The coronavirus disease (COVID-19) brought several challenges in medical education. The aim of our study was to investigate whether virtual distance trainings (VDT) organized during the COVID-19 pandemic at our university were effective in replacing in-person bed-side education in intensive therapy and anaesthesiology among fifth-year medical students, both from students’ and instructors’ perspectives.This was a cross-sectional study consisting of three parts: a 20-item students’ questionnaire filled out by students participating in VDT, a 22-item instructors’ questionnaire filled out by instructors taking part in virtual distance education and a 20-item knowledge test completed by students participating in VDT, as well as by students visiting bed-side trainings (BT) during the same semester, before COVID-19 pandemic. The questionnaires focused on effectiveness, content, self-preparedness, technical background and interactivity of VDT. Instructors’ and students’ responses given to the common questions, as well as the knowledge test results were compared. Mann-Whitney U test was used for group comparisons and binary logistic regression was performed to analyze the influence of previous health-care experience on students’ feeling of self-preparedness.One hundred thirthen students (response rate {RR}: 68%) and 29 instructors (RR: 97%) filled out the questionnaires. The majority of students found our VDT useful and effective; however, a considerable number of participants felt disadvantaged by taking VDT instead of BT sessions and would recommend keeping virtual distance education methods combined with BT. Instructors found VDT overall effective and deemed the transfer of their knowledge satisfactory; however, they described worse interactivity and contact with students during virtual sessions compared to in-person teaching. Instructors showed a clearer consensus that VDT should not replace BT in the future, while students’ answers were more divided in this regard. Previous health-care experience did not influence students’ feeling of self-preparedness.p = 0.015).One hundred and twenty-seven students  completed the end-of-semester knowledge test. Students attending VDT performed better than students visiting BT (median score VDT:83.5 vs BT:77.3; Virtual distance learning incorporating virtual practice sessions was effective in maintaining continuous education of intensive therapy and anaesthesiology among fifth-year medical students during the COVID-19 outbreak.The online version contains supplementary material available at 10.1186/s12909-021-02826-1. The COVID-19 (coronavirus disease 2019) pandemic brought several challenges not only for health care, but for economics, education and everyday life in general. Several countries, including Hungary, chose to introduce drastic measures to slow down the spread of the virus and to “flatten the curve” based on the recommendations of the World Health Organization (WHO) [Intensive therapy and anaesthesiology (ITA) education faces several difficulties during the COVID-19 outbreak. Countries affected by a large number of COVID-19 cases suffer from lack of human resources in the intensive care units (ICU), diminishing teaching capacity. In addition, this is a field requiring not only theoretical knowledge, but clinical skills and hands-on experience as well, hence the transition of the curriculum to a fully e-learning platform is quite difficult even at the undergraduate level , 4.Hungary declared a state emergency due to the COVID-19 pandemic on March 11, 2020. The national regulations restricted all levels of in-person education at Hungarian universities from March 11 to May 25, 2020. In an effort to manage this unexpected emergency situation and continue the intensive therapy and anaesthesiology education among fifth-year medical students, we modified the presentation of our curriculum and introduced online virtual distance trainings (VDT) instead of bed-side trainings (BT). In addition, face-to-face lectures were transformed to online virtual lectures. However, the efficacy of this rapidly introduced educational method was uncertain.The aim of our study was to investigate whether VDTs as a part of our intensive therapy and anaesthesiology virtual distance education were able to replace in-person bed-side education methods among fifth-year medical students during the COVID-19 pandemic. Additionally, we also analyzed if virtual sessions could be incorporated into our course in the future based on our students’ and instructors’ impressions and opinions.We performed a cross-sectional study . Questionnaires and tests were completed anonymously. Potential participants were informed that by completing the questionnaire and/or test they would provide consent for anonymized data analysis and potential publication.The traditional ITA compulsory course contained five 90-min long thematic BTs and four 90-min long simulation trainings  were adapted to virtual sessions. The “Trauma” session was cancelled due to its specific patient population and lack of human resources during the pandemic. Simulation trainings and one BT in the ICU were postponed and then held after the withdrawal of restrictions at the end of the semester, in the same manner as before the restrictions.Virtual lectures took place through Zoom®  platform in the same time points, utilizing the same structure as previous face-to-face lectures. All students participating in the ITA course this semester, regardless of the type of trainings they visited, had an opportunity to take part in six face-to-face and eight virtual lectures.The four thematic VDTs were conducted at specific time points for a given group through the internet using Zoom® platform. A maximum of 21 students took part in one virtual 90-min long training session. A brief PowerPoint®  presentation summarizing the principles of the trainings’ topic, videos and images showing patients and patient management composed the main framework of a virtual practice with the opportunity of further discussion with students. Furthermore, case reports were incorporated into the VDTs to enhance the practical aspect of a session. The same topics were covered as during traditional BTs , two open-ended questions and four multiple-choice questions . The questions were reviewed by three experts and educators in the field of anaesthesiology and