Leishmania infantum and the characteristics of the immune response to this infection remains limited, contrary to the dog. This study aimed to compare the rate of L. infantum infection and parasite-specific humoral and cell-mediated immune responses in cats and dogs living in an area endemic to canine leishmaniosis . About one-third of the dogs and cats studied were positive for at least one molecular or serological diagnostic test. The immunopathogenesis of L. infantum infection in cats showed similarities when compared to dogs, although the parasite-specific immune response level in dogs was generally higher than in cats. This study shows that stray cats are exposed to L. infantum infection similarly to dogs in endemic areas, are able to mount a specific anti-Leishmania humoral and cell-mediated immune response as dogs, and can contribute to the endemicity of infection.The role of the cat as a reservoir of Leishmania infantum and display different immunological patterns correlating with the progression of infection to disease. Data about feline L. infantum adaptive immune response are scant. This study aimed to compare the prevalence and immune response in cats and dogs from the same endemic area of canine leishmaniosis. Stray cats (109) and rescued dogs (59) from Córdoba (Spain) were enrolled. Data about their exposure to L. infantum were analyzed by detection of parasite DNA, measurements of Leishmania-specific interferon-γ (whole blood assay in 57 cats and 29 dogs), and antibodies (enzyme-linked immunosorbent assay and immunofluorescence antibody test). An overall L. infantum prevalence of 30.5% in dogs and 30% in cats were found according to serology and PCR tests. Prevalence was 44.8% in dogs and 35.1% in cats tested also for interferon-γ production. Dogs showed higher anti-L. infantum antibody levels compared to cats. More than one-third of cats had contact with or were infected by L. infantum and they may contribute to the endemicity of leishmaniosis in the investigated region. The immunopathogenesis of feline L. infantum infection has similarities with dogs but cats show a lower level of adaptive immune response compared to dogs.Dogs are the main reservoir of  Leishmania infantum is the most common species of the Leishmania genus in Europe, responsible for a neglected vector-borne zoonosis [Phlebotomus and dogs are considered the principal reservoir of this infection [L. infantum (Li) is endemic with a reported prevalence of dogs with anti-Leishmania antibodies ranging from 0 to more than 50%. Higher rates of positivity have been reported in studies where the parasite DNA was investigated in various canine tissues by polymerase chain reaction (PCR) [Leishmania antibodies has been found to range between 0 and 68.5% [Leishmania-specific interferon-γ (IFN-γ). This immune response enables the host to fight the parasite and control the progression of the disease [L. infantum antibody and parasite DNA detection in blood [L. infantum exposure in cats [L. infantum rates of infection or exposure in dogs and cats in the same period and area [zoonosis . Vectorinfection . In manyon (PCR) . Other don (PCR) ,5, horseon (PCR) , and haron (PCR) ,8,9. Thend 68.5% ,11,12,13nd 68.5% . Furthernd 68.5% ,16,17. Dnd 68.5% . Briefly disease . Convers disease ,23,24,25 disease ,27. In pand area . Accordiand area . The maiFrom May to June 2017, 168 rescued and stray pets (109 stray cats and 59 rescued dogs) from the province of Córdoba (Spain) were enrolled. Stray cats were included during a trap-neuter-release program organized by Córdoba City Council and examined by veterinarians before the elective surgery. Dogs were enrolled in three different kennels located in the suburban area of Córdoba during the annual check-up program for leishmaniosis. Inclusion criteria for enrollment aimed to study dogs and cats at risk of exposure to sand fly bites focusing on animal age, outdoor lifestyle, and treatment against sand fly bites. Therefore, we studied only adults, exposed to at least one (cats) or two (dogs) sand fly seasons, and they had not been treated with ectoparasiticides effective against sand fly bite at least in the last two years. Moreover, as they were kennel dogs and stray cats, all enrolled animals shared an outdoor lifestyle.L. infantum PCR. Blood smears were immediately made and afterward stained by May Grünwald-Giemsa stain  and evaluated microscopically at oil immersion ×1000 magnification for the detection of hemoparasites including Li amastigotes [g for 10 min to obtain serum for serological investigations . The WBA and CBC were performed within 24 h after sampling. EDTA blood was stored at +4 °C until used and was brought at room temperature before analyzing. Blood serum and residual EDTA blood were stored at −20 °C until processed for serological  for IFN-γ release whole blood assay (WBA) and EDTA tubes (1 mL) for complete cell blood count (CBC) (29 dogs and 57 cats) and ical see  and PCR ical see . The CBCA full clinical examination was performed by veterinarians on 57 cats and 29 dogs tested for WBA. Data about body condition score (BCS = 5/5) ,32, muscL. infantum antigens (LSA) and (3) medium with mitogen concanavalin A (ConA) . Concentrations of IFN-γ were determined using canine and feline commercial sandwich enzyme-linked immunosorbent assay (ELISA) for IFN-γ  as previously described [The WBA was performed in dogs (n = 29) and cats (n = 57) as previously described ,23. Threescribed ,23. Accoescribed  and dogsL. infantum antigen were evaluated by both immunofluorescence antibody test (IFAT) and ELISA. A Leishmania IFAT was performed as previously described for dogs [Serum antibodies against for dogs ,36 and cfor dogs . The cutfor dogs , and 1:1for dogs . The endfor dogs  and catsfor dogs . All sam®, INGEZIM FIV, Madrid, Spain) was used and the manufacturer instructions were followed. A ThermoScientific Multiskan FC spectrophotometer was used for optical density readings.A commercial ELISA  that were negative by serological, parasitological, and molecular methods was used as a negative control. The PCR test was targeted at the constant region in the minicircle Kinetoplast DNA (NCBI accession number AF291093). Real Time PCR was developed by the CFX96 Real-time System  using TaqMan Master Mix (Applied Biosystems by ThermoFisher). The procedure was performed as previously described [Total DNA was extracted from EDTA whole blood using the DNA Gene extraction kit  following the manufacturer’s instructions with the following modification: 40 μL of proteinase K solution and 400 μL of whole blood were used for all extractions. DNA from the reference escribed .L. infantum infection among cats and dogs based on the diagnostic test used were analyzed by using the odds ratio (OR) as a measure of association and the associated 95% confidence interval (CI). Spearman’s correlation coefficient was calculated to evaluate relationships between ELISA, IFAT, and PCR results and between levels of IFN-γ, anti-Leishmania antibodies, and L. infantum DNA in samples of animals studied. Finally, Cohen’s kappa coefficient was measured to evaluate agreement between IFAT and ELISA.Statistical analysis was conducted using GraphPad Prism version 7.0 for Windows  and STATA software . After confirming the non-normality of the data through the Skewness/Kurtosis test, continuous data were reported as median value, range, 25° and 75° percentile and categorical data were expressed as frequencies. Comparison between the distribution of studied variables , between cats and dogs was conducted using Fisher’s Exact test for categorical variables. In animals tested for IFN-γ- release whole blood assays (29 dogs and 57 cats) clinical status, and presence of clinical signs or hematology abnormalities compatible with leishmaniosis were also investigated using Fisher’s Exact test. Statistical analysis of the clinical status was performed comparing two groups of animals: those in “very good” and “good” health status versus the animals in “poor” and “very poor” conditions. Mann–Whitney’s U-test was used to compare unmatched continuous variables  between cats and dogs. Wilcoxon’s signed-rank test was used to compare paired continuous variables (LSA and ConA IFN-γ) in the same species. The risk factors for the development of p values were <0.05.Differences were considered significant if p = 0.0004; OR: 5.6944, 95% CI: 2.1456–15.1132). All blood smears of cats and dogs tested with CBC were found negative for Li amastigotes and hemoparasites.The breed, sex, and age class of both cats and dogs are reported in p = 0.0020; Rho = 0.8988) between ELISA and IFAT where the Cohen’s kappa coefficient (0.708) found a substantial good agreement. No agreement between IFAT and ELISA was detected in cats and correlation was not evaluated considering that only one cat was positive by both techniques. In clinically evaluated dogs and cats, no differences in antibody positivity were found based on their health status and the presence of clinical signs compatible with leishmaniosis.Results of serological tests and their differences among dogs and cats are displayed in p = 0.0009; OR = 23.5; 95% CI = 3.108–127.8) or IFAT negative  dogs. No statistical difference was found between IFAT-positive cats compared to the negative ones and all PCR-positive cats were ELISA-negative. In clinically evaluated dogs and cats, no differences in PCR positivity were found based on their health status and the presence of clinical signs compatible with leishmaniosis.Results of PCR tests of dogs and cats are shown in Results of IFN-γ release whole blood assay are shown in No differences were found in antibody levels (IFAT) between dogs and cats that did not produce IFN-γ after LSA stimulation.As reported in Moreover, no differences in parasite loads were found between IFN-γ producer and non-producer dogs and cats. Data about serological and molecular results of IFN-γ producer and not producer dogs and cats are represented in No differences were found in IFN-γ production after LSA stimulation in dogs and cats according to their health status and the presence of clinical signs compatible with leishmaniosis.p = 0.0113) in ELISA negative dogs  when compared to ELISA positive dogs . Conversely, the difference was not significant in cats. No more differences were found in IFN-γ production after ConA stimulation according to IFAT titer, parasite load, LSA-IFN-γ, health status, and the presence of clinical signs compatible with leishmaniosis. The levels of ConA-IFN-γ were statistically higher compared to those obtained after LSA stimulation in dogs . All FIV-positive cats were L. infantum ELISA or PCR-negative and only three cats were IFAT-positive (titer 1:80) but no significant difference was found between FIV antibody-positive and negative cats about L. infantum positivity at IFAT. Among the ten FIV-positive cats, LSA and ConA-IFN-γ production were evaluated in only four cats. Production of IFN-γ was observed in only one cat after LSA stimulation and in all four cats after ConA stimulation.Ten cats were antibody-positive to FIV (9.2%). All FIV-positive cats were adult and more frequently male cats  compared to female positive