The incidence of pertussis shows an increasing trend in recent years, but some clinicians often lack sufficient understanding of the clinical characteristics and risk factors for severe pertussis, and more effective measures should be taken to reduce the incidence and mortality of pertussis in young infantsA retrospective study was conducted, and 184 infants and children with pertussis who had been hospitalized in the Department of Pediatrics of Beijing Ditan Hospital affiliated with Capital Medical University from January 2016 to December 2017 were included. Clinical data of the patients were collected and the clinical characteristics were statistically analyzedP<0.05)Among the 184 patients, 41.85% were infants < 3 months of age, and 65.22% of the total patients were not vaccinated against pertussis. There were 22 critically ill children, among whom 4 died, and compared with mild cases, they had a higher proportion of children younger than 3 months of age and infants not vaccinated against pertussis ; a higher proportion of children with severe pneumonia (100% vs. 0%); higher leukocyte count; and a higher proportion of children with severe hyperleukocytosis  (1. Infants aged <3 months not vaccinated for pertussis appear more likely to become infected and have more severe disease. 2. Severe pneumonia and hyperleukocytosis are the main mechanisms underlying severe pertussis. With the development of modern medicine, the incidence of many infectious diseases has significantly decreased, along with their threat to human health. However, the incidence of pertussis (whooping cough) shows an increasing trend. This phenomenon is known as “pertussis resurgence”. Severe pertussis can cause sudden infant death, which is not completely avoidable , 2. CurrIn the last few years, the number of pediatric patients with pertussis treated by our hospital has increased. This has included many severe cases and deaths. In this study, we employed a retrospective study design to analyze the clinical data of pediatric patients with pertussis treated by the department of pediatrics at Ditan Hospital in the 2 years of 2016 and 2017 in order to examine the clinical characteristics of pertussis and provide a clinical basis for the prevention and treatment of severe pertussis.This was a retrospective study which had been approved by the ethics committee of Beijing Ditan Hospital, Capital Medical University . 184 pediatric patients with pertussis, aged 0-6 years, who were treated at the Department of Pediatrics at Ditan Hospital from January 2016 to December 2017, were included as study subjects. All these children met the diagnostic criteria for pertussis (see below).Bordetella pertussis was cultured in the laboratory, Bordetella pertussis was detected by polymerase chain reaction (PCR), or paired serological test was positive (immunoglobulin G (IgG) titer of single enzyme linked immunosorbent assay (ELISA) test was significantly increased > 80–100 U/ml). In this study, the pathogenic diagnosis was performed using a Bordetella pertussis nucleic acid detection kit, and deep sputum samples of the infants were collected for testing.1) 0–3 months of age: no fever or low fever, cough with increasing frequency and severity, plus one of the following: cough with the characteristic whoop, apnea, vomiting after cough, cyanosis, convulsions, pneumonia, and close contact with patient with long-term fever-free cough ; or only paroxysmal apnea, cyanosis, and convulsions without a cough. 2) 4 months to 9 years old: no fever or low fever, paroxysmal cough ≥ 7 days, non-purulent rhinitis, and one of the following: cough with the characteristic whoop, vomiting after coughing, apnea, convulsions, pneumonia, exacerbated symptoms at night, and close contact with patient with a long-term fever-free cough. The patient should also meet at least one of the following laboratory diagnostic criteria: Infants with pertussis who also had hypoxemia (oxygen saturation<92%), recurrent apnea, cardiovascular dysfunction, or pertussis encephalopathy were considered to have severe pertussis. The diagnostic criteria for cardiovascular dysfunction was as follows: increased heart rate ; increased breathing rate; cardiomegaly (by type-B ultrasonic or X ray checks); irritable, feeding difficulties, oliguresis, edema, idrosis, and breath with difficulty (satisfying at least two items). Children satisfying the above four conditions accompanied with at least one of the following conditions, or the above two conditions accompanied with at least two of the following conditions: hepatomegaly, pulmonary edema, and gallop rhythm. The diagnostic criteria for pertussis encephalopathy: infants with pertussis had hyperspasmia, and examination of cerebrospinal fluid, head MRI, and electrolytic examination of blood serum excluded central nervous system infection and other diseases that could lead to hyperspasmia.Pertussis was treated with intravenous azithromycin at a dose of 10 mg/kg body weight, once a day for 3 days. After a 4-day interval, a second treatment course was administered if spasmodic cough was still severe with the same dosage and treatment course. Azithromycin was administrated orally to neonates less than 28 days old, with the same dosage and course of treatment as above. If the infants were found to have other bacterial infections, other antibiotics were added as needed. If the infants had respiratory failure, myocardial failure, pertussis encephalopathy, or other organ damage, appropriate symptomatic supportive treatments were administrated.The patients’ age, sex, and vaccination status were noted, and blood and sputum samples were collected under sterile condition. When the sputum was too viscous for the infants to cough out, aerosol inhalation therapy with sterile saline, patting back and suctioning sputum from the trachea was performed to promote sputum discharge and to cough out, and to keep respiratory tract unobstructed. Sputum samples were collected when the above therapeutic measures were performed. The infant was fixed by parents in dorsal position with its head back, and its nasal fossae was washed twice with sterile saline. The back end of suction sputum tube was connected with vaccum extractor, and the front end was inserted into the nasal cavity of the patient, and when reaching the trachea, 0.5-1ml of sputum was extracted. The sputum sample was collected in sterile specimen-box before it was sent to the laboratory within 30 min, and the sample was used for Bordetella pertussis testing and bacterial culture, and the results might guide antibiotic treatment. Routine blood test (the test detected the count and percentage of each type of blood cell), alanine aminotransferase (ALT) and creatine kinase isozyme (CK-MB) were tested. When clinically indicated, arterial blood gas analysis was performed. Chest computed tomography (CT) and X-rays were used to evaluate the pulmonary infection status of the infants, and the complications  were evaluated using clinical manifestations and laboratory tests. Antibiotic use, length of hospital stay, and clinical data of routine blood test, C-reactive protein, procalcitonin, and sputum bacterial culture results were collected.SPSS 22.0 software was used for data analysis. Quantitative data such as age, leukocyte count, platelet count, and length of hospitalization were expressed as the mean ± SD . For comparison between moderate and severe cases, the t-test was used for normally distributed data, while the rank sum test was used for non-normally distributed data. Qualitative data such as pulmonary consolidation, fever, non-vaccination with pertussis vaccine, and C-reactive protein elevation were expressed as rates. The chi-square test was used for comparison between the two groups.mild cases, and 22 (11.96%) were severe cases who were complicated with at least one of the following: hypoxemia, heart failure, recurrent apnea, or pertussis encephalopathy were < 3 months of age; 120 (65.22%) were not vaccinated against pertussis. 23(12.50%) were premature babies at birth, 21(11.41%) were with a low birth weight(<2500g), and 10(5.43%) were macrosomia (birth weight>4000g). 5(2.72%) had congenital heart disease, and no other co-morbidities were found. 162(88.04%) were 9/L), and the increase in white blood cells was mainly made up of lymphocytes. Over the course of the disease, 28.8% of the patients had fever, and 13.59% of them showed increased neutrophil percentage. C-reactive protein and procalcitonin increased in 16.85% and 5.98% of the patients, respectively, suggesting the presence of other bacterial infections. In addition, 12.5% had positive sputum bacterial culture. Patients using 3 or more antibiotics and special-grade antibiotics (carbapenems and glycopeptides) accounted for 14.13% and 10.33% respectively, 14 cases (7.61%) required ventilator-assisted breathing, and 8(4.35%) required oxygen inhalation with nasal catheter or head cover . 4 patients died (2.17%), which would be described in detail below. The average hospital stay was 15.68 ± 8.11 days , and 2 and more types of bacteria were cultured in the specimens of 3 severe cases.Of the 184 children with pertussis, 23 had positive sputum bacterial culture (12.50%), 15 in the mild group and 8 in the severe group. The pathogens found in the mild group were common community infectious pathogens, such as 9/L, with the highest count of 106.68 × 109/L. All 4 cases were complicated with severe pneumonia, the percentage of neutrophils was high (44.00–81.54%), C-reactive protein was higher than normal in 3 cases , and procalcitonin was high in 2 cases . All had fever during the course of the disease. One case had positive sputum bacterial culture, which was Enterobacter aerogenes. The drug sensitivity test of the Bordetella pertussis isolated from a 56-day-old infant showed resistance to macrolides. The causes of death of 2 were respiratory and heart failure, 1 was heart failure, and 1 was acute respiratory distress syndrome (ARDS). , and the proportion of unvaccinated infants was significantly higher in the severe cases than in the mild cases, which was consistent with the results of previous studies –9. TherePneumonia is the most common complication of pertussis. In this study, more than 80% of hospitalized infants with pertussis also had pneumonia. Some infants had fever, with increased neutrophils, C-reactive protein, and procalcitonin levels, suggesting the presence of other bacterial infections. All severe cases had severe pneumonia, and all of them required some form of respiratory support. In this way, pneumonia is not only the most common complication of pertussis but also the leading cause of severe disease and death from pertussis –17.9 leukocytes/L) is an independent risk factor for malignant pertussis (life-threatening severe pertussis) [9/L in pertussis patients, and no measures to lower leukocyte counts were carried out, death resulted in all cases [Increased white blood cell count is a characteristic manifestation of pertussis. The white blood cell count of children in the severe cases was significantly higher than that of the mild cases, and the white blood cell count was particularly high in fatal cases. Due to the poor ability of white blood cells to change shape, they tend to obstruct stenosed alveolar capillary beds. White blood cells usually take 10-15 times longer than erythrocytes to pass through these vessels, resulting in embolism due to leukocyte clumps, causing hypoxemia and pulmonary hypertension , 19. Thirtussis) , 21. Manll cases , 23. In ll cases , 25.Pertussis not only seriously endangers the life and health of infants, but also creates a heavy burden on families and society. Severe pneumonia and hyperleukocytosis are key issues in the prevention and treatment of severe pertussis. Adjusting the immunization strategy to vaccinate pregnant women has a clear effect on reducing the incidence and mortality of pertussis in young infants, which should be considered by the government.As a retrospective study, the study has some kind of uncontrollability, some significative data may be absent. For example, hyperleukocytosis causes pulmonary hypertension which affects cardiac function and may leads to life-threatening severe pertussis, but pulmonary arterial pressure has not been measured in these infants. In addition, a proportion of infants with pre-existing co-morbidities were included in the study, such as preterm or low birth weight infants, macrosomia, and infants with congenital heart disease, and they were at a higher risk to develop severe disease when having an infection due to the lower amount of maternal antibodies that were mainly transferred in the last trimester of pregnancy, or the immaturity or low function of their immune system. But the proportion of infants with these co-morbidities had no statistical difference between infants with severe and mild pertussis in the study, this was probably because the number of these infants was too small. The issues not covered in this study need to be studied later.1. Infants aged <3 months not vaccinated for pertussis appear more likely to become infected and have more severe disease. Adjusting the immunization strategy to vaccinate pregnant women to protect the infants should be considered.2. Severe pneumonia and hyperleukocytosis are the main mechanisms underlying severe pertussis.


