Eimeria spp. The challenge was accomplished by inoculating broiler chicks with sporulated oocysts of Eimeria tenella, Eimeria acervulina, and Eimeria maxima via oral gavage. A total of 864, day-old male broiler chicks (Cobb) were randomly assigned to six treatments  in a 3 × 2 factorial arrangement, composed of three additives , with or without Eimeria challenge. Intestinal contents was collected at 28 days of age for microbiota analysis by sequencing 16s rRNA in V3 and V4 regions using the Illumina MiSeq platform. Taxonomy was assigned through the SILVA database version 132, using the QIIME 2 software version 2019.1. No treatment effects (p > 0.05) were observed in the microbial richness at the family level estimated by Chao1 and the biodiversity assessed by Simpson’s index, except for Shannon's index (p < 0.05). The intestinal microbiota was dominated by members of the order Clostridiales and Lactobacillales, followed by the families Ruminococcaceae, Bacteroidaceae, and Lactobacillaceae, regardless of treatment. When the controls were compared, in the challenged control group there was an increase in Erysipelotrichaceae, Lactobacillaceae, Bacteroidaceae, Streptococcaceae, and Peptostreptococcaceae, and a decrease in Ruminococcaceae. Similar results were found for a challenged group that received monensin, while the blend partially mitigated this variation. Therefore, the blend alleviated the impact of coccidiosis challenge on the microbiome of broilers compared to monensin.The objective of this study was to evaluate the effect of supplementation with 100ppm sodium monensin or 0.15% of a blend of functional oils (cashew nut oil + castor oil) on the intestinal microbiota of broilers challenged with three different  Eimeria spp. parasites, which are responsible for coccidiosis, a disease that seriously affects the global poultry industry, are one of the factors that cause changes in intestinal microbiota Coccidiosis causes damage to the intestinal epithelium, changes in the immune response of broiler chickens, reduces the digestion of nutrients, and consequently reduces performance indices [Clostridium perfringens. Although this bacterium is part of the normal microbiota of the cecum of broilers, when there is a significant increase in its proliferation in the small intestine it can cause necrotic enteritis [The more balanced the microbiome, the more resilient the intestine becomes when challenged ,2. Eimer indices –5. Cocci indices . For exanteritis ,4.E. coli, Lactobacillus spp., and Enterococcus spp. However, the continuous use of monensin can cause parasite resistance [Ionophores have been the main choice of drug for controlling coccidiosis. Monensin is an ionophore that forms fat-soluble complexes with sodium and potassium, causing increased permeability of the coccidial membrane, which leads to toxic effects and death due to depletion of cellular energy . This mesistance , which hsistance –13.Eimeria spp. [Phytogenic feed additives can have antimicrobial and anti-inflammatory properties, because they are composed of a complex mixture of volatile substances, such as terpene hydrocarbons, simple alcohols, and aldehydes, amongst other pharmacologically active compounds . Of the ria spp. .The objective of this study was to evaluate the effects of supplementation with either monensin or blend functional oils on the intestinal microbiota of broilers challenged with coccidiosis.The work described here was conducted under protocol number 29814 approved by the Ethics Committee on Animal Use of the Federal University of Rio Grande do Sul, following the legislation for the protection of animals used for scientific purposes . Birds were monitored twice a day, systematically observed by trained people who evaluated possible coccidiosis clinical signs, like: occurrence of mucus-like or bloody diarrhea; dehydration diagnosed by responses of wrinkled skin, dull eyes; decreased appetite detected by decrease in feed intake; occurrence of ruffled feathers; listlessness, detected by activity lack of the bird and; stunted growth, visually lower than the group average. In case of early evidence associated with possible death risk or specific signs of severe suffering or distress, the birds could have euthanized. But these specific situations (euthanasia) were not necessary in this study.2 pen equipped with two nipple drinkers and one tubular feeder. Food and water were provided ad libitum throughout the 28 days of the experimental period. The nutritional program consisted of three phases: pre-starter (1 to 7 d), starter (8 to 21 d), and grower (22 to 28 d), formulated to provide the nutritional requirements recommended by the Brazilian Tables of Poultry and Swine [A total of 864, day-old male broiler chicks (Cobb 500) were obtained from a commercial hatchery and housed in two identical experimental facilities with a controlled-temperature room, one for the challenged, and one for the unchallenged birds, thus avoiding cross-contamination. The rooms were composed of 48 pens with an initial density of 18 birds per pen. Each group was housed in a 1 mnd Swine .The experimental design was completely randomized in a 3 × 2 factorial arrangement, composed of feed additives  and sanitary challenge . Both feed additives, the oil blend  and monensin sodium , were included by replacing an inert ingredient (kaolin) in the basal diet for all phases.E. tenella (10 × 103 oocysts), E. acervulina (200 × 103 oocysts), and E. maxima (80 × 103 oocysts) were inoculated by oral gavage. The oocysts were acquired from the Laboratório de Biologia Molecular de Coccídias . To similarly stress all treatments, unchallenged broilers received 1 mL of saline per os.At 14 days of age, 1 mL of a saline solution containing sporulated oocysts of At 28 days of age, a homogenized sample of the small intestinal digesta from three broiler chickens per replicate (three replicates) was obtained, totaling 18 samples. A 10 cm portion of each segment: duodenum segment , jejunum , and ileum  was homogenized, pooled, and immediately stored at -20°C until further analysis.5′-CCTACGGGRSGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primers, with Illumina adapters . The amplification was performed in 35 cycles at an annealing temperature of 50°C. Sequencing was performed by Illumina MiSeq  using V2x300 kits, with a single-end 300 nt run.The small intestine samples were placed in a 1.5 mL sterile tube and sent to Neoprospecta Microbiome Technologies . All procedures were performed according to the method previously described . Sample Read quality was evaluated using FastQC software (version 0.11.5), and poor quality reads and adapters were removed . All of the following steps were implemented using QIIME 2 (version 2019.1) software . The reahttps://www.R-project.org/), and plyr (v. 1.8.4) [Relative abundance, alpha rarefaction, alpha , and beta diversity indices were determined using the program R, version 3.6 (. 1.8.4) , reshape. 1.8.4) , and phy. 1.8.4)  packages. 1.8.4)  and heat. 1.8.4)  packages. 1.8.4)  R packagA total of 728,462 quality trimmed sequences were produced with an average number of sequences per sample of 40,470.11 ± 7,580.40 . The reaRarefaction curves generated from ASVs  showed hThe Chao 1 index (minimum number of ASVs present in a sample) was based on the richness of families present in the sample. The Shannon index considered uniformity in family abundance, and the Simpson index was based on dominance of abundance .However, only the Shannon index showed significant differences between the six groups after infection (p <0.05). While the Shannon index increased in the monensin and challenged control groups, it did not change in the blend group .To analyze the inter-individual differences, the Bray-Curtis dissimilarity index (CB) was used. which showed that the treatments had a different microbial compositions . Despite the lack of homogeneity in the dispersion, the PCoA graph  based onA paired comparison of the microbial similarity between treatments as well as an analysis of common ASVs, shown in the Venn diagram, was conducted to investigate the microbial community. A total of three ASVs were common to all groups, the number of ASVs present in only one group ranged from 59 for the unchallenged blend to 88 for challenged monensin. Challenging with coccidiosis increased the number of ASVs present only in one group, going from 86 to 88 for Ch, and from 59 to 81 for the blend, monensin, and control treatments, respectively  for Un.The dominant families in the samples were Ruminococcaceae and Lachnospiraceae, followed by Lactobacillaceae, Bacteroidaceae, and Erysipelotrichaceae . A complAt the class level, Clostridia was the most abundant class upper to 75% of the sequences of the 18 samples, regardless of the additive. The challenged broilers showed lower levels of this class, except for those supplemented with blend, which showed difference between challenged and unchallenged broilers . At the An increase in Bacteroidia (~ 0.80% vs. ~ 2.05%) was observed for the blend and monensin challenged groups. There was an increase in the abundance of Erisypelotrichia and Mollicutes, regardless of the treatment, when broilers were challenged, with the exception of the treatment receiving the blend, in which the Mollicutes class was reduced. The class Melainabacteria was identified in low abundance (<0.1%) only in the challenged birds.Thirty families were identified, of which seven had a relative abundance > 1% . The RumEimeria spp. In the absence of parasites, there was no stimulation of genes involved in the inflammatory response, demonstrating the blend was an effective tool to specifically modulate the immune system of birds afflicted with coccidiosis [C. perfringens and S. aureus [The commercial blend of cashew nut liquid and castor oil has been shown to modulate the inflammatory response against cidiosis . Monensi. aureus . In geneIntestinal health