cats ,  and sampled during the same sand fly season. In the current study, the level of dog’s parasite-specific immune response was generally higher compared to cats. In particular, although the proportions of antibody-positive individuals were similar (24.3% of cats and 27.1% of dogs), anti-L. infantum antibody levels were significantly higher in dogs than in cats for both IFAT and ELISA. Moreover, although the difference was not significant, 20% of dogs produced IFN-γ compared to 16% of cats, and the median concentration of IFN-γ produced by dogs was up to four times higher compared to cats. At the same time, parasite DNA was found in 11.9% of dog and 8.3% of cat blood samples studied. Similarly, Otranto et al. (2017) found a higher prevalence of antibody and/or PCR positivity in dogs compared to cats examined in the Eolian islands [L. infantum direct analysis more frequently than cats and they also displayed a higher parasite load. On the other hand, cats showed antibody prevalence (ELISA test) and levels of antibody positivity higher than dogs [This cross-sectional study compared for the first time, the adaptive humoral and cell-mediated immune response to  islands . In Israhan dogs .L. infantum using IFN-γ assays in dogs [L. infantum IFN-γ production and progress to more severe disease [To date, few field studies investigated the role of cellular immunity against  in dogs ,23,25,41 disease ,23,41,42Another important finding of this study is the occurrence of PCR-positive but antibody and IFN-γ negative animals. This pattern can be explained as an early phase of infection that occurred during the sand fly season when we performed the study and adaptive immune response was not yet elicited, or the infection did not progress in these hosts as it was already reported in dogs . At presL. infantum infection in cats shows similarities when compared to canine response, and cats can mount a specific Th1 immune response against L. infantum. Cats with high antibody levels or with positive blood PCR are less able to produce specific IFN-γ [In summary, the immunopathogenesis of ic IFN-γ . Howeveric IFN-γ . UnfortuL. infantum was found in both species, with 30.5% of shelter dogs and 30% of stray cats antibody-positive and/or blood PCR positive. Importantly, the rate of exposure was higher when L. infantum-specific IFN-γ production was measured in both dogs (44.8%) and cats (35.1%). In fact, some dogs (n = 5) and cats (n = 5) negative for anti-L. infantum antibodies and parasite DNA in the blood produced L. infantum specific IFN-γ in the WBA. Interferon-γ is a marker of Th1 immune response, which is prevalent in “resistant” dogs. Some of these dogs do not produce antibodies or have very low levels . However, parasite DNA could be detected in some of these individuals when other tissues are tested, such as bone marrow and lymph nodes [Li [Li in dogs and cats, the combination of results from parasitological (PCR) and multiple immunological markers (specific antibodies and IFN-γ) increase diagnostic sensitivity. This result confirms the endemicity of leishmaniosis in the investigated region and put in evidence that more than one-third of stray cats studied had contact with or were infected by L. infantum. The high prevalence found in cats from the Córdoba area is very similar to those found in the previous study we conducted in Sicily (35%) and Catalonia (36%) [A high prevalence of exposure to ph nodes ,44. We mL. infantum was studied in different Spanish areas and other European countries (Stray cats can potentially be more exposed than owned cats due to the outdoor lifestyle and the lack of preventive sand fly bite treatment and for these reasons their exposure to ountries  [45,46,4Agreement between the two serological methods used was found in dogs but not in cats, and this discrepancy is not unusual and has already been reported by us and also in other studies ,10,53.L. infantum infection [L. infantum infection in cats [Leishmania ELISA, while three were border-line positive at IFAT. We did not find significant differences in IFN-γ production after stimulation with LSA in FIV-positive cats, but the sample size of examined cats was small and we had the opportunity to evaluate IFN-γ production in only four FIV-positive cats, and three of them did not produce IFN-γ. On the other hand, in a previous study, we tested a FIV and FeLV sick cat with positive PCR to L. infantum which had also high levels of antibodies and production of LSA-IFN-γ [L. infantum infection in cats and more extensive investigations are needed [Retroviral infections caused by FIV and FeLV have been associated in some studies with nfection ,59,60,61 in cats ,62,63. ISA-IFN-γ . No defie needed .L. infantum infection endemicity. Furthermore, the lack of preventive treatments due to financial restrictions and particularly long sheltered periods that became years, increased the endemicity as was observed in this study [L. infantum infection similarly to dogs in endemic areas. In light of some considerations, cats can contribute to endemicity since they are exposed to sand fly bites as much as dogs [In an urban context, shelter dogs play a crucial role in the maintenance of is study ,65,66. T as dogs ,28,67,68 as dogs ,69 causiL. infantum DNA in dogs and probably in cats too, from blood is less sensitive than from other tissues [This study presents some limitations that could influence results and preclude a more robust interpretation. It was a cross-sectional study, and the number of dogs studied was small, as well as the number of animals tested for IFN-γ production. Another limitation of this study was that most dogs studied were Greyhound, and we cannot exclude breed differences in the levels of IFN-γ production. Additionally, we could not make a full clinical and clinicopathological evaluation of enrolled dogs and cats. Moreover, parasite load was measured using blood samples; thus, some infected animals were possibly missed since the detection of  tissues ,2. As coL. infantum and are able to mount a specific anti-L. infantum cell-mediated immune response which is similar to dogs. However, a higher proportion of kennel dogs had contact with the parasite. More extensive clinical investigations evaluating immunological markers of the innate and adaptive immune response are needed to better understand the susceptibility of cats to develop the severe disease compared to dogs and the role of retroviral infections.In conclusion, this study proved that in an endemic area, a consistent proportion of stray cats are exposed to


This study aimed to evaluate the association between birth weight (BW), childhood and adolescent BMI, with reproductive capacity in men.A prospective, population-based cohort study (Northern Finland birth cohort 1966).n = 2041), 4128 men were included in the final study population. Results were adjusted for BW, BW for gestational age (GA), mother’s smoking status, marital status, educational level and smoking status.Around 6196 men born in 1966 were followed from birth to age 50 years. Weight and height were measured repeatedly by professionals. Reproductive capacity  was evaluated by questionnaires at ages 31 and 46 years. The number of children by the age of 50 years was recovered from registers. After excluding the men who reported never having attempted to have children or not answering the question at age 31 or 46 years (P = 0.012) or having a lower BW  were associated with childlessness. The association was however no longer significant after adjusting for marital status. Being underweight in early childhood was associated with an increased risk of infertility assessment ) and childlessness (aOR: 1.47(1.01–2.17)) compared to the normal weight group. Conversely, overweight or obesity in early childhood was associated with a decreased risk of infertility assessment (aOR: 0.60 (0.41–0.87)), treatment (aOR: 0.42 (0.25–0.70)) and male factor infertility (aOR: 0.45 (0.21–0.97)). BMI in mid-childhood or puberty had no association with infertility or childlessness.Being small for GA (10.5% vs 8.2%, In boys, an optimal growth trajectory during pregnancy and early childhood seems to be very important for life-long fertility. Obesity is a worldwide pandemic, and the increasing rate of childhood obesity is especially concerning as it increases the risk of long-term health problems , 2. AdulTo our knowledge, only two previous studies have been conducted in adolescent boys, showing an inverted J-shaped relationship between BMI and later reproductive capacity , 14. StuThis study is an extension of our previous study on women’s childhood growth and fertility . The aimThe study population was drawn from a general population birth cohort, the Northern Finland Birth Cohort 1966 (NFBC1966), recruited at gestational week 24 from the two northernmost provinces of Finland. The study included a total of 12 058 live births , covering 96% of all births in this area . The stuThe questionnaires for ages 31 and 46 years included questions on lifestyle, education, family history, health and fertility , male factor infertility (n = 111), infertility treatment (n = 259), childlessness (n = 969) and the reference group which comprised men without any fertility problem (n = 2856) (The outcomes concerning infertility were defined based on the answers to the questionnaires at 31 and 46 years (Supplementary Table 1). The number of childbirths recorded up to the end of 2016 (when the men turned 50 years) was obtained from the Finnish Medical Birth Register, which was established in 1987. Birth data before 1987 were collected from the Population Register Center live births to make the birth data complete. The final study population was divided into five groups: infertility assessment ( = 2856) . Men witn  = 2725) and 12 months (n  = 3513) was analyzed separately. Data on weight and height from infancy to adolescence were collected from measurements reported by the child health and welfare nurses, and later by the school nurses, as part of the national child health program, which is free for all children in Finland.The weight and height of the children aged 6 and 12 months were measured by experienced nurses. The BMI of children aged 6 months (n  = 2603), mid-childhood  and puberty , according to the literature regarding childhood growth  and AR (n = 2922) was made only for the children having at least three measurements during childhood.The timing of AP and AR was derived from fitted growth curves, as described previously, and for each participant, predicted BMI at AP and AR was calculated using the estimated fixed and random coefficients . The norn = 3644) and used only for continuous analyses. There was no statistical difference between measured (n = 2401) and self-reported BMI at age 14 years . Height and weight at age 31 years were measured at the clinical examination. If measurements were not available, self-reported data were used. There was no statistically significant difference between self-reported (25.51 kg/m2) and clinically measured  BMI.Height and weight at age 14 were all measured and reported by the parents (www.dagitty.net).Prenatal factors , birth weight (BW), birth length (BL), BW−BL ratio, BW for gestational age (GA) and GA were included in the background factors as they are known to be associated with childhood growth  and chilt-test, a Mann–Whitney U test, a one-way ANOVA or a Kruskal–Wallis test, as appropriate. To assess differences in the categorical parameters, a chi-square test was used. For these tests, results were reported as means or medians, standard deviations (s.d.) or prevalence (%) and odds ratios (OR) with a 95% CI, respectively. A P-value <0.05 was considered statistically significant. Bonferroni correction was used for additional analyses in different age groups. Multivariable analyses were conducted using binary logistic regression modeling. The results were reported as OR with 95% CI. Linear and quadratic associations between the number of children (the birth of the sixth or any subsequent child was not considered since only 2.9% of the participants had more than six children) and BW and BMI at 14 years were assessed with regression models. IBM SPSS Statistics for Windows, Version 25.0.  