Few studies have explored the optimal examined lymph node count and lymph node density cutoff values that could be used to predict the survival of patients with penile cancer. We further clarify the prognostic value of lymph node density and examined lymph node count in penile cancer.The Surveillance, Epidemiology, and End Results (SEER) database was explored to recruit penile cancer patients from 2010 to 2015. A retrospective analysis of penile cancer patients’ data from the First Affiliated Hospital of Anhui Medical University was performed for verification (2006–2016). The cutoff values of examined lymph node count and lymph node density were performed according to the ROC curve. Kaplan-Meier survival analysis was used to compare survival differences among different groups. Univariate and multivariate Cox proportional hazard regression analyses were used to determine the significant variables. On the basis of Cox proportional hazards regression model, a nomogram was established and validated by calibration plot diagrams and concordance index (C-index).A total of 528 patients in the Surveillance, Epidemiology, and End Results cohort and 156 patients in the Chinese cohort were included in this study. Using the ROC curve, we found that the recommended cutoff values of ELN and LND were 13 and 9.3%, respectively (P <0.001). Kaplan–Meier curves suggested the significant differences of overall survival among different examined lymph nodes and lymph node density. Multivariate analysis indicated ELN and LND were independent prognostic factor for OS of penile cancer patients. Nomogram showed the contribution of ELN and LND to predicting OS was large. The C-index at 3-, and 5-year were 0.744 for overall survival (95% CI 0.711–0.777).The more lymph nodes examined, the lower the density of lymph nodes, and the higher the long-term survival rate of penile cancer. We recommended 13 examined lymph nodes and lymph node density >9.3% as the cutoff value for evaluating the prognosis of penile cancer patients. Penile cancer (PeCa) is a rare disease, but its incidence has been rising slowly in recent years. According to the 2020 Cancer Research UK (CRUK) report, the incidence rate has increased by 15% over the past decades  before its systemic spread , 3. PrevFrom the previous studies we have known that examined lymph node (ELN) count and lymph node density (LND) are the percentage of positive LNs, which have been used as a prognostic factor for other tumors, such as esophageal cancer, non-small-cell lung cancer and bladder cancer , 10–14. Nomogram, a statistical forecasting tool, has the advantages of low cost and strong reliability, which is used to quantify individual risks according to forecasting factors , 17. HowTherefore, in the current study, we analyzed the effect of ELN and LND on OS in patients with PeCa and evaluated the extent of this effect. Moreover, we included the variable ELN and LND to create an accurate and personalized prognostic nomogram for predicting OS in patients with PeCa, in order to further clarify the prognostic value of ELN and LND in PeCa.This is a retrospective study, using the clinical data of two groups of people diagnosed with PeCa: one from the Surveillance, Epidemiology, and End Results (SEER) database as the training cohort (1975–2016) and the other from Blinded for peer review of China as the validation cohort (2006–2016). All patients in both cohorts underwent radical lymphoadenectomy in addition to surgery of primary tumor site. In patients with nonpalpable nodes, a superficial dissection above the fascia lata was performed. In cases with palpable adenopathy or suspicious nodes encountered during superficial dissection, a deep dissection was performed. Pelvic lymphadenectomy was performed in patients with positive deep inguinal lymph nodes or with enlarged pelvic lymph nodes on cross sectional imaging. The demographic information of age at diagnosis, marital status at diagnosis, ELN, LND, surgery of primary site and tumor characteristics of differentiation grade, histological type, T-stage, N-stage, M-stage and tumor size were collected. Incompletely documented variables such as primary surgical site, grade, TNM stage, marital status, tumor size, ELN, and positive lymph nodes were excluded from this study. In the calculation of “examined lymph node count” and “lymph node density”, inguinal and pelvic lymph nodes were included.OS is defined as the time from diagnosis to original death, whatever the reasons. TNM staging and histopathological grading of PeCa were determined according to the American Joint Committee on Cancer (AJCC) 6th edition staging system and SEER cancer grading system, respectively.The SEER database is a publicly available, federally funded cancer reporting system and also the largest publicly available cancer data set. Institutional review committees and ethics committees allow the use of public database data without patient identity information . AdditioP <0.05 values were considered statistically significant for all.All statistical analyses were performed using SPSS version 20.0 . Chi-square, Pearson’s chi-square, and Fisher’s exact tests were used to determine the significance of differences between continuous variables and categorical variables. Kaplan–Meier analysis was used to estimate survival and compare different variables, namely, average survival time, median survival time and 95% confidence interval (95% CI). Based on Cox proportional hazard regression analysis, multivariate and univariate survival analyses were conducted. As for the evaluation of the model performance and the verification of the accuracy of the new scoring system, we use the Harrell concordance index (C-index) and calibration curve, respectively. Moreover, the receiver operating characteristic (ROC) curve was used to evaluate the effectiveness of the nomogram. P <0.001] and 9.3% , respectively , 0.59; ectively Figure 1P >0.05 for all).After screening, 528 patients in the SEER cohort and 156 patients in the China cohort were included in this study. As shown in P = 0.6); however, the association between LND and age at diagnosis (P = 0.003), grade (P <0.001), T-stage (P = 0.001), N-stage (P <0.001), M-stage (P = 0.002), histological type (P = 0.007), ELN (P <0.001), tumor size (P = 0.012) and surgery of primary site (P = 0.012) were significant.With the cutoff value obtained by ROC curve, we divided all the patients of the training group into two groups: LND ≤rain and LND >9.3%, the numbers were 328 (62.1%) and 200 (37.9%), respectively. The connection is displayed in P <0.05 for all). Generally speaking, LND is closely related to the pathological features of tumors.Patients were divided into groups according to LND, and the oncology characteristics of each group were compared shown in Figure 2The distribution and correlation of clinical and pathological characteristics of patients in the training group were represented by the mosaic plot to which area of the nested matrix is proportional to the unit frequency, and the frequency is the frequency in the multi-dimensional contingency table. The residual value of fitted model are represented by color and shading. Patients with LND >9.3% have the characteristics of higher tumor grade, more prone to distant metastasis, higher clinical tumor stage and larger tumor size. Also, their histopathological types are significantly different from LND Figure 3Univariate risk factors of OS are shown in P < 0.001 for all). Patients with LND ≤9.3% had the highest OS (median OS and 95%CI undefined), followed by LND >9.3% . Similarly, patients with ELN >13 have the highest survival rate , followed by ELN ≤39 .In order to evaluate the OS of PeCa patients with different LND/ELN, the Kaplan–Meier survival analysis was performed on all patients. As shown in On the basis of Cox proportional hazards regression model, age, N- and M-stages, ELN and LND were selected as variables to construct nomogram Figure 5As shown in On the basis of the nomogram, we drew 3- and 5-year calibration curves and ROC curves from our single center population for independent verification, and the results of the curves were in high agreement with the results of our training group  ELN and LND are independent predictors for survival of PeCa. (II) A greater number of ELNs and lower LND are associated with better long-term survival of PeCa. We recommended 13 ELNs and LND >9.3% as the cutoff value for evaluating the prognosis of PeCa patients. Therefore, surgeons and pathologists should try their best to explore the LNs and the minimum recommended number for assessing the integrity of LN sampling is 13 and LND needs to be at least 9.3%. Based on real patient data, our research emphasize that surgeons should fully sample and dissect LNs in clinical practice, and carefully explore LNs.Due to the limitation of retrospective and small-scale real data, the prognostic significance of our results may be discounted a little. First, the main limitation is that the universality of our study may be limited by the fact that it is conducted in a single cultural/social context. Our research is carried out in one country, which is probably a relatively homogeneous population. Due to the lack of sample size and stratified sampling, it cannot represent the true situation of all PeCa patients, and the results will inevitably be influenced by local culture. Therefore, this research needs to be carried out in more countries and regions. Second, the results may still be affected by the selection bias inherent in the design of this study, because adjuvant therapy (including adjuvant chemotherapy and/or radiotherapy) and pelvic lymphadenectomy may affect other parameters. Third, we were unable to investigate other important issues, such as the influence of the number of LNs at stations N1 and N2. As the treatment of PeCa progresses, the prognostic significance of our ELN and LND cut-off values may be changed, so this finding needs to be verified in other cohorts. Fourth, SEER databases may include inhomogeneous data about data collection deriving also from different intern protocols adopted by each center enrolled patients coming from.Despite these limitations, our analysis demonstrates that the greater the number of LNs examined, the smaller the LND value, and the higher the long-term OS of patients with PeCa. We recommend checking at least 13 LNs and LND >9.3% as a cut-off point for assessing the prognostic stratification of patients with PeCa. This further proves that ELN and LND are tools for predicting PeCa. More institutional research is needed to further determine the clinically relevant prognosis data of the disease.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.PG and XZ designed the study. JG provided the databases. PG, TZ, HL, and XL assembled and analyzed the data. PG wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by the National Natural Science Foundation of China (No. 82071637) and Natural Science Foundation of Anhui Province, China (No. 2008085QH420).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.