is directly related to the profile of the microbiota that interacts with the host. The microbiota regulate absorption efficiency, antagonize the effects of pathogenic bacteria, improve intestinal integrity, and modulate immunity ,28. A coIn this study, the coccidiosis challenge increased the microbial biodiversity, contrary to what was reported by Zhou et al.  and BortIn humans, weight loss is related to an imbalance in the Firmicutes: Bacteroidetes ratio in the intestinal microbiota, with a decrease in the Firmicutes and an increase in the Bacteroidetes, while the opposite was observed in obese rats . The FirLactobacillus spp. was stimulated by the Eimeria spp. challenge which is in agreement with the results of Kley et al. [Lactobacillus have been associated with beneficial properties to the host, positively contributing to broiler weight gain, reducing injury scores, inhibiting cell invasion, and increasing mucosal integrity [Lactobacillus spp. facing the challenge. It has been demonstrated that many species of Lactobacilli act on the innate and acquired system stimulating immune cells to release pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin-12 (IL-12) [The proliferation of y et al. , M'sadeqy et al. , and Stay et al. . Severalntegrity –39. The  (IL-12) –44.Akkermansia can use mucus as a source of carbon and nitrogen, produce acetate and propionate, and produce better intestinal health, with an inverse relationship between their relative abundance and intestinal disorders [The Aerococcaceae family, another member of the phylum Firmicutes, was observed only in the group supplemented with the blend . This family is one of the bacteria that produce lactic acid ,46, reduisorders –54.Gharib-Naseri et al.  reportedClostridium butyricum and Clostridium perfringens. C. butyricum can produce short-chain fatty acids  and has been studied as a probiotic in broilers, improving intestinal barrier function and inhibiting pathogens [C. butyricum decreased the abundance of C. perfringens in the gut during the development of necrotic enteritis in broilers. C. perfringens is the most important pathogenic strain capable of producing more than 16 toxins with different modes of action [Eimeria infection causes damage to enterocytes releasing cellular proteins, and stimulating mucogenesis, which creates a favorable environment for the reproduction of C. perfringens [C. perfringens compared to the monensin and control groups [In this study it was possible to observe that the non-challenged birds had a greater relative abundance of the Clostridiaceae family, mainly for the monensin and blend groups. Within the Clostridiaceae family, there are different species, including athogens . Huang eathogens  observedf action  toxin isf action –64. Eimefringens ,64,65. Il groups .Lactobacillus and Enterococcus depletion, and when monensin is associated with other antimicrobials, it increases Escherichia coli populations [The use of antimicrobials, including monensin, can delay the maturation of the intestinal microbiota, consequently affecting the development of the bird’s intestinal immune system and negatively affecting the bird’s health ,67. Moneulations . Some stulations –68.Eimeria spp. challenge. The blend acted as a modulator, mainly for gram-positive bacteria, contributing to a better weight gain and feed conversion rate (P > 0.05) after 14 days of the challenge [Based on previous results ,26 and tThe blend of functional oils besides stimulating the beneficial microbiota, mitigated the impact of a coccidial challenge in broilers. This resulted in greater microbial stability when compared to treatment with the ionophore, monensin.S1 FigHorizontal axis: the amount of effective sequencing data (rarefaction depth); vertical axis: the observed number of variations of sequence amplification (ASVs).(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableThe experiment is composed of three feed additives, basal diet (control), sodium Monensin (Mone), or Blend (Blend) and sanitary challenge (CH) or unchallenged (UN) with coccidiosis.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


The mean fungal burden in the air decreased from 221 CFU/m3 to 9CFU/m3. The mean all-surface microbial burden was 79 CFU/100 cm2 after all autopsies, and it decreased to 2 CFU/100 cm2 after NTD. In conclusion, the peroxone-based NTD system was effective for decontamination of the air and surfaces in a dissecting room after autopsies of exhumed bodies.Autopsies of exhumed bodies pose a risk of infections with environmental bacteria or fungi, which may be life-threatening. Thus, it is important to use effective methods of disinfection in forensic pathology facilities. In this study, we investigated the effectiveness of no-touch automated disinfection (NTD) system after autopsies of exhumed bodies. Directly after 11 autopsies of exhumed bodies, we used an NTD system based on a peroxone vapor to disinfect the air and surfaces. We measured microbial burden in the air and on surfaces before and after NTD. The NTD system reduced the mean bacterial burden in the air from 171 colony forming units (CFU)/m Mycobacterium tuberculosis [Aspergillus flavus, often found in corpses, may cause fatal invasive aspergillosis [Eurotium repens, isolated from the surfaces of skin and bones of corpses [Penicillium oxalicum and Cladosporium colocasiae, isolated from corpses preserved in low percentages (4%) of formaldehyde [Forensic pathologists are at an increased risk of various infections, particularly those associated with hepatotropic viruses, coronaviruses, meningococci, or rculosis ,2,3,4,5.rculosis . For exagillosis ,7. A comgillosis . Necrobi corpses  and moldaldehyde . Fungi maldehyde . Pathogealdehyde . Thus, i3/UV); hydrogen peroxide and ozone (O3/H2O2), photocatalytic oxidation with the presence of titanium dioxide (TiO2) and Fenton system oxidation (H2O2/Fe2+). The common feature of these methods is the formation of peroxone, a highly biologically active compound [2O2, and peroxide radicals. In these reactions, the free radical mechanism is dominant, and the most important product is hydroxyl radical •HO having high redox potential  seem to be particularly promising. These methods include ozonolysis in the presence of UV light  systems are gaining increasing popularity in addition to standard surface disinfection ,17,18,19ium spp. . However®2 automated system. All identified microorganisms belong to environmental bacteria and fungi. 3, and it decreased to 3 (0–23) CFU/m3 after decontamination  CFU/m3 after decontamination  colony forming units (CFU)/mmination . The meamination .2 after all autopsies, and it decreased to 2 (0–18) CFU per 100 cm2  CFU per 100 cm 100 cm2 . Table 3This study showed that peroxone-based NTD system was effective in decontaminating the air and surfaces in a dissecting room after autopsies of exhumed bodies. Importantly, the NTD system effectively reduced or eradicated both bacterial and fungal contamination. The literature addressing safety issues in dissection rooms recommend the use of personal protective equipment, such as gloves, goggles or masks during autopsies ,3,4. CurNTD systems are used in so-called clean conditions, i.e., after initial cleaning and disinfection of surfaces. In our study, the level of microbiological contamination was not studied directly after initial cleaning with sodium hypochlorite. However, we found that the microbiological level in the air, where sodium hypochlorite was not used, decreased after the NTD system. The initial cleaning and disinfection followed by using NTD system should be considered as an integrated approach.3 [Aspergillus fumigatus was eradicated after disinfection. In our study, the microbial burden was not detectable after the use of NTD system in 8 of 11 autopsies, and in the remaining autopsies, the residual microbial contamination was low.Decontamination of dissecting rooms is important to reduce the risk of infections, particularly after forensic autopsies of exhumed bodies, which can cause acute life-threatening infectious diseases. In our study, we identified several potentially pathogenic strains of bacteria and fungi after the autopsies ; however3 . In our 3 . In thatAspergillus spp. and Penicillium spp. are often present [Aspergillus flavus is the etiological factor of sinusitis, keratitis, or skin lesions [Aspergillus flavus or Aspergillus fumigatus may cause asthma or allergic pneumonitis [Aspergillus spp. may lead to life-threatening conditions, such as fatal invasive aspergillosis [Aspergillus flavus and Aspergillus fumigatus were identified. This is in line with findings of Schwarz et al., who also reported the presence of Aspergillus fumigatus on decomposed bodies [Fungal contamination is a particular concern after autopsies of exhumed bodies. The growth of fungi is often visible on exhumed bodies, and the spores of different fungi, such as  present ,29. Fung present . Fungi a lesions , and Aspumonitis ,33,34,35gillosis ,7. In ouPenicillium and increased risk of wheeze, persistent cough, and higher asthma severity score [Cladosporium spp. and Penicillium spp., may pose a risk of severe disease [Cladosporium and Penicillium have been re-classified as risk group 3 pathogens, e.g., Cladophialophora bantiana, causing severe infections of central nervous system characterized by high mortality rate [Talaromyces (Penicillium) marneffei, causing severe deep infections. Despite the fact that these species are mostly common in Thailand, Cambodia, Taiwan, and India, the cases of infections with these pathogens have also been reported in other regions [Cladosporium spp. and Penicillium spp. on human corpses had been shown previously [Candida species, which are recognized as possibly allergenic [Similarly to reports by Schwarz et al.  and Łukaty score . Moreove disease ,38. Someity rate , and Tal regions . The preeviously ,42,43,44lergenic ,44.This study has some