions in the yeast  Neurodegenerative diseases are defined by irreversible loss of neurons. Clinically, these diseases manifest as dementias and movement disorders, depending on the subset of neurons that are principally affected. As a whole, the cost of neurodegenerative diseases is staggering. According to the Alzheimer’s Association, in 2018, the total expense of treating dementias in the United States was approximately $277 billion. Unlike cancer and viral diseases, which have seen astonishing advances in treatments, no drugs have been developed that reverse or stop progression of neurodegenerative disease. To develop effective treatments, there is need to better understand the fundamental causes of these diseases and develop tractable models for genetic and pharmacological screening.Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects upper and lower motor neurons. It primarily manifests as a movement disorder, but it has many clinical and pathological features that overlap with frontotemporal dementia (FTD), which primarily manifests as a dementia. A surprising development in recent years is that diseased neurons from ALS and FTD patients frequently feature the accumulation of specific proteins that resemble yeast prion proteins. The intersection of yeast prions and human disease-associated proteins has led to novel experimental systems using tractable yeast models.A common theme among nearly all neurodegenerative diseases is an apparent loss of normal protein quality control, resulting in the accumulation of disease-specific proteins into large aggregates. Increasingly, a prion-like mechanism is being recognized as potentially underlying this protein aggregation  can remain relatively soluble and be passed to progeny yeast cells. In fact, for several neurodegenerative diseases, smaller oligomeric assemblies, which are pre-amyloid or amyloid-like, may in fact be the more cytotoxic and infectious species . They are distinguished by an abundance of polar residues and a paucity of hydrophobic and charged residues  their yeast counterpart Many neurodegenerative disease-linked proteins have been modeled in yeast, including Parkinson’s, Huntington’s and Alzheimer’s diseases Braun, . LikewisFUS is a ubiquitously expressed, predominantly nuclear animal protein originally named for its association to liposarcomas . In this context, they found the D to V mutation dramatically and significantly increased prion formation propensity. Thus, not only could this mutated hnRNPA2 sequence promote Sup35’s nucleation into a prion, the D to V mutation specifically facilitates prion-like aggregation.Kim et al.  discoverIn summary, yeast models reveal that ALS-causing mutations in prion-like proteins often increase the proteins’ intrinsic propensity to form solid cytoplasmic aggregates and/or nucleate aggregation. The advantage to using yeast models is scores of mutations can be assayed rapidly before more expensive model systems are selected for further analysis. High throughput genomic sequencing has revealed numerous low-frequency polymorphisms in ALS-linked proteins, thus yeast could be helpful in determining if certain mutations actually contribute to disease.The prion-like domain of FUS is extensively phosphorylated in human cell models following various types of stress  have been developed to identify proteins with yeast prion-like domains  can be harnessed to evaluate the nucleation potential of human prion-like proteins. Specifically, prion-like domain sequences are substituted for segments within the Sup35 prion domain . To determine if other RRM-containing proteins could be linked to disease, a yeast screen was performed with human RRM proteins . The [PIN+] prion forms cytoplasmic amyloid that can nucleate the aggregation of heterologous proteins, particularly amyloid-forming prion proteins and polyglutamine expansions. Park and coworkers found that [PIN+] slightly enhances TDP-43 and FUS toxicity in yeast models (Park et al., The nucleation of nearly all yeast prion proteins into self-propagating aggregates is influenced by the endogenous yeast prion [PINIn vitro, the large aggregates formed by these proteins are described as “amyloid-like,” which indicates they appear to form fibrous solid aggregates with mild detergent-resistance and weak or no Thioflavin T reactivity (Johnson et al., The question remains about what conformations these ALS-linked prion-like proteins are forming when aggregated in diseased neurons. There are limitations to yeast models. What is true for yeast prion domains is not necessarily true for human prion-like domains, so conclusions must be made carefully. For example, it has been hypothesized that prion formation in yeast is naturally beneficial (True et al., An additional limitation of yeast models is different sets of resident interacting proteins compared to mammalian cells. This is especially important in regards to quality-control proteins like chaperons. Yeast chaperones are integral to propagation of endogenous yeast prions (Masison and Reidy, Yeast models have seen widespread adoption for modeling human neurodegenerative diseases like ALS because they are easily manipulated and recapitulate the major properties of more complex eukaryotic cells. Specifically, these yeast models offer insights into the precise relationship between gain-of-function toxicity and protein aggregation. While they do not recapitulate the numerous mechanisms by which neuronal networks may degenerate, yeast models have proven effective in discovering proteins linked to ALS and FTD. Yeast models of prion-like neurodegenerative disease-linked proteins offer a strategy to inexpensively inform subsequent experiments in higher model systems in the goal of ultimately developing therapeutics.ZM and FS conceived the outline. ZM, SR, DY and FS wrote the article.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.