The purpose of this study was to identify factors associated with HIV-associated neurocognitive disorder (HAND) and symptoms of anxiety and depression in HIV+ Brazilian elderly on antiretroviral treatments. The study included 112 HIV+ elderly who completed a questionnaire, tests for cognitive screening, attention, problem solving, processing speed, visual perception, memory, and anxiety and depression scales. The results showed presence of HAND (89.3%), pathological anxiety (48.2%) and depression (58%) in the sample. Higher income was a protective factor for HAND (OR = 0.33). Waking up well-rested (OR = 0.63) and better diet quality (OR = 0.62) reduced the chance of pathological anxiety. Higher education (OR = 0.74) and waking up well-rested (OR = 0.61) reduced the chance of depression. Being female (OR = 7.73) increased the chance of depression. It can be concluded that it is important to evaluate cognitive and emotional aspects of HIV+ elders and to consider social and educational status, diet, and sleep in interventions, paying special attention to elderly women. The evolution of antiretroviral treatments has contributed to increase the quality and life expectancy of HIV+ people since their emergence, changing their status from rapidly fatal to chronic. But this population still has lower life expectancy than the general population, making HIV+ people to be considered elderly from the age of 50 .Between the emergence of the first case and the year 2020 Brazil registered 1,011,617 HIV+ people, of which 135,907 were elderly . The eldAs one ages there is a greater chance of the emergence of cognitive deficits  and anxiIn HIV+ people, the presence of deficits in two or more cognitive skills simultaneously is the condition for identifying HIV-associated neurocognitive disorder (HAND) . The mosCognitive deficits worsen treatment adherence and increase the chance of early onset of opportunistic diseases and occurrence of risky sexual behavior , which dThe first studies on cognitive deficits and emotional symptoms in HIV+ Brazilian elderly began in the 2000s, but are still scarce . ConsideIn this study, 112 HIV+ elderly people (50 years or older), who had been on ART for 6 months or more in a reference service for infectious diseases in a medium-sized city in the state of Minas Gerais, Brazil, were evaluated. The project followed the ethical precepts for research with human beings and was approved by the Research Ethics Committee (CAAE 01355018.40000.5154). A pilot study was initially conducted with five HIV+ elders to adapt the questionnaire and train the research team that conducted the collection. The evaluations were individual and occurred between August 2018 and March 2019.The elderly had a mean age of 55.7 years (SD + 5.25), HIV+ diagnosis for 12.12 years , and CD4 load of 226.31 (SD = 346.70). Most of the participants were male (50.9%), self-reported as white (55.4%), single (40.2%), with family income of 1 to 2 minimum wages , never studied (55.4%), and did not use other drugs except ART (53.6%) .A questionnaire with demographic and socioeconomic questions, time of diagnosis, use of medication, physical activity, frequency and perception of diet and sleep quality, use of alcoholic beverages and illicit drugs, was used. The following were assessed: functions for basic cognitive screening  adopting Enter Method. For HAND, eight multivariate models were tested. The first model included the variables: sex, marital status, education level, family income, CD4, hours of sleep, waking up rested, drinking alcohol, and depression. For anxiety we tested 12 models. The initial model included: sex, family income, skin color, education level, HAND, CD4, diet quality, waking up rested and the test results for Five Digits Test, and Rey Complex Figures – copy. And for depression seven models were tested. The first model included: sex, wake up rested, family income, meals, diet quality, alcohol beverage, drugs, and Rey Complex Figures – copy. Model fit was based on Nagelkerke’s R2, Wald test, and Hosmer–Lemeshow test. The analyses were performed in IBM SPSS version 22.Descriptive analyses were adopted and in the face of non-normality of cognitive variables (Shapiro–Wilk < 0.001), non-parametric two-way analyses  were used and separate robust logistic regressions were conducted for HAND, anxiety, and depression  and considered their own diet as good/adequate or excellent (57.1%) . Most ofMost of the elderly showed a normal result in cognitive screening (56.3%), but there was inferior performance for processing speed for 82.1% and visual memory (80.4%) . In atte2 = 25.10; p < 0.001) and people with lower income showed more results for depression . People with worse perceptions about their diet showed higher anxiety  and more results for depression . People who slept fewer hours per day  and felt well-rested fewer days per week had higher anxiety  and showed more results for depression .On the emotional aspects , women  and inhibition . People with lower education showed more deficits in cognitive screening , lower result in matrix reasoning  and in the attentional processes of counting , alternating  and inhibition . People with lower income showed worse performance in the attentional process of reading  and higher frequency of HAND .People with CD4 200 > showed worse performance in the attentional processes of choice  and visual perception and planning . People self-referred as brown-skinned performed worse in the attentional processes of reading  and counting . People with worse perceptions about diet showed worse visual perception and planning . People who did not practice physical activity showed worse attentional performance for reading .Females performed worse in problem solving , while higher education (OR = 0.74) and waking up well-rested on more days (OR = 0.61) reduced the chance of the disorder. These three variables explained 40% of the variance in the depression data.This study identified cognitive deficits and emotional symptoms in most of the HIV+ elders investigated. Screening for HAND was present in 89.3% of the sample. The high presence of isolated cognitive deficits or HAND in HIV+ people has been identified independent of age and education  and relaThe worst cognitive scores were perceived in information processing speed, visual memory, attentional alternation, and visual perception. Deficits related to memory, attention, processing speed, and problem-solving ability are the most frequently observed in HIV+ people . Távora Depression is the most common psychiatric complication associated with HIV  and was The high anxiety perceived in the sample corroborates findings of previous studies associating anxiety and depression . In the When assessing the factors associated with HAND, only the income was shown as an independent factor, which went against the study hypotheses, but may be a marker of the profile of the HIV+ population in Brazil. The sociodemographic profile observed in this study shows similarity with the profile of HIV+ Brazilians of other age groups, being marked by people with low education, low income, and a predominance of men . A largeThe perception about better quality of diet and waking up well-rested on more days reduced the chance of pathological anxiety in HIV+ elderly. The relationship between better diet and emotional state has been previously identified in other populations , but is Factors associated with depression were being female, education, and waking up well-rested. The higher risk for depression among women has been previously identified in several populations , and theOur study also has limitations. First, because it was cross-sectional, we were not able to infer causality for the associations found. Another limitation was the absence of a control group, which meant that the results were only compared with the normalized values of the tests and with deficit estimates in the literature for elderly Brazilians. The absence of a control group also prevents us from knowing the specific impact of HIV on the observed deficits, as we do not know what the performance of elderly people with the same socioeconomic conditions without HIV would be when responding to the tests. In addition, our sample size was relatively small and comes from a single service and our analysis will need to be replicated in larger cohorts to confirm the generalizability of our findings. Future research, with larger sample, control group, and that investigate others clinical aspects may help to identify the factors associated with cognitive deficits and emotional symptoms of the HIV+ elderly.The Brazilian HIV+ ages 50 and older increase in the last two decades, thus, more research is needed to identify neuropsychological deficits and emotional symptoms in this population. Our study suggests that elderly HIV+ are at risk for cognitive deficits, pathological anxiety, and depression. The importance of income, education, quality of food and waking up rested on cognitive and emotional aspects, and indicating the greatest risk of women for depression, showing the need to include these aspects in the evaluation and treatment of this population. Future research will be able to identify whether HIV+ elderly people are at greater risk of cognitive deficits or emotional problems when compared to elderly people without this pathology, investigate the influence of other life and health conditions on cognitive abilities and help to understand the role of perceiving oneself rested in emotional symptoms in HIV+ older people.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservations, upon justified request.The studies involving human participants were reviewed and approved by Institutional Review Board of Federal University of Triângulo Mineiro. The patients/participants provided their written informed consent to participate in this study.SB contributed to the study concept and design, data analysis, and study supervision, and wrote the manuscript. KS contributed to the study concept and design, and data collection. Both 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.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.