Aim: This study aimed to identify common variable immunodeficiency (CVID) by high-throughput next-generation sequencing (NGS) in children with refractory immune thrombocytopenia (RITP) to facilitate early diagnosis.Methods: CVID-related genetic mutations were explored in patients with RITP during 2016–2019. They were tested consecutively through NGS by the ITP team of the tertiary children hospital in China. An evaluation system was devised based on the phenotype, genetic rule, and serum immunoglobulins (Igs) of all patients with RITP. The patients were divided into highly suspicious, suspicious, and negative groups using the evaluation system.Results: Among 176 patients with RITP, 16 (9.1%) harbored CVID-related genetic mutations: 8 (4.5%) were highly suspicious of CVIDs. Five had mutations in tumor necrosis factor receptor superfamily 13B (TNFRSF13B), one in lipopolysaccharide responsive beige-like anchor protein (LRBA), one in nuclear factor kappa-B2 (NF-κB2), and one in caspase recruitment domain11 (CARD11). Others were classified into the suspicious group because the clinical phenotype and pedigree were suggestive, yet insufficient, for diagnosis. Repeated infection existed in all patients. Two had an allergic disease. Positive autoimmune serologies were noted in 62.5%. Five had a definite positive family history. The median serum immunoglobulin (Ig)A, IgG, and IgM levels were 0.3875, 6.14, and 0.522 g/L, respectively. Nearly 85.7% of patients had insufficient serum IgA levels, while 37.5% had low IgG and IgM levels.Conclusions: High-throughput NGS and a thorough review of the medical history are beneficial for the early diagnosis of patients without any significant clinical characteristics, distinguishing them from those with primary pediatric ITP. The cases suspicious of CVID need further investigation and follow-up to avoid deterioration. Immune thrombocytopenia (ITP) is the most commonly seen autoimmune thrombocytopenia manifested as different severity of bleeding. A majority of children with ITP have better outcomes compared with adult patients for their spontaneous remission and good response to standard treatments. Yet nearly 20–30% of children with ITP have a frequent relapse or no response to multiple treatments, leading to a chronic duration beyond 12 months and a lower quality of life. The pathogenesis of ITP lies in the destruction and insufficient production of platelets due to the abnormal immune regulation and auto-antibodies. Thus, a disease interrupting the balance of the immune system, such as common variable immunodeficiency (CVID), is more likely to cause ITP. CVID is a primary immune deficiency disease (PID) associated with both immunodeficiency and autoimmune setting. It is one of the possible causes of chronic and refractory ITP (C/RITP) in children with poor prognosis (CVID is a group of highly heterogeneous PIDs. According to the European Society for Immunodeficiencies (ESID)/Pan-American Group for Immunodeficiency (PAGID), the definition of CVID was as follows: patient with a markedly reduced serum levels of immunoglobulin (Ig)G, in combination with low levels of IgA and/or IgM, and meeting the following criteria: (1) the onset of immunodeficiency at more than 2 years of age; (2) insufficient or absent response to immunizations of vaccines; and 3) exclusion of other defined immunodeficiency states  exclusio. The disITP secondary to CVID is hard to diagnose and can be confused with ITP as a consequence of profound heterogeneity in the phenotype and genotype of CVID, different immune statuses in children of different ages, and early administration of intravenous immunoglobulin (IVIG) at the onset of ITP. CVID with ITP was refractory to multiple treatments, with severe bleeding and recurrent thrombocytopenia , 11. FurTherefore, next-generation sequencing (NGS) was used in this study to examine CVID-related genetic mutations in pediatric patients with the primary manifestation as ITP. Also, the clinical characteristics, as well as laboratory features, were summarized to guide clinicians in early identification of pediatric CVID.A retrospective study was conducted on children diagnosed with ITP  in Beiji9/L and 100 × 109/L and twice more than baseline count without bleeding), concentrations of Igs, indicators of immune dysregulation /infection, and imaging results such as abdominal ultrasound and pulmonary computed tomography (CT) scan.The clinical data were collected from admission and follow-ups, including demographics, present and past medical history, bleeding severity evaluated using the Adix–Buchanan bleeding score, family history, therapeutic response to IVIG/corticosteroids and second-line therapy : Patients with RITP who had positive CVID-related mutations fulfilled the following three criteria: (a)–(c).Suspicious CVID (S-CVID): Patients with RITP who had positive CVID-related mutations fulfilled less than three criteria among (a)–(c).Negative (N): Patients with RITP who did not have positive CVID-related mutations.Positive CVID-relevant phenotype , which was self-fulfilling because the study participants were patients with ITP.Decreased concentrations of Igs .Positive family history of autoimmunity, allergic disease, and/or recurrent infections, which accorded with the inheritance law.de novo or inherited.The blood samples of the patients and their biological parents were collected to test whether the mutations were Genomic DNA was extracted from the peripheral blood using a QIAamp DNA Mini Kit .AIRE, C1R, CARD11, CASP10, CBL, CD19, CD27, CD36, CD40LG, CTLA4, FAS, FASLG, GATA2, ICOS, IFIH1, IL2RG, MASTL, MYH9, NFKB2, NHEJ1, NLRP1, NLRP2, NLRP12, PIK3CD, PLCG2, RAG2, SAMD9L, STAT3, STIM1, TCF3, TIRAP, TNFRSF13B, TUBB1, and WAS. The NGS panel was designed referring to the updated classification of PID released by the PID Expert Committee (PID EC) of the International Union of Immunological Societies (IUIS). The biotinylated capture probes were designed to tile all of the exons without any repeated regions. The captured DNAs were eluted and amplified, and then their polymerase chain reaction products were purified with SPRI beads . The enriched libraries were sequenced for 150-bp paired-end reads using Illumina HiSeq X Ten.Targeted genes associated with autoimmunity and thrombocytopenia were selected using a gene capture strategy with a GenCap custom enrichment kit  following the manufacturer's protocol. The diagnosed genes were as follows: http://broadinstitute.github.io/picard). Insertions, deletions, and single-nucleotide polymorphism variants were detected and filtered using the Genome Analysis Toolkit. Then, the identified variants were annotated using ANNOtate VARiation, associated with the following databases: 1,000 g, Exome Aggregation Consortium, and The Human Gene Mutation Database, and predicted using Mutation Taster, Sorting Intolerant from Tolerant, PolyPhen-2 (PP2), and Genomic Evolutionary Rate Profiling (GERP++). The pathogenicity of mutations was assessed in accordance with the American College of Medical Genetics and Genomics (ACMG) guidelines.After sequencing, raw data were saved in the FASTQ file format. Illumina sequencing adapters and low-quality reads (<80 bp) were filtered using Cutadapt. Clean reads were aligned to the University of California Santa Cruz hg19 human reference genome using the Burrows–Wheeler Alignment tool . Duplicated reads were removed using Picard  mutation reads more than five, and the mutation ratio no <30%; (ii) removing the mutation whose frequency was more than 5% in 1,000 g, ESP 6500, and inhouse database; (iii) dropping the mutations if they existed in the normal database (MyGenostics); (iv) removing the synonymous mutations; and (v) after (i), (ii), and (iii), removing the mutations that were synonymous and reported in Human Gene Mutation Database (HGMD). As a result, the remaining mutations should be pathogenic.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The protocol for genetic analysis was approved and performed under the guidelines of the ethics committee of Beijing Children's Hospital (China). Informed consent for the study, including consent for the collection and the use of DNA samples for genetic analysis, was obtained from eligible children and their parents or legal guardians.Data were described as medians (ranges) or numbers (proportions).U test for quantitative variables and the Fisher's exact test for categorical variables. Significant differences were defined as a P-value < 0.05.The IBM SPSS software, version 21.0 was used for statistical analysis. Descriptive statistics were calculated as median (range) for quantitative variables and frequency (percentages) for qualitative variables. Comparisons of the HS-CVID group and the other groups were performed with the Mann–Whitney (TNFRSF13B) (13 patients), lipopolysaccharide-responsive beige-like anchor (LRBA) (1 patient), nuclear factor kappa-B2 (NFκB2) (one patient), and caspase recruitment domain 11 (CARD11) (one patient). Eight patients  were enrolled into the HS-CVID group. Another 4.5% fulfilled the criteria of the S-CVID group. The complete demographics and clinical characteristics are presented in In the present study, 176 children with RITP were enrolled and screened for CVID-related genetic variations using NGS. Further, 16 (9.1%) patients were found with positive gene mutations, including tumor necrosis factor receptor superfamily 13B The positive family history, related to immune defect symptoms , autoimmune cytopenia, and autoimmunity, of participants was significantly higher in the HS-CVID group than in the S-CVID and N groups. Patients in the HS-CVID and S-CVID groups had lower frequencies of response to corticosteroids compared with patients in the N group. The incidence of recurrent infections and the occurrence rate of hypogammaglobulinemia with serum levels of IgG, IgA, and IgM were significantly higher in the HS-CVID group. However, no significant difference was found in the onset age of ITP, sex distribution, baseline of platelet count, Buchanan bleeding score, and rate of temporary response to IVIG and second-line therapy of ITP among different groups.Regarding family history, 25% of patients in the HS-CVID group had a positive family history of recurrent upper respiratory tract infections with levels of IgA/IgG/IgM at or below the lower limit of normal; 12.5% showed a family history related to ITP; and 50% of patients' parents had autoimmunity and allergic diseases, including ankylosing spondylitis (AS), Sjogren's syndrome (SS), and allergic rhinitis (AR). However, in the S-CVID group, one father and two mothers of three patients had allergic diseases. Besides, 12.5% of patients in this group had a positive family history of autoimmune thrombocytopenia. None of the patients in the N group had a related family history. The differences among the three groups were statistically significant.Nearly all patients had a transient response to IVIG in the present study, while the rate of response to corticosteroids showed significant differences among the HS, S, and N groups . Regarding the second-line therapy to ITP, one patient in the HS group failed in treatment with rituximab, recombinant human thrombopoietin (rhTPO), eltrombopag, and mycophenolate mofetil, while the other in this group achieved complete remission after combination therapy of rituximab, rhTPO, eltrombopag, and high-dose dexamethasone. Regarding the other two groups, 93.3% in the N group responded well to rituximab, 50% in the S group and 31.6% in the N group responded to thrombopoietin, and 60% patients in the N group responded to immunosuppressant drugs; however, no one responded in the HS and S groups.Regarding recurrent infections, 62.5% of patients in the HS group had recurrent respiratory infections and 12.5% had a gastrointestinal infection. Identified pathogens were mycoplasma, cytomegalovirus, and tubercle bacillus . Imaging with pulmonary CT showed pulmonary interstitial and parenchymal changes in 37.5% of patients. However, only 25 and 2.2% in the S and N groups had recurrent infections.Regarding allergy, 25 and 