Positive Markers Research Articles

Efficient induction of pluripotent stem cells differentiated into mesenchymal stem cell lineages.

Mesenchymal stem cells (MSCs) have garnered significant attention in the field of cell-based therapy owing to their remarkable capabilities for differentiation and self-renewal. However, primary tissue-derived MSCs are plagued by various limitations, including constrained tissue sources, arduous and invasive retrieval procedures, heterogeneous cell populations, diminished purity, cellular senescence, and a decline in self-renewal and proliferative capacities after extended expansion. Addressing these challenges, our study focuses on establishing a robust differentiation platform to generate mesenchymal stem cells derived from induced pluripotent stem cells (iMSCs). To achieve this, we used a comprehensive methodology involving the differentiation of induced pluripotent stem cells into MSCss. The process was meticulously designed to ensure the expression of key MSC positive markers (CD73, CD90, and CD105) at elevated levels, coupled with the minimal expression of negative markers (CD34, CD45, CD11b, CD19, and HLA-DR). Moreover, the stability of these characteristics was evaluated across 10th generations. Our findings attest to the success of this endeavor. iMSCs exhibited robust expression of positive markers and limited expression of negative markers, confirming their MSC identity. Importantly, these characteristics remained stable even up to the 10th generation, signifying the potential for sustained use in therapeutic applications. Furthermore, our study demonstrated the successful differentiation of iMSCs into osteocytes, chondrocytes, and adipocytes, showcasing their multilineage potential. In conclusion, the establishment of induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) presents a significant advancement in overcoming the limitations associated with primary tissue-derived MSCs. The remarkable stability and multilineage differentiation potential exhibited by iMSCs offer a strong foundation for their application in regenerative medicine and tissue engineering. This breakthrough paves the way for further research and development in harnessing the full therapeutic potential of iMSCs.

Codon Optimization Enables the Geneticin Resistance Gene to Be Applied Efficiently to the Genetic Manipulation of the Plant Pathogenic Fungus Botrytis cinerea.

Botrytis cinerea can infect almost all of the important horticultural crops and cause severe economic losses globally every year. Modifying candidate genes and studying the phenotypic changes are among the most effective ways to unravel the pathogenic mechanism of this crop killer. However, few effective positive selection markers are used for B. cinerea genetic transformation, which limits multiple modifications to the genome, especially genes involving redundant functions. Here, we optimized a geneticin resistance gene, BcNPTII, based on the codon usage preference of B. cinerea. We found that BcNPTII can greatly increase the transformation efficiency of B. cinerea under G418 selection, with approximately 30 times higher efficiency than that of NPTII, which is applied efficiently to transform Magnaporthe oryzae. Using the gene replacement method, we successfully knocked out the second gene BOT2, with BcNPTII as the selection marker, from the mutant ΔoahA, in which OAHA was first replaced by the hygromycin resistance gene HPH in a field strain. We obtained the double knockout mutant ΔoahA Δbot2. Our data show that the codon-optimized BcNPTII is an efficient positive selection marker for B. cinerea transformation and can be used for various genetic manipulations in B. cinerea, including field wild-type strains.

Relationship between the number of positive MSI markers and the efficacy of NIVO+IPI therapy in MSI-H gastric cancer: A subgroup analysis of NO LIMIT study (WJOG13320G/CA209-7W7).