Purpose: To explore the therapeutic effect of a dietary supplement on dry eye with meibomian gland dysfunction (MGD).Methods: Sixty patients with MGD-related dry eye were included in this prospective and randomized, placebo-controlled study. All the subjects were treated with eye hot compress, artificial tears, and antibiotic ointment. After that, the patients received dietary supplementary or placebo daily for 12 weeks. The dry eye signs, function of MG, and visual quality of the patients were assessed at 4, 8, and 12 weeks after the treatment.Results: Twelve weeks after the treatment, patients who received dietary supplement had a significantly better improvement of dry eye symptoms, in terms of ocular surface diseases index and tear breaking-up time (TBUT), than those who received placebo (P < 0.05). The functions of MG, in terms of meibum quality and MG exclusion and MG obstruction scores, were significantly improved in both dietary supplement and placebo groups (P < 0.05). Patients who received dietary supplement had a significantly better improvement in the MG structure, in terms of acinar diameter and acinar density, than those who received placebo (P < 0.05). The number of inflammatory cells near MG was significantly lower in the dietary supplement group when compared with the placebo group (P < 0.05). The objective visual quality was significantly improved in the dietary supplement group, but not in the placebo group (P < 0.05).Conclusion: The dietary supplement can effectively improve the symptoms and signs of MGD-related dry eye, reduce the inflammatory reaction of MG, restore the gland structure, and indirectly improve the visual quality. Meibomian gland dysfunction (MGD) is a chronic, diffuse meibomian gland lesion. The main features of MGD are the obstruction of the terminal duct of the meibomian glands and the abnormal quality or quantity of the meibum secretion . This diThe current treatment of MGD-related dry eye includes physical and oral drug therapies, such as eye drops, hot eye compresses, and MG massage . However® is a new dietary supplement formulation on the market that has recently obtained the national safety standard identification in China. Its main ingredients include omega-3 free fatty acids, lutein, aronia extract, vitamin C, and vitamin E. It has potent antioxidant and anti-inflammatory effects, which play an important role in promoting retinal development and improving visual function  .The inclusion criteria include (1) the age of patients in between 18 and 80 years and (2) the best-corrected visual acuity in both eyes that is higher than 0.1. The exclusion criteria include (1) systemic diseases such as systemic lupus erythematosus, rheumatism, and diabetes; (2) history of eye surgery and trauma; (3) other eye diseases; (4) systemic and ocular medications; (5) pregnant or lactating women; (6) patients who are allergic to the ingredients of the dietary supplement formulation or the drugs involved in this study; and (7) people who cannot take care of themselves and cooperate with treatment and follow-up.All patients have agreed and signed the consent form to be included in the study. This study followed the Declaration of Helsinki, conformed to the principles of medical ethics, and was approved by the Ethics Committee of Xiangya Hospital of Central South University (Document number: 201912528).®  and placebo control group (60 eyes of 30 cases). Both the groups were respectively treated with oral dietary supplementary formulation Retaron®  and placAll subjects were followed up for 12 weeks with a 4-week interval. To avoid systematic errors, all the inspections and operations described below were completed by the same experienced ophthalmologist or technician. All subjects underwent routine examinations, such as vision, optometry, intraocular pressure, and fundus, at each follow-up.Dry eye symptoms and signs were evaluated by Ocular Surface Disease Index (OSDI) Questionnaire Scale,  tear breThe MG obstruction  of each All the five MGs in the upper eyelid of each subject were squeezed, and the function of MG was assessed by the following MG discharge capacity score : 0 pointin vivo confocal microscopy (IVCM)  and evaluated by (1) the diameter of MG opening, (2) acinar diameter, (3) acinar density, and (4) local inflammatory cell count , Strehl ratio (SR), and objective scatter index (OSI). MTFcutoff reflects the resolution of the optical system, and its value is directly proportional to the visual quality. The value below 30 c/deg is considered as abnormal visual quality. SR is the ratio of the light intensity at the Gaussian image point of the actual refractive medium to the light intensity at the Gaussian phase point of the normal refractive medium. The higher the SR value, the closer it is to an aberration-free optical system. Below 0.15, the visual quality is considered abnormal. OSI reflects the degree of turbidity of the refractive medium. Values higher than 2.0 are regarded as abnormal, and higher values represent more turbid refractive media.The visual quality of the patients was objectively assessed using the Optical Quality Analysis System . Several visual quality assessment parameters were assessed, including modulation transfer function cutoff frequency (MTFt-test (two time points). If the data did not meet the “spherical symmetry” assumption, the Greenhouse–Geisser estimate was used for correction. The LSD t-test (multiple time points) and paired t-test (two time points) were used for group comparison at each time point. If the data did not conform to be normally distributed, the Mann–Whitney U-test and the Kruskal–Wallis test were used for pairwise comparisons between groups at each time point. The test level was α = 0.05, and P < 0.05 was considered statistically significant.Statistical analysis was performed using SPASS 19.00 . The Kolmogorov–Smirnov test was used to test the normality of the data. Normally distributed data were expressed as mean ± SD, and non-normally distributed data were expressed as median and interquartile range. If the data conformed to be normally distributed, the comparisons between groups at each time point were tested by repeated-measures ANOVA (multiple time points) and independent-sample P > 0.05 for all indices)  were included in this study, namely, 30 subjects (60 eyes) in the dietary supplement group and 30 subjects (60 eyes) in the control group. Before the treatment, there were no significant differences in age, best-corrected visual acuity, or intraocular pressure between the two groups (indices) .P > 0.05 for all indices). Twelve weeks after the treatment, punctate staining of corneal epithelium disappeared in both groups (P < 0.001) (P < 0.001)   . However< 0.001) . Moreoveindices) .P > 0.05 for all indices). After 12 weeks of treatment, the upper eyelid margin congestion was reduced in both groups, and MG obstruction was significantly improved. The meibum was also clear and easy to be excreted when the MG was compressed . However, there is no significant difference between the two groups at the same time point   .P > 0.05 for all indices). Twelve weeks after the treatment, the number of inflammatory cells  and were significantly better than in the control group  (P > 0.05 for two groups)  -d,h. The groups) -j,l.PMTFcutof, PSR, and POSI, between the two groups . Twelve weeks after the treatment, the MTFcutoff and SR of the dietary supplement group were significantly improved (P < 0.001 for MTFcutoff and SR). In contrast, the improvement of the control group, when compared with the baseline, was not significant (P > 0.05 for MTFcutoff and SR). Moreover, the difference between the two groups was significant  (POSI > 0.05 for two groups)  . However groups) .During the follow-up, no obvious local or systemic adverse reactions were observed in all subjects.The MG is a special exocrine gland. The meibum secreted by the acinar epithelium forms a lipid layer on the tear film surface to make the tear film stable. When the MG is blocked or atrophic, this causes pathological changes in both the quality and quantity of the meibomian ester and its excretion, and finally leads to MGD. At the same time, MGD is often accompanied by local inflammatory reaction, which will eventually lead to lipid deficiency-type dry eye. Current studies have confirmed that MGD is one of the most important causes of dry eye .Dietary supplements have been widely used for long time in all age groups worldwide, especially in developed countries, where dietary supplement use is as high as 11.7–66% . It play® can play an antioxidant role in the human body, thus affecting the retinal dynamic function . The patients/participants provided their written informed consent to participate in this study.KL and YC: data acquisition and/or research execution, data analysis and/or interpretation, and manuscript preparation. XP and ML: data acquisition and/or research execution, data analysis and/or interpretation. RY: data analysis and/or interpretation, manuscript preparation. JL, XT, and YX: data acquisition and/or research execution. YC and QL: data analysis and/or interpretation. HW: research design, data acquisition and/or research execution, data analysis and/or interpretation, and manuscript preparation. All authors contributed to the article and approved the submitted version.This study was supported by National Natural Science Foundation of China (No. 81170823), Natural Science Found of Hunan Province (No. 2019JJ40502), Science and Technology Project of Hunan Administration of Traditional Chinese Medicine (No. 201856), Central Committee Guides Local Science and Technology Development Special Project by Ministry of Science and Technology (No. 2020YDDF0043), and Major Special Project of Key Research and Development Program of Ningxia Province (No. 2021BEG01001).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.