37.5% of patients in the HS and S groups, respectively, showed recurrent allergy during the course. Further, 12.5 and 25% experienced urticaria and AR, respectively. Moreover, 12.5% in both groups complained about the allergic reaction induced by mosquito bites.The incidence of hypogammaglobulinemia (100%), especially IgA levels, was significantly higher in the HS-CVID group. Nearly 85.7% of patients in the HS group had insufficient serum IgA levels, while 37.5% had low IgG and IgM levels. For those in the S and N groups, 25 and 22.8% had hypogammaglobulinemia, respectively. Among these, 12.5 and 2.2% had a low IgG level, 0 and 1.1% had a low IgM level, and 25 and 19.6% had low IgA levels in the S and N groups, respectively.TNFRSF13B, LRBA, CARD11, and NFκB2. A total of 12 different variants were identified, of which four were reported in HGMD; no spontaneous mutation was found in this study  of 176 patients initially diagnosed with RITP could be HS of CVID; another 4.5% (eight patients) in the same population were suspicious of CVID with highly correlated genetic abnormalities and possible phenotype. A definite diagnosis of CVID could not be made because patients were too young for diagnosis, and the testing of Igs was interrupted by some treatments received before the sample was taken.TNFRSF13B, LRBA, NFκB2, and CARD11, which have been widely reported to be associated with CVID  and is vital in the maturation and survival of peripheral B cells  presented with the same monoallelic nonsense mutation: R84T. Three (60%) were detected with biallelic variants, including three nonsense mutations: C104R, V126A, and G76S; and one had stop-gain mutation: Q196X; two had frameshift deletions: F103Lfs*1 and P235Rfs*168. Parents with monoallelic nonsense mutations: C104R, V126A, and G76S, were asymptomatic, while other parents with the monoallelic mutation had features such as recurrent infections, allergy, or autoimmunity, which did not exactly match with those in their kids. The present study reported different mutations in the same gene (TNFRSF13B), sharing distinct clinical phenotypes in children with ITP and late-onset CVID.Most of the known genetic abnormalities are rare, except  B cells . The incith CVID , 30, 31.pulation –34. Besionfirmed , 35. TNF protein , 33, 36. protein , 33, 36.ith CVID . Accordil health . Peng resymptoms . The preNFκB2 mutations were first discovered by whole-exome sequencing in a multiplex CVID pedigree with an autosomal dominant inheritance (NFκB2 causes defective phosphorylation and nuclear translocation of p52 (NF-κB2 deficiency presented with a CVID(-like) phenotype in early childhood and suffered from recurrent respiratory tract infection and autoimmune cytopenia, which could be the predominant autoimmune manifestation in CVID s, Ro-52, and anti-b2 glycoprotein-I (anti-β2-GPI) antibodies, as well as a recurrent infection inside the gastrointestinal tract. The patient shared the mutation (S541T) with his mother diagnosed with SS, suggesting the phenotypic variability between the patient and his/her parent with the same mutation.LRBA protein is a cytosolic protein regulating CTLA-4 expression. It is a potent inhibitory molecule expressed by immune effector cells . The biaRecent studies found that knock-out caspase activation and recruitment domain (CARD)11 in animal models had significant similarities with the phenotype of CVID , 51. CARlowB was also found to increase in CVID and correlated with autoimmunity  –55. ITP immunity –58. Moreimmunity –61. Liu immunity . Liu fouimmunity . A revieimmunity . In summThe main purpose of this study was early and prompt identification of CVID in children with ITP. From the perspectives of the known pathogenesis, ITP is a common autoimmune disease that affects mainly the blood system. No doubt, autoimmunity is one of the characteristics of CVID. Thus, CVID may be the underlying cause of ITP, even for younger children, whose manifestations, such as the absence of severe infection and classical hypogammaglobulinemia, are not specific or recognizable enough to make a definitive diagnosis of CVID. Hence, it is necessary to apply a method for the early detection of CVID in young children. An increasing number of gene defects were discovered in patients with CVID with the development and application of genetic testing. In the present study, the NGS panel was based on the updated IUIS classification for PID, which is the most authoritative guideline in the field of PID. The study found that NGS might be an applicable and useful way to differentiate pediatric CVID from RITP. Rivalta performed a long-term follow-up of pediatric Evans' syndrome and its evolution in PID and showed that NGS evolved into a routine application in evaluating PID in patients with autoimmune cytopenia, especially in those with a nonspecific phenotype . NonetheHowever, this study had some limitations that should not be ignored. The first was the lack of evaluation of the response to vaccines due to the restricted vaccination of patients with ITP. Second, routine treatment of ITP, especially IVIG and corticosteroids, before the testing of Igs might interrupt with the results of IgG, making the Ig levels higher than the actual levels. The aforementioned two issues inevitably led to the difficulty in the evaluation and diagnosis of pediatric CVID. Therefore, an evaluation system that was easier to practice in patients was set up and adopted. The reliability of the evaluation system should be taken into consideration and confirmed in future studies. Besides, medical histories such as recurrent infection and allergic disease of patients, especially those in the negative group, might have had information bias due to the retrospective nature of the study, inevitably affecting the results. Finally, the relatively limited sample size could not explain the entire spectrum of the clinical and immune phenotypes of ITP secondary to CVID. Generally speaking, the sample size should be increased, and the diagnosis of CVID should be standardized in pediatric ITP.In summary, CVID was identified at an early age in this study by examining Chinese children with RITP using NGS, resulting in a detection rate of 4.5%. The study further suggested that physicians should be more aware of the secondary causes of ITP, such as CVID, especially in patients with recurrent and refractory characteristics, tendency to have an infection, and family history of autoimmunity/immunodeficiency. NGS can be applied earlier in the differential diagnosis for suspicious CVID in children with ITP. The findings of this study might have implications for future studies on the pathogenesis and diagnosis of pediatric CVID with ITP.The datasets for this article are not publicly available due to the latest version of Biosafety Law of the People's Republic of China. Requests to access the datasets should be directed to the corresponding author.The studies involving human participants were reviewed and approved by the ethics committee of Beijing Children's Hospital (China). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.JM was involved in the collection and critical analysis of clinical data and also wrote the manuscript. RW participated in concept development, supervision of statistical analysis, and critical review of the manuscript. ZC performed supervision and critical analysis of laboratory data, including the interpretation of NGS results. LF, HG, JZ SZ, XZ, and HL collected and sorted out the clinical data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Reduced muscle mass is frequently observed in older people and can lead to being underweight and/or weight loss (WL), but prediction and screening systems utilizing hematological biochemical parameters are limited. High serum aspartatSe aminotransferase in conjunction with normal serum alanine aminotransferase (HASNAL) can reflect systemic muscle damage. HASNAL and the incidence of being underweight concomitant with WL (UWWL) were investigated in the present 6-year community-based cohort study. Methods: Clinical parameters, lifestyle, the incidence of being underweight, ≥5% WL, and UWWL were investigated in 238,536 Japanese people aged 40–68 years who had normal serum alanine aminotransferase. HASNAL was defined as serum aspartate aminotransferase ≥30 U/L and serum alanine aminotransferase <30 U/L. The subjects were divided into a younger group (<55 years) and an older group (≥55 years). Results: After 6 years, overall body weight had increased by 0.4% in the younger group and decreased by 0.4% in the older group. In logistic regression analysis, in the younger group ≥5% WL was significantly associated with baseline HASNAL compared to baseline low serum aspartate aminotransferase (<20 U/L). In the older group, baseline HASNAL was significantly associated with being underweight, ≥5% WL, and UWWL, even after adjustment for potential confounders, and UWWL was inversely associated with regular exercise and daily alcohol consumption—both of which modified the aforementioned associations. Conclusions: Older subjects with HASNAL were at an increased risk of UWWL, possibly via skeletal muscle damage, which may be affected by common lifestyles. Geriatric people are predisposed to weight loss (WL), and in such patients, WL can be accelerated by reduced skeletal muscle mass, which is a specific feature of sarcopenia ,2,3. BeiIn the past decade, several studies have shown that low blood alanine aminotransferase (ALT) was associated with low fitness, frailty, and sarcopenia ,7,8, altThe present investigation was a composite study using health checkup data acquired in Japan as part of the Kanagawa Investigation of the Total Checkup Data from the National Database (KITCHEN), which is a broader study aimed at elucidating factors associated with cardiometabolic diseases. The design of that study is described in detail elsewhere . Since 2n = 129,077) and “older” . Because it has been reported that in most developed countries peak body weight is observed between the ages of 50 and 59 years, and that body weight begins to decline thereafter [The present cohort study used data derived from 593,517 people who underwent health checks twice, 6 years apart, the first time between April 2008 and March 2009, and the second time between April 2014 and March 2015. Subjects with baseline serum AST ≥ 200 U/L, ALT ≥ 30 U/L, and/or GGT ≥ 200 IU/L, and those with a baseline body mass index (BMI) < 18.5 or ≥ 25.0 were excluded. Subjects with serum ALT ≥ 30 U/L after 6 years were not enrolled, in order to exclude patients with high serum AST due to hepatic disease. A total of 238,536 people  aged 40–68 years and of normal weight with complete data (except for HbA1c and past history of cerebrovascular disease) were enrolled in the present study. To evaluate subject age as a single numeric value, age groups  were transformed into substituted ages (s-age) corresponding to the medians in each age group, which were 42, 47, 52, 57, 62, and 67 years. In the analysis of the effects of age, the subjects were divided into two age groups, “younger” , and 95% confidence intervals (CIs). Because the incidences of being underweight and UWWL were small  , adjusteBaseline characteristics of the younger subjects in the three baseline AST categories are shown in Changes in body weight and the incidence of being underweight, ≥5% WL, and UWWL after 6 years are shown in n = 155) and ≥ 35 U/L (n = 59) in the older group, the ARR of baseline serum AST ≥ 35 U/L for UWWL was 1.51 ((1.21–2.02), p = 0.007) in Model 2 (data not shown). Serum ALT as a continuous variable was positively associated with the incidence of ≥5% WL in younger subjects , whereas in older subjects it was negatively associated with being underweight  and UWWL  (both p < 0.05), and positively associated with ≥ 5% WL . The results of logistic regression analysis of relationships between baseline serum AST and being underweight, ≥5% WL, and UWWL in the two age groups are shown in The results of logistic regression analysis of baseline factors for UWWL are shown in Generally, there is an increase in body weight