AUCTORES
Globalize your Research
Research Article
*Corresponding Author: Dazhi Jin, Department of laboratory Medicine, Hangzhou Medical College. No. 481 Binwen Rd, Hangzhou, Zhejiang, 310053, China.
Citation: Shuangshuang Wan, Guangzhong Song, Hui Hu, Yaqing Xu, Peng Zeng, et al. (2021). Intestine Epithelial cell-Derived Extracellular Vesicles alleviate Inflammation induced by Clostridioides difficile TcdB THROUGH the Activity of TGF- β1, J. Immunology and Inflammation Diseases Therapy. 4(1); Doi:10.31579/2637-8876/018
Copyright: © 2021 Dazhi Jin. This is an open-access article distributed under the terms of The Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received: 11 October 2021 | Accepted: 11 November 2021 | Published: 17 November 2021
Keywords: extracellular vesicles; Clostridioides difficile; TGF- β1; TcdB; regulatory T cells; inflammatory cytokines; immunotherapy 36
Background: Clostridioides difficile infection (CDI) has been primarily associated with the toxin B (TcdB), which can activate the intestinal immune system and lead to pathological damage. Even though the biological functions of intestine epithelial cell- derived extracellular vesicles (I-Evs) have been well documented, the role of I-Evs in the process of CDI is still unknown.
Results: We isolated I-Evs ranging from 100–200 nm in mean diameter, with a density of 1.09-1.17 g/mL. These I-Evs expressed the extracellular vesicle-associated specific surface markers, CD63 and TSG101. In vitro, 50 µg I-Evs decreased the expression of IL-6, TNF- β, IL-1β, and IL-22 in MC38 induced by 0.8 ng/mL C. difficile TcdB, and increased expression of TGF- β1. In vivo, I-Evs also promoted regulatory T cell induction, which improved inflammation of mice up to 80% relative to C. difficile TcdB infected mice, depending on the TGF- β1 signal pathway.
Conclusion: Our study firstly demonstrated that I-Evs originated from intestine epithelial cells is potentially a novel treatment endogenous candidate to effectively reduce the local infection induced by C. difficile TcdB.
In recent decades, with the excessive application of broad-spectrum antibiotics, diseases related to intestinal flora disorders have precipitously increased. Clostridioides difficile (C. difficile) is one of the main pathogens leading to antibiotic-associated diarrhoea and hospital-acquired infections in the United States and other developed countries [1]. Toxin A (TcdA) and B (TcdB) are the major pathogenic factors leading to diarrhoea, pseudomembranous colitis, toxic megacolon, and other intestinal symptoms [2]. The mechanism lies in the inactivation in the host epithelial cells of proteins from the Rho family of GTPases-including Rho, Rac, or Cdc42 by glycosylation, and upregulation of a series of pro-inflammatory cytokines such as interleukin IL-1, IL-6, and TNF-α [3]. Meanwhile, toxins recruit neutrophils and other inflammatory immune cells to induce intestinal mucosal cell apoptosis, necrosis, shedding, and increased permeability, triggering a widespread loss of intestinal barrier function, and initiating imbalance of flora and intestinal epithelial damage. According to the American Infectious Society, and the European Society of Clinical Microbiological Infections, in addition to other practical guidelines, oral metronidazole or vancomycin are the best methods to treat Clostridioides difficile infection (CDI) [4]. In addition, some new narrow-spectrum antibiotics such as fidaxomicin [5] and rifaximin have little impact on the intestinal flora and reduce the risk of drug resistance. In recent years, a number of immune-based agents [6] have entered clinical trials, and however their efficacy needs to be further validated. Faecal microbiota transplantation (FMT) has been recognised in the United States as an optional treatment method to restore normal intestinal flora and prevent recurrent attacks. However, a meta-analysis of randomised clinical trials in 2019 showed that the cure rate of FMT was only 76.1%. Furthermore, there are still many unanswered questions about FMT, including the optimal timing, preparation methods, and the patients who are likely to benefit most from this procedure. As its standard protocol is relatively complicated and involves approval of ethical reviews, FMT has not yet been widely used in China. Extracellular vesicles (Evs) are small vesicle-like substances secreted by cells, which possess various biological activities when released outside of the cell. They have a diameter ranging from approximately 30 nm to1 µm, and are generally classified into exosomes, microvesicles, and apoptotic bodies based on their size, biogenesis, and mechanism of secretion [7]. It is difficult to determine the functional differences between these three types of Evs, due to the lack of specific markers with which to distinguish them. Although once thought to be cellular debris, Evs are now recognized as vital vehicles involved in the communication between cells. Research has confirmed that Evs contain a wide range of biologically active components, and their corresponding functions depend on the source tissue or cell type. Evs also exist in body fluids such as serum, alveolar lavage fluid, and breast milk, carry messenger RNAs, microRNAs, and DNA [8, 9]; this suggests potential applications as biomarkers