limitations. Firstly, it was limited to a single dissecting room, and our observations need to be confirmed in other facilities. We used sodium hypochlorite for pre-cleaning and the residual chlorine could affect the microbial load at the time of assessment. The level of microbiological contamination was not studied directly after this procedure. Moreover, we used only one device for NTD, and other systems available on the market would require similar investigations. Nevertheless, because there is limited data on the use of NTD systems in dissecting rooms, our study presents useful information for facilities that conduct autopsies, particularly forensic ones.In conclusion, NTD system seems promising for the decontamination of dissecting rooms after autopsies of exhumed bodies. Standards for the use of NTD systems in dissecting rooms are yet to appear, and until then the instructions delivered by manufacturers of specific devices should be followed. TM system.We carried out 11 autopsies of exhumed bodies. Deaths occurred in 2010 as a result of injury; exhumations and autopsies were performed in 2018. We used the NTD system directly after autopsies of exhumed bodies. Before NTD was applied, the dissecting room was washed with sodium hypochlorite, according to the manufacturer’s instructions. Sodium hypochlorite is active against bacteria, viruses, spores, fungi, and mycotoxins . The micTM NTD system  in a dissecting room of a volume of 78 m3. The system sprays a peroxone vapor (a combination of hydrogen peroxide and ozone) to decontaminate all surfaces. The duration of decontamination was 1 h each time. Decontamination cycle consisted of the following phases: hydrogen peroxide phase, ozone phase followed by peroxone forming, and one-hour contact phase during which peroxone decomposed. For safety reasons, the device detects residual hydrogen peroxide level after the decontamination procedure. We used the Airdecon 2003. In our study, we calculated the mean CFU/m3 values from three air samples. The device was placed 1.5 m from the floor, all doors and windows were closed, and 300 m3 of air was sampled thrice according to the PN—EN 13098 standard approved by the Polish Committee for Standardization [We used the MicroBio MB 1 PLUS air sampler  to measure the microbiological burden in the air. The MicroBio MB1 air sampler collects airborne micro-organisms on the surface of Petri dishes layered with the malt extract agar and the tryptic soy agar. After exposure, the dishes were removed and incubated under aerobic conditions, and the colony growths were counted. The count and the volume of air sampled were used to calculate the number of CFU per mdization .2, convex RODAC plates (Replicate Organism Detection and Counting) layered with tryptic soy agar with inactivators of inhibitory substances to measure contamination of surfaces . The plates were pressed (500 g/cm2) against the surface for 10 s, with no side movements, and were later incubated at 35 °C under aerobic conditions. The growth of microorganisms was measured in CFU per 100 cm2, and then expressed as a risk level according to the 1993 Draft European Standard CEN/TC 243/WG2 [2; moderate risk, 10–100; high risk, >100–1000, very high risk, >1000.We used 25 cm 243/WG2 ; this wa®2 , according to the manufacturer’s instructions. VITEK®2 is an automated mass spectrometry microbial identification system that uses Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) technology. Fungal species were identified by evaluation of their macroscopic and microscopic morphological features basing on the Atlas of Clinical Fungi [The identification of bacteria was carried out with an automatic detection system Vitekal Fungi .


Toll-like receptors (TLRs) are a pivotal component of the innate immune system that seem to have a role in the pathogenesis of psychosis. The purpose of this work was to compare the expression and functionality of 9 TLRs in three peripheral blood mononuclear cells (PBMCs)  between 33 drug-naïve first-episode psychosis (FEP) individuals and 26 healthy volunteers, at baseline and after 3-month of antipsychotic treatment. The expression of TLRs 1–9 were assessed by flow cytometry. For the assessment of the TLR functionality, cells collected in sodium heparin tubes were polyclonally stimulated for 18 h, with different agonists for human TLR1–9. The results of our study highlight the role that TLR5 and TLR8 might play in the pathophysiology of psychosis. We found a lower expression of these receptors in FEP individuals, regarding healthy volunteers at baseline and after 3-month of treatment on the three PBMCs subsets. Most TLRs showed a lower functionality  in patients than in healthy volunteers. These results, together with previous evidence, suggest that individuals with psychosis might show a pattern of TLR expression that differs from that of healthy volunteers, which could vary according to the intensity of immune/inflammatory response. The role of infection and immunity in psychosis etiology was investigated for more than a century . ExposurToll-like receptors (TLRs) are a pivotal component of the innate immune system that are expressed by various subsets of immune and non-immune cell types ,5, incluThe production of proinflammatory cytokines and free radicals of oxygen and nitrogen, as well as the sustained activation of microglia during the first stages of life, and especially following maternal infection and inflammation, would lead to a reduction in glutamatergic neurons in some brain areas ,14,15,16It appears that TLR level clearly influences the intensity of immune/inflammatory responses, indicating that decreased TLR expression protects against excessive cell stimulation via exogenous or endogenous ligands, and is a counterbalancing mechanism that limits the excessive development of inflammation ,20.Up to now, there are few publications that have studied TLR expression or functionality in psychosis, most of them on chronic schizophrenia individuals ,22,23,24The aims of the present study were (i) to investigate the expression and functionality of 9 TLRs in three different peripheral blood mononuclear cells (PBMCs), monocytes, B cells and T cells, in FEP individuals and healthy volunteers, at baseline and after 3-month of antipsychotic treatment in the patient group; and (ii) to examine both the patterns of TLR expression and functional changes after 3 months of antipsychotic treatment within the FEP individuals group.Our hypothesis is that we will find differences both in the expression pattern of TLRs and in their functionality between subjects with FEP and healthy volunteers. After 3-month of antipsychotic treatment, we will see changes in the expression and functionality of TLRs in patients. Considering that the level of expression of TLRs and the intensity of the immune/inflammatory response influence each other , the patp values > 0.1). Out of 33 patients, 19 (57.6%) were initially assigned to the aripiprazole group, whereas 14 (42.4%) were assigned to the risperidone group; this was a non-statistically significant random distribution .As shown in t = 3.71; p = 0.002), although no differences were found regarding BMI between healthy volunteers and FEP individuals at 3-month. We observed a general improvement in the total scores of clinical scales after 3-month of treatment  except in the SANS .Within-subject analysis showed that BMI increased significantly, following 3-month of antipsychotic treatment . However, healthy volunteers had a higher expression of TLR5 and TLR8 than the FEP individuals on the three PBMCs subsets—TLR5 on B cells , monocytes , and T cells ; TLR8 on B cells , monocytes , and T cells .Regarding TLR expression between healthy volunteers and patients at baseline, U = −4.0; p = 0.000) and T cells , as well as a higher expression of TLR8 on B cells , monocytes , and T cells  than FEP individuals, after 3-month of antipsychotic treatment.F = 12.77; p = 0.003) and we observed a trend toward a significant higher expression levels of TLR8 on monocytes , B cells , and T cells , as well as TLR9 on B cells , as shown in Within-subject analysis shows that after 3-month of antipsychotic treatment FEP individuals had statistically significant higher expression level of TLR6 on monocytes , TLR2 , TLR3 , TLR4 , TLR5 , TLR6 , TLR7 , TLR8 , and TLR9 . In addition, significantly higher levels of TNF-α were found in healthy volunteers following TLR2 stimulation on B cells . No differences were found in the intracellular production of IL-1β, neither in IL-6 between both groups at baseline .U = −2.92; p = 0.003), TLR2 stimulation on B cells , TLR3 stimulation on B cells , TLR4 stimulation on B cells , and TLR8 stimulation on B cells . U = −2.57; p = 0.010) and TLR4 stimulation on B cells  in healthy volunteers. U = −3.18; p = 0.001) in healthy volunteers and a significantly higher intracellular production of IL-6 in the FEP individuals group, after TLR5 stimulation on monocytes .After the FEP individuals were under antipsychotic treatment for 3-month, we found higher intracellular levels of the following cytokines in healthy volunteers as compared to patients a—signifip values > 0.1).Within-subject analysis did not find statistically significant differences in the intracellular cytokine production after 3-month of antipsychotic treatment  in patients than in healthy volunteers, mainly on the monocytes at baseline and on B cells after medication. Both cell types, but not T cells, are antigen-presenting cells in which the altered expression and function of TLRs could have impacted in the inflammatory response of FEP. Within-subject analysis showed a tendency of an up-regulation of the expression of some TLRs after 3-month of antipsychotic treatment.Considering our findings, we focused on the possible pathophysiological implications of impaired expression and functionality of TLR5 and TLR8. TLR5 is expressed in lung and intestinal epithelial