was used to assess differences between the study groups and to perform the regression analyses.Differences in the continuous variables were analyzed by an independent The number of participants varied between each age group and different fertility outcomes, owing to variations in the number of answers to each question and/or participation in the clinical examination . In all,SGA: 1.39 (1.06–1.83), P = .019) (BW: 0.98 (0.97–1.01), P =.124), between BW−BL ratio and childlessness (aORBW–BL ratio: 0.99 (0.98–1.01), P = 0.345) and between SGA and childlessness (aORSGA: 1.23 (0.88–1.72), P = .210) lost their significance. A linear trend for a positive association between BW and number of children remained, even after adjusting for marital status in the regression analysis  . BW and  = .019)  and BW w = .019) . After aanalysis .Figure BMI at ages 6 and 12 months did not associate with infertility assessment, treatment, male factor infertility or childlessness by age 50 (Supplementary Table 2).Lower BMI at AP, but not age, was associated with childlessness, but the significance was lost after adjusting for marital status. BMI at AR did not associate with infertility variables or childlessness. Age at AR did not associate with infertility variables, but AR at an earlier age associated with an increased risk of childlessness after adjustments .Table 2Only 5.4% of boys who were underweight in early childhood and 23% of boys who were obese in early childhood remained underweight or obese in adulthood, respectively.P = .002; 0.45 (0.21–0.97), P = .040 and 0.42 (0.25–0.70), P = .001, respectively). There was neither association with childlessness (aOR: 0.93 (0.69–1.25), P = .643) nor any significant difference in number of children when compared to normal weight boys .Being underweight in early childhood was associated significantly with childlessness  and withAmong boys in mid-childhood, 4.5% of those who were underweight and 31.7% of those who were obese remained underweight or obese in adulthood, respectively. Weight class in mid-childhood had no significant association with infertility assessment, male factor infertility, infertility treatment, childlessness  or numbeAmong boys in puberty, 3.5% of those who were underweight and 41.1% of those who were obese remained underweight or obese in adulthood, respectively. Weight class in puberty did not associate with infertility assessment, male factor infertility or infertility treatment, childlessness  or numbeIn this population-based cohort study, our main observation was that boys who had a lower BW, who were born SGA and those who were underweight during early childhood had an increased risk of involuntary childlessness as adults. Furthermore, these boys had fewer children in adulthood. Especially association between BW/SGA and childlessness was highly influenced by the decreased probability to have a partner. In contrast, being overweight or obese during early childhood was associated with a decreased risk of infertility assessment, male factor infertility and infertility treatments in adulthood. According to our results, BMI after mid-childhood was not associated with infertility.Our study involved a large, population-based cohort and incorporated data from birth until age 50 , as such, provided a unique opportunity to investigate associations between childhood growth and several fertility parameters in adulthood. The study population was ethnically homogeneous. The data on births were reliable as they were derived from the Finnish Medical Birth Register and the Population Register Centre, which together account for all births in Finland. All measurements from birth until adolescence – except BMI at age 14, which was used for curve estimation – were performed by trained professionals at every stage. Additionally, men who reported never having attempted to have children were excluded. Another novel contribution of the present work was that, in addition to analyzing specific childhood and adolescent age groups, childhood growth trajectory data and important physiological landmarks (AP and AR) were also assessed.in utero, especially men born SGA, were more likely to have decreased sperm counts  and the lack of longitudinal data from AR onwards is a limitation of this study. As underweight and obesity were defined by the presence of at least one BMI value under or over the 95th pc in any age group, respectively, there was likely to be an over-diagnosis, especially in early childhood. Also, BMI does not remain constant throughout life and is especially prone to fluctuations from birth until the age at which AR occurs , Center of Excellence in Complex Disease Genetics and SALVE, the Sigrid Juselius Foundation, Biocenter Oulu, University Hospital Oulu and University of Oulu (75617), Jalmari ja Rauha Ahokkaan säätiö¨, The Finnish Medical Foundationhttp://dx.doi.org/10.13039/100001236, Medical Research Center Ouluhttp://dx.doi.org/10.13039/100016332, National Institute for Health Researchhttp://dx.doi.org/10.13039/100005622 (UK), The European Union’s Horizon 2020 research and innovation program (under Grant agreement no. 633595 for the DynaHEALTH action and GA 733206 for LifeCycle). The funders had no role in study design, in the collection, analysis and interpretation of the data, in the writing of the article.NFBC1966 received financial support from University of Oulu Grant no. 65354, Oulu University Hospital Grant no. 2/97, 8/97,


The PWS group that maintained recombinant human growth (rhGH) treatment in adulthood had a lower probability of having a BMI ≥ 30 at the last follow-up (odds ratio = 0.106 (0.012–0.948), p = 0.045). Endocrine and metabolic illnesses in individuals with PWS may have already started in the early teens; therefore, appropriate screening and early intervention are important. Better understanding of the natural history of PWS and age-related complications will lead to better-quality medical care for individuals with PWS.Prader–Willi syndrome (PWS) is a rare genetic disorder characterized by an insatiable appetite that leads to morbid obesity. Previous studies reported health problems in adults with PWS. However, studies on younger adults are lacking, and there are no specific studies of endocrine and metabolic illness in this age group. We performed a retrospective cohort study of 68 individuals with PWS aged 19 to 34 years at Samsung Medical Center. The prevalence of endocrine and metabolic illnesses were compared with those in an age-, sex-, and BMI-matched healthy control group. Young adults with PWS had a higher prevalence of metabolic syndrome (35.3% vs. 4.4%), type 2 diabetes mellitus (50.0% vs. 5.4%), hypertension (30.8% vs. 16.1%), dyslipidemia (38.2% vs. 14.7%), decreased bone density (26.4% vs. 0.9%), and sleep apnea (32.3% vs. 4.4%) than controls (all  Prader–Willi syndrome (PWS) is a rare genetic disorder characterized by muscular hypotonia, dysmorphic features, short stature, low lean body mass, mental retardation, behavioral abnormalities, and progressive obesity owing to hyperphagia . The benWe reviewed the medical records of adults with PWS diagnosed by clinical criteria and methylation PCR between March 1994 and May 2021 at Samsung Medical Center. Adults aged 19 to 34 years at the last follow-up were selected; 68 subjects were included in this study. All subjects were Korean individuals with PWS who visited our hospital on a six-monthly basis. To compare the prevalence of endocrine and metabolic illnesses, we collected a healthy control group with matching age-, sex-, and BMI groups. A total of 204 individuals (aged 19–34 years) were selected from among patients who visited the Department of Family Medicine or the Examination Center. None of these individuals had genetic syndromes or chronic disorders. In addition, the group with history of rhGH treatment, the group that continued to receive rhGH treatment after adulthood, and the group that had never received rhGH treatment were compared within our PWS cohort.2; overweight, 23.0–24.9 kg/m2; obese class I, 25.0–29.9 kg/m2; obese class II, 30.0–34.9 kg/m2; obese class III ≥ 35 kg/m2. Blood samples were collected in the morning in the fasting state for the measurement (in serum) of lipids, glucose, insulin, hemoglobin A1c (HbA1C), free T4, T3, TSH, ACTH, and cortisol. All laboratory data were analyzed at Samsung Medical Center.Medical and biometric data, including height, weight, vital signs, physical examination findings, blood tests, spine X-ray findings, dual energy X-ray absorptiometry (DXA) findings, and polysomnography (PSG) findings were collected from medical records. Subjects were categorized into one of five groups according to body mass index (BMI) following the World Health Organization’s recommendations for Asians: normal weight, 18.5–22.9 kg/mADA guidelines were used to diagnose diabetes mellitus. Hypertension was defined as a systolic blood pressure (SBP) ≥ 140 mmHg or diastolic blood pressure (DBP) ≥ 90 mmHg or on an anti-hypertensive medication. Dyslipidemia was defined as taking a medication for uncontrolled lipid abnormalities despite lifestyle changes. Individuals who met any of the following lipid abnormalities were defined as having dyslipidemia: (1) Triglycerides ≥ 200 mg/dL, (2) LDL-C ≥ 160 mg/dL, and (3) HDL-C ≤ 40 mg/dL .2; (ii) dyslipidemia; and (iii) hypertension or on an anti-hypertensive medication.Metabolic syndrome was defined as a requirement for fasting glucose ≥110 mg/dL (impaired fasting glucose or diabetes mellitus), plus at least two of the following: (i) obesity, defined as a BMI; ≥25 kg/mPrimary hypothyroidism was defined as a TSH level ≥ 10 μIU/mL with a free T4 level of <0.75 ng/dL. Central hypothyroidism was defined as a free T4 level of <0.75 ng/dL and non-elevated TSH levels. Secondary hypothyroidism was indicated by a low basal serum free T4 level with an inappropriately normal or low TSH level (not increased by >5 mU/L) in a thyrotropin-releasing hormone stimulation test. Hyperthyroidism was defined as a TSH level of ≤0.10 μIU/mL and elevated free T3 and/or free T4 levels. Decreased bone density was defined as a T-score  below −2.5 on