for the diagnosis of diseases, as part of a liquid biopsy technology [10]. Recently, it has been reported that Evs can be designed to function as effective carriers in the treatment of various diseases, including in the delivery of long non-coding RNAs [11, 12]. In addition, Evs play a significant therapeutic role in regulating complex intracellular pathways in certain diseases, such as inflammatory bowel disease (IBD) [13, 14], and osteoarthritis [15]. Furthermore, it has been discovered that Evs derived from mesenchymal stem cells possess important immunomodulatory effects in areas such as neurodegenerative diseases, ageing, and inflammation [16-18]. Previously, we have reported that CD8α+CD11c+ Evs derived from lungs reduce the allergic reaction of asthmatic mice through TGF-β1 and IL-10, thereby maintaining the immune balance of the respiratory tract [19]. In the context of the recent outbreaks of COVID-19 around the world, mesenchymal stem cells and their Evs could be used as potential drug candidates for the treatment of severe cases, mainly through the induction of anti-inflammatory macrophages, regulatory T and B cells, and regulatory dendritic cells [20]. Strikingly, infection with TcdB-producing strains alone, but not TcdA+B- strains, can cause severe CDI symptoms [21]. Our work using purified C. difficile TcdB, together with cell lines and mice, confirmed that TcdB can induce expression of the inflammatory genes IL-6, TNF- β, IL-22, and IL-1 β, and upregulation of TGF-1 in vitro. Intestine epithelial cell-derived extracellular vesicles (I-Evs) rescue this phenomenon in vivo by inducing proliferation of regulatory T cells, dependent on TGF- β1 and the corresponding downstream molecules Smad2/3. Here, we studied the role of I-Evs on inflammation induced by C. difficile TcdB and evaluated biological functions of I-Evs in alleviating pathological damage led by CDI in mice.
Results
Isolation and identification of intestine epithelial cell-derived extracellular vesicles We used electron microscopy to visualise the morphology of the purified I-Evs; combined with Nanoparticle tracking analysis this showed that the isolated I-Evs had a mean diameter of 100–200 nm (Figure 1A, B). To further explore the I-Evs, sucrose density gradient centrifugation was used to detect the density range of I-Evs, which was 1.09–1.17 g/mL (Figure 1C). Protein analysis identified that I-Evs were positive for universal surface markers of extracellular vesicles, including CD63 and TSG101, and the intestinal epithelial cell-specific protein A33, but negative for GRP94, as detected by western blot (Figure 1D).
In addition, high levels of TGF-β1 were expressed in I- Evs, implying a role in immunoregulation. The results showed that we successfully isolated and identified I-Evs. I-Evs attenuated the down-regulation of TGF- β1 induced by purified C. difficile TcdB in vitro Real-time PCR results showed that, compared to the control group, the expression of pro-inflammatory genes (IL-6, TNF- β, IL-1β, and IL-22) was increased in the 0.4 ng/mL C. difficile TcdB group, but significantly decreased in the 0.8 ng/mL I-Evs group. In contrast, the expression of the anti-inflammatory genes TGF- β1 and IL-10 was statistically increased in the I-Evs group compared to the TcdB groups (Figure 2A).
Western blot results showed that protein levels of the immunosuppressive cytokine TGF- β1 were decreased in MC38 murine colon carcinoma cells, and LOVO human colon carcinoma cells, stimulated by C. difficile TcdB (Figure 2B). The concentration of C. difficile TcdB used was 0.1 ng/mL, 0.2 ng/mL, 0.4 ng/mL, or 0.8 ng/mL. This decrease could be rescued by I-Evs when TcdB concentration was 0.4 ng/mL (Figure 2C). Altogether, these results indicate that the I-Evs containing TGF- β1 had anti- Inf ammatory effects in vitro.
I-Evs alleviate C. difficile TcdB-induced local colon inflammation in mice
Intestinal epithelial damage caused by C. difficile TcdB, generally confined to the local intestine, is a severe inflammatory intestinal lesion. We sought to explore whether I-Evs can be applied in this condition as a type of anti-inflammatory immunotherapy. I-Evs contain more TGF- β1 than intestinal lysates as determined by western blot, which indicated a likely strong immunosuppressive effect. Next, we established a murine local colon infection model to investigate the treatment effect of I-Evs (Figure 3A). As shown in Figure 3B, the survival rate of mice after C. difficile TcdB injection was only 50%, while I-Evs increased the survival rate of mice up to 80%. The intestinal tissues displayed marked leukocyte infiltration and sections of glandular structure damage; consistently, histopathological analysis showed only slight leukocyte infiltration and epithelial cell damage after application of I-Evs (Figure 3C, D). Moreover, intestinal epithelial damage, congestion and mucosal oedema were significantly increased in the C. difficile TcdB mice when compared with the control mice (Figure 3E, F), however, less intestinal damage and limited leukocyte infiltration were observed when mice were treated with I-Evs. These findings implied that I-Evs attenuated pathological changes occurring as a result of C. difficile TcdB-induced inflammation, thereby protecting mice from local colon inflammation.