cells, human endometrium, bladder, granulosa cells leukocytes, adipocytes, and some cancer cells, among others. Recent research studies described that the correct expression of TLR5 during neonatal period influences the long-term gut microbiota composition , and theTLR8, an endosomal sensor of RNA degradation products in human phagocytes, is involved in the recognition of viral and bacterial pathogens through the modulation of the suppressive activity of regulatory T cells . SeveralThus far, only a handful of studies evaluated the expression and functionality of TLRs in schizophrenic individuals. Chang et al.  describeSome of the differences between these studies with each other and with respect to our findings could be explained if we consider the methodological limitations described in the introduction section. In addition, we suggest that the immune response probably could not be defined categorically (elevated or decreased), but should be understood as a spectrum in which the expression and functionality of TLRs could vary, depending on various factors, such as the clinical situation (compensation vs. relapse), the presence of an infection or autoimmunity, the intensity of the inflammatory response, the time under antipsychotic treatment, etc. We speculated that decreased TLR expression and functionality might be a consequence of an excessive cell stimulation via exogenous or endogenous ligands, through a counterbalancing mechanism limiting the excessive development of inflammation ,20. WithThis hypothesis would help to understand why several studies found a different pattern of TLR expression, as their samples could be constituted by individuals in different stages of the disease and under different antipsychotic treatments, in which the immune response could thus vary, considering all clinical and immunological variables described above . TherefoThe fact that we observed an alteration in the innate immune response (monocytes) and in the adaptive immune response (B cells and T cells), both at the onset of the disease and after 3 months of antipsychotic treatment see , suggestOur findings, together with previous evidence, might be consistent with the neurodevelopmental model of schizophrenia, in which a prenatal infection or inflammation might produce a change in both brain areas and immune system, which increase the future risk to develop both psychosis disorders and immune disturbances, like autoimmunity ,41. It iThis study has several limitations that should be considered—first, stress level, which is known to influence inflammatory status, was not measured. Second, we did not consider the consumption of classical analgesic or anti-inflammatory drugs as possible confounding factors. However, it is important to remark that the subjects in this study were young, healthy individuals without chronic diseases or chronic treatments. Third, analysis was performed on peripheral blood. We assumed that these findings were an indicator of what occurred in the CNS and, therefore, we would expect to find a greater disturbance of TLR expression and intracellular cytokine production in the CNS.ClinicalTrials.gov ID: NCT02897167 (13/09/2016). “Study of the Activation of Proinflammatory Pathways of Toll-like Receptors in Schizophrenia Patients (P AFIP_TLR)”. https://clinicaltrials.gov/ct2/show/NCT02897167.The data for the present study were obtained from a large epidemiological cohort of patients who were treated in a longitudinal intervention program of FEP  conducted at the University Hospital Marqués de Valdecilla in Cantabria, Spain. The main procedures that were carried out in this program are described elsewhere . The stuFrom October 2016 to March 2019, 33 individuals meeting the following inclusion criteria—age between 15 and 50; residency in the catchment area; experiencing their first episode of psychosis; no prior treatment with antipsychotic medication (not even a single dose of antipsychotic medication before blood test), and DSM-IV criteria for brief psychotic disorder, schizophreniform disorder, schizophrenia, schizoaffective disorder, or psychotic disorder not otherwise specified, were selected as entry in the present investigation. Patients were excluded if they met any of the following criteria—DSM-IV criteria for drug dependence; DSM-IV criteria for mental retardation; history of neurological disorder or brain injury; history of inflammatory diseases or immunodeficiency, and recent (1 week) use of glucocorticoids or vaccination less than 3 months ago. The diagnoses were confirmed by an experienced psychiatrist, applying the Structured Clinical Interview for DSM-IV (SCID-I), 6 months after the baseline visit.In the same period of time, we also selected 26 healthy volunteers with similar characteristics to patients, according to the following variables—sex, age, body mass index (BMI), and cannabis and tobacco consumption. Healthy volunteers were excluded if they had—DSM-IV criteria for drug dependence; DSM-IV criteria for mental retardation; first-degree relatives with a diagnosis of psychosis; history of neurological disorder or brain injury; history of inflammatory diseases or immunodeficiency, and recent (1-week) use of glucocorticoids or vaccination less than 3-month ago.This was a prospective, randomized, flexible dose, open-label study. At baseline blood sampling, all patients were antipsychotic naïve. A computer generated a randomization list based on a simple randomization procedure to select at baseline the antipsychotic used between aripiprazole or risperidone. At the treating physician’s discretion, the dose and type of antipsychotic medication could be changed, based on clinical efficacy and the profile of side effects during the follow-up period. Concomitant medication, such as antimuscarinics, benzodiazepines, antidepressants, and mood stabilizers, was considered as possible confounding factors in the analyses.Fasting venous blood samples were collected at baseline and three months following the starting of treatment. In addition, height, weight, and BMI were measured at baseline and 3-month after medication.We compared the expression of 9 TLRs and their functionality  between healthy volunteers and patients at baseline and at 3-month. Likewise, we performed within-subject analyses to study the threshold change of TLR expression and function after 3 months of antipsychotic treatment.To evaluate the antipsychotic efficacy for reducing psychotic symptoms we compared the total scores of the following clinical scales at baseline and following 3-month of antipsychotic treatment—Scale for the Assessment of Positive Symptoms (SAPS), the Scale for the Assessment of Negative Symptoms (SANS), the Brief Psychiatric Rating Scale (BPRS), and the Clinical Global Impression (CGI).The cell-surface expression of TLR1, TLR2, TLR4, TLR5, and TLR6 and the intracellular expression of TLR3, TLR7, TLR8, and TLR9 were assessed on distinct PBMCs subpopulations  using flow cytometry, as previously described . Cells cFor this purpose, we decided to quantify the intracellular expression of three of the most important proinflammatory cytokines , after TLR stimulation. Cells collected in sodium heparin tubes were polyclonally stimulated for 18 h, with different agonists for human TLR1–9 , in the presence or absence of Brefeldine A  in polypropylene tubes. As a negative control, cells were incubated in identical medium without stimulus. After culture, cells were permeabilized with FACS permeabilizing solution (BD Biosciences) and intracellularly stained with PE-labeled mAb against cytokines  and analyzed by flow cytometry. Percentage of intracellular cytokine-producing B cells, T cells, and monocytes was analyzed using the CellQuest Pro software (BD Biosciences), as previously described .The Kolmogorov–Smirnov test was used to examine the normality of data. Demographics and clinical measures were analyzed using T-Student test, W-Wilcoxon rank-sum test, or Chi-Square test as necessary. T-Student test for dependent data and W-Wilcoxon signed-rank test were used to assess the change of BMI and clinical scales within the patient group after 3-month of treatment. Wilcoxon rank-sum test for independent data was used to compare the expression and functionality of TLRs between healthy volunteers and patients at baseline and after antipsychotic treatment. Repeated measures Analysis of Covariance (ANCOVA) with BMI as covariate was used in the within-subject analysis. BMI was used as covariate as statistically significant changes were found with regards to this variable during the follow-up period within the sample of FEP individuals. In addition, previous studies described the influence of BMI on cytokine levels .Median MFI expression of TLRs and median intracellular levels of cytokines and interquartile ranges were represented by Box Plots after performing a natural logarithmic transformation. Only the statistically significant differences are shown in the figures, because otherwise it would take many figures to show clearly all the data concerning the expression and functionality of the 9 TLRs. However, all the expression and functionality data of the 9 TLRs are available upon request.As many tests were conducted, we assumed that the probability of a Type I error  was increased. Keeping this in mind and considering our research was exploratory, we decided to use a more conservative statistical significance threshold of 0.01, instead of applying multiple-testing correction in order to avoid Type II error .STATA 16.0 was used for statistical analysis. Statistical tests were two-tailed with a 99% confidence interval.Drug-naïve patients with schizophrenia spectrum disorders exhibit a pattern of TLR expression that differs from that of healthy volunteers, and which could vary according to the intensity of immune/inflammatory response. We highlight the role that TLR5 and TLR8 might have in the pathophysiology of psychosis.