with aging, and the peak prevalence of obesity is reportedly observed in individuals aged 50–59 years in most developed countries ,19. AfteIndeed, aminotransferase abnormalities, particularly AST but not ALT, and elevated creatine kinase are common in the setting of rhabdomyolysis and post-exercise ,27, whicBeing underweight or exhibiting WL after skeletal muscle damage indicate a reduction in muscle mass, and can be one of the main etiologies of sarcopenia or frailty ,2,3,4,5,In most developed countries widespread incidences of being underweight, sarcopenia, and frailty reduce the welfare of older members of society due to increasing medical costs and nursing care ,4. GivenAlthough serum albumin is often reduced in patients with malnutrition, the use of hypoalbuminemia is limited in the clinical settings ,33,34 beIt has been reported that lower ALT levels may reflect or predict sarcopenia, although the underlying mechanisms potentially involved remain unknown ,7,8. In Intriguingly, the association between HASNAL and UWWL observed in the current study was modified by adjustment for regular exercise and drinking alcohol daily. In this study, regular exercise and regular physical activities, favorable lifestyle choices in terms of the prevention of sarcopenia ,2,3, werThe present study had some limitations. It is unknown whether the causes of WL in the patients in the study were unintentional or intentional, and this may be of relevance with regard to the outcomes identified in the different age groups 6 years after the initial examination ,22,23. AIn the current study, older subjects with HASNAL were at an increased risk of being underweight, ≥5% WL, and UWWL, which are characteristic features of sarcopenia. These conditions may have involved skeletal muscle damage, which can be modified by lifestyle choices.


This study explored the biopsychosocial predictors of dropout and weight loss during a combined behavioural and surgical weight-reduction program. Behavioural treatment for six months was followed by bariatric surgery and a visit six months after surgery. The success criterion was the loss of ≥50% of excess BMI above 25 kg/m2 (%EBMIL). Thirty-one men and 113 women with BMI 43.5 kg/m2 (SD 4.3) and 41.8 kg/m2 (SD 3.6), respectively, were included; 115 underwent bariatric surgery , and 98 had a follow-up visit six months after surgery. The mean %EBMIL at follow-up was 71.2% (SD 18.5). Treatment success was achieved in 86 subjects. Assuming success in 17 subjects who did not attend the follow-up visit (best possible outcome), 103 out of 144 subjects (72%) achieved successful weight reduction. Cohabitation was the only predictor of accomplishing surgery. Neither weight loss during behavioural therapy nor biopsychosocial factors were found to be clinically significant predictors of weight loss after surgery. The success rate of less than three in four subjects was unsatisfactory. There is a need to improve the regimen and to determine effective alternative interventions.Treatment of subjects with morbid obesity (Body Mass Index (BMI) > 40 kg/m Obesity is a worldwide disease with serious health consequences . SubjectIn most countries, subjects with morbid obesity, especially those who are motivated to undergo bariatric surgery and are referred to specialised surgery units, accomplish combined treatment consisting of a behavioural intervention that aims to achieve a predefined weight loss, followed by bariatric surgery and a follow-up period of variable duration . During The aim of this study was to explore the biopsychosocial predictors of dropout from a combined behavioural and surgical weight reduction program and the predictors of weight loss six months after bariatric surgery.The data used were a subset of data from in a study in subjects with morbid obesity, the MO-BiPS study  ,11,12,13The study had a prospective cohort design. The first part was six months with behavioural interventions aimed at weight loss, and motivation for and information about bariatric surgery. After finishing the first part, the participants were offered bariatric surgery and were followed for six months (the second part).2 or > 35 kg/m2 with obesity-related complications) in the age range of 18–65 years referred from primary care to the specialised obesity unit at Innlandet Hospital Trust, Gøvik, Norway for the evaluation of bariatric surgery. Subjects who had undergone previous major gastrointestinal surgery or who had organic gastrointestinal disorders, problems with alcohol and drug abuse, major psychiatric disorders, or serious somatic disorders not related to obesity were excluded.The study included subjects with morbid obesity , giving a maximum daily energy intake of 4200 kJ/day.Bariatric surgery was performed as Roux-en-Y gastric bypass or Gastric sleeve ,15. A fo2);Demographic and anthropometric data: age (years), gender , height (m), body weight (kg), and body mass index . Treatment success was defined as %EBMIL ≥ 50%;Weight loss was measured as reduction in BMI and relative loss of excess BMI above 25 km/mSocial factors: cohabitation (yes/no); education (number of years); employed (yes/no); smoking (daily/not daily);Dietary habits: The diet was assessed with a validated food frequency questionnaire, and the daily intake was based on the Norwegian food composition table . Use of The physical activity score was calculated as the sum of two scores and classified as follows: easy activity (not sweaty/breathless)—none; <1 h/week, 1–2 h/week, and >3 h/week (scores 0–3); strenuous activity (sweaty/breathless)—none; <1 h/week, 1–2 h/week, and >3 h/week . The total sum of the scores for physical activity was 0–8;Comorbidities: The participants were asked about the presence of 12 current or previous disorders ;Musculoskeletal pain from six parts of the body (scores 0–12);The World Health Organisation – Five Well-Being Index (WHO-5)  ;Hopkins Symptom Checklist 10 to measure psychological distress  ;Fatigue Severity Scale: A validated Norwegian translation of the Fatigue Severity Scale was used  ;Rosenberg Self-Esteem Scale: A validated Norwegian translation of the international questionnaire was used  ,21;Epworth Sleepiness Scale: A validated Norwegian translation was used  ;Sense of Humour Questionnaire: A Norwegian version of the short form SHQ-6: “Attitudes toward humour” was used (scores 6–24) ;Suter’s food tolerance questionnaire  .At inclusion, the following biopsychosocial variables were collected:t-test depending on the type of data. The predictors of weight loss were analysed with linear regression analyses. The analyses were performed with IBM SPSS Statistics for Windows, Version 25.0. IBM Corp: Armonk, NY, USA. p-values < 0.05 were judged as statistically significant.The results are reported as mean (SD) and number (with proportion in percentage). Comparisons between groups were analysed with chi-square tests and The study was approved by the Norwegian Regional Committees for Medical and Health Research Ethics, PB 1130, Blindern, 0318 Oslo, Norway (reference number 2012/966) and performed in accordance with the Declaration of Helsinki. All participants gave written informed consent before inclusion.2 and 41.8 (3.6) kg/m2 respectively (p = 0.024) were included in the analyses; 115 underwent bariatric surgery , and 98 had a follow-up six months after surgery. Thirty-one men and 113 women with BMI values of 43.5 (4.3) kg/mBMI at inclusion was positively associated with a reduction in BMI and negatively associated with %EBMIL. Weight loss was significant after both treatment periods, but there were no significant associations between weight loss in the two periods. Several criteria for weight loss success after bariatric surgery have been used ,7. A comLooking separately at the two treatment periods, the weight loss during the first period was satisfactory with a mean %EBMIL of 19%. For all subjects included in the study, the weight loss was probably less because the weight loss in 29 subjects (20%) who dropped out could have been less than those who accomplished the regimen. Comparisons with other studies are difficult because the behavioural weight loss regimens vary in length and content and are not always completely described. In large, the weight reduction was as reported in other studies ,27,28.Short- and long-term weight loss after bariatric surgery has been thoroughly studied ,26,27,28Are the results satisfactory? More than one out of four subjects did not reach the anticipated weight loss target. Overall, the treatment was challenging, involving the stringent behavioural intervention, which was followed by a surgical procedure that demands drastic changes in dietary habits and lifestyle. Most subjects with morbid obesity lack motivation and therefore desist from such treatments, even if they are free of cost, as they are in Norway. Knowing that the subjects in this study represent a selected and highly motivated group given a treatment not accepted by all subjects with morbid obesity, the combined treatment is not optimal. The study demonstrates the challenges in the treatment of obesity. The combined procedure used in this study, which is probably the most effective therapy, is neither optimal nor accepted by all subjects with morbid obesity. The method needs improvement, and new treatment options that are more acceptable by the participants with less radical interventions are required and better results need to be achieved. However, notably, subjects with a significant weight reduction who withdraw from surgery and those without optimal weight loss after surgery achieve favourable health effects .The high rates of dropouts and treatment failures call for predictors of failures. The dropout rate in this study during behavioural therapy (20%) was high but was as anticipated. A review reports dropout rates above 35% in half of the included studies . The sixAlthough the mean %EBMIL after the combined therapy was judged as satisfactory, a large variation was observed. Valid predictors of weight loss success could improve both the selection of subjects for surgery, the pre-treatment procedures, and per-operative interventions. In this study, BMI at inclusion was positively and negatively associated with the changes in BMI and %EBMIL, respectively ,31. The There are numerous studies of various predictors of weight loss after bariatric surgery, such as personality, anxiety, depression, psychopathology, eating disorders, impulsivity, socioeconomic status, alcohol and drug abuse, cognitive function, cognitive treatment, comorbidity, metabolic diseases, and hormones ,33,34,35Preoperative weight loss has been a requisite for surgery at many obesity units. The purpose is to evaluate compliance with the necessary dietary changes before and after surgery, reduce complications and improve the outcomes . The benThe strengths of the study were the inclusion of unselected, consecutive subjects at the only free of charge public obesity unit in the region, the use of a preoperative period with behavioural therapy according to the national standard and international recommendations, the use of validated questionnaires, the clear separation of the effects of the two treatment modalities, and the study of associations between the effects in the two treatment regimens. Details about the duration of obesity, the presence of comorbidities including psychopathology, the use of alcohol and drugs, biomarkers, complications, etc. were incomplete or unavailable. A longer follow-up period would have been desirable.The overall success rate, defined as %EBMIL ≥ 50%, was less than 72%. Taking into account that the subjects referred to an obesity unit accepted a challenging weight loss treatment and were a selected and highly motivated group, the success rate was judged as unsatisfactory. No clinically useful predictors of success, including the outcomes of the pre-surgical behavioural intervention, were found. The results conform to other studies. However, further improvements are required in the regimen and other effective behavioural, psychiatric, medical, endoscopic and surgical interventions.