This study aimed to explore the effect of enriched rehabilitation (ER) on cognitive function and serum glutamate levels in patients with stroke.n = 20) or conventional medical treatment . In addition, 20 age-matched healthy subjects who were outpatients in our hospital during the same period formed the healthy control (HC) group. In- and between-group differences in cognitive function were assessed during pre-intervention and post-intervention based on the Montreal Cognitive Assessment (MoCA), the Symbol Digit Modalities Test (SDMT), and the Trail Making Test (TMT). The serum levels of glutamate, tumor necrosis factor (TNF), and malondialdehyde (MDA) levels were also detected pre-intervention and post-intervention.Forty patients diagnosed with post-stroke cognitive impairment (PSCI), according to the inclusion criteria, and undergoing inpatient rehabilitation were enrolled in the study. Patients were randomly assigned to receive 8 weeks of ER treatment , but not between the two groups of patients with PSCI (p > 0.05). Significant improvements were observed in cognitive function in both the ER and the CM groups post-intervention compared with pre-intervention, as evidenced by the measured improvement in MoCA, SDMT, and TMT scores. Similar improvements were seen for serum glutamate, the degree of oxidative damage, and the level of inflammation in both the treatment groups (p < 0.05). More enhancements in cognitive function, including MoCA, SDMT, TMT scores, and the serum levels of glutamate, the degree of oxidative damage, and the level of inflammation were shown in the ER group compared with the CM group post-intervention (p < 0.05).Pre-intervention cognitive function and the levels of all the serum parameters assessed significant difference between the HC group and the PSCI group (both ER and CM groups) (ER can improve cognitive function in patients with PSCI. The associated mechanism may be related to the negative regulatory effect of ER on serum glutamate, TNF, and MDA levels, which is likely to enhance synaptic plasticity and alleviate oxidative stress- and inflammation-related damage, at least to some extent. Post-stroke cognitive impairment (PSCI) is one of the most common sequelae of stroke. Over 70% of survivors have cognitive impairment in the first week post-stroke while 37% still display cognitive deficits after 6 months . PSCI isEnriched rehabilitation (ER), a comprehensive strategy that combines environmental enrichment and task-oriented exercises, has shown the potential to improve cognitive function in animal models of central nervous system diseases, including stroke and Parkinson's disease , 6. SeveNeurotransmitters are critical in cognitive function. Glutamate is an excitatory neurotransmitter that has been implicated in cognitive function, especially in memory and learning processes. After a stroke, glutamate levels in the infarcted site were found to be up to 80-fold higher compared with its physiological level, resulting in elevated glutamate contents in the cerebrospinal fluid and blood . HoweverGiven that ER has been associated with improved cognitive outcome brain functions and that serum glutamate levels have been associated with cognition, in this study, we explored the effect of ER on cognitive function, serum glutamate levels, oxidative stress responses, and inflammation-related responses in patients with PSCI.All patients with cerebral infarction at the Yangzhou University Clinical School from January 2020 to June 2021 were assessed for enrollment in the study based on the following inclusion criteria: cerebral infarction confirmed by MRI scan due to the lesion in the internal carotid artery system; meeting the diagnostic criteria for PSCI with a Montreal Cognitive Assessment (MoCA) score between 18 and 23; the absence of sensory aphasia and having the ability to communicate with others, at least simply; primary and unilateral onset and a volume of ischemic necrosis between 20 and 40 ml as determined diffusion-weighted imaging (DWI)  how the; 55–70 yAmong the 217 identified patients, 61 met the inclusion criteria. Of these, 12 were unwilling to participate in the study. As measured by the clinical sample size, a minimum of 20 participants was required for each group . All patients signed an informed consent form. The general process of the study is shown in The patients in the CM group received conventional medication, including drugs for controlling blood pressure, lowering cholesterol, improving cerebral blood flow (nimodipine 20 mg two times a day), promoting cognitive function (huperzine A 100 mg twice a day), and other related medications, according to Chinese Guidelines for diagnosis and treatment of acute ischemic stroke 2018 and other guidelines for diagnosis and treatment of ischemic stroke , 17. In The Internet was used to select the reading materials and images the patients were interested in for reading and viewing one time a day for 10 min each time.The patients smelled two bottles of perfume with different scents and tried to name the scents for 5 min each time.Toys or objects of different shapes, sizes, and textures were placed in black cotton cloth bags such that patients could not see the objects. The patients tried to name the objects or describe their characteristics by touching them with the hand on the paralyzed side. This process lasted for 10 min each time.For lower limb exercise, the patients performed activities such as field walking, mushroom picking in the forest, or skiing in the snow using virtual reality equipment and a flat treadmill. For upper limb exercise, the patients were asked to perform tasks such as painting with a brush or making different items with plasticine. The patients were required to use the paralyzed limb aided by the healthy limb. The task lasted for 15 min each time.Patients in the ER group were required to complete specific tasks by integrating multiple cognitive functions using virtual reality equipment or actual scenarios, including:The patients were given a list before shopping and had to return the list within a specified time. The patients had to purchase the listed items in the supermarket by themselves.Patients had to attend lectures in the room and to recite the content as much as possible, just like student activity. Stimuli such as noise, birds, and other people's voices were added to simulate interference during the class. Therapists asked questions after the class.A set of cards with different colors and graphics was used. The therapist placed three cards according to a specific rule  and asked the patient to identify the rule and select the appropriate fourth card.This task simulated a patient going home from the hospital. The patient was asked to complete planning the route, to choose a station to board and disembark, to enter and exit subway stations on their own, etc.Cognitive activities were performed one time daily for a total of 40 min.Participants were encouraged to communicate with each other during the entire ER training process; to play card games, chess, table tennis, and other sports involving more than one person; and to express their opinions on a hot social topic.Social activities lasted for 40 min each time. All the above activities were undertaken under the guidance of therapists.All the patients completed the assessment of cognitive function and the measurement of serum parameters within 48 h before the first treatment and 48 h after the 8-week rehabilitation process, respectively. The HC group completed the above tests within 24 h after enrollment.The overall cognitive function of patients in each group was determined based on the MoCA score. The assessment comprised eight items, including visual space and executive function, attention, naming, memory, abstract reasoning, language, delayed recall, and orientation ability, up to a maximum of 30 points, with ≥26 points being considered normal .The symbol digit modalities test (SDMT) was used to assess attentional function. The SDMT records the number correctly entered within 90 s as the final score, excluding the number filled in during the practice .The Trail Making Test (TMT) was used to evaluate the executive function, in which part A (TMT-A) is connected in sequence from numbers 1–25. TMT part B (TMT-B) is connected sequentially with numbers and letters alternating. The time spent recording TMT-A and TMT-B represented the evaluation index .All the patients were assessed for serum glutamate levels within 48 h before treatment, within 48 h after 8 weeks of treatment, and 12 h after fasting. In the morning, blood was drawn into a test tube without anticoagulant. Once it had precipitated at room temperature, the serum was collected and stored at −80°C. The glutamate concentration in serum was detected by high-performance liquid chromatography . The expThe serum MDA level is a sensitive index reflecting the degree of oxidative damage in the body . Serum wThe serum TNF content is a sensitive indicator that reflects the level of inflammation in the body . Serum wU test was utilized to compare the between-group differences post-intervention (ER and CM groups) for continuous variables, including MoCA, SDMT, and TMT scores and serum levels of glutamate, TNF, and MDA post-intervention. The significance was set at p < 0.05.Data were analyzed using SPSS 24.0 . Results are presented as means ± standard deviation (SD) or median (quartiles) for continuous variables, depending on the normal or non-normal distribution of data. It was assessed to the normality of data by Shapiro–Wilk, and the variance homogeneity of data by one-way ANOVA test/Levene test. A chi-square test was used to analyze the between-group differences for categorical variables, such as gender and stroke site. A one-way, repeated-measures ANOVA or the Kruskal–Wallis test was performed to detect between-group differences  for continuous variables, which included MoCA, SDMT, and TMT scores as well as serum levels of glutamate, TNF, and MDA pre-intervention and post-intervention. A paired t-test or the Wilcoxon Rank Checking