DXA in patients who underwent DXA at 19 years or older than 19 years, and as a Z-score (adjusted by age- and gender-matched standards for Korean children) below −2.0 in patients who underwent DXA when younger than 19 years of age [PSG was performed in patients who showed symptoms suspicious of sleep apnea and who were cooperative when conducting the test. Sleep apnea was classified based on the apnea–hypopnea index, i.e., the mean number of apneas and hypopneas per hour of sleep. An apnea–hypopnea index of 5–15 per hour was classified as mild sleep apnea, 15–30 per hour as moderate sleep apnea, and >30 per hour as severe sleep apnea.The presence of central adrenal insufficiency was investigated by estimation of serum ACTH and cortisol levels in blood samples obtained in the early morning and by low-dose ACTH stimulation test, when needed. The cutoff level for appropriate cortisol response was accepted as 18 mcg/dL. 2 per day (not exceeding a maximum of 2.7 mg per day). After reaching the final height or epiphyseal closure, if growth hormone deficiency was confirmed through growth hormone provocation test, the treatment for adult growth hormone deficiency was maintained. Adult rhGH doses ranged from 0.4 to 0.5 mg per day [In our cohort, the dose of rhGH in patients receiving rhGH treatment was 1.0 mg/m per day . rhGH trp value < 0.05 was considered statistically significant. Baseline characteristics are presented as the number of frequency (percentage) for categorical variables and the mean ± SD and range for continuous variables. Chi-square test and Fisher’s exact test were used to determine the significance of differences in the proportions of endocrine and metabolic illnesses between the PWS and control group. Comparisons between groups divided by rhGH treatment status were performed using Fisher’s exact test for categorical variables and one-way ANOVA for continuous variables. Crude and adjusted logistic regression analyses were performed to assess the associations between rhGH treatment and each one of the endocrine and metabolic illnesses. All statistical analyses were performed using SPSS 27 . A two-sided Demographic and clinical characteristics are presented in p < 0.05), but there was no significant difference in thyroid disorders between the two groups  years. Hypertension was diagnosed in 21 of the 68 patients. Mean age at diagnosis of hypertension was 22.20 (SD 4.24) years. Medications included calcium-channel blockers (five patients), angiotensin II receptor blockers (seven patients), and angiotensin converting enzyme inhibitors (nine patients).Twenty-six patients took medications for hyperlipidemia. Mean age when medications to treat this were started was 17.74 (SD 3.83) years. There were 16 patients with combined hyperlipidemia, 8 patients with isolated hypercholesterolemia, and 2 patients with isolated hypertriglyceridemia. Among these patients, 11 patients had hypoalphalipoproteinemia. Twenty-four patients took an HMG-CoA reductase inhibitor, and one patient also took omega-3 fatty acids. The other two patients took only omega-3 fatty acids.Most patients in our cohort had normal thyroid function. Two females had primary hypothyroidism with TSH levels ≥ 10 μIU/mL and free T4 levels < 0.75 ng/dL at diagnosis age of 19 years and 14 years, respectively. Their thyroid antibody test was negative. There was no hyperthyroidism in our cohort.DXA was performed in 41 patients. Eight patients (26.4%) had decreased bone density. Mean age at diagnosis of decreased bone density was 15.18 (SD 3.36) years. Among patients with decreased bone density, four males had hypogonadism, but hormone replacement was not performed due to concerns about possible behavioral-related side effects.p < 0.05).PSG was performed in 34 patients. Sleep apnea was confirmed in 22 patients, and the mean age at diagnosis of sleep apnea was 15.35 (SD 6.59) years old. Mean total sleep apnea–hypopnea index was 26.94 (SD 29.57) per hour. Six of the patients with sleep apnea received CPAP treatment. One of these patients was treated with uvulo-palato-pharyngoplasty, radiofrequency tongue base volume reduction, and palatine tonsillectomy due to severe sleep apnea. Echocardiography was performed in 10 patients with sleep apnea. Among them, one patient had an increase in right atrial pressure up to 15 mmHg and RV dilatation, while another patient had decreased RV systolic function and severe pulmonary hypertension. Patients with sleep apnea had a significantly higher BMI than patients without sleep apnea (42.99 (SD 14.15) vs. 30.73 (SD 7.89), respectively, Adrenal function was evaluated in 30 patients (44.1%); all of them were found to have sufficient hypothalamic–pituitary–adrenal axis function and no indication for perioperative hydrocortisone treatment.In our study, 58 patients were treated with rhGH since childhood. Of these, 48 patients discontinued treatment at a mean age of 14 (SD 2.4) years, and 10 patients continued to receive adult rhGH treatment. One patient was diagnosed with PWS at the age of 21 and was subsequently treated with adult rhGH treatment. Among the patients treated with rhGH treatment, six patients discontinued rhGH treatment prematurely due to adverse events. Four patients discontinued due to uncontrolled hyperglycemia, and two patients discontinued due to worsening of the scoliosis.A comparison of demographic, metabolic and endocrine illnesses in young adults with PWS according to rhGH treatment status and comparison of age-, sex-, and BMI-matched healthy control group with each group are shown in p = 0.045).Crude and adjusted logistic regression analyses were performed to assess the associations between rhGH treatment status and each one of the endocrine and metabolic illnesses b. ThyroiThis is the first study to investigate endocrine and metabolic illnesses in adults with PWS in Korea. In addition, this is the first study to focus on young adults with PWS worldwide. In this study, young adults with PWS had a higher prevalence of metabolic syndrome, T2DM, hypertension, dyslipidemia, decreased bone density, and sleep apnea than an age-, sex-, and BMI-matched healthy control group. Mean age at diagnosis of these problems ranged from the late teens to the early twenties, emphasizing the importance of appropriate screening and intervention during this period. The high morbidity and mortality rates associated with PWS are related to severe obesity as a result of hyperphagia . TherefoSevere obesity is a significant risk factor for developing T2DM in individuals with PWS as in other populations. Perturbations in glucose metabolism occur in PWS individuals during the transition phase to adulthood ,13. In oDifficulties in compliance with diet and food compulsivity can explain the severe uncontrolled diabetes in some patients. In our study, there were more cases of glucose control with a combination therapy with insulin or other oral hypoglycemic medications than with metformin monotherapy. Furthermore, 10 patients were treated with a combination therapy of a glucagon-like peptide 1 (GLP-1) receptor agonist due to uncontrolled diabetes. A recent systematic review has shown that GLP-1 receptor agonists are safe in patients with PWS and may potentially help with weight reduction, blood sugar control, and appetite control . In our Hypertension and dyslipidemia were highly prevalent in our cohort, with prevalence rates higher than those reported previously ,16. OldeHypothyroidism was found in only two (2.9%) patients in our cohort, which was not a significant difference in frequency from that in the healthy control group. Thyroid axis dysfunction is a frequent feature during infancy in individuals with PWS . TherefoBoth low BMD and increased fracture risk have been described in adults with PWS . Our stuThe higher risk of central sleep apnea in individuals with PWS is associated with hypothalamic dysfunction . HoweverCentral adrenal insufficiency can be a part of PWS. Clinicians need an evaluation of adrenal function in patients with PWS. However, the frequency of central adrenal insufficiency in patients with PWS is not clear, and the frequency varies widely between studies ,27,28. IrhGH treatment has become the standard of care in PWS children, even in those who do not have proven GH deficiency . rhGH trIn our cohort, 11 adult patients were receiving adult rhGH treatment. Only one patient discontinued rhGH treatment due to uncontrolled hyperglycemia. The rhGH treatment was not associated with the prevalence of endocrine and metabolic illnesses such as metabolic syndrome, diabetes, hypertension, dyslipidemia, decreased bone density, and sleep apnea. Additionally, rhGH treatment is generally well tolerated, and there were no major safety issues in young adult patients in our PWS cohort. If rhGH is used in adults with PWS, this should be managed by a specialist multidisciplinary team.The annual mortality rate of adults with PWS is approximately 3%, and the average age at death is 33 years. Causes of death of adolescents and adults with PWS are related to cardiac disease and failure, pulmonary thromboembolism, accidents, sepsis, and obesity-related complications. In contrast, respiratory failure, aspiration, infection, and choking are causes of death in infants and young children with PWS . Early iThis study had several limitations. This was a retrospective study, and there were missing values related to decreased bone density, sleep apnea, and hypogonadism, both of secondary and primary origin. The strengths of our study are our focus on young adults, which allowed us to exclude aging-associated conditions and diseases. We investigated endocrine and metabolic illnesses in this cohort, and our results emphasize the need for specialized guidance and medical care in the transition from childhood to adulthood. In addition, we found that endocrine and metabolic illnesses were more prevalent in young adults with PWS than in a normal age-, sex-, and BMI-matched healthy control group.In our study, young adults with PWS were more likely to develop endocrine and metabolic illnesses than the age-, sex-, and BMI-matched healthy controls that likely started in their teen years, highlighting the importance of appropriate screening and intervention at this time. The rhGH treatment was not associated with the prevalence of metabolic syndrome, diabetes, hypertension, dyslipidemia, decreased bone density, and sleep apnea. The PWS group that had rhGH treatment in adulthood had a lower probability of having a BMI ≥ 30 at the last follow-up. The rhGH treatment is generally well tolerated, and there were no major safety issues in our young adult with PWS. Better understanding of the natural history of PWS patients and age-related complications will improve the quality of medical care in adults with PWS. Further studies of behavioral and psychiatric problems and the quality of life of patients and parents with PWS, and longer-term controlled studies of the benefits and risks of rhGH treatment in adult with PWS are necessary. Furthermore, we are hopeful that the development of appetite suppressant drugs will help control obesity and reduce the incidence of related diseases in PWS.