386 Background: Although high instability of microsatellite lesion (MSI-H) is an established biomarker for response to immune checkpoint inhibitors, the relationship between the number of MSI markers and the efficacy of immune checkpoint inhibitors in MSI-H tumors has not been well elucidated. NO LIMIT (WJOG13320G/CA209-7W7) is a phase II trial of nivolumab plus low-dose ipilimumab (NIVO+IPI) for MSI-H advanced gastric or esophagogastric junction cancer (GC) in the first-line setting.1 Here, we investigated the relationship between the number of positive MSI markers and the efficacy of NIVO+IPI in NO LIMIT study. Methods: Eligible patients were unresectable advanced, recurrent, or metastatic GC with confirmed as MSI-H with the MSI-IVD Kit (FALCO), where two or more of the five MSI markers are defined as positive. Nivolumab (240 mg) biweekly and ipilimumab (1 mg/kg) every six weeks were given until disease progression or unacceptable toxicity. The primary endpoint was the objective response rate (ORR) assessed by a blinded independent central review. Secondary endpoints included progression-free survival (PFS), overall survival (OS), safety, and the association between number of positive MSI markers and clinical efficacy (ORR, PFS, OS). The analysis was performed 18 weeks after the first dose in the last patient, with a median follow-up of 9 months (range, 4.0-18.0). Results: The breakdown of MSI markers in the 29 patients enrolled in NO LIMIT was as follows: BAT-26 (26/26, 100%), NR-21 (25/29, 86.2%), BAT-25 (25/29, 86.2%), MONO-27 (29/29, 100%), NR-24 (27/29, 89.4%). Since most of the cases were positive for all five of them (the number of cases with 2, 3, 4, and 5 positive markers was 2, 2, 1, and 24, respectively), we dichotomized cases into group A (all five positive) and group B (the others). Response rates in groups A vs. B were 62.5% (15/24) vs. 60.0% (3/5), respectively. There was no difference in PFS or OS between the two groups. Conclusions: The number of MSI markers is not related to the efficacy of NIVO+IPI in NO LIMIT study. Further follow-up is needed to confirm the relationship between survival. 1. Muro K, et al. ESMO Congress 2023. Clinical trial information: jRCT2080225304 . [Table: see text]

BRAF variants and oncogenic fusions to define sporadic MMR-deficient colorectal cancer subtypes.

211 Background: Mismatch-repair deficient (MMRd) colorectal cancers (CRC) are rare tumors exquisitely sensitive to immunotherapy. The molecular events that initiate and sustain MMRd tumors remain controversial. The majority of sporadic MMRd CRCs exhibit BRAF-V600E variants with MLH1 hypermethylation, however BRAF-wild-type(wt) tumors exhibit high rates of rare gene fusions. Methods: We performed an integrated molecular and clinical analysis for 253 patients with MMRd sporadic CRC who received MSK-IMPACT genomic profiling at Memorial Sloan Kettering Cancer Center. Tumors were classified based on BRAF-V600E variants and gene fusion events. We evaluated the tumor burden of molecular alterations and disproportionate co-occurrence of specific driver variants of significance between each subtype. We used allele-specific FACETS analysis and MLH1 promoter hypermethylation assessments to infer the MMR-gene inactivation mechanism per tumor. We also examined differences in tumor phenotype and patient clinical outcomes. Patient progression-free-survival after treatment and overall survival was assessed with the Kaplan-Meier curves, and multivariable analyses were performed with Cox proportional hazards regression modeling. Results: Patients exhibited BRAF-V600E-mutated (mut: n=178), BRAF-V600E-wt/gene-fusion positive (n=42), and BRAF-V600E-wt/fusion-negative (n=33) disease. Gene fusion events were mutually exclusive with BRAF-V600E variants and occurred in NTRK1 (n=13), NTRK3 (n=8; one with co-occurring NTRK1 fusion), RET (n=8), BRAF (n=8), ALK (n=5), and FGFR2 (n=1). BRAF-V600E-mut and fusion-positive tumors display similar predominant MLH1 silencing, WNT-signaling activators, and co-alterations. In contrast, BRAF-V600E-wt/fusion-negative tumors exhibit MLH1 inactivation associated with increased copy number alterations, and exhibit distinct co-occurring APC and TCFF7L2 variants. Both fusion-positive and BRAF-V600E-wt/fusion-negative tumors disproportionately arise in the transverse and left colon. In a multivariable analysis of 73 patients with stage IV MMRd CRC, fusion-positive status was an independently-significant, positive prognostic marker. For patients treated with immune checkpoint inhibitors (ICIs), tumor regression rates were high in BRAF-V600E-mut (63%; n=22/35) and fusion-positive (62.5%, n=5/8) subtypes, with slightly poorer outcomes in BRAF-V600E-wt/fusion-negative (40%, n=2/5) tumors. Patients with fusion-positive tumors exhibited clinical benefit to NTRK inhibition. Conclusions: In sporadic MMRd CRC, gene fusion-positive tumors exhibit an improved prognosis with similar molecular and treatment outcomes to BRAF-V600E-mut tumors. BRAF-V600E-wt/fusion-negative tumors are a unique sporadic MMRd subtype driven by alternative WNT-activation and potentially lower immunotherapy responses in small numbers of patients.