Induction of regulatory T cells by I-Evs alleviated infection caused by C. difficile TcdB through a TGF-β1-dependent mechanism
A previous study showed that EpCAM-dependent I-Evs alleviated IBD by inducing regulatory T cells [22]. I-Evs induced an increase in the proportion of CD4+Foxp3+Tregs in vitro and in vivo (Figure 4A–D); these immunoregulatory cells exhibit immunosuppressive effects in the development of disease. When the activity of TGF-β1, a potent immunosuppressive cytokine, was blocked (using the protocol described in the Materials and Methods), I-Evs immediately lost the ability to induce CD4+Foxp3+Tregs in the spleen. Concurrently, I-Evs were not able to increase the survival rate of mice, and the improvement of pathological effects previously seen was also undetectable (Figure 4E, F,4G).
Together, these results suggest that immunosuppressive regulatory T cells induced by I-Evs attenuated C. difficile TcdB- induced local colon inflammation in a mechanism dependent on TGF-β1. Smad2/3 are the main downstream proteins involved in the TGF- β 1 signalling pathway. The phosphorylation levels of Smad2/3 were decreased after stimulation with C. difficile TcdB, although protein levels of Smad2/3 remained the same; treatment with I-Evs promoted phosphorylation of Smad2/3, and thereby upregulation of TGF- β1 (Figure 4H). These results suggest that Smad2/3 is inhibited by C. difficile TcdB, leading to the down-regulation of TGF- β1 expression. Conversely, I-Evs with high expression of TGF- β1 activate Smad2/3 and contribute to the upregulation of TGF- β1, there by alleviating C. difficile TcdB-induced local colon inflammation in mice.
A common clinical symptom of CDI is local colon infection, which may arise due to intestinal perforation after either infection or intestinal surgery, particularly in high- risk populations, such as patients with IBD; respiratory insufficiency; heart and renal failure; ages over 60 years; and several other underlying diseases. The majority of CDI can be treated with metronidazole and fidaxomicin, in addition to other antibiotics. Surgical removal of necrotic intestinal tissue can reduce mortality rates with severe explosive colitis. Nevertheless, postoperative bleeding, and intestinal stenosis and obstruction, are extremely distressing to the patient. Prevention, management, and non- surgical treatment are the fundamental principles of CDI. However, the most severe toxic colitis cases are unable to benefit from drugs and surgery, and there is an urgent need to establish an effective treatment programme based on immunotherapy. Evs participate in a variety of physiological and pathological processes, including neurological disorders [23], osteoarthritis [24], infection [25], and tumours [26]. Evs have been proven to be involved in immune regulation and antigen presentation, and our research group demonstrated that Evs derived from intestinal epithelial cells alleviate IBD in mice by inhibiting dendritic cell activation and inducing Tregs [22]. In this study, I-Evs isolated from the intestine, with mean diameters of 100–200 nm as detected by electron microscopy scanning and Nanoparticle tracking analysis, expressed the characteristic protein markers of Evs, CD63 and TSG101. Enrichment of the immunosuppressive cytokine, TGF- β 1, in I-Evs inspired us to hypothesise an immunomodulatory function for I-Evs. A recent study verified that Evs derived from human mesenchymal stem cells can relieve colitis by reducing pro-inflammatory responses and increasing anti-inflammatory responses [27]. As is well-established, colitis caused by C. difficile relies on a series of virulence factors, including toxins, which initially target intestinal epithelial cells and subsequently destroys the intestinal membrane integrity. Hosts exposed to intestinal microorganisms trigger immune inflammatory responses. The dominance of either TcdA or TcdB was still controversial in this research field, despite a multi-laboratory follow-up research study pronouncing that TcdB acts as a critical toxin in colonic epithelial injury and mortality in vivo, whereas TcdA caused inflammation in mice to a small extent [28]. In the work presented here, TcdB induced increased gene expression of IL-6, TNF- β, IL-1β, and IL-22. I-Evs were able to rescue this phenomenon, and interestingly, TGF- β 1 and IL- 10 gene actually increased upon co-incubation with I-Evs. Moreover, I-Evs could reverse the decreased expression of TGF-β1 stimulated by C. difficile TcdB, as detected by western blot analysis of MC38 and LOVO lysates. We also report for the first time that I-Evs can improve survival of mice with local colon inflammation induced by C. difficile TcdB. The mechanism lies in the induction of CD4+Foxp3+Tregs, which play an important role in maintaining immune tolerance and homeostasis; the decline or dysfunction of Tregs has previously been shown to increase intestinal inflammation in IBD mice[29]29. Similarly, CD4+CD25+ Treg cells transferred into hosts ameliorated colitis symptoms. The I-Evs in this study contained TGF- β1; IL-10 is also known to be an important immunosuppressive cytokine, but we could barely detect the presence of IL-10 in our isolated I-Evs. Whether IL-10 still performs an important function is unknown. Furthermore, proinflammatory cytokines were undetectable following stimulation with C. difficile TcdB. Indeed, we improved various experimental schemes to optimise the experimental conditions, unfortunately, the corresponding data were still not available. We speculate that the effect of C. difficile TcdB on cells in vitro was different from that in vivo. As for the animal challenge experiment, in order to induce chronic inflammatory intestinal infection, five antibiotic mixtures were fed to mice, in addition to intraperitoneal injection with clindamycin and local injection with C. difficile Tcd B. I-Evs improved both the survival of mice, and intestinal tissue pathological scores, when transferred into mice.