Despite receiving definitive chemoradiotherapy (dCRT) with radiation dose (RTD) of 50.4 Gy, survival of esophageal carcinoma was dismal. The effect of RTD in cancer control and radiotoxicity, and the extent to which local-regional control (LRC) influenced survival remain vague. This study aimed at evaluating RTD-effect relationship in esophageal squamous cell carcinoma (ESCC).  1440 dRT/CRT-treated ESCC patients were enrolled. Restricted cubic spline regression model was applied to reveal nonlinear relationship between RTD and survival/radiotoxicity. Linear regression analysis (LRA) was performed to evaluate correlations between LRC and overall survival (OS) or progression-free survival (PFS).  For 1440 dRT/CRT-treated ESCC patients, with RTD escalating, hazard ratios (HRs) of OS, PFS, LRC declined until RTD exceeded 60 Gy, then increased. HR of treatment-related mortality was stable until RTD exceeded 60 Gy, then increased. HR of LRC was lower for majority of patients treated with RTD≥60 Gy, except for those with KPS<80, T1-2 lesion, or without lymph node metastasis. LRA revealed strong correlations between LRC and OS/PFS. 45.5% and 44.9% of OS and PFS improvements were owing to improved LRC.  RTD of 60 Gy was well tolerated, with favorable survival resulted of LRC improvement in local-advanced ESCC. Further stratification analyses based on radiation sensitivity will be helpful to determine potential beneficiaries of RTD escalation. Nevertheless, because of poor treatment efficacy, EC remains the 9th most common cancer and the 5th leading cause of cancer-related deaths worldwide  or “cancer” [Title] or “carcinoma” [Title] or “neoplasm” [Title]) and (“radiation” [Title] or “radiotherapy” [Title] or “radiotherapy” [Title] or “chemoradiation” [Title] or “chemoradiotherapy” [Title]). All statistical calculations were performed with the In the current study, 1440 patients with pathologically confirmed ESCC who were treated with dRT/CRT from 2002 through 2016 were enrolled. Most patients were male . Almost 30% of the patients were aged ≥70 years, and 85.4% of the patients were diagnosed with disease of stages III to IV according to the 6th edition of the AJCC staging system. Most patients received RT/CRT with the radiation dose being 50–60 Gy  or 60–66 Gy . Patients' clinicopathologic characteristics are listed in p < 0.001), <60 Gy group and ≥60 Gy group . Similarly, the 1-, 3-, 5-year PFS were significantly different between the RT group and CRT group , <60 Gy group and ≥60 Gy group .Both univariable and multivariable analyses indicated that sex, Karnofsky performance status (KPS), the 6th edition of the AJCC TNM staging system, tumor location, radiation dose, and concurrent chemotherapy were related to OS  of esophagitis, pneumonia, myelosuppression, and radiodermatitis were similar in a steady low level till radiation dose approached 60 Gy and then enhanced sharply Figures .p < 0.01) decline in patients treated with a higher RT dose (EQD2 ≥ 60 Gy) compared to that in those treated with a lower RT dose (EQD2 < 60 Gy). In terms of subgroup analysis  and LRC and PFS (p < 0.05). An absolute advancement of 1% in LRC could translate into an improvement of 0.65% in OS and 0.42% in PFS ; 45.5% of the OS improvement and 44.9% of the PFS improvement was attributable to enhancement in the LRC. When the linear regression formula of the current study was used to predict OS or PFS based on LRC information from the 42 studies, the predicted OS and PFS were significantly associated with the actual OS and PFS .OS, PFS, and LRC information from 42 studies were collected . In the current study, Cox proportional hazard regression stratification forest plots analogously showed that improvement in LRC could be obtained with a radiation dose of 60 Gy or higher in most subgroups . Furthermore, the correlation between improved LRC and survival benefit had been demonstrated in several previous studies. However, the extent to which LRC influenced survival outcomes and how gains of LRC were converted to survival improvement still remain vague. The linear regression models of the survival data from our center and published clinical trials revealed a conversion relationship between LRC and survival outcomes, and nearly 50% of the improvement in OS and PFS (48% for OS and 49% for PFS) was attributable to LRC enhancement. Thus, it was rational to assume that escalation in the radiation dose could lead to LRC improvement, and the LRC improvement would subsequently convert to survival benefit. However, along with the survival benefits derived from enhancing LRC, the radiation dose-dependent toxicities emerged gradually. The nonlinear continuous dose-response smooth HR curve showed once the radiation dose exceeded 60 Gy, the risk of radiotoxicities and treatment-related mortality increased sharply. There was a threshold of survival outcomes (OS or PFS) to radiation dose, since the HR descended until the radiation dose reached 60 Gy and ascended subsequently. Escalation of the radiation dose beyond 60 Gy did not yield additional gains in OS or PFS. This could be attributed to the loss of survival benefits resulting from radiation toxicity itself and the effects of the accompanying discontinuous treatment in overwhelming the LRC-associated survival gain achieved with high-dose radiotherapy. The optimal radiation dose was deemed to balance the toxicity-related survival impairment and the tumor control-associated survival gain to the greatest extent.Dose-volume metrics and biological modeling of the trade-off in tumor coverage and OAR sparing in dose escalation for CRT indicated that escalation from 50 Gy to 62.5 Gy provided significant gains of more than 18% in tumor control (from 38.3% to 56.3%), with only a modest increase in predicted toxicity . In termp=0.22, and 59% vs. 52%, p-0.08). There was a marginal significant LRPFS improvement of 8% in the high dose arm. As the sample size of this phase 3 trial was calculated based on the hypothesis of a respectively large enhancement (15%) in local control, for reference, the LRC enhancement of the current study was 11.4% between patients treated with radiation dose of 50–60 Gy and 60–66 Gy. The hypothesis of a respectively large enhancement could lead to respectively small sample size with insufficient power to confirm the local-regional enhancement. Meanwhile, the planning target volume of the integrated boost dose merely consisted of the primary tumor but excluded the involved lymph nodes. As lymph node response to chemoradiotherapy potentially affect the treatment response of primary tumor, and 45.7% (48/105) and 51.4% (54/105) of patients with lymph nodes persistent resulted in partial and no response in the primary tumor [P=0.04) or stage III-IV disease  comparing with patients receiving chemoradiotherapy with radiation dose of 50.4 Gy without an SIB. Analogously, subgroup analysis of the current study revealed that patients with KPS ≥ 80, indicating superior treatment tolerance, and patients with T3-4 lesion or lymph node metastasis, indicating a higher tumor burden, tended to show survival benefit in radiation dose escalation. The negative findings of recently published prospective clinical trials [In contrast to the current study and other studies supporting radiation dose escalation-associated LRC and survival gains, several previous prospective clinical trials failed to reproduce the results presented above. The ARTDECO study  failed iry tumor , dose esry tumor , and surry tumor , 27. Forry tumor . On the ry tumor . For refry tumor . The mix45.7% 48/5 and 51.The main limitation of the current study was its retrospective nature, which may have introduced some bias in the results and conclusion. Thus, the results should be validated in well-designed randomized controlled trials or retrospective studies based on real-world data with a large population. In addition, detailed information for several parameters could not be collected and enrolled in the current study, including clinical details of concurrent chemotherapy intensity, planning parameters of radiotherapy, the incidence of long-termtreatment-related toxicity, and other radiological and histopathological details.Radiation dose of 60 Gy could yield favorable LRC and acceptable toxicities for patients with local advanced ESCC receiving definitive RT/CRT. The LRC gains obtained from radiation-dose escalation could convert to survival benefit to a large extent. In patients with KPS < 80, of early TNM stage and with respectively high radiation sensitivity, 50 Gy might be a sufficient definitive radiation dose. Clinicians should realize radiation-dose escalation is the potential way to improve the unfavorable LRC and survival outcomes in dRT/CRT-treated ESCC patients. The findings of the current study could serve as evidence for formulating treatment plans and designing additional prospective stratification randomized controlled trials.