test was used to detect within-group differences (pre-intervention vs. post-intervention in the PSCI groups) for continuous variables. A least-significant difference test or the Mann–Whitney In this study, the data including the scores of MoCA and TMT-A, and the serum concentration of TNF were not normally distributed nor was variance homogeneity met. There were 10 men and 10 women in the CM group, with an average age of 60.95 ± 5.70 years. The ER group consisted of 9 men and 11 women, with an average age of 61.70 ± 5.59 years. The HC group comprised 11 men and 9 women, with an average age of 61.03 ± 5.42 years. The baseline information for each group is presented in p < 0.05) were found between the HC group and patients with PSCI (both the CM and ER groups) but not between the two groups of patients with PSCI (CM and ER groups). After the intervention, general cognitive function, the attentional function score, and the executive function score of patients in the ER group and the general cognitive function and the attentional function score of patients in the CM group were significantly changed as compared with those before intervention (p < 0.05); however, those cognitive function indicators still differed significantly between the HC group and the PSCI group (both the CM and ER groups) (p < 0.05). Additionally, the overall cognitive function and attentional function scores were significantly higher in the ER group than in the CM group after intervention (p < 0.05).As shown in p < 0.05) from those in the HC group; however, no differences were detected between the two groups of patients with PSCI (p > 0.05) for the above indicators. After the intervention, the serum levels of glutamate, MDA, and TNF in both the PSCI groups were significantly improved compared with the pre-intervention levels (p < 0.05). Additionally, the levels of the above indicators in the ER group were significantly better than those in the CM group (p < 0.05). However, post-intervention, the serum levels of glutamate, MDA, and TNF in patients with PSCI were still significantly different from those in the HC group (p < 0.05)  .Enriched rehabilitation represents a novel therapeutic strategy that has been used to enhance neuroplasticity and improve functional outcomes in animal models with central nervous system diseases, including stroke and perinatal hypoxia–ischemia , 32. In p < 0.05) in the overall cognitive function, attentional function, and executive function in patients with PSCI, as determined by the MoCA, SDMT, and TMT scores.Post-stroke cognitive impairment is a common complication in stroke survivors that requires complex treatment regimens. ER represents a non-pharmacological therapeutic strategy that combines environmental enrichment with task-oriented exercises, in which a multistimuli environment is accompanied by high-quality social and cognitive exercises. Animal studies have suggested that ER may be a viable approach for treating PSCI –34. AddiCognitive function requires the participation of a variety of neurotransmitters, which have to do with synaptic plasticity , 37. GluThe process is as follows: first, excessive glutamate can lead to the overactivation of NMDA receptors in neurons, followed by a massive influx of calcium ions into cells, resulting in mitochondrial damage , 44 and In addition, under physiological conditions, intracerebral nitric oxide (NO) produced by nNOS mediates communication between nerve cells. However, under inflammatory conditions, iNOS overexpression in cerebral vascular smooth muscle cells and macrophages leads to excessive NO production , furtherp < 0.05) in patients with stroke than in HCs, which is consistent with previous findings (p < 0.05). Surprisingly, the serum glutamate level in the ER group was reduced to a greater extent than that in the CM group post-intervention (p < 0.05). The above results suggested that the efficacy of ER in enhancing cognitive function in patients with PSCI may be related to its effect on decreasing serum glutamate levels. However, the underlying molecular mechanisms require further investigation.Metabolites can be transferred between cerebrospinal fluid and blood through meninges , 57. A pfindings . Howeverp < 0.05). We inferred from the above results that ER can improve cognitive function by abrogating oxidative stress and neuroinflammation induced by increased levels of glutamate post-stroke.As previously mentioned, oxidative and inflammatory mechanisms also play a pathogenic role in the process of cognitive impairment post-stroke , 54. In The strengths of our study were that, in addition to the behavioral assessment, we also analyzed serum glutamate levels in patients with PSCI during pre-intervention and post-intervention. Additionally, we investigated oxidative stress- and inflammation-related mechanisms mediated by ER through the measurement of serum MDA and TNF levels. Nonetheless, our study also had several limitations. First, this study was conducted in only one hospital and the sample size was small, which is likely to introduce bias. Additionally, due to the small sample size, we did not stratify participants based on different cognitive levels, which may mask the effect of ER. Furthermore, we could only infer the preliminary mechanisms involved in the effects of ER on PSCI; identification of the precise underlying mechanisms will be explored in a future study.In conclusion, the present study provided clinical evidence that ER can improve cognitive function in patients with PSCI. The associated mechanism may be related to the negative regulatory effect of ER on serum glutamate, TNF, and MDA levels, which is likely to enhance synaptic plasticity and alleviate oxidative stress- and inflammation-related damage, at least to some extent.The original contributions presented in the study are included in the article/This study was approved by the Medical Ethics Committee of Clinical Medical College of Yangzhou University . The patients/participants provided their written informed consent to participate in this study.XW, YP, and JG designed the study and wrote the manuscript. XW, YP, HZ, WD, JuW, JiW, TW, XT, and YL performed the experiments. XW, YP, HZ, WD, JuW, and JG analyzed the data. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (Grant No. 82072533), the Six One Project Scientific Research Project for High-Level Health Talents of Jiangsu Province (Grant Nos. LGY2017028 and LGY2018027), the Key Young Medical Talents in Jiangsu Province (Grant No. QNRC2016339), Yangzhou Science and Technology Development Plan Project (YZ2020201), Huxin fund of Jiangsu Key Laboratory of Zoonosis (Grant No. HX2003), Natural Science Foundation of Shandong Province, China (Grant No. ZR2019MH104), Scientific Research Project of Jiangsu Commission of Health (Grant No. Z2020055), Yangzhou Social Development Funds (Grant No. YZ2021059), the Science Foundation of Subei People's Hospital (Grant No. SBJC21005), and the Science Foundation of Guangzhou First People's Hospital (Grant No. M2019009).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. 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Purpureocillium lilacinum. Our study aimed to evaluate some cell subsets and inflammatory markers involved in the in situ immune response to subcutaneous hyalohyphomycosis by P. lilacinum in C57BL/6 murine models. The fungal isolate was inoculated in mice randomly distributed in immunocompetent/infected (CI) and immunosuppressed/infected (SI) groups. Mice were evaluated on days 1, 3, 5, and 7 after inoculation. Histopathological studies showed several lesions in the site of infection as well as the formation of multifocal and mixed inflammatory infiltrates, which differed between the CI and SI groups. This analysis also revealed conidia and hypha-like structures in subcutaneous tissues of mice of both groups. The immunohistochemical analysis showed lower percentages of macrophages and neutrophils in the SI group compared to those in the CI group. Moreover, the intensity of interleukin (IL)-1β and nitric oxide synthase 2 production by cells of immunosuppressed mice was discreet, compared to immunocompetent mice that ranged from moderate to intense over time. The quantitative interference of dexamethasone in the response to the fungus was also demonstrated. We concluded that our results can be useful not only to broaden the knowledge on P. lilacinum but also, based on this host–parasite relationship, to contribute to the understanding of the mechanisms of infection.Hyalohyphomycosis is a fungal infection characterized by the presence of a hyaline mycelium in the host. It is caused by several agents, such as  Paecilomyces spp., Fusarium spp., Scedosporium spp., Scopulariopsis spp., Penicillium spp., and Purpureocillium lilacinum. Some species, including P. lilacinum, in addition to hyaline mycelium have the ability to produce adventitious forms in the tissue, which are structures morphologically similar to phialides and microconidias that spread through the blood, causing occlusion, infarction, and vascular necrosis  Luangsa-ard, Houbraken, Hywel-Jones and Samson, comb. nov 2011, previously called Paecilomyces lilacinus, is a filamentous, hyaline, anamorphic fungus  and other substances secreted by these cells   . Since cls (DCs) .