We firstly demonstrated that I-Evs can alleviate inflammation induced by C. difficile TcdB in vitro and in vivo, and protect against pathological lesions in the animal intestine. Our data indicated that I-Evs could activate the TGF- β1 pathway and the downstream proteins Smad 2/3, to alleviate local colon inflammation induced by C. difficile TcdB, providing a novel endogenous candidate for treatment of C. difficile infections.
Toxins, antibodies, and reagents
C. difficile TcdB was gifted from the Tao Liang research group (West Lake University, Hangzhou, China) [30]. Primary antibodies against CD63 (ab213090), TGF-1 (ab8227), GRP94 (ab238126), TSG101 (ab125011), and β-Actin (ab8227) were from Abcam (Cambridge, MA, USA). PRMT1 (A33) (#2449), Smad2 (D43B4), Smad3 (C67H9), phospho-Smad2 (Ser465/Ser467) (E8F3R), and phospho-Smad3 (Ser423/425) were from Cell Signalling Technology (Danvers, MA, USA), and the corresponding secondary antibodies were from BBI (Shanghai, China). Fluorescent- labelled antibodies against CD4 (GK1.5) and Foxp3 (PCH101) were from eBioscienc (San Diego, CA, USA).
Real-time fluorescence quantification PCR
The classic TRIzol (Gibco, USA) method was used to extract RNA, using a reverse transcription kit (TOYOBO) to acquire cDNA. Real-time, fluorescence quantification PCR (qRT-PCR) was performed in a Step One Plus Real Time PCR System (Roche) to detect gene expression. The mouse-specific primers used are shown in Appendix A.
Mouse and cell lines
The MC38 cell line was purchased from Wuhan Fine Biotech Co., Ltd. (Wuhan, China). The cells were negative for mycoplasma as detected by fluorescence and culture methods. Human LOVO cells were kindly provided by Jia Jing (Hangzhou Medical College, Hangzhou, China). Male C57BL/6J mice (6–8 weeks old) were purchased from Shanghai Laboratory Animal Co., Ltd. (Shanghai, China). The mice were housed in a specific pathogen-free animal facility, and experimental protocols were approved by the Animal Care and Use Committee of Hangzhou Medical College, all animals were treated according to the guidelines for animal experimentation of Hangzhou Medical College in Hangzhou, China. The animal experiments were also performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines [31].The mice were sacrificed 5 days after anesthetized with intraperitoneal injection chloral hydrate (375 mg/kg of body weight)
Isolation and quantification of I-Evs
Mouse large intestines were surgically extracted and ground in a sufficient volume of PBS. They were then digested with 1 mg/mL collagenase type Ⅱ from Clostridium histolyticum (Gibco) for 2 h at 37 ˚C. The resulting suspension of intestinal tissue fragments was centrifuged at 400 g for 10 min, and the supernatant carefully removed for further centrifugation at 10,000 g for 30 min, to remove larger vesicles. The resulting supernatant was then filtered by a 0.22-µm screening and ultracentrifuged at 100,000 g for 1 h. Crude pellets of I-Evs were washed in sterile PBS and centrifuged at the same speed for an additional 1 h. The harvested I-Evs were resuspended in PBS. A BCA assay was used to detect the concentration of I-Evs (ThermoFisher, Waltham, MA, USA).