Oral Mucositis (OM) is a common adverse effect of head and neck squamous cell carcinoma (HNSCC) treatment. The purpose of this study was to investigate the significance of early changes in tissue electrical parameters (TEPs) in predicting the development of OM in HNSCC patients receiving radiation therapy (RT). The current study combined two study designs. The first was a case-control study. The control group comprised of RT patients who did not receive head and neck RT, and patients with HNSCC who received RT comprised the case group. In the second part of the study, the case group was included in a parallel cohort. A total of 320 patients were assessed for eligibility, and 135 patients were enrolled. Double blinding was performed, and neither the patients nor the care providers knew the measured parameters. The primary outcome was the detection of between-group changes in local TEPs over the follow-up period. The secondary outcome was the appearance of OM grades II, III, or IV and the predictive value of local TEPs in determining the incidence of OM after RT. The variables, impedance module, resistance, reactance, phase angle, and capacitance, were analyzed by the receiver operator curves (ROC). The case and control groups did not differ in demographic and clinical characteristics. Radiation therapy increased the local impedance module, resistance, reactance, and phase angle and reduced the local tissue capacitance in both groups. Evaluation of TEPs in the first week of RT correlated with the development of OM lesions during cancer therapy. ROC analysis showed that local impedance module and resistance presented higher specificity than did other parameters in predicting OM. In conclusion, local tissue electrical parameters measured at the first RT week can be useful tools to predict oral mucositis. Moreover, HNSCC treatment is generally associated with high costs for patients and public health services7.Therapeutic approaches for head and neck squamous cell carcinoma (HNSCC) include surgery, radiation therapy (RT), chemotherapy alone, or in combination, based on clinical staging9. Oral mucositis (OM), dry mouth, sticky saliva, difficulty in swallowing solid foods, and loss of taste sensation are frequent complications of HNSCC RT treatment10. Oral mucositis is classified into 4 grades according to severity12. Grade 2 lesions are associated with pain, but the patient can still swallow solids. In grades 3 and 4, the discomfort interferes with food intake10. Oral mucositis may necessitate modifications in the RT fraction regimen as high grades of OM need treatment interruption13. Discontinuation of RT is associated with a decrease in local control rates and reduced survival15. Despite the impact of OM in the patient’s quality of life, there is no test to predict the incidence of OM during cancer therapy.Treatment of HNSCC has some side effects that are painful and difficult to manage16, and some of these electrical parameters have proved to be useful in differentiating between normal and pathological processes21. TEPs are different and useful identifying tissues such as adipose and muscular tissue20. The majority of the studies have attempted to investigate only impedance as a marker of biological processes23. For example, impedance module and phase angle are widely used to evaluate body adiposity24 or, in a few cases, to detect neoplasms21.Many physiological processes are associated with electrical activity25, thus changing TEPs during RT26. However, no study has attempted to evaluate the local changes in impedance module, resistance, reactance, phase angle, and capacitance caused by RT. Moreover, there is no information on the association between TEPs and OM. Thus, the current study aimed to evaluate the significance of early changes in TEPs in predicting the development of OM in HNSCC patients receiving RT.It is known that RT alters the tissue concentrations of free radicalshttp://www.ensaiosclinicos.gov.br/, and ethical approval can be evaluated at http://plataformabrasil.saude.gov.br/. All patients signed the informed consent forms.All performed procedures were conducted following the ethical standards of the institutional and national research committees, the 1964 Helsinki declaration, and its later amendments, or comparable ethical standards. Ethical approval for this study (Number 80731617.0.0000.5146) was obtained from the Universidade Estadual de Montes Claros Institutional Review Board. The study was also registered in the National Clinical Trials Network (UTN: U1111-1214-7398/RBR-7yygb2). Data were collected at Dilson Godinho Hospital in Brazil from March 2017 to July 2018. The trial protocol can be assessed at 27. The study was reported following the Standards for Reporting Diagnostic Accuracy (STARD) criteria28  with confirmed histopathological diagnosis of squamous cell carcinoma of the base of the tongue, malignant neoplasms of other or unspecified parts of the tongue, unspecified, squamous cell carcinoma of the gum, and squamous cell carcinoma of the floor of the mouth, the palate, other or unspecified parts of the mouth, the tonsil, the oropharynx, the piriform sinus, the hypopharynx, and its variants, other or ill-defined sites in the lip, oral cavity, or pharynx receiving RT alone or in combination with chemotherapy. All patients treated with RT received 3D-RT. Patients were excluded if they had previously received head and neck RT, presented with OM, received pacemakers or declined enrollment.in situ, malignant neoplasms of the penis, unspecified, squamous cell carcinoma in situ of the mucocutaneous epithelium of the penis, other specified malignant neoplasms of the anus and anal canal, squamous cell carcinoma of the cervix uteri, adenocarcinoma of the cervix uteri, malignant neoplasms of the esophagus, unspecified, ductal carcinoma in situ of the breast, other specified malignant neoplasms of the breast and unspecified carcinoma of unspecified sites receiving RT alone or in combination with chemotherapy. All patients treated with RT received 3D-RT. Patients were excluded if they had previously received head and neck RT, presented with OM, received pacemakers or declined enrollment.The control group included patients who received RT for prostate, urinary bladder, rectum anus, breast, and cervical cancer. The inclusion criteria for the control group were adults over 18 years of age (both sexes) with confirmed histopathological diagnosis of malignant neoplasms of the prostate, adenocarcinoma of the prostate, metastasis in other or unspecified organs of the urinary system, prostate carcinoma 12.The case group was followed during the RT treatment (approximately 60 days). Both primary and secondary outcomes were evaluated in the case group weekly right after the start of RT treatment until the end of RT. In the case of OM occurrence, treatment was initiated following standard protocols as described elsewhereThe primary outcome was a change in local TEPs . The examination was done weekly right after the start of RT treatment until the end of RT.12. The presence of pain and the clinical appearance of mucositis lesions were detected during clinical examination, which was also performed weekly right after the start of RT treatment until the end of RT.The secondary outcome was the incidence of OM grades II, III, or IV, identified as previously described24. Impedance (Z) is an expression of the opposition that a system offers to alternating electric current31. Resistance (R) is a measure of the extent to which a substance opposes the movement of electrons among its atoms31. Reactance (X) is the amount of energy that a circuit stores31. When an alternating current passes through a body that contains reactance, the energy is released in the form of an electric field, in which case the reactance is capacitive (denoted -jX_C)31. Reactance is conventionally multiplied by the positive square root of −1, which is the imaginary unit number called the j operator, to express Z as a complex number of the form R- jX_C (when the reactance is capacitive)31. The impedance Z can also be represented in the form of impedance module (|Z | ) and phase angle (θ), where |Z | = √(R^2 + X^2) and θ = tan^(−1) (X/R)31. The capacitive reactance is given by X_C = (1/(2πfC)), where f is the frequency of the alternating current, and C is the capacitance. Capacitance is the ratio of the change in the electric charge of a system to the corresponding change in its electrical potential31. The concepts are summarized in Table The electrical resistance properties of the tissues were evaluated based on the following variables: impedance module, resistance, reactance, phase angle, and capacitanceLocal TEPs  were measured in the irradiated area. Differently, systemic TEPs were collected from the whole body. Local and systemic TEPs were collected from case and control groups.24. Briefly, a calibrated Bioelectrical Impedance Analysis Analyzer Unit  using an electric current (400 mA) at a high frequency (50 kHz), between 0 and 1 ohms, was used to measure bioimpedance. Electrodes  were placed on the right hand, wrist, foot, and ankle  and reactance (Xc), were used to derive mathematical equations to determine the phase angle (θ = tan^(−1) (Xc/R)), impedance module (Z = √(R^2 + X^2)), and capacitance (Xc = 1/((2π *f *C))).Systemic tetrapolar bioimpedance was evaluated as described elsewhere32. The same dentist performed all OM gradation.Patients were evaluated weekly, and mucositis was graded according to the World Health Organization (WHO)Data are shown as the mean ± standard deviation for continuous variables and as frequency and proportion for categorical variables. To determine if the data were well-modeled by a normal distribution, the Kolmogorov-Smirnov test and the Shapiro-Wilk test were performed. These analyses revealed that the data were non-parametrically distributed. Therefore, the Mann-Whitney test was performed. Pearson correlation was performed to evaluate the changes in local and systemic tissue electric parameters.The variables, impedance module, resistance, reactance, phase angle, and capacitance, were tested by plotting the empirical Receiver Operating Characteristic (ROC) curves using the gold standard method for comparison. The definition of areas should fill predictive indicators and classifications under the ROC curve. The Youden index was used to define the ROC cut-off values. The significance of each analysis was increased by 95%. Data were applied through MedCalc Statistical Software version 16.4.3 . A chi-square test was used for the statistical analysis of distribution differences of categorical data between groups. All statistical analyses were performed with PASW® v 18.0 for Windows®. The results had statistical significance at p < 0.05.All patients signed informed consent.The case group comprised 75 patients (60 men and 15 women) with a mean age of 63.15 ± 11.64 years. Seventy (55.10%) patients of the case group received less than 2 Brazilian minimum wages (250 dollars) per month. Most patients  did not complete elementary school (9 years of schooling). Regarding RT doses, 68% (N = 51) of the patients from the control group received more than 64 Gy during the RT. In the case group 58 (77.3%) patients presented OM grade 2, 8 (10.7%) presented grade 3 and 3 (2.7%) presented grade 4 , slightly older than the case group, with a mean age of 69.32 ± 12.94 years. The