Low-dose fluconazole as a useful and safe prophylactic option in patients receiving allogeneic hematopoietic stem cell transplantation.

Invasive fungal infections (IFIs) represent a potentially fatal complication in patients who undergo allogeneic hematopoietic stem cell transplantation (HSCT) if the initiation of therapy is delayed. Some guidelines recommend antifungal prophylaxis or preemptive therapy for these patients depending on the risk of IFIs following allogeneic HSCT. This retrospective study aimed to identify the group of patients who safely undergo allogeneic HSCT with low-dose fluconazole (FLCZ) prophylaxis (100 mg/day). We retrospectively reviewed 107 patients who underwent their first allogeneic HSCT at Nagoya City University Hospital from January 1, 2010, to December 31, 2019. We analyzed the efficacy of low-dose FLCZ prophylaxis and investigated the relationship between major risk factors and antifungal prophylaxis failure (APF) within 100 days post-transplant. Of the 107 patients, 70 received low-dose FLCZ prophylaxis, showing a cumulative incidence of APF of 37.1% and a proven/probable IFI rate of 4.3%. There were no fungal infection-related deaths, including Aspergillus infections, in the FLCZ prophylaxis group. In a multivariable analysis, cord blood transplantation (CBT) (subdistribution hazard ratio (SHR), 3.55; 95% confidence interval (CI), 1.44-8.77; p = 0.006) and abnormal findings on lung CT before transplantation (SHR, 2.24; 95% CI, 1.02-4.92; p = 0.044) were independent risk factors for APF in the FLCZ prophylaxis group. Low-dose FLCZ prophylaxis is a useful and safe option for patients receiving allogeneic HSCT, except in those undergoing CBT or having any fungal risk features including history of fungal infections, positive fungal markers, and abnormal findings on lung CT before transplantation.

The positive impact of targeted modulating food intolerance on immune-related recurrent pregnancy loss

Background It has been proven that immune disorders are one of the vital risk factors of recurrent pregnancy loss (RPL), and the presence of food intolerance seems to play an essential role in this. However, the impact of immune status induced by food intolerance on RPL has not been reported. This study utilized a targeted diet avoiding food intolerance as much as possible for each participant to investigate their effects on pregnancy outcomes in RPL patients with positive autoimmune markers. Methods From January 2020 to May 2021, fifty-eight patients with RPL were enrolled. They were divided into two groups based on the presence of autoantibodies: the autoantibody-positive group (AP, n = 29) and the autoantibody-negative group (AN, n = 29). Their food-specific immunoglobulin (Ig) G antibodies for 90 foods were tested using enzyme-linked immunosorbent assay (ELISA). The levels of immune parameters and the presence of gastrointestinal discomforts (diarrhea or constipation, eczema, and mouth ulcers) were recorded before and after dietary conditioning, followed by the analysis of pregnancy outcomes. Results Compared to the AN group, the patients in the AP group showed immune disorders at baseline, such as reduced levels of IL-4 and complement C3, and increased levels of IL-2 and total B cells. These parameters within the AP group were significantly improved after dietary conditioning that avoided food intolerance, while no significant changes were observed in the AN group. Patients in the AP group had significantly higher food-specific IgG antibodies for cow’s milk (89.66% vs. 48.28%, p < .001), yolk (86.21% vs. 27.59%, p < .001), bamboo shoots (86.21% vs. 44.83%, p < .001) compared to those in the AN group. Additionally, gastrointestinal discomforts including diarrhea or constipation, eczema, and mouth ulcers were more common in the AP group than in the AN group. After 3-month dietary conditioning, these significantly improved characteristics were only observed in the AP group (p < .001). Finally, the baby-holding rate was higher in the AP group compared to the AN group (p < .05). Conclusions The RPL patients in the AN group did not exhibit immune disorders, whereas those in the AP group experienced immune disorders and gastrointestinal discomforts. For patient with positive autoantibodies, dietary intervention may mitigate immune disorders and gastrointestinal discomforts, presenting a promising approach to enhance pregnancy outcomes.