Electron microscopy scanning and Nanoparticle tracking analysis
Suspensions of I-Evs were loaded onto a coated copper grid, and a drop of 2% phosphotungstic acid added as a negative staining method. The sample was then allowed to dry at room temperature and transferred to a transmission electron microscope (Hitachi H7650, Hitachi, China) to take pictures and record at a voltage of 80 kV. To detect size distribution, I-Evs were diluted with PBS, and 0.3 mL analysed by NanoSight Nano instruments (Malvern, UK).
Western blot and flow cytometry analysis
For western blot analysis, 40 µg I -Evs or protein lysates extracted from intestinal tissues were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 5% milk in phosphate buffered solution-Tween 20 (PBS-T) and incubated with the corresponding primary antibodies at 4 ˚C overnight. The next day, membranes were incubated with an HRP-coupled secondary antibody for 1 h at room temperature and scanned using a Canon 4500 imaging system (Shanghai, China). For cytometry analysis, cells were washed with cold PBS and incubated with a fluorescent antibody for 30 min at 4 ˚C in the dark. Cells were analysed by fluorescence-activated cell sorting (BD,Franklin Lakes, NJ, USA).
CD4+Foxp3+Tregs induction assay
Murine CD4+ T cells were isolated with the EasySep Mouse CD4+ T Cell Isolation Kit (Stemcell), and labelled with an anti-CD62L antibody for flow cytometry. Magnetic sorting was then performed using the EasySep Mouse Biotin Positive Selection Kit (Stemcell). Cells were then incubated with 1 µl anti -CD3/CD28-coated beads and 200U/mL IL-2 for 72 h (2 x 105 cells/well), with or without 50 µg/mL I -Evs. To block the TGF- β1 signal, 0.6 µg/mL TGF- β1 inhibitor was applied to cells (in vitro), or 15 µg/mL anti–TGF- β1–neutralising antibody was injected into mice (in vivo). The percentage of CD4+Foxp3+Tregs was analysed by flow cytometry.
Induction and treatment of murine local colon inflammation induced by C. difficile Tcd B
C57BL/6J male mice were randomised into groups and given antibiotics through their drinking water for 5 days. The antibiotic mixture consisted of gentamicin (0.035 mg/mL), kanamycin (0.4 mg/mL), colistin (850 U/mL), metronidazole (0.215 mg/mL), and vancomycin (0.045 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA). The following day, mice were injected with clindamycin (10 mg/kg). After this, purified TcdB was surgical injected into local colon of mice (0.5 µg/kg); this was noted as day 0. Functional I-Evs (50 µg/100 µL PBS) were intraperitoneal injection after 5 h, and on day 1. After sacrificing the animals, the intestinal tissue in different groups was collected and prepared for H&E staining.
Data are presented as the mean ± SEM. Data were compared using a Student’s t- test with GraphPad Prism 8 (San Diego, CA, USA). P＜0.05 was considered statistically significant.
C. difficile: Clostridioides difficile; CDI: Clostridioides difficile infection; FMT: Faecal microbiota transplantation; Evs: Extracellular vesicles
Clearly Auctoresonline and particularly Psychology and Mental Health Care Journal is dedicated to improving health care services for individuals and populations. The editorial boards' ability to efficiently recognize and share the global importance of health literacy with a variety of stakeholders. Auctoresonline publishing platform can be used to facilitate of optimal client-based services and should be added to health care professionals' repertoire of evidence-based health care resources.
Journal of Clinical Cardiology and Cardiovascular Intervention The submission and review process was adequate. However I think that the publication total value should have been enlightened in early fases. Thank you for all.
Journal of Women Health Care and Issues By the present mail, I want to say thank to you and tour colleagues for facilitating my published article. Specially thank you for the peer review process, support from the editorial office. I appreciate positively the quality of your journal.
Journal of Clinical Research and Reports I would be very delighted to submit my testimonial regarding the reviewer board and the editorial office. The reviewer board were accurate and helpful regarding any modifications for my manuscript. And the editorial office were very helpful and supportive in contacting and monitoring with any update and offering help. It was my pleasure to contribute with your promising Journal and I am looking forward for more collaboration.
We would like to thank the Journal of Thoracic Disease and Cardiothoracic Surgery because of the services they provided us for our articles. The peer-review process was done in a very excellent time manner, and the opinions of the reviewers helped us to improve our manuscript further. The editorial office had an outstanding correspondence with us and guided us in many ways. During a hard time of the pandemic that is affecting every one of us tremendously, the editorial office helped us make everything easier for publishing scientific work. Hope for a more scientific relationship with your Journal.
The peer-review process which consisted high quality queries on the paper. I did answer six reviewers’ questions and comments before the paper was accepted. The support from the editorial office is excellent.