family income was lower than 2 Brazilian minimum wages (250 dollars) per month for 57 (95%) patients of the control group. Only six patients (10%) completed elementary school. Similar to the case group, 41 (68.33%) patients of the control group received more than 64 Gy. The groups did not differ in demographic and clinical characteristics  to predict OM (Fig. The local electrical resistance parameters were changed by RT. Moreover, the evaluation of local TEPs in the first week of RT was able to predict the development of OM grade Table . The fir OM Fig. . Despite33, with developing countries showing increased incidence rates in the last year29. Treatment modalities include surgery, RT, chemotherapy1, or combination therapy, depending on the TNM classification2. Treatment of HNSCC is associated with sequelae such as OM, radiodermatitis, and xerostomia34. The incidence of severe OM is responsible for RT interruptions12, that may affect RT fraction regimen13, resulting in decreased local control rates and reduced survival15. Strategies to prevent oral mucositides include cryotherapy35, low-level laser therapy36, and two-phase intensity-modulated radiation therapy37. Currently, there is no diagnostic tool to predict OM in cancer patients12.Head and neck squamous cell carcinoma, an aggressive neoplasm, poses a significant public health issue1 Thus, we aimed to use initial tissue electrical parameters to estimate OM in HNSCC patients receiving RT. Tissue electrical parameters are widely used to evaluate body composition, lymphedema, and obesity38. Most of the studies on tissue electrical parameters in HNSCC were performed to study body composition and used only bioimpedance22. Recently, a study examined an association between the status of hydration and systemic impedance with OM26. However, other tissue electrical parameters such as resistance, reactance, phase angle, and capacitance have not yet been evaluated in the literature26. In the current study, the local measure of impedance module resistance, reactance, phase angle, and capacitance changed during RT.Similarly, there are no diagnostic tools to predict the RT side effectsThe ROC curve demonstrated that all local tissue electric parameters were useful to differentiate cases from control. Furthermore, the tissue electrical parameters predicted post HNSCC treatment OM. Moreover, the values of the tissue electrical parameters measured in the first week of RT predicted the appearance of OM. This suggested that local tissue electrical parameters could be useful in stratifying patients with an increased risk for OM and determining preventive strategies to avoid poor prognosis. It is essential to highlight that data of the current study suggest that the tissue electrical parameters of the first week could be used to predict future side effects of RT. Currently, there is no available tool to predict OM during cancer therapy. The current study is the first trial to demonstrate that local tissue electrical parameters can predict oral mucositis in HNSCC. Prediction of OM will identify patients needing preventive interventions or treatment.44. However, studies have demonstrated that RT reduces the in vivo and in vitro levels of HIF-1α, miR-210, and LDH44. It is well-known that RT promotes ischemia-reperfusion injury25. In the current study, local impedance module, resistance, reactance, and phase angle were higher in irradiated anatomical sites. In contrast, local capacitance reduced with RT. The divergence in capacitance was expected because, by definition, impedance is directly proportional to frequency, while capacitance is inversely proportional to frequency. In corroboration with the current study, it was observed that there was an increase in the impedance parameters under ischemia and ischemia-reperfusion conditions19. Currently, there is no clinically approved tool to predict the success of RT1. In the future, the electrical parameters may also be useful to predict the effects of RT.HNSCCs are known to contain abundant hypoxic areasThe main limitation of the current study was that it was conducted at a single center. Single-center studies usually present small and homogeneous patient populations. At the same time, the current study used two groups with similar clinical and demographic characteristics.In conclusion, local tissue electrical parameters measured at the first RT week can be useful tools to predict oral mucositis.Supplementary material.


Prevotella. The abundances of the Fibrobacter, Campylobacter and RFP12 were all higher in ALNA than in ileal PPs. While, the abundances of the 5-7N15, Clostridium, and Escherichia were all higher in ileal PPs than in ALNA. The results suggested that the host’s intestinal microenvironment is selective for the symbiotic bacteria colonizing the corresponding sites, on the contrary, the symbiotic bacteria could impact on the physiological functions of this local site. In ALNA and ileal PPs of Bactrian camel, the bacteria which colonized different immune inductive sites have the potential to stimulate different immune responses, which is the result of the mutual selection and adaptation between microbial communities and their host.The microbial communities colonize the mucosal immune inductive sites could be captured by hosts, which could initiate the mucosal immune responses. The aggregated lymphoid nodule area (ALNA) and the ileal Payer’s patches (PPs) in Bactrian camels are both the mucosal immune inductive sites of the gastrointestinal tract. Here, the bacteria community associated with the ALNA and ileal PPs were analyzed using of 16S rDNA-Illumina Miseq sequencing. The mutual dominant bacterial phyla at the two sites were the Bacteroidetes, Firmicutes, Verrucomicrobia and Proteobacteria, and the mutual dominant genus in both sits was  They can even reach 10contents , and thecontents . These cdent IgA , 7. Furtdent IgA , and impdent IgA . Also, s6 (IL-6) , 12.Nodule-likePPs, an organized lymphoid tissue, mainly distributed in the ileal mucosa and submucosa, are the classic inductive site for gastrointestinal mucosal immunity to capture antigens, induce mucosal immune responses and regulate mucosal immune homeostasis , 14. IntAt present, the studies on the bacterial community in the gastrointestinal tract of camels mainly focused on rumen, intestines, and/or feces, and the results suggested that the composition of microbial community in the rumen of a camel was similar to other ruminants with differences in the abundance, meanwhile, there were different metagenomic profiles in different digestive tract regions , 20. HowAll experimental procedures were approved by the Animal Care and Use Committee (IACUC) of College of Veterinary Medicine of Gansu Agricultural University . All efforts were made to minimize animal suffering.Agriophyllum pungens, Ceratoides latens, and Nitraria tangutorum etc. when grazing time. All the six healthy pubertal Alashan Bactrian camels  were randomly selected, anaesthetised intravenously with sodium pentobarbital (20 mg/kg), and exsanguinated until death. After the abdominal cavity was opened, the whole abomasum (from cardia to pylorus) and ileum were cut, and their food residues were gently washed with sterilized saline, then the mucus was quickly scraped from the mucosal surface of ALNA (including RMFR and LMFR) and ileal PPs in each Bactrian camel under a sterile operation. A total of 18 samples were collected from the above three locations of six Bactrian camels, and quickly put into the 2.5 ml cryopreserved tube and kept in liquid nitrogen for further use.All Bactrian camels were distributed in Minqin county, Gansu province, China. They mainly eat The total genome DNA was extracted from samples using the TIANamp Stool DNA Kit  according to manufacturer’s instructions. 1% agarose gels were used to detect the integrity and impurity in the extracted bacterial DNA samples. The purity and concentration were tested by NanoPhotometer spectrophotometer  and Qubit 2.0 Flurometer , respectively. According to the concentration, DNA was diluted to 1 ng/μL using sterile water.5’-CCTACGGGNGGCWGCAG-3’) and 805R (5’-GACTACHVGGG-TATCTAATCC-3’) [® High-Fidelity PCR Master Mix ; 0.2 μM of forward and reverse primers, and about 10 ng of template DNA. Thermal cycling was consisted of initial denaturation at 95°C for 3 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 30 s, finally 72°C for 5 min. The same volume of 1× loading buffer (contained SYB green) was mixed with PCR products and electrophoresis on 2% agarose gel was operated for detection. Samples with bright main strip about 460 bp (V3-V4) were chosen for further experiments. PCR products were mixed in equidensity ratios. Then the mixture of PCR products were purified with GeneJET Gel Extraction Kit . Sequencing libraries were generated using NEB Next® UltraTM DNA Library Prep Kit for Illumina  following manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer  and Agilent 2100 Bioanalyzer . At last, the library was sequenced on an Illumina MiSeq platform and 250 bp paired-end reads were generated. All the sequencing data were deposited in the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) under accession no. PRJNA624714.The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 341F (ATCC-3’)  on the G(v0.9.10) software [The raw data were filtered to obtain clean reads by removing adapter-polluted and low-quality  reads and the reads with N bases > 5% etc. The Q30 percentages of the clean reads were all greater than 85% after filtering. According to overlap of the clean data, we spliced the paired reads using the PEAR software . After msoftware  based onsoftware . Alpha dsoftware . Rarefacsoftware . The hiesoftware .After the quality control, a total of 872,650 sequence reads were obtained from all the samples and grouped into 2,017 OTUs at the 97% similarity cut-off level. There was a similar pattern in the rarefaction curves: the number of OTUs of all samples gradually increased with the increase of the number of measured sequences, furthermore the curves became more gentle and the increasing trend became smaller, indicating that most of species existing in each sample were observed .The alpha-diversity of the microbial communities in ALNA and ileal PPs of Bactrian camels was estimated by Chao, Shannon and Simpson indices which are listed in Tables In order to understand the similarities of microbial community in the LMFR, RMFR and ileal PPs of Bactrian camels, hierarchical clustering analysis was performed based on Bray-Curtis distances generated from the OTU abundance. The results showed that the all samples could be grouped into two clusters: ALNA (LMFR and RMFR) and ileal PPs clusters . Among tVenn diagram was made according to the overlapping OTUs among the LMFR, RMFR and ileal PPs, then the mutual and unique OTU numbers were calculated in each group. The results showed that the OTU counts were 1207 in LMFR, 1178 in RMFR, and 1086 in ileal PPs; 520 OTUs were shared among the above three sites and accounted for 43.1%, 44.1% and 47.9%, respectively. Between LMFR and RMFR, there were 901 identical OTUs accounted for 74.6% and 76.5%, respectively. Between LMFR and PPs, there were 529 identical OTUs accounted for 43.8% and 48.7%, respectively. Besides, between RMFR and PPs, there were 544 identical OTUs accounted for 46.2% and 50%, respectively . We can All sequences were identified and