To determine atrial fibrillation (AF) prevalence and temporal trends, and examine associations between AF and risk of adverse health outcomes in older care home residents.Retrospective cohort study using anonymised linked data from the Secure Anonymised Information Linkage Databank on CARE home residents in Wales with AF (SAIL CARE-AF) between 2003 and 2018. Fine-Gray competing risk models were used to estimate the risk of health outcomes with mortality as a competing risk. Cox regression analyses were used to estimate the risk of mortality., the overall prevalence of AF was 17.4%  between 2010 and 2018. There was no significant change in the age- and sex-standardised prevalence of AF from 16.8% (15.9–17.9) in 2010 to 17.0% (16.1–18.0) in 2018. Residents with AF had a significantly higher risk of cardiovascular mortality , all-cause mortality , ischaemic stroke  and cardiovascular hospitalisation .There were 86,602 older care home residents (median age 86.0 years [interquartile range 80.8–90.6]) who entered a care home between 2003 and 2018. When the pre-care home entry data extraction was standardisedOlder care home residents with AF have an increased risk of adverse health outcomes, even when higher mortality rates and other confounders are accounted for. This re-iterates the need for appropriate oral anticoagulant prescription and optimal management of cardiovascular co-morbidities, irrespective of frailty status and predicted life expectancy. The prevalence of atrial fibrillation remained stable in older care home residents from 2010 to 2018.Residents with atrial fibrillation had a significantly higher risk of all-cause and cardiovascular mortality.The risk of stroke and cardiovascular hospitalisation was also significantly higher, even when mortality was adjusted for as a competing risk.Optimal management of atrial fibrillation in older care home residents is critical to improve health outcomes.Older care home residents are a high-risk group of people with atrial fibrillation (AF) who are underrepresented in clinical trials. Improved understanding of AF epidemiology and management in this population is paramount. The health and social care burden of AF and AF-associated adverse health outcomes presents as a major public health issue, but the burden of AF in the care home setting is unclear. Estimates of AF prevalence in care homes have been reported to range from 7 to 38%, likely as a result of diversity and variability across residents and facilities . FurtherThis study aims to use anonymised, individual-level population-scale routinely linked data for older people aged ≥65 years living in care homes in Wales to determine the prevalence of AF and temporal trends by year of care home entry, and associations between AF and adverse health outcomes including stroke, transient ischaemic attack (TIA), major bleeding, myocardial infarction (MI), cardiovascular hospitalisation and mortality.The Secure Anonymised Information Linkage (SAIL) Databank project on CARE home residents with AF (SAIL CARE-AF) was a retrospective cohort study of anonymised, individual-level data on care home residents in Wales, provisioned from 1 January 2003 to 31 December 2018 and conducted following the REporting of studies Conducted using Observational Routinely-collected health Data (RECORD) 2015 guidelines  Appendi.This study utilised data available from the SAIL Databank . The SAIThe care home dataset (CARE) within the SAIL Databank relies on care home information available from the Care Inspectorate Wales (CIW) registry. In Wales, care homes must be registered with CIW who are responsible for inspection and quality improvement. Care homes include residential and nursing homes, which provide different levels of assistance to residents. Nursing homes provide a higher level of care and greater assistance to residents with the support of qualified nurses. The CIW registry 2017/18 was used in this study . This wa2DS2-VASc and modified HAS-BLED score for each individual, respectively ) of each outcome using mortality as a competing risk. All participants were followed up until they died, moved out of Wales or until the end of study (31 December 2018). Analyses were adjusted for covariates known to be associated with the outcomes of interest. These were pre-agreed amongst authors . The main analyses were adjusted for age, sex, WIMD, AF, frailty, smoking history, dementia, pulmonary disease, cancer diagnoses, peptic ulcer disease, prescription of OAC (with or without antiplatelet therapy) within 6 months prior to care home entry, CHA overlap . When co overlap  [18]. Mu overlap . If cova overlap . All ana The SAIL Databank consumer panel includes member of the public and provided feedback that the study outcomes were of interest.Between 2003 and 2018, 86,602 people aged ≥65 years who had at least 12 months of primary care data captured within the SAIL Databank became new residents in care homes in Wales . The medn = 3,155 vs. 16.6% [16.2–17.0], n = 5,351), and increased with advancing age  but stabilised in people ≥95 years (18.2% [17.1–19.5]). There was a non-significant increase in age- and sex-standardised prevalence (95% CI) of AF recorded within 10 years prior to care home entry from 16.79% (15.9–17.9) in 2010 to 17.0% (16.1–18.0) in 2018 .There were 48,767 people aged ≥65 years who had at least 10 years of primary care data captured within the SAIL Databank prior to care home entry between 2010 and 2018, and 8,506  had AF recorded 10 years before care entry. Prevalence [95% CI] of AF was higher in males than females  of 538 days . Residents with AF on care home entry had significantly higher incident rates per 1,000 person-years of all adverse outcomes . When individual stroke subtypes were examined, the risk of haemorrhagic stroke or stroke of unknown origin was not significantly different between residents with or without AF, but there was a significantly greater risk of ischaemic stroke . Having AF on care home entry did not significantly increase the risk of TIA, major bleeding or MI, but those with AF were at a significantly higher risk of cardiovascular hospitalisation , cardiovascular mortality  and all-cause mortality  . The Varsted for . In thissted for .2DS2-VASc and HAS-BLED risk assessments were adjusted for, the risk of TIA and sustaining a stroke of unknown origin was also significantly higher in residents with AF  overall crude prevalence of AF in older care home residents was 17.4% (ii) over time, there was no significant change in crude or age- and sex-adjusted AF prevalence; (iii) AF prevalence was higher in men and increased with advancing age from 65 years onwards, stabilising in residents aged ≥95 years; and (iiii) AF was associated with a significantly higher risk of adverse health outcomes, including ischaemic stroke, cardiovascular hospitalisation, cardiovascular and all-cause mortality after adjusting for potential confounding factors.P = 0.012, respectively) and found to increase with advancing age in 2004 [The Global Burden of Disease study in 2019 concluded that there has been a rise in the number of AF cases globally . This wa in 2004 , therebyStroke and TIA are the major adverse health outcomes associated with AF . AF has The study’s findings reinforce the need for a holistic and integrated approach to optimise AF management in the care home population. The current gold-standard AF management strategy is the Atrial Fibrillation Better Care pathway . Howevern = 674) and demographic data on ethnicity were unavailable.This study provides a pooled estimate of crude and age- and sex-adjusted AF prevalence that is inclusive of all types of care homes and residents by using a nationwide data linkage resource. Despite limiting our analyses to cases of AF recorded within 10 years prior to care home entry, we captured 91% of AF cases from 2010 to 2018. The main limitations pertain to the use of routinely collected data. First, international normalised ratio data were unavailable. Second, it is possible that some diagnoses (e.g. dementia) were missed using Read or ICD-10 codes by relying on positive recordings of diagnoses, or classified incorrectly. Third, it was not possible to provide a further breakdown of AF outcomes such as major bleeding based on the prescription of OAC or not because prescription data were not available for the entire study period, or based on falls because data were unavailable. Fourth, deprivation data were missing for a small proportion of residents (0.8%, This study provides an accurate estimation of AF prevalence, incidence and risk of adverse health outcomes exclusively in a population of older care home residents. It highlights the need for appropriate OAC prescription for stroke prevention and optimal management of cardiovascular co-morbidities, irrespective of frailty status and predicted life expectancy. Future research efforts should focus on improving the quality of routinely collected data in care home populations to help define ‘optimal’ AF management in older care home residents, and encompass AF classifications, prescription refill and falls data. Algorithms to identify care home populations from other routinely collected datasets would be valuable.aa-22-0771-File002_afac252Click here for additional data file.