Detection and evaluation of positive acute phase reactant protein markers in neonates and adults with sepsis

. Introduction and Aim: Septicemia is a clinical condition caused due to bacterial infection of the blood. Septicemia leading to sepsis could be life-threatening leading to tissue damage, organ failure, and death. The current study aimed to determine the impact of various biomarkers on patients suffering from sepsis, including Serum amyloid A, C-reactive protein, HS- Troponin, fibrinogen, haptoglobin and alpha1-antitrypsin, as well as identify the common bacterial types associated in septic patients. Materials and Methods: This case-control study conducted during October 2022 to May 2023 involved 100 participants that were divided into groups which included patients diagnosed with septicemia due to bacteria (Group A), patients with septicemia with non-bacterial infection (Group B) and healthy individuals as controls (Group C). Results: Serum amyloid A, C-reactive protein, HS-Troponin, Fibrinogen, Haptoglobin, and Alpha-1 antitrypsin levels in neonates and adults with sepsis were found to be elevated when compared to healthy individuals. No significant difference was observed for these parameters between Group A patients (positive for blood culture) and Group B patients (negative for blood culture) among neonates and adult sepsis patients. Conclusion: Neonates and adult sepsis patients exhibited higher levels of serum amyloid A, C-reactive protein, HS-Troponin, fibrinogen, hemoglobin, and Alpha-1 antitrypsin in comparison to healthy controls. Hence, these biochemical parameters could be used as biomarkers to assess a patient's level of sepsis for further therapeutic action.

Progress in vaccines against peste des petits ruminants virus

Peste des petits ruminants (PPR) is a highly contagious and economically important viral disease of both domestic (goats and sheep) and wild small ruminants. Due to the devastating effect of this disease on livestock and livelihoods, the Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (WOAH) endorsed the Global Strategy for the Control and Eradication of PPR (PPR GCES) and launched the PPR Global Eradication Programme (PPR GEP) to eradicate PPRV by 2030. In order to achieve this goal, a potent, safe and efficacious live-attenuated PPR vaccine with long-lasting immunity is available for immunoprophylaxis. However, the live-attenuated PPR vaccines are thermolabile and require maintenance of an effective cold chain to deliver to the field. In addition, infected animals cannot be differentiated from vaccinated ones (DIVA). To overcome these limitations, some new generation PPR vaccines have been developed: poxvirus vaccine, positive and negative marker vaccine through reverse genetic approach, chimeric vaccine, anti-idiotypic vaccine, subunit vaccine, virus-like particles vaccine, edible vaccine and combined vaccines. Novel recombinant PPR DIVA vaccines were evaluated in goats for safety and efficacy, and all vaccinated animals were clinically protected against an intranasal PPRV challenge. Furthermore, newly developed ELISAs were capable of differentiating between infected and vaccinated animals. Therefore, these DIVA vaccines and the associated tests can facilitate the serological monitoring process and speed up global PPR eradication through vaccination.