intensive therapy. Each question consisted of five possible answers and maximum 100 points could be reached. Time for the knowledge test was maximized in 30 min. Both students participating in the traditional BTs and students taking part in VDTs received the test at the end of the semester, when all students completed the thematic trainings, before the oral exams. Taking the test was voluntary and the results did not influence final grades.The results of the questionnaires were analyzed using descriptive statistics. We determined the medians and interquartile ranges of the grades at each Likert-scale question, which were graded from 1 to 5. Additionally, continuous variables  were described as medians and interquartile ranges (IQR). Categorical variables  were described as numbers and percentages.The open-ended responses went through a content-analysis and were summarized into categories.Less than 5% of data were missing; the missing data were excluded from the analysis.Mann-Whitney U test was applied to compare the answers to four Likert-scale questions between students and instructors regarding the effectiveness and future application of VDTs.In addition, Mann-Whitney U test was used to compare the knowledge test results of students who participated in the traditional bed-side practices and the ones who underwent virtual practice sessions.Moreover, a binary logistic regression analysis was performed to determine if previous job experience with critically ill patients has an influence on students’ self-reported preparedness after completing the course . Figures were created by GraphPad Prism version 8.3.0. .Table Demographic data of responders are presented in Tables As can be seen in Table Most of the responders reported a positive feeling of self-preparedness in recognizing respiratory failure, shock and in being able to manage perioperative situations after completing the VDTs. Previous experience with critically ill patients did not influence the students’ feeling of self-preparedness . However, satisfactory content, structure and interactivity were achieved during the VDTs based on the answers . However, as Table p = 0.162). Although this difference is not significant, there is some shift in IQR, indicating that students tended to be more satisfied with the VDT. Additionally, instructors showed a clear consensus that VDTs should not replace in-person education in the future, while students’ answers were more divided in this regard (Table p < 0.001), as well as VDTs are useful in a combination with BTs .As can be seen in Table rd Table . Instrucp = 0.015).The knowledge test was completed by 34% of students participating in VDTs and by 67% of students taking part in BTs Table . The demThe objective of our study was to examine the effectiveness of virtual distance learning and VDTs held instead of in-person bed-side ITA trainings for fifth-year medical students at our university during the COVID-19 pandemic. Furthermore, we explored the students’ and instructors’ opinions about introducing at least some elements of virtual distance education into our course in the future.The restrictions implemented to control the COVID-19 outbreak resulted in several challenges in education and particularly in health-care education . A numbeIn-person education was suspended from the middle of March 2020 until the end of May 2020 in Hungary, with the option of virtual distance education or postponing courses. Our institute changed the presentation of ITA curriculum and chose to develop a hybrid course with VDTs and virtual distance lectures during the restrictions combined with in-person simulation sessions and one visit at the ICU delayed until restrictions were lifted. Intensive therapy and anaesthesiology is a medical field requiring a complex curriculum even in undergraduate education covering cognitive and technical skills, as well as teaching valuable communication skills and overall attitude in a specialized environment . ConveyiStudents participating in in-person practices met critically ill patients with the previously mentioned conditions  as filling out the test was voluntary. Moreover, only 34% of students taking part in VDTs completed the knowledge test compared to 67% of students participating in the conventional BTs. In addition, the multiple-choice test measures theoretical knowledge and does not give enough information about practical skills and attitudes acquired during the practices. We also need to highlight that more time elapsed between the traditional BTs and the knowledge test than between VDTs and the knowledge test. The knowledge test was accessible when all students completed the thematic practices to avoid wide distribution of the test questions within the student body.Despite these limitations our investigation gives new information about the implementation of VDT to keep undergraduate intensive therapy and anaesthesiology education continuous during a pandemic.Our cross-sectional study demonstrates that virtual distance education is effective in maintaining education of intensive therapy and anaesthesiology continuous among fifth-year medical students during the COVID-19 outbreak. Students and instructors participating in our rapidly introduced VDTs during the restrictions due to COVID-19 outbreak found the new method acceptable. However, BTs, personal communication, interactivity and experiencing ICU atmosphere play a crucial role in teaching intensive therapy and anaesthesiology and should still be included in the undergraduate educational practice.Additional file 1 STROBE Statement—Checklist of items that should be included in reports of cross-sectional studies.Additional file 2: Table 1. Practical curriculum of traditional undergraduate education in intensive therapy and anaesthesiology at Semmelweis University. Table 2. Students' questionnaire. Table 3. Instructors' questionnaire. Table 4. Steps of questionnaire development.Additional file 3: Table 1. Description of binary logistic regression analysis performed to determine if students' previous job experience with critically ill patients has an influence on their self-reported preparedness. Fig. 1. The relationship between previous job experience with critically ill patients regularly and self-preparedness in recoginzing patients with respiratory failure, shock or being aware of perioperative management.