Regulatory T cells (Treg) have been identified as a key modulator of neuroinflammation in stroke. However, little is known about the association of Treg subpopulations with clinical outcome in patients with acute ischemic stroke (AIS).Patients within 1 week from stroke onset were prospectively enrolled in this study. Healthy controls were sex-and age-matched 1:1 to AIS patients. The frequencies of Treg and Treg subsets were analyzed by flow cytometry and compared with nonstroke control. Univariate and multivariate logistic regression analysis was performed to investigate the prognostic value of Treg subsets in stroke outcomes.p < 0.001; integrated discrimination improvement 22.38, 95% CI 17.16–27.59%, p < 0.001).A total of 328 patients and 328 controls were included in the study. Compared with controls, patients with AIS had higher levels of Treg frequency and memory Treg (mTreg) frequency, but lower levels of naïve Treg (nTreg) frequency and nTreg/mTreg ratio. One hundred twenty-six (38.4%) patients experienced unfavorable outcome (modified Rankin score 2–6). Multivariate regression analysis showed that nTreg/mTreg ratio was negatively associated with unfavorable 90-day outcome . The risk estimation of unfavorable 90 day outcome can be significantly improved by adding nTreg/mTreg ratio to the conventional clinical parameters (continuous net reclassification improvement 91.26, 95% CI 69.04–113.5%, This study showed that patients with AIS had elevated Treg frequency and mTreg frequency, but reduced nTreg frequency and nTreg/mTreg ratio. Admission nTreg/mTreg ratio was an independent predictor of unfavorable 90 day outcome in AIS. However, large sample-size cohort studies are needed to confirm our findings. Acute ischemic stroke (AIS) is a leading cause of death and disability worldwide. Stroke-triggered neuroinflammation is crucial in the pathogenesis of brain injury and recovery . Infiltrin vitro, generating a homogeneous Treg population . Patients with AIS onset within 1 week and with Treg subpopulation data available who was treated medically were included in analysis. Clinical diagnosis of ischemic stroke was confirmed by CT or MR imaging. Each patient was sex-and age-matched with a non-stroke healthy control (the same sex as the matched patient and a difference of age within ±2 years old). Participants with known autoimmune or inflammatory diseases, clinical signs of acute infection on admission, and that who underwent endovascular treatment were excluded. Blood was obtained from participants between 5:00 a.m. and 6:00 a.m. the next morning after they were hospitalized. This study was approved by the Ethics Committee of Tongji Hospital, and written informed consent was obtained from patients or their surrogates before enrollment.Baseline demographic and clinical data, including age, sex, medical history, blood pressure, serum glucose, and treatment with intravenous thrombolysis were all prospectively collected. The stroke severity was evaluated with the NIHSS score (range from 0 to 42) score at their admission by a stroke neurologist. In this study, ischemic stroke was classified into 3 subtypes according to the TOAST criteria: large artery atherosclerosis (LAA), cardiogenic embolism (CE), and other causes.Heparinized peripheral blood was collected for lymphocyte phenotyping. One hundred μL of whole blood were added with the following monoclonal antibodies: anti-CD3, anti-CD4, anti-CD25, anti-CD127, anti-CD45RA, and anti-CD45RO . Isotype controls with irrelevant specificities were used as negative controls. Cell suspensions were incubated for 20 min at room temperature. After lysing red blood cells, the cells were washed and resuspended in phosphate buffer saline. Then, the cells were analyzed with FACSCanto flow cytometer. The gating strategies for Treg phenotype analysis were as follows: CD4+ T cells (CD3 + CD4+), Treg (CD3 + CD4 + CD25 + CD127low), nTreg (CD3 + CD4 + CD25 + CD127lowCD45RA+), and mTreg (CD3 + CD4 + CD25 + CD127lowCD45RO+) . The freU rank sum test were used for categorical variables and continuous variables with nonnormal distribution, respectively. Univariate and multivariate binary logistic regressions were performed to assess the associations of admission Treg and differential Treg subsets  with 90 day outcomes . Variables with p < 0.1 on univariate analyzes, as well as age and NIHSS score, were included in the next multivariate analyzes. Tests for linear trend in risk across Treg subsets tertiles were performed. Subgroup analyzes were also performed according to age, NIHSS score and TOAST classification. Optimal cut-off points were calculated utilizing receiver operating characteristic curve analysis.Categorical variables were represented as percentage and frequency. Continuous variables were expressed as mean ± SD if distributed normally and otherwise, as median and interquartile range (IQR). For variables with normal distribution, differences between groups were compared using Student’s t-test. Chi-square test and Mann–Whitney p < 0.05 was considered significant.Moreover, we calculated C statistics, net reclassification improvement (NRI) and integrated discrimination improvement (IDI) to investigate the added prognostic values of Treg subsets. Calibration plot and its slope was used to evaluate the calibration of the new model. Statistical analysis was conducted using SPSS Statistics version 24.0  and R Studio 4.2.1 . A two-tailed value of A total of 328 patients with AIS and matched 328 controls were included in the analysis. The mean ages were 55.9 ± 11.0 and 55.7 ± 11.5 years, respectively. The male proportion was 80.8% for each group. The two groups were comparable in age, sex, proportions of hypertension, diabetes mellitus, dyslipidemia, coronary heart disease, atrial fibrillation, and current smokers, alcohol consumption, and levels of SBP, DBP and serum glucose. For the AIS group, the median NIHSS score on admission was 3 points ; and 236 (72.0%) and 10 (3.0%) participants had large artery atherosclerotic and cardioembolic ischemic strokes, respectively. The main baseline characteristics of the 2 groups are shown in p < 0.001) and mTreg frequency , but lower levels of nTreg frequency  and nTreg/mTreg ratio   .Of the 328 patients with AIS, 126 (38.4%) had an unfavorable outcome. The main baseline characteristics according to 90 day neurological outcome are shown in p = 0.011) and lower nTreg/mTreg ratio   .P for trend = 0.01 and 0.001, respectively) while Treg and mTreg frequency were not   . nTreg fC statistic: 0.79, 95% CI: 0.73–0.85, p < 0.001).To minimize the influence of collinearity between these three parameters on the regression analysis, separate models were run for them. After adjustment for age, SBP, NIHSS score and TOAST classification, only nTreg/mTreg ratio remained significantly associated with unfavorable 90 day outcome . Comparep = 0.003) (p < 0.001). IDI was 22.38%   . The con< 0.001) . The calIn this study, we observed an elevation of circulating Treg and mTreg frequencies, in parallel with the reduction of nTreg frequency and nTreg/mTreg ratio in patients with AIS. Moreover, our results suggest that peripheral nTreg/mTreg ratio was an independent predictor of unfavorable stroke outcome.Treg play a crucial part in the modulation of immune responses and the control of detrimental immune activation due to their immunoregulatory and immunosuppressive functions . Most prin vitro expansion compared to nTreg  can be further divided into naïve Treg  and memory Treg  according to the expression of CD45RA and CD45RO . Most ofto nTreg . The obsto nTreg , which sThe imbalance of Treg subpopulations (decreased nTreg and increased mTreg) has been observed in chronic obstructive pulmonary disease , non-ST In this study, we found that circulating nTreg/mTreg ratio was negatively associated with unfavorable outcome in AIS, which means impaired immunomodulatory activity at the early stage of AIS may contribute to unfavorable outcome. To the best of our knowledge, this is the first report evaluating the prognostic value of Treg subsets in stroke outcome. As mentioned above, nTreg is the main subpopulation with suppressive activity while mTreg have limited suppressive activity, but can produce pro-inflammatory cytokines . The redThere are several limitations in our study. Firstly, the sample size may be not sufficient to perform more comprehensively adjustment for other confounders, such as laboratory and imaging parameters. Secondly, Treg can be further subdivided into other biomarkers functionally , which allows a more precise evaluation of the role of Treg in stroke. But this did not influence our clinical observation, though the simple characterization of Treg subsets limit the exploration of related pathophysiological mechanisms of stroke. Thirdly, the temporal change of Treg subsets in AIS was not observed. Finally, the symptoms of the included participants were generally mild–moderate stroke with low NIHSS score, so the applicability of our results to the whole population with AIS should be questioned. Thus, the prognostic role of nTreg/mTreg ratio in patients with more severe stroke needs further verification in larger prospective trials.In conclusion, we found nTreg/mTreg ratio was an independent predictor of unfavorable 90 day outcome in patients with mild–moderate AIS. Larger sample and multicenter studies are needed to confirm our results, and interventions targeting nTreg/mTreg ratio may provide a potential treatment strategy for stroke in the future.The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The patients/participants provided their written informed consent to participate in this study.GD and YT designed and conceived the study and wrote the first draft of the manuscript. JX, XC, Y-HC, KS, and L-QZ collected clinical and radiological data and performed laboratory analysis. GD, YT, and JX conducted the statistical analysis. CQ, FW, and D-ST supervised the study and critically revised the manuscript. D-ST obtained funding. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (Nos. 81873743 and 82071380).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Toll-like receptor 4 (TLR4) identifies Gram-negative bacteria or their products and plays a crucial role in host defense against invading pathogens. In the intestine, TLR4 recognizes bacterial ligands and interacts with the immune system. Although TLR4 signaling is a vital component of the innate immune system, the influence of TLR4 overexpression on innate immune response and its impact on the composition of the intestinal microbiota is unknown.S. Typhimurium) in macrophages. Meanwhile, we characterized the complex microbiota inhabiting the stools of TLR4 transgenic (TG) sheep and wild-type (WT) sheep using 16S ribosomal RNA (rRNA) deep sequencing.Here, we obtained macrophages from sheep peripheral blood to examine phagocytosis and clearance of Salmonella Typhimurium  and upregulating the abundance of Bacteroidetes population and short-chain fatty acid (SCFA)-producing bacteria, including Prevotellaceae. These dominant bacterial genera changed by TLR4 overexpression revealed a close correlation with the metabolic pathways of TG sheep.The results showed that TLR4 overexpression promoted the secretion of more early cytokines by activating downstream signaling pathways after stimulation by S. Typhimurium invasion as well as resist intestinal inflammation in sheep by regulating intestinal microbiota composition and enhancing anti-inflammatory metabolites.Taken together, our findings suggested that TLR4 overexpression can counteract  Salmonella Typhimurium (S. Typhimurium), a zoonotic pathogen causing public health hazards, is endemic in many countries, where it causes significant economic losses due to the gastrointestinal infections in ovine hosts  and Bacteroidetes (Gram-negative) are the dominant phyla of sheep gut microorganisms that produce various metabolites capable of influencing the physiology of the host . WhereasIntestinal bacteria not only promote nutrients absorption by secreting enzymes to break down other indigestible substances, but they are also involved in inflammation, immune system development, and the gut-brain axis regulation . DisruptS. Typhimurium (a food borne pathogen) invasion and