Cellular therapies, including those based on T cells, are becoming approved options for clinicians treating a range of diseases. Cytotoxic T lymphocytes (CTLs) can be modified ex vivo to express receptors such as chimeric antigen receptors (CARs) or T cell receptors, allowing them to target tumour cells when infused back into patients with particular cancers. CTLs specific for viruses can be purified ex vivo and reinfused into patients transplanted with haematopoietic stem cells to help combat viral reactivation. Regulatory T cells (Tregs) can be expanded ex vivo for infusion into patients with autoimmunity or allergy, or into those at risk of rejecting transplanted cells or tissues, or suffering graft versus host disease. Effector and regulatory T cells can also be generated by infusion of patient-derived dendritic cells (DCs) conditioned in ways to elicit anti-tumour immunity (CTLs) or Tregs. All such therapies are resource-heavy (particularly in process regulation) and so must be initially targeted to patients that have limited treatment options, but also where they have a chance of being effective. Cells, entitled Emerging Cellular Therapies: T Cells and Beyond, aims to highlight these approaches and applications, touch upon their technical and mechanistic bases, and give an update on their current status and efficacy. Future paths that such cellular therapies might take will also be discussed. This editorial signposts readers to the key areas of cellular therapies relating mainly to T cells, but does not extensively review them.Cellular therapies are fast becoming a viable option for the treatment of numerous diseases, thanks to the progress of technologies that enhance capabilities and reduce costs. They are typically used only when standard treatments have failed, since they are intensive in terms of skilled labour, reagents and facilities; are usually tailored to the patient; and thus remain expensive and limited in availability compared to off-the-shelf medications. Therapies that have their basis in immune cells benefit from the potency of such cells, together with a mechanistic understanding of their often complex action. Immunologists have always recognized the key role of T cells in health and disease. Indeed, the importance of T cells clinically is highlighted when they are absent or dysfunctional, such as in certain primary (genetic) or secondary (induced) immunodeficiencies, where viral infections are prevalent . Less obT cell-based cancer therapies have had a recent resurgence through the approval and use of immune checkpoint inhibitors . These iAnother approach that has recently received approvals from the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) is the use of chimeric antigen receptor (CAR)-T cells, with two products on the market—axicabtagene ciloleucel (Yescarta) and tisagenlecleucel (Kymriah). These T cells, derived either from patient (autologous) or from healthy donor , are modified by introduction  of anti-cancer receptor genes, a single chain variable scFV fragment, and additionally carrying intracellular signalling domain CD3ζ and costimulatory domains 41BB or CD28 . Anti-CDTransplantation of organs, tissues or cells presents the recipient with several issues that clinicians need to manage. A common factor is the immune suppression given to avoid T cell-mediated rejection of the foreign cells. Recipients are invariably at risk of viral infection or reactivation. In the case of Haematopoietic Stem Cell Therapy for cancer, the recipient’s immune system is ablated with drugs pre-transplant since the malignancies it is treating are usually immune cell-derived. Reconstitution of the recipient with donor T cells gives rise to unwanted GvHD together with the desired graft versus leukaemia effect. These outcomes must be carefully managed with immune suppression. Pre-emptive antiviral drugs are also given but patients suffer from toxicity, and a proportion of viral infections are refractory to drug treatments. The application of virus-specific T cells generated from the donor allows a more targeted approach that gives rise to 70% efficacy in recipients with minimal side effects . An issuα (CD25) and intracellular transcription factor FOXP3, are vital to down-regulating unwanted harmful immunity, often via CTLA-4 and TGFβ. Autoimmune diseases are characterized by dysregulated immune response towards self-antigens, whereas in organ transplantation the host’s immune system can recognize and attack the transplanted allogeneic organ. In both indications, current standard treatment involves prolonged, often life-long, treatment with immunosuppressive drugs. Regulatory T cells have shown much promise in their ability to inhibit immune responses in vitro and in the animal models and can be utilized to reduce the need for pharmacological immunosuppression. Recent advances in the understanding of Treg biology, isolation and expansion of human regulatory T cells, and in the development of Good Manufacturing Process (GMP)-compliant cell production methods, have led to a series of phase I clinical trials using Tregs in autoimmunity and transplantation , cell survival and stability in vivo, as well as production costs. Newer approaches include the development of antigen-specific Tregs, including CAR-Tregs, improvements in the initial purity and phenotypic stability, and genetic modifications to increase cell potency  [Tregs, typically defined by high levels of surface IL-2Rand Tang  and Kawaand Tang , respectd FOXP3) . Most ofd FOXP3) . The nexBeing classed as Advanced Therapy Medicinal Products by the EMA, T cells for adoptive therapy present a range of regulatory hurdles that require negotiation . These rThe power of T cells is starting to be harnessed in T cell adoptive therapies. Such therapies, that are often personalized, require high-tech production that is resource-heavy. Thus, they are currently reserved for situations where other, usually standard, treatments have failed. It may be more challenging to demonstrate efficacy in such patients, but accumulating data will likely identify situations where these Regenerative Medicine treatments can become mainstream.