subjected to different taxonomic levels. The results showed that 14 phyla and 46 genera in RMFR, 14 phyla and 41 genera in LMFR, and 16 phyla and 59 genera in PPs were detected  Tables. Prevotella  was the highest, and there were no differences among the three sites. Sixteen genera showed significant differences in abundances among the three sites: the abundances of Fibrobacter, RFN20, Campylobacter, BF311, Ruminococcus, Desulfovibrio and Pyramidobacter in the RMFR and LMFR were significantly higher than in ileal PPs, but the abundances of Escherichia, Bacteroides, 5-7N15, Akkermansia, Epulopiscium, Phascolarctobacterium, rc4-4 and Turicibacter in ileal PPs were higher than in RMFR and LMFR  and Escherichia in ileal PPs. It was mainly related to the following reasons. First, the effects from the pH, bile salts and oxygen content, for example, the pH is about 5.55 (acidic) in the camel’s abomasum, but it is about 7.73  in the ileum [At the genus level, there were significant differences in the relative abundance of the bacteria colonized ALNA and ileal PPs, which were mainly caused by Fibrobacteres in ALNA and he ileum . The oxyhe ileum . And in he ileum . TherefoF. succinogenes was an important symbiotic bacterium that could degrade cellulose in the rumen of ruminants [Bacteroides could produce a weak agonistic activity of LPS (LPSBV), which could induce the semi-mature of the CD11c + bone marrow-derived dendritic cells, and this response did not induce the pro-inflammatory factors expression [Clostridium (SMB53) and Escherichia in ileal PPs were significantly higher in ALNA. Therefore, the colonization of the local differential bacteria makes the whole digestive tract mucosal immune homeostasis different among different intestinal segments.As well, the main differential bacteria could impact on the physiological functions. For example, the uminants , and itsuminants ; and thepression . Moreovepression . And in In addition, some intestinal bacteria were not identified in this study, so the biological information hided in them needs further research and exploration. However, the results of the present study will lay a foundation for further research the role of microbial flora on gastrointestinal mucosal immunity.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Although PVE is safe and effective, it does not always induce sufficient hypertrophy of the future remnant liver, even after a significant time. Consequently, several other approaches have been proposed as alternatives to PVE. The most recently developed of such approaches is liver venous deprivation (LVD), a technique that combines hepatic vein embolization of the right hepatic vein (RHV) and accessory RHV, if present and PVE. Among the various interventional procedures that could be used for this purpose, LVD has been shown to promote the fastest and greatest increase in the future remnant liver volume, the reported growth at 14 days being 62.5% after LVD, compared with 8-27% after PVE-4.In many cases, hepatectomy is the only option for long-term survival in patients with primary or secondary liver malignancies. When a tumor proves unresectable because the resection will result in insufficient remnant liver volume, portal vein embolization (PVE) can be performed to stimulate future liver growth, the objective being to avoid post-hepatectomy liver failureHere, we describe the case of a 70-year-old patient diagnosed with Bismuth type IIIa cholangiocarcinoma. The patient was treated successfully with LVD prior to hepatectomy.The patient underwent computed tomography (CT) with angiography and volumetric analysis of the liver, processed with specific software , 30 days before the procedure in order to determine the remnant liver volume, which was found to be insufficient. Hepatectomy was also contraindicated because the patient showed an elevated bilirubin level, and an internal-external biliary catheter was therefore inserted for. The patient received antibiotic prophylaxis with a single dose (3 g) of ampicillin/sulbactam. Because of the long duration of the procedure and need for cooperation, as well as the possible benefit of apnea to facilitate punctures, general anesthesia was administered.The intervention begins with embolization of the right portal vein (RPV). A decision must be made as to whether access should be gained through the RPV, with the disadvantage of requiring a countercurrent catheter to deliver the embolization materials, or through the left portal vein (LPV), our preferred method, which, despite the manipulation of the future remnant liver, grants direct access to the RPV. The LPV was accessed under ultrasound guidance with a percutaneous access set, containing a 15-cm 22-gauge Chiba needle, a 0.018-in. nitinol guidewire, and a 0.038-in. introducer. The distal part of the LPV was punctured with the Chiba needle and opacified. The nitinol guidewire was then introduced through the needle to gain initial access. The Chiba needle was then removed, and the access set introducer was advanced over the wire, after which a stiff 0.035-in. hydrophilic guidewire was advanced over the introducer. A 5 Fr angiographic catheter was then advanced over the introducer and the stiff guidewire into the portal trunk, where portography was performed. We then performed selective catheterization of the segmental RPV and posterior embolization of the portal branches of segments V, VI, VII, and VIII, one by one with n-butyl-2-cyanoacrylate  and iodized oil , at a ratio of 1:2 or 1:3 when injected via a 4 Fr or 5 Fr catheter and 1:4 or 1:5 when injected via a microcatheter. That was followed by selective microcatheterization of the portal branches of segment IVa, distal embolization with microspheres, and proximal embolization with a metal coil .. A control CT with volumetric analysis of the liver (After RPV embolization, the hepatic vein embolization was initiated. The hepatic veins can be accessed via a transparietal approach or a transjugular approach. At our institution, the transjugular approach is preferred. An ultrasound-guided right internal jugular vein puncture was performed with a micropuncture access set kit. After the introducer was in place, a 5 Fr diagnostic catheter and hydrophilic 0.035-in. guidewire were passed through, RHV was catheterized, and pre-embolization venography was performed. A stiff Teflon guidewire was then introduced, and 7 Fr Multiport guide catheter was introduced up to the RHV. Through that guide catheter, an 18 mm × 22 mm vascular plug was deployed with its distal part 10 mm before the junction with the lower superior vena cava, to facilitate subsequent surgical ligature of the hepatic vein, and the RHV was embolized  and 3. Ihe liver  and 4 wa


Saliva plays a crucial role in oral cavity. In addition to its buffering and moisturizing properties, saliva fulfills many biofunctional requirements, including antibacterial activity that is essential to assure proper oral microbiota growth. Due to numerous extra- and intra-systemic factors, there are many disorders of its secretion, leading to oral dryness. Saliva substitutes used in such situations must meet many demands. This study was design to evaluate the effect of core-shell magnetic nanoparticles (MNPs) adding (gold-coated and aminosilane-coated nanoparticles NPs) on antimicrobial , rheological  and physicochemical  properties of three commercially available saliva formulations. Upon the addition of NPs (20 µg/mL), antibacterial activity of artificial saliva was found to increase against tested microorganisms by 20% to 50%. NPs, especially gold-coated ones, decrease the adhesion of Gram-positive and fungal cells by 65% and Gram-negative bacteria cells by 45%. Moreover, the addition of NPs strengthened the antimicrobial properties of tested artificial saliva, without influencing their rheological and physicochemical properties, which stay within the range characterizing the natural saliva collected from healthy subjects.  Increasing the use of drugs against hypertension, diabetes, allergies, and depression  reflectsAdditionally, current progress in cancer treatment is associated with an increasing number of immunocompromised patients, characterized by a higher risk of opportunistic infections. Aggressive chemo- and radiotherapy cause inescapable oral toxicity, manifested by disruption of the mucosal barrier, leading to the development of inflammation in the oral cavity such as mucositis, gingivitis, periodontitis and leukoplakia . In accoStaphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida spp, and Aspergillus spp. The main reasons for an increase of pathogenic bacteria content possessing the ability to form biofilm are irradiation-induced histological changes within oral mucosa as well as changes in salivary glands function [Imbalance in the microbiota and oral infections caused by opportunistic pathogens were reported as well. As described by Panghal et al. , the lisfunction . Indeed,function , which pfunction . It is wfunction . bacilli and Candida spp. with the number of chemotherapy cycles and combined cancer therapy. Additionally, apart from the secretion function of salivary glands, it is established that radio- and chemotherapy might cause the reduction of phagocytic activity of salivary granulocytes [A recent study  indicateulocytes . In effeIn light of the above-listed problem, the development of artificial saliva preparations to reduce symptoms of dry mouth and to improve the quality of patients’ life have been proposed by several investigators ,15,16,17Recent progress in the application of nanotechnology has efficiently accelerated the development of different industries ,24. AmonP. aeruginosa and C. tropicalis, where proliferation was classified at the level of 30% in comparison to control. The 50% inhibition was noted after incubation of evaluated substitute B with S. aureus, E. coli, and C. albicans. In other tested conditions, it was indicated that the addition of preparation A has a lower ability to restrict the proliferation of the oral pathogens, while in the case of preparation C, a lack of impact on cell division process has been noted. Importantly, in the case of S. mutans, which are known as crucial bacterial pathogens that are responsible for dental plaque formation, it was noted that the addition of all tested substitutes has no influence on the microorganisms‘ proliferation process. In turn, the evaluation of anti-adhesion properties of tested substitutes indicated that, among them, the best protective effect was noted for preparation B  might form the natural protective layer on the mucosal surfaces, preventing microbial colonization. Other components  have antimicrobial properties, prevent growing of harmful flora. Results of this study show that preparation B, based on xylitol, hydroxyethylcellulose and system of enzymes, exhibits the best antimicrobial activity, confirming previous reports ,34, indiHowever, to increase the activity of tested formulations, and to prevent microbial adhesion, processes that are required to achieve colonization and formation of dental plaque, and modification of the preparation content by the addition of core-shell magnetic nanoparticles was conducted (working concentration of 20 g/mL). Proposed nanostructures possess well-defined physicochemical properties and proven antibacterial activity. According to published literature ,36,37, tE. coli, and ~40% in the