英汉赞同类肯定性应答标记对比研究——以“就是”及其英译形式“Exactly”“I Know”为例

英汉赞同类肯定性应答标记对比研究——以“就是”及其英译形式“Exactly”“I Know”为例

Segurança clínica à administração intravenosa de células mesenquimais alógenas em cães saudáveis

ABSTRACT Despite the significance of adipose-derived stem cells in complementary and regenerative medicine, studies regarding clinical safety for allogeneic intravenous administration in healthy dogs remain insufficiently conducted. This study aimed to assess cellular viability through laboratory and microbiological evidence, along with genetic markers, in addition to evaluating clinic-laboratory safety for allogeneic intravenous administration of adipose-derived stem cells in healthy dogs. To achieve this, two cell batches and eight dogs were included in the study. The results revealed positive genetic markers for CD29, CD44, CD105, SOX2, and OCT3.4. Both batches exhibited positive cell differentiation into adipocytes, chondrocytes, and osteoblasts. Microbiological evidence showed negative results, and cell viability after thawing indicated 92% and 88.5% viable cells after 30 minutes, and 86% and 83.5% after 24 hours, respectively, for both batches. Hematological cell counts and serum biochemical enzyme levels, before and after intravenous treatment, did not exhibit statistical differences between the time points (p&gt;0.05). Median values remained within the reference range for the species during and after 30 days of treatment. Based on the cellular viability results, observed patterns, and the absence of hematological side effects, it can be concluded that intravenous therapy with allogeneic adipose-derived stem cells is clinically safe.

Vitamin D and celiac disease.

Celiac disease (CD) is an immune-mediated condition affecting the small intestine. Its reported global prevalence falls within the range of 0.7% to 1.4%. Notably, historically, higher rates, reaching 1% in Western Ireland, have been documented. Recent research has even revealed prevalence rates as elevated as 2% in northern Europe. These findings underscore the urgency for swift and cost-effective diagnosis, especially in individuals identified through screening efforts. At present, the diagnosis of CD relies on a multifaceted approach involving positive serological markers such as IgA anti-tissue transglutaminase (anti-TTG) and anti-endomysial antibodies (anti-EMA). These serological findings are assessed in conjunction with classical histological alterations, as outlined in the Marsh classification. CD is an inflammatory condition triggered by the consumption of gluten, resulting from intricate interactions between genetic, immunological, and environmental factors. CD is linked to malabsorption, leading to nutritional deficiencies. Individuals with CD are required to adhere to a gluten-free diet, which itself can lead to nutrient deficiencies. One such deficiency includes vitamin D, and there is substantial experimental evidence supporting the notion of a bidirectional relationship between CD and vitamin D status. A low level of vitamin D has a detrimental impact on the clinical course of the disease. Here we summarize the key characteristics of CD and explore the prominent roles of vitamin D in individuals with CD.

Development and validation of nomograms based on pre-/post-operative CEA and CA19-9 for survival predicting in stage I-III colorectal cancer patients after radical resection.

Carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) are the predominant serum tumour markers (STMs) for predicting the prognosis of colorectal cancer (CRC). The objective of this research is to develop clinical prediction models based on preoperative and postoperative CEA and CA19-9 levels. 1,452 consecutive participants with stage I-III colorectal cancer were included. Kaplan-Meier method, log-rank test, and multivariate COX regression were used to evaluate the significance of preoperative and postoperative STMs. Patients were grouped into a discovery cohort (70%) and a validation cohort (30%). Variables for the nomograms were selected according to the Akaike information criterion (AIC). Subsequently, two clinical predictive models were constructed, evaluated, validated, and then compared with the AJCC 8th TNM stage. The overall survival (OS) rate and disease-free survival(DFS) rate declined progressively as the number of positive tumour markers(NPTMs) before and after surgery increased. For both OS and DFS, age, sex, pN stage, and NPTMs before and after surgery were independent prognostic factors, and then clinical prediction models were developed. The Concordance index (C-index), Receiver operating characteristic (ROC) curve, calibration curve, Decision curve analysis (DCA), and risk score stratification all indicated that the models possessed robust predictive efficacy and clinical applicability. The Net reclassification index (NRI) and Integrated discrimination improvement (IDI) indicated that the performance of models was significantly superior to the TNM stage. Nomograms based on pre-and postoperative CEA and CA19-9 can accurately predict survival and recurrence for stage I-III CRC patients after radical surgery, and were significantly better than the AJCC 8th TNM stage.

Identifying the seeds of heterotic pools for Southern and Eastern Africa from global elite spring wheat germplasm.