The worldwide prevalence of allergic diseases has drastically increased in the past decades. Recent studies underline the importance of microbial exposure for the development of a balanced immune system. Consequently, probiotic bacteria are emerging as a safe and natural strategy for allergy prevention and treatment. However, clinical probiotic intervention studies have so far yielded conflicting results. There is increasing awareness about the importance of host-associated factors that determine whether an individual will respond to a specific probiotic treatment, and it is therefore crucial to promote a knowledge-based instead of an empirical selection of promising probiotic strains and their administration regimen.In this Review, we summarize the insights from animal model studies of allergic disease, which reveal how host-related factors – such as genetic makeup, sex, age and microbiological status – can impact the outcomes of preventive or curative probiotic treatment. We explore why and how these factors can influence the results of probiotic studies and negatively impact the reproducibility in animal experiments. These same factors might profoundly influence the outcomes of human clinical trials, and can potentially explain the conflicting results from probiotic intervention studies. Therefore, we also link these host-related factors to human probiotic study outcomes in the context of airway allergies. Summary: This Review discusses the host factors that influence the outcomes of in vivo probiotic interventions in allergy, focusing on genetics, sex, age and microbiological status in mouse models of allergic disease. Allergic diseases present a compelling challenge for public health owing to their increasing prevalence in both developed and developing countries. Approximately 1 billion people worldwide currently suffer from allergies, and these numbers are estimated to increase to 4 billion in the next 30-40 years .Adjuvant: substance capable of boosting the immune response to an antigen.Airway hyperreactivity (AHR): increased response of the airways to nonspecific stimuli, resulting in easily triggered airway constriction typical of asthma.Allergic sensitization: process during which the immune system becomes hypersensitive to an allergen, linked to allergen-specific antibody production.Aluminium hydroxide : adjuvant often used in vaccines or to boost sensitization in animal models.Atopy: predisposition of an individual to develop allergic disease.Bronchoalveolar lavage fluid (BALF): saline solution collected after irrigation of the airways containing cells and fluid from the bronchi and alveoli.Colony-forming unit (CFU): a commonly used measure of microbial load.Commensal: organism exhibiting commensalism, a form of symbiosis between two species.Dendritic cell (DC): immune cell capable of antigen presentation that forms a link between the innate and adaptive immunity.DNA fingerprint analysis: analysis of variable DNA regions to determine the DNA profile unique to an individual.Enhanced pause (Penh): dimensionless measurement reflecting airway obstruction through analysis of an airflow pattern in a live animal, e.g. during whole-body plethysmography.Fc receptor (FcR): antibody receptor located on the surface of immune cells and capable of binding antibodies.Haematoxylin and Eosin (H&E) staining: a common tissue staining method used to distinguish cellular structures.Innate lymphoid cell (ILC2): innate immune cell that is lymphoid in morphology, but lacks antigen-specific receptors.Interleukin (IL): signal protein belonging to the group of cytokines and involved in immune response regulation.Intestinal epithelial barrier: property of intestinal mucosa responsible for containment of intraluminal molecules and regulation of their passage into the underlying lamina propria.Intragastrically (i.g.): administered into the stomach.Intranasally (i.n.): administered into the nostrils.Intraperitoneally (i.p.): administered into the peritoneal or abdominal cavity.Jaccard similarity index: statistical measure of similarity between two sample sets expressed as the intersection of the sets divided by their union.Mast cell: white blood cell that contains granules with chemical mediators responsible for the typical symptoms of allergy.Major histocompatibility complex class II (MHCII): molecular complex located on antigen-presenting cells that mediates antigen presentation and immune response initiation.Mesenteric lymph nodes (MLNs): lymph nodes located in the mesentery .Microbe-associated molecular patterns (MAMPs): molecular structures of bacteria recognized by receptors on host cells, resulting in immune effects on the host.Ovalbumin (OVA): abundant egg white protein, one of the major egg allergens.Ovariectomized: a term used to describe a woman or a female animal whose ovaries have been surgically removed.Pattern recognition receptors (PRRs): receptors on innate immune cells of the host that recognize microbe-associated molecular patterns.Periodic acid–Schiff (PAS) staining: tissue staining method for detection of polysaccharides and mucosubstances.Peyer's patches: isolated lymphoid follicles found in the small intestine.Prebiotic: food ingredient promoting development and activity of beneficial organisms within the host.Regulatory T cell (Treg): a type of white blood