Association of temporomandibular disorders (TMD)-related pain with severe headaches (migraine and tension-type headaches [TTH]) was studied over a follow-up period of 11 years.The data used was from two nationally representative health surveys in Finland—the Health 2000 Survey (baseline) and the Health 2011 Survey (follow-up) (Bioresource Research Impact Factor [BRIF] 8901)—conducted by the Finnish Institute for Health and Welfare (THL). The primary dataset of the current study included a subset of the population undergoing a clinical oral examination, including TMD examination, at baseline, and answering the questions related to severe headaches, both at baseline and at follow-up (n = 530). From the primary dataset, two datasets were created to study the onset of migraine (dataset 1) and TTH (dataset 2) separately. Dataset 1 included participants healthy of migraine, but not other headaches, at baseline (n = 345), and dataset 2 participants healthy of TTH and other headaches, except migraine, at baseline (n = 464). Bayesian logistic regression models with weakly informative priors were utilized to assess the association of muscle-related TMD pain (mTMD) at baseline and temporomandibular joint-related TMD pain (jTMD) at baseline with the presence of migraine and TTH at follow-up.Neither of the baseline TMD-related pain variables were associated with the presence of migraine at follow-up , whereas mTMD at baseline , but not jTMD at baseline , was associated with the presence of TTH at follow-up. Bayesian sensitivity analyses revealed that the estimates of the regression models were stable, demonstrating sufficient validity and consistency of the estimates.These results indicate that diverse mechanisms may exist behind the associations of TMD-related painful conditions with different types of severe headaches.TMD-related pain is a frequent comorbidity of severe primary headaches. Therapy of severe primary headaches may thus benefit significantly with the incorporation of a multi-disciplinary clinical team. The prevalence of temporomandibular disorder (TMD) symptoms varies significantly between populations. A recent systematic review indicated that in general populations the prevalence of having at least one clinical sign of TMD ranges between 5 and 60% . NonethePrimary headaches (migraine and tension-type headaches [TTH]), on the other hand, affect more than 2.5 billion individuals worldwide. A recent global study ranked headaches as the second leading cause of years lost due to disability after lower back pain . GloballTMD-related pain (mTMD and jTMD) seldom occurs in isolation; approximately only 17% of the TMD-related pain cases occur with no other comorbid pain condition . TMD-relTo the best of the authors’ knowledge, all the previous studies of the association of TMD-related pain with headaches have been based on the Frequentist statistics. Compared to the Bayesian approach, the Frequentist statistics suffer from some limitations, most importantly the dependence on large sample sizes for effect sizes to be accurately determined . AdditioThe current study—utilizing the Bayesian methodology—aimed to examine the association of TMD-related pain with severe headaches (migraine and TTH) over a follow-up period of 11 years. The hypothesis was that a prospective association exists between TMD-related pain and severe headaches.The current study utilized data from the Health 2000 (baseline) and the Health 2011 (follow-up) Surveys (Bioresource Research Impact Factor [BRIF] 8901), conducted by the Finnish Institute for Health and Welfare (THL) .n = 9125) (participation in at least one phase of the survey) [The Health 2000 Survey, conducted in the years 2000 and 2001, included 9922 invited participants aged 18 years or older living in mainland Finland. The data for this survey were collected through self-administered questionnaires and interviews, and for those aged 30 years or older also by clinical oral and health examinations as well as by laboratory analyses. The participation rate of the Health 2000 Survey was 92% ( survey) .n = 8135), participation rate in at least one phase of the survey being 73% (n = 5903). The main reason for non-participation was the refusal to participate, following inability to contact the participant, the death of the invited participant, and the invited participant living abroad. The Health 2011 Survey included many smaller studies specific to a common disorder/group of disorders. Participants reporting to ever suffer from severe (moderate to intense) headaches during the health interview of the Health 2011 Survey proper (n = 875) were invited to participate one of the smaller studies, namely Migraine Sub-study. It included questions on headache types, severity, and their impact. Of the eligible participants, 832 eventually participated (95%) [The Health 2011 Survey was a follow-up study of the Health 2000 Survey, fieldwork of which was conducted in the years 2011–2012. The invited participants of the Health 2011 Survey included those participants of the Health 2000 Survey sample who were alive, living in Finland, had contact details available, and not refused to participate in further surveys previously (ed (95%) .n = 530). From this subset, two datasets were created to study the onset of migraine (dataset 1) and TTH (dataset 2), and were analyzed separately. Dataset 1 