Hybrid breeding can increase the competitiveness of wheat (Triticum aestivum L.) in Sub-Saharan Africa by fostering more public-private partnerships and promoting investment by the private sector. The benefit of hybrid wheat cultivars in South Africa has previously been demonstrated but due to the high cost of hybrid seed production, hybrid breeding has not received significant attention in the past decade. Considering the renewed commitment of the private sector to establish wheat as a hybrid crop globally, coupled with significant research investment into enhancement of outcrossing of wheat, hybrid wheat breeding in Southern and Eastern Africa should be revisited. Our study aimed to identify genetically distinct germplasm groups in spring wheat that would be useful in the establishment of heterotic pools targeting this region. Multi-environment yield testing of a large panel of F1 test hybrids, generated using global elite germplasm, was carried out between 2019 and 2020 in Argentina, Africa, Europe, and Australia. We observed significant genotype by environment interactions within our testing network, confirming the distinctiveness of African trial sites. Relatively high additive genetic variance was observed highlighting the contribution of parental genotypes to the grain yield of test hybrids. We explored the genetic architecture of these parents and the genetic factors underlying the value of parents appear to be associated with their genetic subgroup, with positive marker effects distributed throughout the genome. In testcrosses, elite germplasm from the International Maize and Wheat Improvement Center (CIMMYT) appear to be complementary to the genetically distinct germplasm bred in South Africa. The feasibility of achieving genetic gain via heterotic pool establishment and divergence, and by extension the viability of hybrid cultivars in Sub-Saharan Africa, is supported by the results of our study.

Atypical Manifestation of Zoster Meningitis Presented with Headache and Multiple Cerebral Vasoconstrictions

Reversible cerebral vasoconstriction syndrome (RCVS) is characterized by headaches and multiple transient cerebral vasoconstrictions. Central nervous system (CNS) infections related to the varicella zoster virus (VZV) can complicate various neurological disorders, including cerebral vasculopathy. We present the case of a patient who complained of a severe headache without any other symptoms or focal neurological signs. Brain magnetic resonance angiography showed multiple focal stenoses, especially in the right middle cerebral artery (MCA), and transcranial Doppler sonography revealed increased velocities in the MCA and right anterior cerebral artery. Initially, the headache was attributed to RCVS, and nimodipine infusion was initiated; however, the headache worsened. A cerebrospinal fluid study showed pleocytosis, increased protein levels, and positive polymerase chain reaction markers for VZV. No skin lesions or fever were observed during treatment. Our case emphasizes considering VZV meningitis as a cause of cerebral vasoconstriction, even in the absence of typical signs of CNS infection.

Object Play as a Positive Emotional State Indicator for Farmed Spotted Paca (Cuniculus paca).

We aimed to assess whether object play can be used as a positive emotional state indicator for farmed spotted pacas (Cuniculus paca) by examining its association with other positive welfare markers including affiliative behavior and low-amplitude vocalizations. We submitted six groups of spotted pacas (one male/two females per group) (N = 18) to an ABA experimental design (A1/A2: without ball; B: with three boomer balls). Object play behavior occurred only during phase B (mean = 35.5 s, SE = 6.4). The spotted pacas spent more time in affiliative and exploratory behaviors and less time engaging in agonistic interactions during phase B than in both control phases (A1 and A2) (p < 0.05). Moreover, the spotted pacas emitted more low-amplitude bark vocalizations during phase B than during either control phase (p < 0.05), and such vocalizations have previously been shown to indicate a positive affective state and low arousal level. Because the expression of object play was associated with a decrease in aggression, an increase in affiliative behavior, and an increase in low-amplitude barking, we suggest that object play can be used as a non-invasive indicator of positive emotional state in this species.