cases of P. aeruginosa and C. albicans, regardless of the kind of nanoparticles  has been observed for gold-coated nanoparticles. Importantly, both the NPs, at a concentration of 20 μg/mL, significantly inhibited the formation and viability of microorganisms’ biofilm in the presence of oral bacteria, if compared to preparation without magnetic nanoparticles. The results of microscopic observations of 48 h C. tropicalis biofilm shown in In physiological conditions, saliva plays a major role in determining the nature and activity of the oral microbiota. For this purpose, different mechanisms are engaged. Saliva controls the pH around neutrality, in effect creating an environment that is adequate for the growth of many oral microorganisms, and provides a relationship developing between the host and the resident microbiota. However, any perturbation in the flow of saliva, including xerostomia, might cause dysbiosis to occur rapidly . Oral cabacteria . As publIt is established that three major mechanisms of nanomaterials are responsible for their antimicrobial properties, including membrane damage and cellular uptake; releasing toxic metals, which can react with macromolecules, causing a loss of their function, and in effect restricting grow or killing microbes; and generating reactive oxygen species (ROS), which cause DNA, RNA, and proteins damage . Many reTaking into account that those saliva preparations play a different role in the oral cavity, their complex properties are important from a practical point of view. In the aspect of the dental practice, saliva is also responsible for the proper use of prosthetics and orthodontics elements and dental fillings, especially in the field of neutral pH and lubrication ability. Thus, in the second part of the work, the physicochemical and rheological properties were evaluated. As shown in 2 or MNP@Au nanoparticles was measured. As is shown in 2, and to the value of −30 mV for the substitute B modified with MNP@Au, was obtained. Simultaneously, some increase in particle size was observed—the addition of nanoparticles caused the formation of some aggregates (1400 nm for preparation B with MNP@NH2 and 750 nm for preparation B with MNP@Au). However, it does not cause the destabilization of saliva preparation, as a high value of zeta potential was preserved. The aggregation can be explained by the formation of new hydrogen bonds between saliva and MNP@NH2 or MNP@Au nanoparticles. The samples still possess free –OH groups that come from the magnetic core. Apart from the formation of some aggregates, the discussed samples are stable.The stability of the saliva and saliva modified by MNP@NHThe next of the tested physicochemical parameters, surface tension, in addition to rheological properties, are responsible for lubrication in the oral cavity. The surface tension of natural saliva was reported ,49 to raThe role of saliva in the oral health system is important. In addition to biological functions or related to maintaining homeostasis in the oral cavity, it is also responsible for reducing friction between surfaces in contact , due to The shearing curves of tested solutions at different shear rates, between 0.01 1/s to 200 1/s, are presented in The second stage of rheological tests involved viscoelasticity tests, with the goal to assess the elastic modulus. This storage module provides information on the conditions in which the molecular structure of biomolecules remains unchanged, due to the possibility of stress absorption. On the other hand, it represents the ability of a substance to accumulate energy during deformation. For natural saliva, this module is 3.5 Pa at 1 Hz . At workIn summary, it should be emphasized that the addition of NPs strengthened the antimicrobial properties of tested artificial saliva preparations. However, their physicochemical and rheological parameters relevant to the functional activity have been preserved.Magnetic nanoparticles with gold- or aminosilane shells were obtained as described previously, with some modification . In brieCore-shell nanoparticles were characterized using the following physicochemical methods, including FT-IR spectroscopy , differential scanning calorimetry  and thermogravimetric analysis , dynamic light scattering and zeta potential analysis , and transmission electron microscopy TEM/EDX . Data are already published . Three commercial saliva substitutes were tested. All preparations, according to the manufacturer’s information, are characterized by moisturizing and soothing dryness, as well as antimicrobial and refreshing properties. Next to inorganic compounds affecting physicochemical properties, in each preparation, there are organic active compounds responsible mainly for lubricating and antimicrobial properties. Especially in composition of substitute A, there are xylitol, poloxamer, hydroxyethylcellulose, and a system of three enzymes; in the composition of substitute B there are xylitol, glycerine, and cellulose gum; in the composition of substitute C there are sorbitol, xylitol and Yerba Santa extract. To evaluate the effect of tested artificial saliva preparations on the growth of oral pathogens, cells were suspended to an optical density at 600 nm of 0.1 in LB or BHI growth medium. Then, the cells (100 μL) were placed into 96-well microtiter plates containing tested preparations (100 µL) and resazurin was added at a final concentration of 5 µg/mL. Fluorescence was recorded using Varioskan LUX multimode microplate reader  for 6 h at 37 °C, with excitation/emission wavelengths of 520/590 nm.To determine the anti-adhesion activity of tested artificial saliva preparations with or without core-shell magnetic nanoparticles, the CV-staining method was used. We compared the abilities of tested strains to adhere to wells of polystyrene microtiter plates in the presence of tested artificial saliva preparations. In brief, pathogen cells were suspended to an optical density at 600 nm of 0.1. In the next step, the cells (100 μL) were placed into 96-well microtiter plates containing tested preparations (100 µL) and incubated for 2 h at 37 °C. Then, 1% crystal violet (CV) (25 μL) was added to each well, and the plates were incubated at room temperature for 15 min. After that, each well was carefully rinsed with water, and ethanol (200 μL) was added to extract the CV. Optical density (OD) was measured at a wavelength of 580 nm, using a Varioskan LUX multimode microplate reader .To assess the ability of the tested preparations to restrict the formation of biofilm, representative of oral pathogens were grown for 48 h at 37 °C in the presence of artificial saliva. After incubation, each well was washed with PBS to eliminate unbounded planktonic cells. Biofilm mass was assessed using the crystal violet CV-staining (0.1%) method. The unbound stain was rinsed off with deionized water, and then ethanol  was added and the optical density (OD) was measured at 580 nm using a Varioskan LUX multimode microplate reader .In other sets of the experiment, to evaluate the viability of cells embedded in biofilm matrix during the formation of biofilm in the presence of tested preparations, MTT test, which was based on the reduction of tetrazolium salt to unsoluble formazan, was performed. Biofilm was formed over the course of 48 h, then unbonded planktonic cells were carefully rinsed using PBS. In the next step, after 3 h of incubation, dye was resolved in DMSO and the plates were spectrophotometrically measured at a wavelength of 540 nm, using a Varioskan LUX multimode microplate reader .Streptoccocus mutans. Dental plaque was obtained from volunteers/patients. The trial was approved by the Human Research Ethics Committee of the Medical University of Bialystok, Poland . For both studies, all subjects provided informed written consent and collected samples were anonymous.To determine the ability of the tested artificial saliva preparations to restrict adhesion and formation of the biofilm in physiological conditions, measurements were performed in the presence of 50% human dental plaque containing Candida tropicalis 48 h biofilm were observed using confocal laser scanning microscopy (CLSM) LEXT OLS 4000 . Samples were observed just after being rinsed three times in pure water to remove free molecules from the polymer surface. Then, 2D surface reconstruction with biofilm was obtained in automatic mode. To obtain three-dimensional reconstructions of the biofilm structure, a sample scan was performed at a frequency of 0.05 micrometers in the Z-axis.Surfaces of the polymer samples with 2 nanoparticles in the presence of the following strains: Staphylococcus aureus, Streptococcus mutans, Pseudomonas aeruginosa, Escherichia coli, Candida glabrata, Candida tropicalis, and Candida albicans, was carried out, measuring the latter strains using 2′,7′-dichlorofluorescin diacetate (DFCH-DA) as a fluorescent probe. In the experiment, bacterial cells (OD600 = 0.1) and fungal cells (OD600 = 0.7) were pipetted into 96-well black plates. Then, nanoparticles  at concentrations 5, 20 and 50µg/mL were added to each well in the presence of artificial saliva. After that, the solution of 20 µM DFCH-DA in PBS was prepared. Finally, fluorescence was measured for 60 min, immediately after the addition of the dye at excitation/emission wavelengths of 488/535 nm.The assessment of ROS generation by MNP@Au and MNP@NHThe SevenMulti  multifunctional ionoconductometr with special electrodes was used for measuring pH and conductivity. Tests of surface tension were conducted with a platinum ring using the STA1 tensiometer . Both tests were performed at room temperature (21 ± 1 °C). The stability of saliva-MNP was studied using a Zetasizer Nano-ZS  equipped with a 4 mW helium/neon laser (l = 633 nm) and a thermoelectric temperature controller. All measurements were performed at 25 °C with a backscatter detection system at 173°. Rheological tests were carried out using the HAAKE Rheostress 6000 rheometer, with an automatic temperature module recognition Peltier system . Viscosity measurements of saliva preparations were performed in a cone-plate arrangement, where the diameter of the cone was 35 mm, and the angle between the slant height and the cone radius was 1 degree. The shear rate range in viscosity measurements was 0.1–200 1/s. The viscoelastic properties of the preparations were examined by the oscillation method using a system of two parallel plates, in which the diameter of the upper plate was 35 mm. Using the control strain mode, in the first step, the deformation range, in which the viscoelasticity modules are constant, was determined. On this basis, a frequency sweep in the range 0.1–10 Hz was carried out for the determined strain amplitude equal to 0.1. Rheological tests were carried out at 37 °C, where the maximum allowable temperature deviation was ±0.1 °C.Our results showed that the addition of NPs modulates the antibacterial and antifungal activity of saliva substitutes by decreasing microorganism adhesion and biofilm formation. Additionally, nanoparticles restrict the proliferation of bacterial planktonic cells, as well as the viability of cells embedded in the biofilm matrix. Moreover, the physicochemical and rheological properties of tested saliva preparations with the addition of nanoparticles were not changed, indicating that nanoparticles will likely not interfere with the biophysical homeostasis of the oral cavity.