included only the participants healthy of migraine, but not other headaches, at baseline  (n = 345), and dataset 2 only the participants not suffering from TTH or other headaches, except migraine, at baseline  (n = 464)  and answering the questions related to severe headaches both at baseline and at follow-up  (The outcome variables were the presence (yes/no) of migraine or TTH at follow-up . These were asked by questions: “Did a physician ever diagnose you for migraine?” and “Did a physician ever diagnose you for tension-type headaches?” .m. temporalis and m. masseter) and the temporomandibular joint (TMJ). The pain response during the inspection of the temporomandibular area was recorded dichotomously (yes/no). Further details regarding the TMD examination methodology and protocol can be found at Suominen-Taipale et al. (2008). TMD-related pain variables, mTMD (yes/no) and jTMD (yes/no), were used as the predictor variables. The percentage of agreement for recording pain responses during the TMD examination between the examiners and the reference examiner was 95%  for mTMD and 92%  for jTMD [In the Health 2000 Survey, five experienced and calibrated dentists performed a standardized clinical oral examination. This included TMD examination partly based on the Research Diagnostic Criteria for TMD (RDC-TMD) Axis I criteria , focusinfor jTMD .Other covariates and potential confounders of the current study were utilized from the baseline data . These included gender and age taken from the population registers. Variables recorded during the home-visit interview included the level of education , and the use of anti-inflammatory drugs (yes/no). Body mass index (BMI) was calculated using the weight and height of the study participants assessed during the health examination . BMI was used as a continuous variable in the analyses .vs. those not reporting migraine at follow-up, as well as between the TTH and non-TTH study participants at follow-up, were analyzed using chi-square test. Regarding the continuous covariates, the normality of the distributions in different categories of the outcome variables was assessed visually through distribution histograms and tested with Kolmogorov–Smirnov and Shapiro–Wilk tests. Based on these, the Mann–Whitney U test was used to observe differences amongst them in the different outcome variable categories. The p-values for the descriptive statistics of the study population were based on two-tailed tests of significance.Differences in the frequencies of the categories of the predictor variables and the categorized covariates between those participants reporting The current study employed directed acyclic graphs (DAG) for the evaluation of the study hypothesis, based on a priori knowledge Fig. . AssociaMultivariate Bayesian logistic regression analyses were performed for analyzing the effect estimates between the predictor and the outcome variables. Weakly informative priors  were utilized for the regression models in both datasets due to the lack of substantial longitudinal evidence regarding the association of TMD-related pain with severe headaches. Another reason was the better computational stability of the Bayesian algorithm incorporating weakly informed priors in the case of logistic regression models . The effhttp://www.R-project.org/).SPSS  version 27 was used for the descriptive data analyses. Bayesian regression and sensitivity analyses were performed through Mplus version 8.4 . DAGs and flow charts for the current study were drawn through DigrammeR package of R software  and without (n = 261) migraine at follow-up, and participants with (n = 105) and without TTH (n = 425) at follow-up are presented in Tables Basic characteristics of the participants with . However, null value was included in the 95% CI of the posterior effect estimate of jTMD at baseline in the association with the presence of TTH at follow-up suggesting no association  in the TMD examination. However, this was taken into consideration by adding the anti-inflammatory drug usage as a covariate, since a correlation has been reported between the anti-inflammatory drug usage and the psychological status of migraine  and TTH The current study is, according to the authors’ knowledge, the first prospective study reporting the effect estimates between different TMD-related pain variables and the presence of migraine and TTH through Bayesian methodology. Bayesian methodology, which compared to its Frequentist counterpart, provides better estimations in situations of non-random or nested study samples . UtilizaThe Bayesian methodology employed in the present study offers several advantages over the traditional Frequentist methodology. This analytical approach produces a range of values (Bayesian 95% CI) as posterior distributions that reflect the uncertainty inherent for the multiple unknown parameters, rather than a fixed value for parameter estimates as in Frequentist approaches. Also, the Bayesian statistical analyses allow for the sub-analyses without the need for the classic statistical adjustments for multiple comparisons. The Bayesian parameter estimates may remain less biased and hence more appropriate and stable with moderate and even smaller sample sizes . Additiovs. population-based sample in this study. Another difference was the clearly larger proportion of chronic migraineurs in the study