cell capable of regulating or suppressing immune responses and promoting immunological tolerance.Specific-pathogen-free (SPF):  free of certain microorganisms and parasites belonging to a defined list.Subcutaneously (s.c.): administered under the cutaneous skin layer.Steroids : medication used to treat inflammation, such as asthmatic inflammation of the airways.T helper (Th) cell: a type of white blood cell capable of recognizing antigens presented by antigen-presenting cells and releasing cytokines that regulate the immune response.Tolerogenic: leading to induction of immunological tolerance.Toll-like receptor (TLR): a type of PRR characterized by the presence of a toll-IL-1 receptor (TIR) domain.Tumor necrosis factor (TNF): a cytokine involved in inflammation signalling.Whole-body plethysmography: technique used for measuring respiratory function in live subjects placed in a sealed chamber by recording pressure changes within the chamber.Allergy is defined as a hypersensitivity reaction caused by an immunological response to a specific antigen, referred to as an allergen . WidesprThe currently rising incidence of allergic disease has tentatively been explained by the ‘hygiene hypothesis’, which links a general decrease in microbial exposure of children in developed countries to reduced immune system stimulation and subsequent Th1/Th2 imbalance, which predisposes to allergic diseases . MammalsThese organisms are thought to have been part of our microbiota during human evolution and to have co-evolved with the human immune system to promote immune tolerance. This includes regulatory T cell (Treg)  inductioLactobacillus or Bifidobacterium, or mixes thereof (7 to 1010 or more colony-forming units (CFU)/day, and the treatment might last from several months to several years  to the allergen, through either aerosol inhalation  or intra6 , higher doses (1011 CFU) led to a robust Th1 response regardless of the host genetic background  and an increased relative abundance of Enterobacteriaceae in the intestinal microbiome (Similarly to mouse studies, the influence of the human genotype has also been suggested to play an important role in the outcomes of probiotic interventions, including those performed in the context of allergic disease. For example, administration of a combined or atopy  (Lau et or atopy . Also, schildren . Individchildren . An assocrobiome . These fThe age of the host can likewise influence probiotic study outcomes in humans. In fact, the perinatal period was suggested to represent a window of opportunity for effective probiotic intervention in allergic disease in humans . As suchL. rhamnosus GG in combination with L. rhamnosus LC705, B. breve Bb99, Proprionibacterium freudenreichii ssp. shermani JS and prebiotic galactooligosaccharides to pregnant mothers, and subsequently to the infants, reduced eczema in children at 2 years of age (in vivo and in vitro research are thus required to promote detailed selection of probiotic strains for prevention and treatment of allergic disease.Both the age of the individual during probiotic administration and the influence of their individual microbiota have been suggested to play a role in human trials. For instance, administration of s of age . Howevers of age . Althougs of age , its impIn conclusion, the outcome of studies regarding the influence of probiotic interventions on various immune system functions can be greatly influenced by host parameters, which should be taken into consideration, both in animal and human studies. An adequate design of animal experiments will result in an improved predictive value of probiotic interventions in future clinical studies, and a more accurate assessment and extrapolation of the immune mechanisms involved. It would therefore be advisable to conduct experimental probiotic interventions in animal models closely resembling the envisioned human target group in regard to the variety of host-related factors. For example, by including different sex and age groups during experimental design in animals, it would be possible to avoid a skewed view of probiotic effects in the general population. Furthermore, accounting for sources of variation stemming from experimental protocol designs as well as the host would greatly contribute to experimental reproducibility. Once the promising effects of a probiotic strain are thoroughly described in different settings, it is then possible to focus on the most effective approaches, such as those targeting the neonatal window of immune development.When certain host-related parameters, such as host genetics or resident microbiota, cannot be standardized as a result of study design, thorough investigation of these factors in the study subjects with subsequent subject stratification for data analysis should be considered. We therefore believe that conflicting results obtained in probiotic clinical trials could largely be explained by a lack of attention to these potential sources of variation. As heterogeneity in clinical studies with probiotics considerably complicates subsequent meta-analysis, increased awareness of the influence of host-related parameters and their acknowledgement in the context of probiotic interventions can aid appropriate patient stratification and greatly contribute to knowledge-based probiotic applications in future trials.