Journal of Neuroscience and Neurological Surgery. I had the experience of publishing a research article recently. The whole process was simple from submission to publication. The reviewers made specific and valuable recommendations and corrections that improved the quality of my publication. I strongly recommend this Journal.
Dr. Katarzyna Byczkowska My testimonial covering: "The peer review process is quick and effective. The support from the editorial office is very professional and friendly. Quality of the Clinical Cardiology and Cardiovascular Interventions is scientific and publishes ground-breaking research on cardiology that is useful for other professionals in the field.
Thank you most sincerely, with regard to the support you have given in relation to the reviewing process and the processing of my article entitled "Large Cell Neuroendocrine Carcinoma of The Prostate Gland: A Review and Update" for publication in your esteemed Journal, Journal of Cancer Research and Cellular Therapeutics". The editorial team has been very supportive.
Testimony of Journal of Clinical Otorhinolaryngology: work with your Reviews has been a educational and constructive experience. The editorial office were very helpful and supportive. It was a pleasure to contribute to your Journal.
Dr. Bernard Terkimbi Utoo, I am happy to publish my scientific work in Journal of Women Health Care and Issues (JWHCI). The manuscript submission was seamless and peer review process was top notch. I was amazed that 4 reviewers worked on the manuscript which made it a highly technical, standard and excellent quality paper. I appreciate the format and consideration for the APC as well as the speed of publication. It is my pleasure to continue with this scientific relationship with the esteem JWHCI.
This is an acknowledgment for peer reviewers, editorial board of Journal of Clinical Research and Reports. They show a lot of consideration for us as publishers for our research article “Evaluation of the different factors associated with side effects of COVID-19 vaccination on medical students, Mutah university, Al-Karak, Jordan”, in a very professional and easy way. This journal is one of outstanding medical journal.
Dear Hao Jiang, to Journal of Nutrition and Food Processing We greatly appreciate the efficient, professional and rapid processing of our paper by your team. If there is anything else we should do, please do not hesitate to let us know. On behalf of my co-authors, we would like to express our great appreciation to editor and reviewers.
As an author who has recently published in the journal "Brain and Neurological Disorders". I am delighted to provide a testimonial on the peer review process, editorial office support, and the overall quality of the journal. The peer review process at Brain and Neurological Disorders is rigorous and meticulous, ensuring that only high-quality, evidence-based research is published. The reviewers are experts in their fields, and their comments and suggestions were constructive and helped improve the quality of my manuscript. The review process was timely and efficient, with clear communication from the editorial office at each stage. The support from the editorial office was exceptional throughout the entire process. The editorial staff was responsive, professional, and always willing to help. They provided valuable guidance on formatting, structure, and ethical considerations, making the submission process seamless. Moreover, they kept me informed about the status of my manuscript and provided timely updates, which made the process less stressful. The journal Brain and Neurological Disorders is of the highest quality, with a strong focus on publishing cutting-edge research in the field of neurology. The articles published in this journal are well-researched, rigorously peer-reviewed, and written by experts in the field. The journal maintains high standards, ensuring that readers are provided with the most up-to-date and reliable information on brain and neurological disorders. In conclusion, I had a wonderful experience publishing in Brain and Neurological Disorders. The peer review process was thorough, the editorial office provided exceptional support, and the journal's quality is second to none. I would highly recommend this journal to any researcher working in the field of neurology and brain disorders.
Dear Agrippa Hilda, Journal of Neuroscience and Neurological Surgery, Editorial Coordinator, I trust this message finds you well. I want to extend my appreciation for considering my article for publication in your esteemed journal. I am pleased to provide a testimonial regarding the peer review process and the support received from your editorial office. The peer review process for my paper was carried out in a highly professional and thorough manner. The feedback and comments provided by the authors were constructive and very useful in improving the quality of the manuscript. This rigorous assessment process undoubtedly contributes to the high standards maintained by your journal.
International Journal of Clinical Case Reports and Reviews. I strongly recommend to consider submitting your work to this high-quality journal. The support and availability of the Editorial staff is outstanding and the review process was both efficient and rigorous.
Thank you very much for publishing my Research Article titled “Comparing Treatment Outcome Of Allergic Rhinitis Patients After Using Fluticasone Nasal Spray And Nasal Douching" in the Journal of Clinical Otorhinolaryngology. As Medical Professionals we are immensely benefited from study of various informative Articles and Papers published in this high quality Journal. I look forward to enriching my knowledge by regular study of the Journal and contribute my future work in the field of ENT through the Journal for use by the medical fraternity. The support from the Editorial office was excellent and very prompt. I also welcome the comments received from the readers of my Research Article.