population of that study compared to the current study population . Increased pain sensitivity and lowered pressure pain thresholds have been reported along with higher frequency of migraine headaches [The current study found no association between TMD-related pain (mTMD and jTMD) at baseline and the presence of migraine at follow-up. Only one study , to our eadaches . These dvs. neurology), vigilance should also be exercised regarding the distinction between these two disease entities during their treatment.The prospective association of mTMD at baseline with the presence of TTH at follow-up found in the current study is in line with previous epidemiological, clinical, and physiological evidence. Previous epidemiological studies have shown an association between TMD-related pain and TTH . ClinicaIn terms of autonomic dysfunction, both mTMD  and TTH Current research reports a significant role of central sensitization in the pathogenesis of chronic TTH . ContinuThe finding of no association between jTMD and TTH in the current study is in accordance with the findings of a cross-sectional study by Gonçalves et al. (2011) . This maThe current study reported a prospective association of mTMD, but not jTMD, with the presence of TTH. However, TMD-related pain was not found to be associated with the presence of migraine. These results were found reliable after the application of Bayesian sensitivity analyses. An interpretation of these results may be the existence of diverse mechanisms playing a role in the associations between different TMD-related painful conditions and the presence of severe headaches.


Background: A growing body of evidence is emerging suggesting testosterone can affect all cells involved in the immune response to both bacterial and viral infections, and the testosterone effect on the immune response could explain the greater susceptibility of men to infections including COVID-19. We aimed to explore the predictive role of male serum total testosterone (TT) levels on the time till viral negativity testing among hospitalized COVID-19 patients. Methods: The univariate effect of risk factors for the duration of COVID-19 viral positivity was evaluated using the log-rank test and Kaplan–Meier estimates. A multivariable Cox regression model was developed to test the role of TT levels and the subsequent odds for shorter viral positivity intervals. Results: Increasing serum TT levels and the need for an oxygen administration strategy were independently predictive for respectively reduced and increased days to negativization . Conclusion: Baseline higher TT levels for male COVID-19 patients at hospital admission are associated with shorter durations of positive COVID-19 testing and thus viral clearance. Our preliminary findings might play a relevant to help pandemic control strategies if these will be verified in future larger multicentric and possibly randomized trials. The pandemic caused by the respiratory syndrome coronavirus 2 (SARS-CoV-2), which leads to COVID-19, is devastating the health systems around the world with geographical variations . SeveralAfter Institutional Review Board (IRB) approval, we performed a retrospective analysis of our prospectively maintained database of patients who were hospitalized due to COVID-19 infection between March 2020 and June 2020. Patients were considered eligible if they had a positive SARS-CoV-2 test by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) at the moment of hospital admission. Daily viral testing  was performed with RT-PCR during the range of hospital stay. A return to negative results for SARS-CoV-2 testing  was determined with at least two consecutive RT-PCR negative results. The COVID-19 positivity duration was defined as the timeframe between the first confirmatory result at admission and the day of the two consecutive RT-PCR negative tests during the range of hospitalization. Patient medical history and routine laboratory test data, including counts of peripheral white blood cells, immune cell subsets, cytokine interleukin (IL) 6, and TT levels were matched with the day of the primary confirmatory RT-PCR result. The severity of clinical manifestations was assessed at admission on the basis of oxygen requirements  and clinical symptoms such as fever and respiratory tract pneumonia (identified on imaging).p ≤ 0.05. A multivariable stepwise regression model (forward selection) was further generated, by selecting those predictive variables that were significant upon univariate analysis, with entering and removing limits set at p = 0.05 and p = 0.10, respectively. Given that impaired TT levels are a function of different confounders rather than a pre-established threshold, we also modeled the multivariate effect of serum TT according to the Age-Adjusted Charlson Comorbidity Index (ACCI), history of hypertension, dyslipidemia, and smoking status. Finally, the Locally Weighted Scatter-Plot Smoother (LOWESS) function was used to graphically depict the predicted probability of a median longer positivity duration and the variables of interest. Statistical analysis was performed using Stata version 16.1 .Descriptive statistics were used to summarize pertinent study information. The association between variables was tested by the values according to Fisher’s Exact test or Mann–Whitney U test when appropriate. The differences in duration