Chronic pelvic inflammatory disease (CPID) is a difficult-to-treat gynaecological disorder, which has complex etiologies, among married women. In recent years, moxibustion has gradually shown its clinical advantages and been more and more widely used In China. The protocol is try to synthesize and assess the effectiveness and safety of moxibustion for patients with CPID.Seven databases as following: PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure, WangFang Database, Chinese Scientific Journal Database, Chinese Biomedical Literatures Database will be searched from their inception to May 2020. No restrictions about language and status. Study selection, data collection, and quality assessment will be respectively conducted by 2 researchers. Based on the heterogeneity test results, the fixed-effects or random-effects model will be selected to synthesize data. The effective rate, Pelvic inflammatory mass diameter and Pelvic fluid depth will be the primary outcomes. Patient reported outcome scale, visual analog scale, C-reactive protein, transforming growth factor β1 =  transforming growth factor β, incidence of any adverse events will be the secondary outcomes. Revman 5.4 software will be implemented for data synthesis. Dichotomous data will be represented by risk ratio for efficacy and safety of CPID treated with moxibustion, while continuous data will be represented by mean difference with a 95% confidence interval.The results of this study will be published in a peer-reviewed journal. This study will provide a comprehensive review of the available evidence for the treatment of moxibustion with CPID.This study expects to provide high-quality, evidence-based recommendations on further treatment for clinical guidance of CPID.CRD42020158744 in PROSPERO 2020. It is a leading cause of infertility and ectopic pregnancy, and is related to an increased risk of ovarian tumor.Chronic pelvic inflammatory disease (CPID) and the regional hospitalization rate is about 63.3/100,000 . At the same time, 18% of PID patients may have chronic pelvic pain, and 10% ∼ 20% may have secondary infertility, which seriously affects the reproductive health and quality of life of women of childbearing age.,6 The incidence of pelvic infectious disease is 4.4% among sexually active women aged 18 to 44 years in the United States. In the UK, Chronic pelvic pain costs an estimated £158 million/year to the NHS. Studies have shown that the incidence of PID among women of childbearing age in developing countries is as high as 40%. The average annual medical expenditure per case is about $3,025 in the US and about £163 per patient in Hong Kong.At present, the global incidence of PID is about 2% ∼ 12%, It results from bacterial infection including chlamydia trachomatis, neisseria and genital mycoplasmas with many predisposing factors including instrumentation of the female genital tract such as uterine curettage, insertion of intrauterine contraceptive devices and hysterosalpingography and vaginal douching, or secondary to adjacent inflammation as appendicitis or some distant focus as pulmonary tuberculosis. Chlamydia trachomatis is the most common cause of the pelvic inflammatory disease (PID) in the United States.The etiological factors are complex, which include polymicrobial infection, pelvic musculoskeletal disorders, and psychoneurological causes. In the case of surgery, the patients need surgical indications, which are usually found to be resistant to antibiotics. Besides, surgery may cause iatrogenic uterine damage. As a result, many patients with CPID migrate from the above therapy and seek alternatives which are effective and safe.Western medicine in the treatment of CPID is given priority to antibiotic treatment against infection, and surgical treatment when necessary. However, as the course deferment, illness becomes increasingly complex, of which often by a variety of pathogenic bacteria mixed infection of the disease. Combination of broad-spectrum antibiotics is not targeted therapy, so the effect is not very ideal. What's more, using antibiotics in large doses over long periods leads to a double infection, as well as the resistance easily. It is explained that chronic pelvic inflammation is mostly caused by an invasion of pathogenic heat or dampness and blood circulation disorder leading to lower abdominal pain and other symptoms according to the TCM theory of differential diagnosis and treatment. The pathogeny of it includes qi stagnation and blood stasis staying in Chong Meridian, Ren Meridian, uterus, and uterine collateral, which are mostly caused by the evil invasion of heat-dampness and cold-dampness, liver stagnation and kidney-Yang deficiency. Moxibustion is a TCM therapy for some chronic and debilitating diseases, which igniting the heat of moxa to stimulate the acupuncture points. It stimulates the blood circulation through the warm, promotes the inflammation to subside, and has the effect of bidirectional regulation on the body function state. At present, it has been found that moxibustion can adjust the function of zangfu, promote metabolism, change the amount of blood composition, increase the phagocytosis of red blood cells, white blood cells, haemoglobin and white blood cells, enhance immunity and improve health.Pelvic inflammation is unrecorded in ancient Books of Traditional Chinese medicine, and its clinical characteristics lump it as “heat discharge into the blood chamber”, “abdominal pain”, “carrying the disease”, “postpartum fever”, “infertility”.Nowadays, a large number of clinical studies in moxibustion regulating CPID have been carried out. Nevertheless, no systematic reviews has reported the effectiveness of moxibustion on CPID. Thus, we conducted this protocol of moxibustion as an intervention for CPID patients in order to achieve a better therapeutic effect.2This study is aimed at summarizing the clinical evidence about the effectiveness and safety of moxibustion for CPID and provide doctors, patients, policy decision-makers with reliable recommendations.33.1 It is registered on the International Prospective Register of Systematic Reviews , which is stated the “randomization” phrase and without blinding restriction, will be included. The animal mechanism studies, case reports, or non-RCTs are excluded.3.2.2Patients diagnosed as CPID regardless of race, age, status or education, marital status, and economic status. Trials will be included which applying validated diagnosed criteria, for instance, WHO/NIH/OSHK guideline, Chinese guidelines for diagnosis, and treatment of CPID.3.2.3RTCs that involved any form of moxibustion  as the sole treatment or as a part of combination therapy with another intervention  will be included. While combined with any complementary therapy will be excluded, for example, Chinese herb decoction, acupuncture, and other complementary therapy.3.2.4There is no limit to the treatment of the control group, including no treatment, or placebo, orThe following treatment comparisons will be included in the analysis:(1)Moxibustion compared with no treatment;(2)moxibustion compared with placebo or sham moxibustion therapy;(3)moxibustion compared with other active therapy;(4)moxibustion plus other active therapy compared with the same active therapy.3.2.53.2.5.1The effective rate, pelvic inflammatory mass diameter and Pelvic fluid depth.3.2.5.2Secondary outcome measures will include:(1)Patient reported outcome scale(2)Visual analog scale.(3)C-reactive protein.(4)transforming growth factor β1 =  transforming growth factor β1.(5)incidence of any adverse events3.3The exclusion certain contain the following items:(1)Patients with acute medical conditions or pregnancy(2)The literature related to the same study, and duplicated publications(3)Unable to get literature for available data or full text through various means3.4Seven databases as following: PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure, WangFang Database, Chinese Scientific Journal Database, Chinese Biomedical Literatures Database will be searched from their inception to May 2020. To describe moxibustion for CPID, the initial search strategy Table  was condAlso, we will search for eligible trials, which are ongoing or unpublished, in the WHO International Clinical Trial Registration Platform and the China Clinical Trial Registration Platform to make a comprehensive literature search.3.53.5.1To better understand the inclusion and exclusion criteria of each systematic review, we will conduct a pre-test, which is to randomly select some literature for screening. As for the differences of literature screening results, we will exchange and discuss to make the future literature selection more uniform.3.5.2Each literature of title and abstract is scanned by 2 reviewers (QSH and JX) respectively to eliminate some ineligible articles. Then all relevant articles of full text are investigated. When the 2 reviewers cannot agree on the selection process through consultations, the third reviewer (XHZ) will ultimately make the decision. If there is an article with unclear information or missing data, HFH will attempt to contact the original author for the full text and data. The primary selection process is shown in a Preferred Reporting Items for Systematic Reviews and Meta-Analyses flow chart Fig. .3.5.3Two independent reviewers (QSH and HFH) shall design a standardized data extraction form, which will be performed by Microsoft Excel, independently extract data and cross-check. The extracted