A Case of Posterior Scleritis with Negative Rheumatoid Factor and Positive Anti-Cyclic Citrullinated Peptide Antibody Status: Case Report

Background: Posterior scleritis is a potentially vision threatening condition which is often underdiagnosed due to varied clinical presentation, several non-infectious inflammatory diseases can cause this disorder, although infectious diseases should be ruled out. Case presentation: A 56 years-old-female with acute unilateral decreased vision in right eye and positive history for rheumatoid arthritis (RA) was referred, based on ophthalmic examination the diagnosis of posterior scleritis was considered. The only positive inflammatory marker was found is high serum titer of Anti-Cyclic Citrullinated Peptide (Anti-CCP). Conclusion: This is the first case of posterior scleritis with only positive Anti-CCP. Elevated serum Anti-CCP titer may be considered as risk factor for inflammatory scleritis in patient with RA.

Association of typical atrial flutter and cavotricuspid isthmus ablation on clinical recurrence after cryoballoon ablation for atrial fibrillation.

Typical atrial flutter commonly occurs in patients with atrial fibrillation (AF). Limited information exists regarding the effects of concurrent atrial flutter on the long-term outcomes of rhythm control. This study investigated the association between concurrent typical atrial flutter and cavotricuspid isthmus (CTI) ablation and the recurrence of atrial arrhythmia. The data were obtained from a multicenter registry of cryoballoon ablation for AF (n = 2,689). Patients who were screened for typical atrial flutter were included in the analysis (n = 1,907). All the patients with typical atrial flutter underwent CTI ablation. The primary endpoint was the late recurrence of atrial arrhythmia, including AF, atrial flutter, and atrial tachycardia. Among the 1,907 patients, typical atrial flutter was detected in 493 patients (25.9%). Patients with concurrent atrial flutter had a lower incidence of persistent AF and a smaller size of the left atrium. Patients with atrial flutter had a significantly lower recurrence rate of atrial arrhythmia (19.7% vs. 29.9%, p < 0.001). In patients with atrial flutter, the recurrence rate of atrial tachycardia or atrial flutter was more frequent (7.3% vs. 4.7%, p = 0.028), but the recurrence rate of AF was significantly lower (17.0% vs. 29.4%, p < 0.001). Atrial flutter has been identified as an independent predictor of the primary endpoint (adjusted hazard ratio, 0.704; 95% confidence interval, 0.548-0.906; p = 0.006). Typical atrial flutter in patients with AF may serve as a positive marker of the recurrence of atrial arrhythmia, and performing CTI ablation in this population is associated with a reduced likelihood of AF recurrence. Performing routine screening and ablation procedures for coexisting atrial flutter may improve the clinical outcomes of AF.

Media matters: culture medium-dependent hypervariable phenotype of mesenchymal stromal cells

BackgroundDespite a long history of investigation and sustained efforts in clinical testing, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration. Several barriers are impeding the clinical progression of MSC therapies, to the forefront of these is a lack of standardised manufacturing protocols which is further compounded by an absence of biologically meaningful characterisation and release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, among other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action.MethodsThis work analysed the influence of five widely used culture media formulations on the colony-forming potential, proliferation kinetics, trilineage differentiation potential and immunomodulatory potential of human bone marrow-derived MSCs (BM-MSCs). The surface marker expression profiles were also characterised using a high-content flow cytometry screening panel of 243 markers.ResultsSignificant differences in the biological attributes of BM-MSCs including clonogenicity, proliferation, differentiation propensity and immunomodulatory capacity were revealed in response to the composition of the culture medium. Despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs: CD73, CD90 and CD105. Immunophenotypic profiling revealed that the culture medium also had a significant influence on the surface proteome, with one-third of tested markers exhibiting variable expression profiles. Principal component analysis demonstrated that BM-MSCs isolated and expanded in a proprietary xeno- and serum-free medium displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate and foetal bovine serum-containing media.ConclusionsThese data suggest that media composition has a highly significant impact on the biological attributes of MSCs, but standard surface marker tests conceal these differences. The results indicate a need for (1) standardised approaches to manufacturing, with an essential focus on defined media and (2) new biologically relevant tests for MSC characterisation and product release.

A modified animal-free serum technique for efficient isolation and proliferation of mesenchymal stem cells from Hoffa fat pad.