++ T lymphocytes, and to a lesser extent CD8+ T lymphocytes, in addition to monocytes, macrophages, and polymorphonuclear cells. In the murine model of systemic infection, The impact of immunosuppression by glucocorticoids on the immune response is due to their mechanisms, which promote the inhibitory effect of the synthesis of pro-inflammatory cytokines such as interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α, and prostaglandins . AccordiP. lilacinum due to its emerging and opportunistic potential in an immunocompetent and immunosuppressed murine model, characterizing phenotypically and functionally some cells involved in this process.Thus, this study aimed to evaluate some cell subsets and inflammatory markers involved in the skin immune response in subcutaneous hyalohyphomycosis by This study was performed in strict accordance with the Brazilian College of Animal Experimentation (COBEA). All experimental procedures involving animals followed the regulations for animal experiments of the Ethics Committee for Animal Study of FIOCRUZ (license number L-031/2015-CEUA/IOC-FIOCRUZ) and were performed under anesthesia. All efforts were made to minimize the suffering of the animals.Ninety-six male C57BL/6 mice, aged 6–8 weeks, weighing approximately 21 g were used in the experiments. The number of animals used in this study was calculated to guarantee statistically valid results obtained with as few individuals as possible without compromising the degree of reliability and avoiding the unnecessary use of animals.3/h. Breeding room was managed at room temperature; 23–25°C, humidity; 30–70%, light/dark cycle; 12 h, and water and food were given ad libitum. We took the utmost care to alleviate any pain and suffering on the part of the mice. Mice were submitted to euthanasia by CO2 exposure prior to analysis.Mice were randomly allocated to each experimental group and were kept under specific pathogen-free conditions at the Laboratory of the Animal Experimentation Center (IOC FIOCRUZ) and maintained in micro-insulators made of polypropylene, autoclavable, containing pine beds. Micro-insulators were disposed in ventilated racks (ALESCO) with air exchange measures of 350 mP. lilacinum was used in this study: S2, kindly provided by Dr. Annette Fothergill , isolated from skin biopsy on the left foot of a female patient. The strain was molecularly authenticated and deposited in GenBank® sequences database with access number MF590109 (One human strain of MF590109 .The isolate was subcultured on potato dextrose agar (PDA) medium at room temperature for 12 days, and then, conidia were collected by scraping the colonies, suspended in phosphate buffered saline (PBS), followed by thermal shock protocol to separate conidia from hyphae, according to ad libitum in the drinking water for 3 days before fungal inoculation and during all the experiments  was also added to the drinking water in parallel to prevent bacterial infections. The route of administration of the drug had already been demonstrated by our group as safe and effective in inducing immunosuppression without, however, causing stress in animals as do invasive methods like gavage  administered eriments . We chose gavage , 2017.5 conidia of P. lilacinum with more than 80% viability. Control groups (CC and SC) were similarly inoculated with PBS. Mice were checked daily for 7 days to observe abnormal behaviors and pathological changes, with the aim of minimizing suffering. On each observation point , four animals from the CI and SI groups and two from the CC and SC groups were euthanized.The fungal isolate was inoculated subcutaneously by a syringe at the base of the tail in 32 mice randomly distributed into two groups: immunocompetent (CI) and immunosuppressed mice (SI). Sixteen mice were distributed into immunocompetent (CC) and immunosuppressed mice (SC) control groups and similarly inoculated with PBS. In dorsal–ventral position, mice were inserted in an acrylic container, and after trichotomy and asepsis of the tail base region, the CI and SI groups were inoculated with 0.02 ml of the cell suspension containing 1 × 102 exposure. After asepsis of the region, two skin fragments of approximately 3 mm in diameter around the point of inoculation were removed. One skin fragment was submitted to histological analysis and the other to immunohistochemistry.Mice were submitted to anesthesia with 10% ketamine hydrochloride associated with 2% xylazine hydrochloride  and submitted to euthanasia by COThe skin fragment destined for histopathology was fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. Sections were cut and stained with hematoxylin–eosin (H&E), periodic acid-Schiff (PAS), and Grocott (methenamine silver), and the photographs were taken with a Leica DM 1000 . The inflammatory infiltrate in tissues was classified into: granulomatous, predominance of macrophages, and non-granulomatous, predominance of other types of inflammatory cells . The intensity of the inflammatory infiltrate was classified as follows: absent or mild (cellular infiltrate absent or mild and dispersed foci) and moderate to intense (cellular infiltrate dense and diffuse).Biopsy fragments frozen in OCT resin  were cut into 3-μm-thick sections and mounted on microscope silanized slides . The slides were fixed in acetone PA  and hydrated in PBS, pH 7.4. Blockage of endogenous peroxidase  and non-specific staining  were performed before the use of specific antibodies.+ macrophages, anti-neutrophilic elastase, nitric oxide synthase type 2 (NOS2), and IL-1β , followed by incubation with the biotinylated secondary antibody (Zymed) and the streptavidin–biotin–peroxidase complex . Aminoethylcarbazole  was used as the substrate–chromogen system, and the slides were counterstained with Mayer’s hematoxylin (Dako). The slides were examined under a light microscope , and the percentage of stained cells was determined by counting 500 cells as standard. The intensity of IL-1β and NOS2 was scored in 10 microscope fields. + = discreet (at least one positive site per field); ++ = moderate (two or three positive sites per field); +++ = intense (four or five positive sites per field).The specimens were then incubated with the primary antibodies directed to surface receptors: CD68p-value cutoff for statistical significance was 0.05, and the n value for each group on each time point = 4.Data were analyzed using GraphPad Prism R software, version 8 . The Kolmogorov–Smirnov test was used to evaluate the distribution of variables. Mann–Whitney test was used to perform comparisons between groups. Data are expressed as mean ± standard deviation. The All the experiments were carried out twice to ensure reproducibility.Mice were observed for 7 days. No clinical alterations as apathy and weight and/or hair loss were observed in the immunocompetent (CI/CC) group. The inflammatory response at the site of the infection was discreet, non-suppurative, and non-ulcerative . On the P. lilacinum.P. lilacinum (CI group) presented dermatitis, panniculitis, and skin ulcerations with diffuse inflammatory infiltrate reaching superficial and deep dermis  had more severe lesions and more fungal structures than the immunocompetent ones (CI Group) from the first day after inoculation. In all the sampling days, the histological analysis of the tissue collected from these animals revealed the formation of diffuse and/or multifocal inflammatory infiltrate, which intensified until the end of the experiment. Extensive areas of ulcer covered by crusts, dermatitis, and suppurative panniculitis were also observed (data not shown).In general, immunosuppressed mice inoculated with P. lilacinum. As expected, all control mice (Groups CC and SC) were negative.On day 1, similarly to the immunocompetent group, numerous conidia in the interior of macrophages and hyphae in the tissue were revealed . On the P. lilacinum (CI group), the percentages of macrophages  and control (SC) groups. Approximately 280 cells were quantified in these animals and, as observed in immunocompetent mice, there was a higher percentage of neutrophils than macrophages , especiaP. lilacinum , and memory T cells, of immunocompetent and immunosuppressed mice infected with P. lilacinum for a period of up to 45 days. It was possible to observe the impact of dexamethasone in the course of the infection, as presented here, allowing us to observe that the administration of dexamethasone can quickly (within 2 h) modify the immune response  and immunosuppressed (SI) groups with respect to severity—more pronounced in SI. It is interesting to highlight that in the study published by The results of this work go in line with those obtained by in situ. In general, percentages of neutrophils in immunocompetent and immunosuppressed mice were higher than those of macrophages, except for the seventh day after inoculation, in the immunocompetent group. We know that P. lilacinum is capable of destroying macrophages that phagocytose the conidia, as well as forming germ tubes and hyphae in the first 24 h after infection in vitro  and immunosuppressed (SI and SC) mice allowed us to identify and quantify macrophages, neutrophils, and two inflammatory markers, IL-1β and NOS2, involved in the immune response in vitro . Thus, tin vitro . This hye tissue . These rSporothrix spp. in the host acquire the yeast form against which the macrophage-mediated immune response may be effective. The difference in the number of macrophages found in this study between the immunocompetent and immunosuppressed groups is also worth noting, demonstrating once again the action of dexamethasone. We know that the drug, by interfering with IL-2 synthesis, can suppress the production of inflammatory markers, such as IL-1β .DC-M, JO-F, and CB designed the study. DC-M and AS carried out the experiments. DC-M, RM, and FM analyzed the data. DC-M wrote the manuscript. DC-M, RM, FM, CB, and JO-F revised and approved the final manuscript. 