information includes descriptions of studies, characteristics of participants, interventions of both the observation group and control group, quality, randomization, allocation concealment and blinding methods, outcome measures, primary outcomes, adverse effects, duration of follow-up, type and source of financial support, and the Standards for Reporting Interventions in Controlled Trials of Moxibustion (STRICTM) checklist. In case of any discrepancies, we will resolve by discussion and reach a consensus, and if necessary, seek arbitration from a third reviewer (JX). For RCTs lacking important information or data, the reviewers will track back to the primary studies of them.3.6 the Consolidated Standards of Reporting Trials and Standards for Reporting Interventions in Clinical Trials of Moxibustion checklist. The risk of bias in included studies will be evaluated according to the seven aspects: randomly generated sequence number, allocation sequence concealment, blinding of participants and personnel, blinding of outcome assessors, incomplete outcome data, selective outcome reporting and other sources of bias. Each of them will be classified into low risk, high risk, and unclear risk based on information provided by the trials. Inconsistency will be consulted with the third review author (HLL). For the data which is missing or ambiguous, we will try to contact the author as possible.The assessment will be conducted by 2 reviewers (QSH and JX) with the risk-of-bias assessment method from Cochrane Reviewer's Handbook 5.0.24,3.7We will be conduct statistical analyses by RevMan 5.4 software. Weight Mean difference (WMD) with 95% CIs will be used to analyze continuous data. Other forms of data will be changed into WMD values. As for the results of dichotomous data, risk ratio (RR) and its 95% CI will be used.3.8Data from parallel-group studies will be collected for analysis. If multiple nonmoxibustion control groups are included, pooled analyses of the control groups against the intervention group will be used. We will gather individual data of each result index for evaluation.3.9If there are missing or incomplete data for the primary results, we will contact the corresponding authors for the missing data to get specific information by telephone or email. If we can’t obtain that missing information, we will exclude from the analysis.3.10 which called heterogeneity, is quantified by the value of I2. The magnitude of heterogeneity was categorized by the I2 statistic with I2 of 0% to 24% = no heterogeneity, I2 of 25% to 49% = moderate heterogeneity, I2 of 50% to 74% = substantial heterogeneity, and I2 of 75% to 100% = considerable heterogeneity. When the results are substantial heterogeneity or considerable heterogeneity, sensitivity analysis or subgroup analysis will be made to look for the possible causes.The difference among studies in the systematic review,3.11If more than 10 trials are included, the funnel plots will be used to assess reporting biases. If funnel plot asymmetry is detected, the reasons will be analyzed.3.12I2≥50%) or fixed-effects model (I2 < 50%). If the substantial heterogeneity cannot be identified or there are not enough RCTs, we will perform a narrative, the qualitative summary or subgroup analysis.We will use RevMan for all statistical analyses. There are 2 modes to choose from: the random-effects model .The grading of evidence quality will be conducted by 2 independent reviewers using the grading of recommendations, assessment, development and evaluation system instrument.4 What's more, PID is a risk marker for CHD that is independent of traditional CHD risk factors. Therefore, it is meaningful to resort to complementary approach to manage CPID.CPID is a major gynecological health problem affecting more than 1 million women yearly. It begins with cervicitis and progress leading to serious clinical consequences, which including endometritis, salpingitis, pelvic peritonitis, tubal infertility, ectopic pregnancy, pelvic adhesions, pelvic abscess and chronic pelvic pain.This review, which will be the first summary of existing RTCs of moxibustion treatment of CPID, can help medical workers choose effective and safe interventions. The full discussion section of the report is planned to include the following: main findings, advantages and limitations, comparison with other studies and opinions, interpretation of results and conclusion.All authors have read and approved the publication of the protocol.Conceptualization: Fanghui Hua, Jun Xiong.Data curation: Jie Xiang, XiaoHong Zhou, Shouqiang Huang.Formal analysis: Shouqiang Huang, Jie Xiang.Investigation: Jun Xiong, Honglian Li, XiaoHong Zhou.Methodology: Fanghui Hua, Shouqiang Huang, Jie Xiang.Software: XiaoHong Zhou, Honglian Li.Supervision: Jun Xiong, Honglian Li.Writing – original draft: Jun Xiong, Fanghui Hua, Shouqiang Huang, Jie Xiang.Writing – review & editing: XiaoHong Zhou, Honglian Li.


This study aimed to compare the effects of an acute Pilates program under hypoxic vs. normoxic conditions on the metabolic, cardiac, and vascular functions of the participants. Ten healthy female Pilates experts completed a 50-min tubing Pilates program under normoxic conditions  and under 3000 m (inspired oxygen fraction = 14.5%) hypobaric hypoxia conditions  after a 30-min exposure in the respective environments on different days. Blood pressure, branchial ankle pulse wave velocity, and flow-mediated dilation (FMD) in the branchial artery were measured before and after the exercise. Metabolic parameters and cardiac function were assessed every minute during the exercise. Both trials showed a significant increase in FMD; however, the increase in FMD was significantly higher after the H trial than that after the N trial. Furthermore, FMD before exercise was significantly higher in the H trial than in the N trial. In terms of metabolic parameters, minute ventilation, carbon dioxide excretion, respiratory exchange ratio, and carbohydrate oxidation were significantly higher but fat oxidation was lower during the H trial than during the N trial. In terms of cardiac function, heart rate was significantly increased during the H trial than during the N trial. Our results suggested that, compared to that under normoxic conditions, Pilates exercise under hypoxic conditions led to greater metabolic and cardiac responses and also elicited an additive effect on vascular endothelial function. Pilates is a series of around 500 exercises inspired by calisthenics, yoga, and ballet. Pilates is a system of exercises that emphasizes control, breathing, flow, accuracy, centering, and concentration, by which the person perceives the body and activates the muscles . In partHowever, Pilates programs have been proposed to have low positive impact on cardiopulmonary and vascular functions due to the low exercise intensity ,7. ThereNowadays, hypoxic conditions are utilized to achieve a greater metabolic effect while lowering the mechanical loads during physical exercise ,15. The Hypoxic conditions elicit a reduction in arterial stiffness and increase in the blood flow within the skeletal muscle vascular beds ,18,19. IAs mentioned earlier, owing to its lower intensity, Pilates program is not effective in enhancing cardiovascular function. Therefore, it is crucial to examine whether a combination of Pilates and hypoxic condition exerts additive effects on cardiac and vascular functions. The combination of Pilates program and hypoxic conditions might be an effective approach to improve musculoskeletal function and cardiovascular function while reducing the risk of injury by reducing the exercise load and mechanical stress. Although the exercise type is different, many previous studies have reported that exercise intervention under hypoxic conditions led to a greater improvement in the body composition, metabolic function, and cardiovascular function with lower exercise intensity than that induced by exercise intervention under normoxic conditions ,24,26,27However, the present study presents a new insight into combination of Pilates program and hypoxic conditions by examining the differences in the metabolic, cardiac, and vascular functions responses under hypoxic and normoxic conditions in Pilates participants who can perform Pilates programs with accurate movements. It is necessary to examine these effects, before employing this combined approach for sedentary/overweight/obese people.Therefore, we conducted a randomized crossover trial to compare the effects of an acute Pilates program under hypoxic conditions vs. normoxic conditions on metabolic, cardiac, and vascular function in experienced Pilates participants.The study subjects included 10 healthy women who were experienced in Pilates , were non-smokers, and had no history of musculoskeletal, cardiovascular, or pulmonary disease. The consolidated standards of reporting trial (CONSORT) flow diagram is shown in iO2 = 20.9%) prior to the main trial for adaptation to the Pilates program. On the second and third occasions, the subjects underwent a randomized crossover experimental trial under either hypoxic conditions  or normoxic conditions . All subjects had a washout period of at least 7 days between each trial. The order of the conditions for each experimental trial was randomized, and each subject underwent the protocol at the same time at each visit. However, the subjects did not undergo blinded experiments under environmental conditions.The study design is shown in During all