Dear Erica Kelsey, Editorial Coordinator of Cancer Research and Cellular Therapeutics Our team is very satisfied with the processing of our paper by your journal. That was fast, efficient, rigorous, but without unnecessary complications. We appreciated the very short time between the submission of the paper and its publication on line on your site.
I am very glad to say that the peer review process is very successful and fast and support from the Editorial Office. Therefore, I would like to continue our scientific relationship for a long time. And I especially thank you for your kindly attention towards my article. Have a good day!
"We recently published an article entitled “Influence of beta-Cyclodextrins upon the Degradation of Carbofuran Derivatives under Alkaline Conditions" in the Journal of “Pesticides and Biofertilizers” to show that the cyclodextrins protect the carbamates increasing their half-life time in the presence of basic conditions This will be very helpful to understand carbofuran behaviour in the analytical, agro-environmental and food areas. We greatly appreciated the interaction with the editor and the editorial team; we were particularly well accompanied during the course of the revision process, since all various steps towards publication were short and without delay".
I would like to express my gratitude towards you process of article review and submission. I found this to be very fair and expedient. Your follow up has been excellent. I have many publications in national and international journal and your process has been one of the best so far. Keep up the great work.
We are grateful for this opportunity to provide a glowing recommendation to the Journal of Psychiatry and Psychotherapy. We found that the editorial team were very supportive, helpful, kept us abreast of timelines and over all very professional in nature. The peer review process was rigorous, efficient and constructive that really enhanced our article submission. The experience with this journal remains one of our best ever and we look forward to providing future submissions in the near future.
I am very pleased to serve as EBM of the journal, I hope many years of my experience in stem cells can help the journal from one way or another. As we know, stem cells hold great potential for regenerative medicine, which are mostly used to promote the repair response of diseased, dysfunctional or injured tissue using stem cells or their derivatives. I think Stem Cell Research and Therapeutics International is a great platform to publish and share the understanding towards the biology and translational or clinical application of stem cells.
I would like to give my testimony in the support I have got by the peer review process and to support the editorial office where they were of asset to support young author like me to be encouraged to publish their work in your respected journal and globalize and share knowledge across the globe. I really give my great gratitude to your journal and the peer review including the editorial office.
I am delighted to publish our manuscript entitled "A Perspective on Cocaine Induced Stroke - Its Mechanisms and Management" in the Journal of Neuroscience and Neurological Surgery. The peer review process, support from the editorial office, and quality of the journal are excellent. The manuscripts published are of high quality and of excellent scientific value. I recommend this journal very much to colleagues.
Dr.Tania Muñoz, My experience as researcher and author of a review article in The Journal Clinical Cardiology and Interventions has been very enriching and stimulating. The editorial team is excellent, performs its work with absolute responsibility and delivery. They are proactive, dynamic and receptive to all proposals. Supporting at all times the vast universe of authors who choose them as an option for publication. The team of review specialists, members of the editorial board, are brilliant professionals, with remarkable performance in medical research and scientific methodology. Together they form a frontline team that consolidates the JCCI as a magnificent option for the publication and review of high-level medical articles and broad collective interest. I am honored to be able to share my review article and open to receive all your comments.
“The peer review process of JPMHC is quick and effective. Authors are benefited by good and professional reviewers with huge experience in the field of psychology and mental health. The support from the editorial office is very professional. People to contact to are friendly and happy to help and assist any query authors might have. Quality of the Journal is scientific and publishes ground-breaking research on mental health that is useful for other professionals in the field”.
Dear editorial department: On behalf of our team, I hereby certify the reliability and superiority of the International Journal of Clinical Case Reports and Reviews in the peer review process, editorial support, and journal quality. Firstly, the peer review process of the International Journal of Clinical Case Reports and Reviews is rigorous, fair, transparent, fast, and of high quality. The editorial department invites experts from relevant fields as anonymous reviewers to review all submitted manuscripts. These experts have rich academic backgrounds and experience, and can accurately evaluate the academic quality, originality, and suitability of manuscripts. The editorial department is committed to ensuring the rigor of the peer review process, while also making every effort to ensure a fast review cycle to meet the needs of authors and the academic community. Secondly, the editorial team of the International Journal of Clinical Case Reports and Reviews is composed of a group of senior scholars and professionals with rich experience and professional knowledge in related fields. The editorial department is committed to assisting authors in improving their manuscripts, ensuring their academic accuracy, clarity, and completeness. Editors actively collaborate with authors, providing useful suggestions and feedback to promote the improvement and development of the manuscript. We believe that the support of the editorial department is one of the key factors in ensuring the quality of the journal. Finally, the International Journal of Clinical Case Reports and Reviews is renowned for its high- quality articles and strict academic standards. The editorial department is committed to publishing innovative and academically valuable research results to promote the development and progress of related fields. The International Journal of Clinical Case Reports and Reviews is reasonably priced and ensures excellent service and quality ratio, allowing authors to obtain high-level academic publishing opportunities in an affordable manner. I hereby solemnly declare that the International Journal of Clinical Case Reports and Reviews has a high level of credibility and superiority in terms of peer review process, editorial support, reasonable fees, and journal quality. Sincerely, Rui Tao.