We focus on this study in designing an alternative technique for obtaining mesenchymal stem cells (MSCs) from residual tissue, Hoffa fat, in arthroscopic procedures. Two males and two females were included, and underwent knee arthroscopy; a sample of infrapatellar adipose tissue was obtained with basket forceps. The primary culture was made using the explant method and the culture media: DMEM-high glucose, supplemented with 10% of inactivated human allogeneic serum. All the cellular cultures remained under culture conditions for three weeks, after that by flow cytometry the cells were characterized by MSCs antibody panel: CD105, CD73 and CD90. Subsequently, in the first pass, the MSCs were cultured in commercial human chondrogenic, osteogenic and adipogenic mediums, respectively. After primary culture, we obtained on average 95,600.00 ± 7,233.26 cells/cm2, and the duplication time of MSCs isolate from Hoffa fat pad was established in 39 hours. By flow cytometry, we found that surface markers percentage for expanded MSCs (CD105, CD73, CD90) in primary culture significantly increased and its morphology was fibroblastic-like. After differentiation culture which was made in the first pass, by immunofluorescence, we obtained positive cell markers for three lineages of differentiation, adipocytes: LPL protein, osteocytes: RUNX2, Osteopontin, chondrocytes: SOX9, Aggrecan and COL2A1. We managed to isolate a significant number of MSCs from this source using an easy method to implement and minimal nutrient supplementation, with high potential for differentiation to mature mesenchymal tissues and potential use in basic experimental, preclinical and even clinical research.

ARIA‐E, ARIA‐H, and CAA‐related inflammation: time of reconsiderations?

AbstractBackgroundEvidence from published clinical trials suggests that high‐dose of monoclonal antibodies (mAbs) are needed to significantly remove parenchymal‐deposited Aβ plaques. However, high‐dosing mAbs resulted in a 25‐35% increased risk of amyloid‐related imaging abnormalities‐edema (ARIA‐E) and ARIA‐hemorrhage (ARIA‐H) side events. Although most ARIA‐E are incidentally recognized as clinically silent during pre‐planned safety monitoring MRI scans and suggested to be managed by dose reduction or suspension, it is noticeable that half of ARIA‐E progressed to symptomatic at continued dosing and to associate with late occurrence of ARIA‐H. Of particular importance, a few of them aggravated to potentially severe brain injury or death due to fatal CAA‐related events. Increased evidence from natural history studies suggests that ARIA‐E is the iatrogenic manifestation of cerebral amyloid angiopathy‐related inflammation (CAA‐ri), a rare autoimmune encephalopathy due to raised anti‐Aß autoAbs levels in the cerebrospinal fluid (CSF).MethodNatural history study of CAA‐ri demonstrated that immunotherapy‐related ARIA‐E share several commonalities with the spontaneous occurrence of autoAbs‐related ARIA‐E, including fluid, MRI, and PET biomarker changes and the potential response to treatment outcomes.ResultThe prospective longitudinal monitoring of patients with CAA‐ri provide evidence for a regional and temporal association of ARIA‐E on MRI with focal peaks of neuroinflammation on microglial‐PET imaging. This association was strong in patients with positive CSF markers of AD (ATN positive) and comorbid CAA pathology on MRI, while it was negligible in patients with CAA disease w/o AD (ATN negative). Raised CSF concentrations of anti‐Aß autoAbs were found in all patients at (sub)acute presentation, and reduced within 5 months from the ARIA‐E index event, in parallel with evidence of radiographic resolution of ARIA‐E. At follow‐up MRI scan, the occurrence of new hemorrhagic markers (aka ARIA‐H in trials) were found only in patients with coexisting CAA and AD comorbid pathology. The MRI scales for ARIA commonly used in clinical trials only partially paralleled the clinical, biological, and MRI findings.ConclusionThis suggests that to mitigate and monitor the risk of ARIA, more sensitive, specific, cost‐effective and easy to interpret diagnostic tools and biomarkers than MRI alone will be needed for precision drug‐dose tailoring.