on the intestinal microbiota composition of sheep. We analyzed the microbiota of TLR4 overexpressing sheep and their WT controls using high-throughput sequencing of 16S rRNA genes amplified from fecal samples. An objective of the current study was to determine whether TLR4-overexpressing alters the composition of the intestinal microbiota in sheep.Given that the immune system has an impact on the gut microbiota, in this study, we were concerned about the role of TLR4 signaling in defense against Salmonella All sheep were housed in accordance with the national feeding standard NT/T815--2004. All procedures performed for this study were consistent with the National Research Council Guide for the Care and Use of Laboratory Animals. All experimental animal protocols in this study were approved and performed in accordance with the requirements of the Animal Care and Use Committee at China Agricultural University .ovis aries TLR4 . Designed Southern blotting probes and prepared PCR products. The primer used are following: forward 5′-ACTGGTAAAGAACTTGGAGGAGG-3′ and reverse 5′-CCTTCACAGCATTCAACAGACC-3′, and the 671-bp PCR product labeled with digoxigenin . There were five The peripheral blood (10 mL) of sheep was collected from the jugular vein, and peripheral blood lymphocyte separation solution  was added promptly. After density gradient centrifugation , the peripheral blood mononuclear cells were washed with PBS. Mononuclear cells were resuspended in RPMI 1640 medium  supplemented with 10% fetal bovine serum  and 1% penicillin–streptomycin, then cultured in 6-well culture plates at 37°C with 5% CO2. After 2 h of incubation, the wells were washed with PBS to remove non-adherent cells, and single adherent mononuclear cell was differentiated into spindle-shaped macrophages, giving rise to proliferating clones after 1 week .S. Typhimurium was preserved in our laboratory. The bacterial strain was cultured in Luria broth (LB) medium at 37°C to the logarithmic growth phase for later using. To detect the phagocytic capability of the TG and WT macrophages, cells were infected with live S. Typhimurium CVCV541. The bacteria were suspended in RPMI-1640 medium  with 10% FBS. Then the adherent macrophages were infected with S. Typhimurium at two different levels of MOI (5 and 10) for 5, 15, and 30 min, and were washed with PBS including 100 μg/ml gentamicin. Then the cells were lysed by 0.25% tritonX-100 to release intracellular bacteria and the number of intracellular survival bacteria was quantified by counting CFUs after serial dilution.The S. Typhimurium CVCV541 for 30 min. Extracellular bacteria were removed by washing the macrophages with PBS including 100 μg/mL gentamicin for three times. Then add RPMI-1640 containing 10% FBS to the cells for 30 min, which was recorded as the time point 60 min. The cells were washed and lysed with 0.25% Triton X-100 (in PBS). Calculate the phagocytosis time from the time of adding bacteria. The data on the number of cleared bacteria were accessed by subtracting intracellular bacteria number in 60 min from the bacteria number in 30 min.In order to determine the bacterial clearance capacity in macrophages, macrophages were first infected with S. Typhimurium, the cells were treated with S. Typhimurium SL1344 for 0, 0.5, and 4 h at a MOI of 10, and the mRNA expression of related genes was detected and quantified by RT-PCR. Total RNA from sheep macrophages was extracted using RNA extraction kit  according to the manufacturer’s instructions. RNA was treated with DNAse I to remove any residual DNA, and the quantity and quality of RNA were assessed with a NanoDrop 2000 spectrophotometer . The extracted 1 μg of RNA was reverse transcribed into cDNA using PrimeScript RT kit (TaKaRa). The cDNA was analyzed by RT-qPCR. The gene primers shown in −ΔΔCT method) by averaging the Ct values from three replicates, and the expression levels of mRNA were normalized to housekeeping gene GAPDH mRNA levels.In order to compare the expression of factors between the TG and WT groups in sheep under the attack of Cell proteins were extracted with RIPA lysis buffer . The protein concentrations were determined by bicinchoninic acid (BCA) protein assay kit , and then boiled at 95°C with 6 × loading buffer for 8 min. Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immediately transferred onto 0.22 μm polyvinylidene fluoride membranes , the membranes were blocked with Tris-buffered saline with Tween 20 (TBST) containing 5% nonfat milk at room temperature for 60 min. After washing three times, the membranes were incubated overnight at 4°C with anti-TLR4 . Then blots were bound with horseradish peroxidase (HRP)-conjugated secondary antibodies  for 60 min at 37°C. After washing again with TBST three times, experimental results were obtained by the pierce enhanced chemiluminiscent (ECL) western blotting kit (Thermo Scientific), and imaged by chemiluminescence system .Ten adult female sheep were used for this work. They were divided into two groups, five TLR4 overexpressing sheep for the experimental group (TG group), along with five WT sheep for the control conditions (WT group). Fecal samples were taken from the TG and WT groups with clean plastic gloves in autumn. Fresh and individualized samples were collected from two groups of sheep. Samples are transported on dry ice and stored at −80°C for subsequent experiments.Total bacterial genomic DNA samples were extracted from 10 sheep fecal samples using the Fast DNA SPIN extraction kits , according to the manufacturer’s protocol (n = 10). The quantity and quality of isolated DNA quantity and quality were determined by a spectrophotometer  and agarose gel electrophoresis, respectively . The extThe prokaryotic 16S rRNA gene libraries were generated from the extracted DNA using amplicon PCR with variable V3–V4 region  and the Illumina 16S Sample Preparation Guide. Preparation of 25 μL PCR components for amplification. The amplification procedure consists of an initial denaturation at 98°C for 2 min, followed by 25 cycles consisting of denaturation at 98°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min, hold at 10°C. Unbound primers, other contaminants, and primer dimer fragments were removed and quantified. The purified samples were collected in equimolar amounts, and paired-end sequencing was performed with the IllluminaMiSeq platform at Shanghai Personal Biotechnology Co., Ltd. .The Quantitative Insights into Microbial Ecology (QIIME) pipeline was employed to carry out the quality control of the sequencing data . SpecifiSequences were classified from the phylum level to the species level. To classify and identify sequence reads using Silva version 132 as a reference database . This waStatistical and bioinformatics analysis of 16S rRNA gene sequencing. The within-community diversity (α-diversity), including Chao1 richness, Shannon diversity index, and Simpson index, were detected using the ASV table in QIIME. ASV-level abundance curves were generated to compare the richness and homogeneity of ASVs in samples. Changes in community composition (β-diversity) was performed by the principal coordinates analysis (PCoA), weighted and unweighted UniFrac NMDS analysis, as well as hierarchical clustering analysis based on weighted UniFrac distances. They were performed by QIIME and R software. The Gram-positive/Gram-negative ratio was calculated by dividing the number of Actinobacteria and Firmicutes sequences by the number of Gram-negative bacteria  . The relp < 0.05, **p < 0.01).All the bar and box plots were generated using GraphPad Prism version 8.0 . All data were expressed as mean ± SD. One-way ANOVA was used for statistical analysis, followed by the Turkey test for multiple comparisons  than WT macrophages at different times after live S. Typhimurium treatment, regardless of the MOI 5 or 10  in the TG macrophages than in the WT macrophages at 0.5 h after S. Typhimurium stimulation . As shown in Venn’s plot in PCoA based on unweighted UniFrac distances was used to show the effect of TLR4 overexpression on bacterial community patterns. As seen in It is worth noting that the control groups samples showed less bacterial diversity than the TG group. This low diversity could explain why the samples belonging to WT were similar according to NMDS, while individuals in the TG group were more dispersed from one another.Despite this study indicated that alpha diversity was not notably affected by TLR4, the TLR4 overexpression changed the abundance of the intestinal bacteria in sheep. In practice, by using the difference analysis of the DESeq2 package, we further investigated the composition of the intestinal flora affected by TLR4 in TG vs. WT at the phylum levels. Ten different bacterial phyla were determined in two groups, which dominated in most individuals: Firmicutes, Bacteroidetes, Spirochaetes, Proteobacteria, Patescibacteria, Cyanobacteria, Fibrobacteres, Tenericutes, Actinobacteria and Verrucomicrobiota . Phylum p = 0.044), which suggests the influence of TLR4 overexpression at this phylum. The Proteobacteria phylum was also decreased through TLR4 expression in TG sheep (p = 0.870), although not significantly compared to the WT group. Interestingly, however, overexpression of TLR4 increased the number of Spirochaetes (p = 0.397). The last phylum, Bacteroidetes, was the one in which TG resulted in significant population growth compared to WT (p = 0.039)  . Supplemp < 0.05)  .p < 0.05) (p < 0.05), Christensenellaceae  , which mellaceae . They prp < 0.05). The high expression of TLR4 also improved the abundance of Ruminococcaceae_UCG-005, Rikenellaceae_RC9_gut_group, p-251-o5, and Bacteroides (p > 0.05)  .The resup < 0.05), with Bacteroidia, Bacteroidetes and Bacteroidales being the main microbiota. WT group also displayed 16 dominant microorganisms, and the primary microbiota were Ruminococcaceae_UGG_010, Ruminococcaceae_UGG_013, Ruminococcaceae_NK4A214_group . In the KEGG function analysis, the top three raised functional abundances were K03088 , K02529 , K02014 (iron complex outer membrane receptor protein), whereas the top three reduced functional abundances were K01990 (BC-2 type transport system ATP-binding protein), K06147 , K01992 (ABC-2 type transport system permease protein) in the top 50 highest abundances of KEGG orthology  analysis, the findings reflected that the three most increased functional abundances of microbial community were cell wall/membrane/envelope biogenesis; post-translational modification, protein turnover, and chaperones; carbohydrate transport and metabolism, and the three most decreased in microbial community functional abundances were transcription, cell motility and signal transduction mechanisms . In concS. Typhimurium, capable of stimulating downstream signaling cascades that activate NF-κB or the production of cytokine secretion. Nevertheless, the defense mechanisms of TLR4 overexpression in sheep against invading pathogens are still incompletely clear. In the present research, using macrophages isolated from the venous blood of TG and WT sheep, we examined the cytokines expression downstream of TLR4 under S. Typhimurium stimulation. Our data indicated that the production of pro-inflammatory cytokines, like IL-6, IL-1β, IL-8, and TNFα, was notably higher in TG group than the control group at different time points. These data illustrated that S. Typhimurium induces a stronger natural immune response in TLR4 overexpressing TG sheep. The TG group exhibited higher phagocytosis levels than the control group, in other words, more S. Typhimurium could be phagocytosed in the TLR4 overexpressing TG sheep. This result is coherent with other studies  under accession number PRJNA891502.The datasets presented in the study are deposited in the NCBI Sequence Read Archive .X-LX: writing original draft preparation. YZ, J-LZ, and X-SZ: collecting the samples. YL, M-MC, and S-JW: editing and technical review. YL: visualization. KY and Z-XL: supervision. All authors have read and agreed to the published version of the manuscript.This work was supported by the National Transgenic Creature Breeding Grand Project (2016zx08008-003) and the National Key Research and Development Project of China (2021YFF1000704).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.