trials, all subjects were exposed to each environmental condition for 30 min, and then, a tubing Pilates program was performed for 50 min under each environmental condition. The Pilates program using tubing was composed of 25 types of motion, each performed for 2 min. The Pilates motion was configured as follows: roll up and down, biceps, arm circles, teaser, rolling like a ball, spine twist and arm extension, tubing mermaid, cobra, swimming, double kicks and arm circles, swan, cat, thigh stretching, hug a tree, squat, row, saw, hip pull, the hundred, lats pull three way, leg arc, scissor, helicopter, right side leg pull, and left side leg pull .2, carbon dioxide excretion: VCO2, respiratory exchange ratio: RER, carbohydrate oxidation: CHO, fatty acid oxidation: FAO, and energy expenditure: EE) and cardiac function  were measured every minute during the Pilates program. The sum of the values measured for 50 min was used as the result . Blood pressure (BP), branchial ankle pulse wave velocity (baPWV), and flow-mediated dilation (FMD) in the branchial artery were measured as indicators of vascular function before and after the Pilates program. Metabolic function  × 7 m (length) × 3 m (high) chamber at a temperature of 23 ± 1 °C and humidity of 50% ± 5% regulated by an environmental control chamber .Height, weight, body mass index, fat-free mass, and percent body fat were analyzed as the body composition parameters using bioelectrical impedance analysis equipment . All subjects fasted overnight prior to the body composition measurements. The subjects wore lightweight clothing and were asked to remove any metal items.2, VCO2, RER, CHO, FAO, and EE) were measured every minute. VE, VO2, VCO2, and RER were measured using the K5 auto metabolism analyzer  and a breathing valve in the form of a facemask during the 50-min Pilates program. The measured rates of whole body VO2 and VCO2 were used to estimate the rates of EE  and the disappearance rates (g/min) via CHO and FAO substrates. The following formulae were used to estimate CHO, FAT, and EE during 50-min Pilates program: CHO = 4.210 × VCO2 − 2.962 × VO2, FAT = 1.695 × VO2 − 1.701 × VCO2, EE = 4.07 × CHO + 9.75 × FAT [All metabolic function parameters .The cardiac function parameters, HR, SV, CO, EDV, ESV, and EF, were assessed noninvasively every minute using a thoracic bioelectrical impedance device  during the 50-min Pilates program. The sum of the values measured during the 50-min program was used as the result .BP, baPWV, and FMD in the branchial artery were measured as indicators of vascular function before and after the Pilates program. BP was measured twice using an autonomic BP monitor  before and after the Pilates program, and the average value was used as the result. Resting BP was measured after 30 min of exposure to each environmental condition, and post-exercise BP was measured immediately following the Pilates program for each environmental condition.Pulse wave velocity is the best available parameter for assessing arterial stiffness. Therefore, baPWV was measured using an automatic oscillometric device  before and after the Pilates program. Resting baPWV was measured after 30 min of exposure to each environmental condition, and post-exercise baPWV was measured within 10 min after completion of the Pilates program for each environmental condition.FMD refers to dilation (widening) of an artery when blood flow increases in that artery. The primary cause of FMD is the release of NO by endothelial cells. Therefore, FMD of the branchial artery was measured to evaluate the vascular endothelial function using noninvasive Doppler ultrasound  before and after the Pilates program. The resting FMD was measured after 30 min of exposure to each environmental condition. After fixing the ultrasound equipment to the brachial artery region 3–5 cm above the elbow, the diameter of the medial muscular artery was measured by Doppler. After the measurement, blood was removed for 5 min by increasing 50 mmHg based on resting BP. After 5 min, deflation was automatically recorded for next 2 min to evaluate the diameter and blood flow rate, and the calculated values of FMD (FMD = (reactive hyperemia diameter - baseline diameter) × 100%) were used. Post-exercise FMD was measured in the same way within 20 min after completion of the Pilates program for each environmental condition.t-test was used to compare the metabolic function and cardiac function during the N trial vs. those during the H trial. Second, two-way analysis of variance (ANOVA) with repeated measures was used to assess the presence of interactions  in vascular function before and after the Pilates program under each environmental condition. When ANOVA revealed a significant interaction, the Bonferroni test was performed as a post-hoc analysis to identify the differences. We used Cohen’s d where the term effect size reflects the value of a statistic calculated from a sample of data and standardized mean differences. A significance level of p < 0.05 was used to determine statistical difference for the mean of effect size and the confidence interval (CI) was reflected at a confidence level of 95%.All statistical analyses were conducted using the SPSS software version 25.0 for Windows . Data are presented as means ± standard deviations. The assumption of normality and homoscedasticity was verified using the Shapiro-Wilks W-test prior to the parametric tests. First, paired 2, RER, CHO, and FAO during the 50-min Pilates program between the two trials. The H trial showed a greater increase in VE , VCO2 , RER , and CHO  than the N trial. The H trial showed a greater decrease in FAO  than the N trial. However, there was no significant difference in the values of VO2 and EE between the H trial and N trial.As shown in d, 0.9; 95% CI, 0.0, 1.7 bpm; p = 0.011) than the N trial. The values of other cardiac function parameters  showed no significant differences between the H trial and N trial.p < 0.01, η2 = 0.692). Post-hoc analyses found significant improvement in the values of FMD  during the Pilates program in both trials. In addition, the H trial showed a greater increase in FMD after the Pilates program than the N trial .As shown in 2 perfusion [2max at high altitudes [2 consumed  when glucose is fully oxidized compared to complete oxidation of fatty acids [Hypoxic conditions reduce arterial oxygen saturation, which is more affected during exercise due to increased pulmonary blood flow, which limits gas exchange at the alveoli . Exerciserfusion . Howeverltitudes . The absltitudes ,33. Replty acids . This shty acids ,32. Therty acids .2, RER, CHO, and HR and decreased FAO during Pilates program compared to that under normoxic conditions. However, no significant difference between the values of VO2, SV, CO, EDV, ESV, and EF were observed during the Pilates program performed under hypoxic conditions and normoxic conditions. Moon et al. [iO2 = 16.5%, 14,5%, 12.8%, and 11.2%) vs. normoxic conditions (FiO2 = 20.9%) on metabolic parameters and cardiac function during constant load  submaximal bicycle exercise. They reported that acute exercise under hypoxic conditions (FiO2 = 14.5% and below) might be associated with decreases in EDV and ESV and increases in blood lactate level, VE, HR, EF, and CO during submaximal exercise. These changes are an acute compensation response to reduced aerobic exercise capacity due to decreased oxygen delivery and utilization capacity under hypoxic conditions.We examined the effect of an acute 50-min Pilates program under hypoxic conditions on metabolic and cardiac functions of Pilates participants. We observed that the acute Pilates program under hypoxic conditions led to greater increase in VE, VCOn et al.  evaluate2 between hypoxic and normoxic conditions during submaximal exercise; this is likely because the exercise intensity was fixed at a constant level across simulated environment conditions, resulting in the expenditure of same amount of energy. In addition, Hill et al. [2 during submaximal exercise between both environmental conditions is due to an increase in VE, HR, and CO because of acute hypoxic conditions. Furthermore, exercise under hypoxic conditions results in a decrease in the partial pressure of oxygen in the arterial blood, which leads to metabolic acidosis due to increase in ATP synthesis by the anaerobic metabolic process, increase in hydrogen ion, and decrease in pH [3− and increased blood lactate level, which ultimately decreased muscular performance. Consequently, metabolic changes in ATP synthesis led to a shift in fuel selection, resulting in increased carbohydrate oxidation. Indeed, Peronnet et al. [Corroborating the results of Moon et al. , we obsel et al.  and Mazzl et al.  reportedse in pH . Ramos-Cse in pH  investigt et al.  demonstrt et al. . Mazzeo t et al.  stated tt et al. ,39. 