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 and Aim: Studies on hematological parameters in the differential diagnosis of idiopathic granulomatous mastitis (IGM) and breast cancer (BC) are limited. This study investigated whether preoperative fibrinogen and hematological indexes can be used in the differential diagnosis of patients with IGM and early-onset BC. Methods: Fifty patients with BC, 55 patients with IGM, and 50 healthy volunteer women were included in the study. Results: There was a statistically significant difference between the IGM and the BC with respect to fibrinogen, fibrinogen/albumin (Fib/Alb) ratio, C-reactive protein (CRP), white blood cells (WBC), neutrophils, neutrophil–lymphocyte ratio (NLR), platelet–lymphocyte ratio (PLR), and monocyte values. When fibrinogen (p < 0.001), the Fib/Alb ratio (p < 0.001), CRP (p < 0.001), WBC (p < 0.001), neutrophil (p < 0.001), NLR (p < 0.001), monocyte (p = 0.008), and 2-hour sedimentation rate (p < 0.001) were compared between the groups, the highest levels were found in the IGM group. There was a negative relationship between CRP and albumin, and a positive relationship was observed between CRP and WBC, NLR, PLR, and 2-h sedimentation rate. CRP had the highest sensitivity (95%), whereas the Fib/Alb ratio (86%) had the highest specificity. Patients with recurrent IGM had increased fibrinogen, Fib/Alb, CRP, neutrophils, NLR, and 2-h erythrocyte sedimentation rate (ESR) and decreased lymphocyte levels compared to non-recurrent patients. Conclusions: Preoperative CRP, albumin, fibrinogen, Fib/Alb, WBC, neutrophil, NLR, monocyte, and 2-h ESR have considerable potential to be early and sensitive biomarkers of IGM caused by inflammation compared to BC. These parameters also have a significant effect on the recurrence of the disease, suggesting their potential as a practical guide for the differential diagnosis of BC from IGM. Idiopathic granulomatous mastitis (IGM) is a rare, chronic inflammatory breast lesion of unknown etiology that clinically and radiologically mimics breast cancer . IGM, whBC is the second most commonly diagnosed cancer worldwide, behind lung cancer . The IntNLR and fibrinogen levels, which are among the new indicators of systemic inflammation, have been associated with the prognosis of tumors such as gastric cancer , colon cStudies on hematological parameters in the differential diagnosis of IGM and BC are limited . The curThe study was approved by the Ethics Committee of the Cerrahpasa Faculty of Medicine . Fifty patients with solid tumors who were followed by a diagnosis of BC in the general surgery outpatient clinic of Cerrahpasa Medical Faculty, 55 patients diagnosed with IGM, and 50 healthy volunteer women with similar demographic characteristics were included in the study. Women over the age of 18, whose microbiological culture samples were confirmed to be sterile and found to have cancer and IGM in the histopathological evaluation, were included in the study. We excluded women who received chemotherapy or radiotherapy and were diagnosed with old or new BC or IGM, with another history of malignancy , and were pregnant or lactating. In addition, according to core needle biopsy taken from the suspicious lesion in the breast or pathological examination of the tissue taken from the abscess wall, patients with tuberculosis capable of granulomatosis reaction, infection such as fungal and parasitic reactions, sarcoidosis, Wegener granulomatosis, and non-infectious agents such as foreign bodies were excluded from the study.g. Plasma and serum were transferred to Eppendorf tubes and was studied immediately.Venous blood samples were collected from control subjects and patients with BC and IGM after 12 h of fasting. The samples were centrifuged for 15 min at 3000× Demographic data , biochemical parameters, cancer antigens (Carcinoembryonic antigen (CEA), CA 19-9, CA 15-3, CA-125), and pathology reports were obtained from the subjects’ medical records.Biochemical parameters were measured on an Olympus AU 800 analyzer by enzymatic methods using commercial kits  at the Central Biochemistry Laboratory of Cerrahpasa Medical Faculty. Tumor markers were measured by immunometric assay using an IMMULITE 2000 .p values were used to conduct the post-hoc test after the Kruskal–Wallis test. The Mann–Whitney U Test was used to compare two independent groups. Spearman’s correlation was used to show an association between two continuous variables. Receiver operating characteristic (ROC) curve analysis was performed to determine the sensitivity and specificity values of biochemical and hematological indexes that can be used to define BC and IGM. The statistical significance level was set at p < 0.05.SPSS 21 for Windows was used to conduct the statistical analyses. The Kolmogorov–Smirnov test was used for normal distribution. The Kruskal–Wallis test was used to compare data of three independent groups; adjusted The demographic characteristics and tumor markers of the study groups are shown in p < 0.01). The two-hour erythrocyte sedimentation rate (2-h ESR) was significantly higher in the IGM group compared to the control (p < 0.01). A comparison of fibrinogen (p < 0.001), the fibrinogen/albumin (Fib/Alb) ratio (p < 0.001), C-reactive protein (CRP) (p < 0.001), white blood cells (WBC) (p < 0.001), neutrophil (p < 0.001), NLR (p < 0.001), monocyte (p = 0.008), and 2-h ESR (p < 0.001) within the groups revealed the highest levels in the IGM group.As shown in Correlation analysis of the biochemical variables of the IGM group is shown in The recurrence rate of IGM was 20% at 2 years of follow-up. Biochemical laboratory findings and hematological indexes of patients with non-recurrent and recurrent IGM are shown in IGM continues to be an important problem for both surgeons and patients due to the difficulty in the diagnosis and treatment of the disease, as well as acute and chronic exacerbations and long-term effects on quality of life. The prognosis of IGM is not as clear as its treatment. IGM may be misdiagnosed as breast cancer.BC is still a worldwide public health dilemma. It is the most common female malignancy in the world and the primary cause of mortality due to cancer in women. Early diagnosis provides the best survival rate; thus, novel diagnostic markers are essential for reducing mortality and morbidity. Inflammatory factors such as the preoperative CRP, interleukin (IL)-6 and -8, and tumor necrosis factor-α are associated with BC prognosis ,26,27. TIn our study, there was a statistically significant difference between the IGM and the BC with respect to fibrinogen, Fib/Alb, CRP, WBC, neutrophil, NLR, PLR, and monocyte values. Some of these variables are components of IGM (WBC and CRP) and are, therefore, not surprising. More importantly, there was a negative relationship between CRP and albumin and a positive relationship between CRP and WBC, NLR, PLR, and ESR.Systemic inflammation and infection can promote thrombosis by increasing the serum levels of fibrinogen, leukocytes, coagulation factors, and cytokines, and by altering the metabolism and function of certain cells, monocytes, and macrophages . AlthougCRP, a member of the pentraxin family and a marker of inflammation, constitutes the prototype of acute phase proteins. CRP is a major acute phase reactant in humans, with an acute and rapid rise in response to infection and tissue damage. In response to all types of tissue damage and infection, the human body activates various mechanisms to correct this damage. The most important of these mechanisms is the acute phase response. In addition to radiological data and breast biopsy, circulating CRP levels may be non-invasive biomarkers that can help differentiate IGM from BC. CRP levels were found to be highest in the IGM group in the current study. A positive correlation was also found between 1-h ESR, 2-h ESR, WBC NLR, and PLR and CRP in IGM. ROC analysis results in the IGM group showed that the cut-off values for the best sensitivity and specificity for CRP were > 3.625 . This is consistent with the results of our other study  and thosn = 7) and without (n = 52) recurrence and found that NLR was significantly higher in patients with a recurrence, but they did not perform a ROC analysis to support their observations. In our study, according to the ROC analysis for the BC and IGM groups, the sensitivity (65%) and specificity (74%) of the NLR were found to be low. According to the results of the ROC analysis, NLR might not be a good biomarker for differentiating BC and IGM. The reason for the low sensitivity and specificity may be due to the different inflammation statuses of cancer patients. The routine collection of the CBC in clinical practice is simple and cost-effective to the patient, making the NLR a highly promising indicator for monitoring the systemic inflammatory status of IGM.Chronic inflammation is thought to be a predisposing factor for cancer formation by initiating the carcinogenesis process . ChangesSurgical treatment of IGM is controversial due to delays in wound healing, high local recurrence rates, and poor cosmetic results. In a study by Yau et al. , surgicaThe study has several limitations. Genomic whole exome or targeted exome sequencing of samples from IGM lesions and breast cancer patients could have been beneficial in identifying the difference. However, due to economic reasons, we could not perform sequencing. Most of these markers are acute phase reactants and we have aimed to check these acute phase reactants during the most acute time when these patients were newly diagnosed. Both diseases are progressive; however, we have taken samples from the patients at the time of the diagnosis at their first admission. In addition, in the results the cut off values for both diseases show a statistically significant difference. This constitutes a limitation for our study, and it would be reasonable to compare the severity of these diseases and markers according to the stage.If inflammation is treated or self-limiting, acute phase protein levels return to normal levels within days to weeks. Therefore, acute phase proteins such as albumin, fibrinogen, and CRP can be used as markers in monitoring the treatment of IGM, which is an inflammatory disease. However, breast biopsy is the gold standard for the differential diagnosis of IGM, and is also widely used in other situations. The diagnosis of IGM is not yet supported by any serum marker. CRP, fibrinogen, and complete blood count used in daily practice, which are cheap, easy, and non-invasive, can be useful in guiding follow-up and treatment. These parameters also play a significant role in the recurrence of the disease. The relatively low number of cases in both groups is another limitation of our study. Nevertheless, our findings provide important insights for larger studies that will examine IGM patients’ responses to treatment and the effects of inflammation-induced changes in these responses.