Clinical Cardiology and Cardiovascular Interventions I testity the covering of the peer review process, support from the editorial office, and quality of the journal.
Clinical Cardiology and Cardiovascular Interventions, we deeply appreciate the interest shown in our work and its publication. It has been a true pleasure to collaborate with you. The peer review process, as well as the support provided by the editorial office, have been exceptional, and the quality of the journal is very high, which was a determining factor in our decision to publish with you.
The peer reviewers process is quick and effective, the supports from editorial office is excellent, the quality of journal is high. I would like to collabroate with Internatioanl journal of Clinical Case Reports and Reviews journal clinically in the future time.
Clinical Cardiology and Cardiovascular Interventions, I would like to express my sincerest gratitude for the trust placed in our team for the publication in your journal. It has been a true pleasure to collaborate with you on this project. I am pleased to inform you that both the peer review process and the attention from the editorial coordination have been excellent. Your team has worked with dedication and professionalism to ensure that your publication meets the highest standards of quality. We are confident that this collaboration will result in mutual success, and we are eager to see the fruits of this shared effort.
Dear Dr. Jessica Magne, Editorial Coordinator 0f Clinical Cardiology and Cardiovascular Interventions, I hope this message finds you well. I want to express my utmost gratitude for your excellent work and for the dedication and speed in the publication process of my article titled "Navigating Innovation: Qualitative Insights on Using Technology for Health Education in Acute Coronary Syndrome Patients." I am very satisfied with the peer review process, the support from the editorial office, and the quality of the journal. I hope we can maintain our scientific relationship in the long term.
Dear Monica Gissare, - Editorial Coordinator of Nutrition and Food Processing. ¨My testimony with you is truly professional, with a positive response regarding the follow-up of the article and its review, you took into account my qualities and the importance of the topic¨.
Dear Dr. Jessica Magne, Editorial Coordinator 0f Clinical Cardiology and Cardiovascular Interventions, The review process for the article “The Handling of Anti-aggregants and Anticoagulants in the Oncologic Heart Patient Submitted to Surgery” was extremely rigorous and detailed. From the initial submission to the final acceptance, the editorial team at the “Journal of Clinical Cardiology and Cardiovascular Interventions” demonstrated a high level of professionalism and dedication. The reviewers provided constructive and detailed feedback, which was essential for improving the quality of our work. Communication was always clear and efficient, ensuring that all our questions were promptly addressed. The quality of the “Journal of Clinical Cardiology and Cardiovascular Interventions” is undeniable. It is a peer-reviewed, open-access publication dedicated exclusively to disseminating high-quality research in the field of clinical cardiology and cardiovascular interventions. The journal's impact factor is currently under evaluation, and it is indexed in reputable databases, which further reinforces its credibility and relevance in the scientific field. I highly recommend this journal to researchers looking for a reputable platform to publish their studies.
Dear Editorial Coordinator of the Journal of Nutrition and Food Processing! "I would like to thank the Journal of Nutrition and Food Processing for including and publishing my article. The peer review process was very quick, movement and precise. The Editorial Board has done an extremely conscientious job with much help, valuable comments and advices. I find the journal very valuable from a professional point of view, thank you very much for allowing me to be part of it and I would like to participate in the future!”
Dealing with The Journal of Neurology and Neurological Surgery was very smooth and comprehensive. The office staff took time to address my needs and the response from editors and the office was prompt and fair. I certainly hope to publish with this journal again.Their professionalism is apparent and more than satisfactory. Susan Weiner
My Testimonial Covering as fellowing: Lin-Show Chin. The peer reviewers process is quick and effective, the supports from editorial office is excellent, the quality of journal is high. I would like to collabroate with Internatioanl journal of Clinical Case Reports and Reviews.
My experience publishing in Psychology and Mental Health Care was exceptional. The peer review process was rigorous and constructive, with reviewers providing valuable insights that helped enhance the quality of our work. The editorial team was highly supportive and responsive, making the submission process smooth and efficient. The journal's commitment to high standards and academic rigor makes it a respected platform for quality research. I am grateful for the opportunity to publish in such a reputable journal.
My experience publishing in International Journal of Clinical Case Reports and Reviews was exceptional. I Come forth to Provide a Testimonial Covering the Peer Review Process and the editorial office for the Professional and Impartial Evaluation of the Manuscript.