2 = 12.8% and FiO2 = 11.2%) than under normoxic conditions. They showed that these results were probably because of the activation of the sympathetic nervous system and alteration in the catecholamine level of the blood, which was triggered by increased levels of calcium ions in cardiac muscle cells during exercise under severe hypoxic conditions [2 = 16.5%) and those under normoxic conditions; these results were consistent with our findings. However, unlike previous studies, our study did not show an increase in CO even though HR increased to a greater degree during constant load exercise under hypoxic conditions than under normoxic conditions [However, our results did not show any differences in SV, EDV, ESV, and CO during the 50-min Pilates program between the two trials. With respect to the cardiac function, EDV and ESV are major determinants of SV, and an increase in EDV and decrease in ESV result in an increase in ventricular contractility and SV . Moon etnditions . Howevernditions . Therefonditions  showed tExercise induces augmented blood flow and subsequently increases vasodilation and upregulates endothelial NO synthase ,44. The 2 = 12%) vs. normoxic conditions (FiO2 = 21%) on FMD. They reported that the %FMD and normalized FMD were significantly higher following exercise under hypoxic conditions  than under normoxic conditions. These findings suggested that exercise with light intensity under hypoxic conditions had a significant impact on endothelial-mediated vasodilation. The effects of acute exercise under hypoxic conditions on blood pressure and arterial stiffness need to be further validated in future studies while considering subject characteristics , exercise type, and exercise intensity.The present study demonstrated improved branchial artery vasodilation and higher FMD following the Pilates program under hypoxic conditions compared to the Pilates program under normoxic conditions; however, there was no significant difference in BP and baPWV. These results indicated that an acute Pilates program, with light exercise intensity, under hypoxic conditions enhanced endothelium-dependent vasodilation but did not reduce the arterial stiffness in Pilates participants with clinically normal blood pressure and baPWV. Katayama et al.  investigIn summary, an acute Pilates program under hypoxic conditions leads to a greater metabolic and cardiac response and also elicits an additive effect on vascular endothelial function in the participants compared to the Pilates program under normoxic conditions.There are some limitations of the current study. Although our study is the first to combine a Pilates program and hypoxic conditions within a randomized crossover design, the small sample sizes limit our ability to efficiently compare the effects of an acute Pilates program under hypoxic conditions vs. normoxic conditions on metabolic, cardiac, and vascular functions in experienced Pilates participants. Thus, a higher number of subjects may be needed in future studies to accurately assess the efficacy of this combined technique in clinical practice. In addition, the subjects’ dietary habits and physical activities were not regulated.The present study is the first to combine a Pilates program and hypoxic conditions. We performed a randomized crossover trial to compare the effects of a Pilates program under hypoxic vs. normoxic conditions on metabolic, cardiac, and vascular functions in Pilates professionals. Consequently, our study confirmed that a Pilates program under a moderately hypoxic condition led to a greater metabolic response by relative higher exercise intensity . In addition, a Pilates program under hypoxic conditions led to an additive effect on vascular endothelial function compared to that under normoxic conditions in experienced Pilates participants.


Episodic ataxias comprise a group of inherited disorders, which have a common hallmark—transient attacks of ataxia. The genetic background is heterogeneous and the causative genes are not always identified. Furthermore, the clinical presentation, including intraictal and interictal symptoms, as well as the retention and progression of neurological deficits, is heterogeneous. Spells of ataxia can be accompanied by other symptoms—mostly from the central nervous system. However, in some of episodic ataxias involvement of peripheral nervous system is a part of typical clinical picture. This review intends to provide an insight into involvement of peripheral nervous system in episodic ataxias. Episodic ataxias  are a group of inherited disorders with a common denominator in the form of transient attacks of incoordination. However, the duration and triggers of these attacks vary between different types of EAs. Moreover, the neurological symptoms present during attacks and/or between them are different. In some patients, the resolution of the neurological deficits is incomplete, and these deficits increase with the duration of the disease. This heterogeneity can be observed even within families with the same mutation and factors  that modify the clinical presentation remain largely unknown. In some of the EAs symptoms from the peripheral nervous system (PNS) are part of the clinics.v1.1 and calcium Cav2.1 voltage-gated channel, respectively. These two EAs remain relatively best characterized on the molecular level when compared to the rest of EAs. However, even in their case, the genotype–phenotype correlation is unknown. So far, all described EAs have an autosomal dominant pattern of inheritance.The catalogue of EAs seems to be incomplete. Causative genes were identified in some of them, but in some EAs we know only the approximate gene location. The most common types are EA1 and EA2. Their causative genes encode the alpha subunits of potassium KThis review aims to present an overview of PNS involvement in EAs and the underlying pathophysiology. A brief summary presents The causative gene for EA1  is KCNA1 on chromosome 12p13.32. This ataxia is also known as episodic ataxia with myokymia, paroxysmal ataxia with neuromyotonia, and hereditary myokymia with periodic ataxia. Its clinical presentation varies between and even within families. Its onset is typically in childhood or early adolescence with brief attacks lasting seconds to minutes of different frequency—from about a dozen per day up to less than once a month. These attacks can be provoked by exertion, abrupt postural change, stress, emotions, fatigue, vestibular stimulation, caffeine, alcohol, ischemia, temperature changes, food rich in salt, bitter oranges, or chocolate. Attacks manifest in generalized ataxia with loss of balance, incoordination of hands, dysarthria, dizziness, blurry vision/diplopia, nausea, vomiting, diaphoresis, dyspnea, muscle weakness, twitching, stiffening, neuromyotonia, myokymia in the limbs, facial muscles , or hands . The phenotypic spectrum of EA1 is very broad with a substantial both inter- and intrafamilial variability without a defined genotype-phenotype correlation. In some patients, seizures of various type  were reported. Symptoms from the PNS, i.e., myokymia and neuromyotonia can be also present between ataxic spells. Some patients present only peripheral symptoms without ataxia such as neuromyotonia alone or muscle cramps .v1.1 channels. These are Shaker-related delayed-rectifier channels present both in the CNS and PNS, where the channel is especially enriched in basket cells in the cerebellum and in the juxtaparanodal regions as well as at branch points of myelinated motor axons, respectively .Administration of acetazolamide is effective in prophylaxis of EA5 attacks ,3,69.SLC1A3) gene on chromosome 5p13.2. The gene product is excitatory amino acid transporter 1 (EAAT1) protein—a glutamate transporter expressed throughout the CNS.EA6  is caused by mutations in solute carrier family 1 member 3  but also in vertigo, seizures, hemiplegia, hemianopia, and migraine.There are no peripheral symptoms .Brain MRI may show mild cerebellar atrophy and during attacks a hyperintense signal in FLAIR sequence may be visible. Routine laboratory test with electrolytes and CK levels within the normal range. NCS and EMG is normal.Treatment with acetazolamide is effective ,3.The causative gene for EA7  is unidentified, so far. The associated locus is on chromosome 19q13.Onset is the first–second decade of life. Attacks last hours to days and may be provoked by exertion or excitement. They present with ataxia, dysarthria, weakness, and vertigo in some patients. Symptoms from the PNS were not reported, so far ,70.Effective pharmacotherapy remains unknown ,3.UBR4) encoding E3 ubiquitin-protein ligase UBR4. UBR4 is known to interact with calmodulin, a Ca2+ regulating protein in the cytoplasm, and thus may be involved in the regulation of neuronal excitability [The causative gene for EA8  is mapped to chromosome 1p36.13-p34.3 and the suspected gene is ubiquitin protein ligase E3 component N-recognin 4  is caused by heterozygous mutation in the SCN2A gene on chromosome 2q23. Furthermore, the allelic variants comprise the less severe phenotype of benign familial infantile seizure type 3 (BFIS3) and the more severe phenotype epileptic encephalopathy, early infantile, 11 (EIEE11). Despite wide range of clinical symptoms, the PNS involvement was not reported, so far.FGF14 gene. No symptoms from the periphery were reported [In the report published in Annals of Clinical and Translational Neurology, Piarroux et al. described four patients with atactic attacks lasting minutes to days, with a childhood onset. This phenotype, the authors proposed to name EA9. The identified mutation was in reported .In addition, there are a number of genetic disorders that can present with some EA-like phenotypes. Many of their causative genes encode proteins important for the ion transport  ,72. TheiIn summary, the involvement of PNS is typical part of EA1—in the form of myokymia and neuromyotonia. Also, EA2 patients can present symptoms related to PNS involvement—as a consequence of disturbed NMJ transmission. As for the other rare types of EA—myokymia was also reported in EA3 and EA4 interictally and in EA8.