Introduction: The transition from pediatric health care and school systems presents enormous challenges for young adults with cerebral palsy (CP). The lack of strong societal support during this seminal life event is well-documented and leads many adults with CP to struggle with independence, higher education, and employment. Despite the relatively high prevalence of CP, information about the experiences and function of adults with CP in our society continues to be limited. The purpose of this project was to describe well-being by assessing education, employment, physical function, walking activity, and utilization of health care in an ambulatory adult cohort with CP who received specialized pediatric care at our center.Method: In this Institutional Review Board-approved prospective study, we invited former patients from our tertiary care pediatric CP center to complete a set of patient-reported outcomes including (1) the Patient-Reported Outcomes Measurement Information System domains of physical function and pain interference, (2) the Satisfaction with Life Scale, and a project-specific demographic questionnaire about education, employment, income, independence, pain, and health care utilization. Participants also wore a pedometer for 8 days to monitor community walking activity. Chi-squared pairwise or t-tests were used as appropriate to compare survey responses and walking activity data between three groups: participants who self-reported, those who reported by proxy, and published normative data from age-matched typically developing adult (TDA) samples.Results: One hundred twenty-six adults with CP consented to participate; 85 self-reported  and 41 reported by proxy . For the group who self-reported, high school graduation rate (99%) was similar to TDA  but bachelor's degree achievement rate (55%) was higher than TDA . Despite more advanced education, the unemployment rate in this group was higher than national levels at 33% and was associated with high utilization of Social Security Disability Insurance (33%). Within the self-reporting group, 13% required a caregiver. For the group who reported by proxy, educational levels  were lower than the general population (p < 0.001) and unemployment was higher than the national level, at 64%. Unemployment in this group was associated with high utilization of Social Security Disability Insurance (85%). Within the proxy-reporting group, 71% required a caregiver. The full cohort demonstrated lower levels of physical function according to the Patient-Reported Outcomes Measurement Information System and less community walking activity compared with TDA references (p < 0.001). This cohort of adults with CP reported significantly higher frequency of chronic pain , but less pain interference with daily activities than TDA based on Patient-Reported Outcomes Measurement Information System results (p < 0.001). This cohort reported good to excellent overall health (93%) and high utilization of primary care (98%), but limited utilization of specialty care, specifically orthopedic care (21%) and physical therapy (15%).Discussion: This cohort of adults with CP had similar levels of education as the general population, but had relatively high rates of unemployment, caretaker need, and Social Security Disability Insurance utilization. Although chronic pain was frequent, the impact of pain on work and independent living did not exceed reports from a typically developing reference. Better targeted societal resources for adults with physical disabilities are urgently needed to allow equitable access to employment, promote opportunities for independence, and enable full participation in community life. Cerebral palsy (CP) describes a group of permanent disorders of the development of movement and posture, which cause activity limitation and are attributed to non-progressive disturbances that occurred in the developing fetal or infant brain. The motor disorders of CP are often accompanied by disturbances of sensation, perception, cognition, communication, and behavior; by epilepsy; and by secondary musculoskeletal problems . CerebraCurrently, limited research is available on self-reported outcomes of adults with CP, whether functional in nature or related to quality of life , 3. FromConflicting evidence exists related to achievements in education and employment for adults with CP –11. RepoAccess to primary and specialized health care is important for well-being. There is conflicting evidence in the literature regarding adequate access to health care for adults with CP in the United States. For instance, a study by Gannotti et al.  found thA few studies have investigated quality of life (QoL) in adults with CP –16. SimiThe purpose of this project was to assess independence, education, employment, walking activity, pain, utilization of health care, and physical function in an adult cohort of former patients from a pediatric CP specialty center.Potential study participants were identified from a historical database from the authors' institution. Inclusion criteria were individuals who (a) were between the ages of 25 and 45 years; (b) had a diagnosis of CP; and (c) were functioning at Gross Motor Function Classification System (GMFCS) level I, II, or III. The study was approved by our institutional review board (# 1115672).Participants were asked to complete, with or without caregiver assistance, an online demographic survey and two domains of the Patient-Reported Outcomes Measurement Information System (PROMIS) tool, including (1) physical function v2.0 and (2) pain interference v1.1. The PROMIS is a rigorously constructed, generalizable, and clinically relevant set of patient-reported outcomes developed by the National Institutes of Health . AdditioTo monitor community-based walking activity, participants were given a Food and Drug Administration approved class-2 activity monitoring device , to track the participant's walking activity for 8 consecutive days. The device was calibrated to each adult's specific gait pattern in our gait analysis laboratory following the guidelines of the manufacturer to verify accuracy .Whenever possible, we compared our data with published results for age-matched TDA in the United States. Education was compared with data collected through the United States Census in 2017  for adult-tests were used as appropriate to compare responses between self-reporting adults (n = 85), proxy-reporting adults (n = 41), and normative data for TDA. Statistical significance was set at p < 0.0167 to account for multiple (three-way comparisons). All statistical analyses were performed using R-Studio version 1.4.1106 .A total of 645 adults who met the inclusion criteria were identified from historical databases and invited to participate in the study. Of these, we successfully contacted 171, and a total of 152 adults with CP agreed to participate in the study see . Twenty-A total of 41 participants were included in the proxy-reported (PR) group . This group self-identified ethnically as 73% White, 20% Black or African American, 2% Korean, and 5% as “Other.” The GMFCS distribution was I (10%), II (68%), and III (22%).p = 0.0173) graduated from high school and 48  obtained a bachelor's degree or higher. Only 30 participants in the PR group (73%) were found to have graduated from high school and none had attained a level of higher education .Participants in the SR group were found to have similar or higher levels of education compared with the TDA population, in contrast with the PR group who were found to have lower levels of academic achievement see . Eighty-p < 0.001). In terms of unemployment rate, both groups in this study showed higher levels of unemployment compared with the 4.4 ± 0.2% unemployment rate reported for adults in the United States through 2017 (p < 0.001) (p < 0.001).Similarly, participants in the SR group were found to have a comparable level of personal and household income as TDA, while participants in the PR group had significantly reduced personal income compared with the SR group (< 0.001) . Additiop < 0.001) (see p < 0.001), but demonstrated less pain interference in daily activities than TDA reported according to PROMIS results (p < 0.001).Regarding levels of functional independence, the PR group showed a greater need for a caregiver than the SR group (001) see . Adults p-value < 0.001) (p < 0.001).In terms of walking activity, 75 individuals from the SR group and 39 from the PR group had valid data from the StepWatch device. Walking activity was significantly lower in both groups compared with a published cohort of 193 TDA in the United States (< 0.001) . These rp > 0.1)  see . Our resThe transition from adolescence to adulthood is a seminal life event. For youth with CP, the loss of pediatric health care and school-based support systems presents enormous challenges. A lack of strong societal support mechanisms is well-documented for people with disabilities and leads many young adults with CP to struggle with independence, higher education, and employment –11, 30. From a socioeconomic perspective, there were clear differences in this cohort between those who self-reported and those who did not. Educational achievement and employment rates were higher in the SR group compared with the PR group. In terms of work “quantity,” full-time employment was more prevalent than part-time in the SR group, whereas part-time employment was more commonly reported in the PR group.When compared with the TDA reference, both the SR and PR groups had significantly lower employment rates. While adults with CP in the SR group had similar high school and higher post-secondary education attainment rates as TDA, there was a significantly lower employment rate than in the TDA reference. This suggests the presence of societal barriers to employment opportunities for adults with CP.Our results on educational attainment compared with the TDA population are in contrast with other studies where lower levels of advanced educational attainment were found , 9. SomeIt has been reported that adults with CP face declining health with aging related to increased levels of pain , 33, 34.Utilization of specialized musculoskeletal care by the young adults with CP in this study was much lower compared with the highly specialized pediatric services provided during childhood, specifically, orthopedics, and physical therapy. While we could not confirm that low utilization was related to lack of access, participants frequently reported that they could not find adult providers specializing in CP care.It has been hypothesized in the literature that access to specialty health care for adults with CP is limited because CP is considered to be a static condition with a focus on care during childhood , 30, 37.Children with CP have higher utilization of specialty care, the focus of which is optimizing mobility function and musculoskeletal health, compared with typically developing youth. The lack of quality rehabilitation and orthopedic care for adults with CP could hinder the maintenance of functional mobility since orthopedic sequelae persist throughout the lifespan and continue to impact motor performance. Compounding this on the medical side, a high prevalence of comorbidities in persons with CP is associated with decreased overall health in adulthood , 33, 34.This study was not population-based and included ambulatory adults with CP who were previously treated at the authors' institution. Therefore, our results should be interpreted with caution when extrapolating to adults with CP in other locales. GMFCS scores were given at the adult visit, but the classification has not been validated in that population. Another limitation in this study was the inability to distinguish between participants in the PR group who had a substantial cognitive or fine motor skill impairment as the reason for a proxy requirement and those who did not. Future research into the health-related outcomes and community participation of adults with CP should focus on identifying and removing barriers as well as developing effective care and support mechanisms throughout the lifespan for people with CP.The cohort of young adults with CP in this study had similar levels of higher education as the general population and had good access to primary health care. Despite this, there were relatively high rates of unemployment, caretaker need, and SSDI utilization. Although reports of pain were frequent, the pain did not interfere with physical activities and did not limit satisfaction with life compared with an age-matched reference. Improvements in societal and medical resources for adults with childhood onset physical disabilities such as CP are urgently needed to allow equitable access to employment and independent living opportunities and promote full societal integration.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Nemours Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.MS, CC, NL, JH, and FM contributed to the conception and design of the study. MS, CC, NL, TS, JS-T, JH, and FM contributed to the methodology of the study. JS-T and JH contributed to software design and procurement for the study. CC, NL, TS, JS-T, and JH participated in the validation of the data. MS, CC, NL, TS, JS-T, and FM participated in the formal analysis of the data. MS, CC, TS, JS-T, and FM participated in investigation of the data and provided resources for the study. MS, CC, TS, JS-T, and JH curated the data for the study. MS and TS wrote the original draft of the manuscript. MS, CC, NL, and FM provided visualization for the study and acquired the funding for the study. MS, CC, and FM provided supervision for the study. CC provided project administration for the study. All authors contributed to manuscript revision, read, and approved the submitted version.This study was funded by the Pedal with Pete Foundation.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. The reviewer HG declared a past collaboration with two of the authors MS and FM to handling Editor.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.