of viral positivity were initially explored using the Log-rank test and graphically presented as a Kaplan–Meier curves estimate. A univariate Cox proportional hazard model was developed by testing each potential risk factor (both dichotomized and continuous variable) as a covariate for time to RT-PCR negative result with significance set at n = 9; CPAP, n = 7) were associated with a significantly longer median viral positivity duration  and lower median TT serum levels . Patients’ hemato-chemical and clinical characteristics, stratified according to impaired TT levels  at the moment of hospital admission, are presented in A total number of 25 patients with a median age of 70 years (IQR: 57–78) and a median ACCI value of 3 (IQR: 2–5) were considered eligible for final analysis. The median duration of positive viral RT-PCR for the whole cohort was 20 days . Patients requiring invasive oxygenation support at hospital admission  as well as increasing TT levels were associated with a significantly shorter viral positivity duration . The probability of having a longer duration  showed a linear and inverse relationship with the increasing values of serum TT and IL-6 , which represents the major limitation of our study, we believe that these data suggest that more complex interactions exist for viral positivity duration including the immune system and hormonal constitution.In the current study, the median viral positivity duration for the whole cohort was 20 days . This duration is longer than some previous studies on this topic. For example, Young BE et al. observed a median duration of viral positivity of 12 days  in a population of 18 patients with COVID-19 infection ,22. Morep = 0.011). To our knowledge, our study was the first to demonstrate an association between TT levels and the duration of viral positivity.In this study, we were able to evaluate the association of testosterone levels with the duration of viral positivity. Interestingly, in our analysis, increasing serum TT levels (ng/mL) were independently associated with shorter viral positivity duration . Recently, testosterone has received great attention even in the context of the COVID-19 pandemic for its effects on the general health status and for modulating the transmembrane serine protease 2 (TMPRSS2), which plays a crucial role in the entry of the SARS-CoV-2 virus into the respiratory epithelial cells leading to the COVID-19 disease ,25. TMPRWe believe that the results of our study should lead to better defining the role of testosterone levels in the pathogenesis of the COVID-19 disease. It is known that hypogonadism is associated with male health status and recently great attention has been given to its role in the context of COVID-19, given the sex differences in disease outcomes ,38,39. IOur findings suggest that testosterone values may be associated with the duration of viral positivity and thus could be used to predict viral clearance to help pandemic control strategies. In particular, baseline higher total testosterone levels for male COVID-19 patients at hospital admission are associated with shorter durations of positive COVID-19 testing and thus more rapid viral clearance. Importantly, further studies are needed to understand the role of testosterone in the pathogenesis of the disease.


Previous studies have revealed that current secondhand smoke exposure showed highly suggestive evidence for increased risk of simultaneous sleep problems in children. Data on the associations between early-life exposure to SHS with subsequent sleep problems in children were scarce. We aimed to evaluate the associations of early-life SHS exposure with sleep problems in children.In this cross-sectional study, children were recruited from elementary and middle schools in Liaoning Province, China between April 2012 and January 2013. We assessed early-life SHS exposure (pregnancy and the first 2 years of life) via questionnaires. Sleep problems and different types of sleep-related symptoms were measured based on the validated tool of the Sleep Disturbance Scale for Children (SDSC). Generalized linear mixed models were applied to estimate the associations of early-life SHS exposure with sleep problems.We included a total of 45,562 children  and 6167 of them (13.5%) were exposed to early-life SHS during both pregnancy and the first 2 years of life. Compared with unexposed counterparts, children exposed to early-life SHS had higher total T-scores of SDSC  and higher odds of increased sleep problems . When considering different sleep-related symptoms, the associations between early-life SHS exposure and symptom of sleep-wake transition disorders  were the strongest in all analyses.Early-life SHS exposure was associated with higher odds of global sleep problems and different sleep-related symptoms in children aged 6–18 years. Our findings highlight the importance to strengthen efforts to support the critical importance of maintaining a smoke-free environment especially in early life.The online version contains supplementary material available at 10.1186/s12940-021-00793-0. In 2019, China still accounted for more than one-third of the global tobacco use according to the latest Global Burden of Disease Study –5. RecenMost of the previous observational studies across different countries have evaluated current SHS exposure and simultaneous sleep problems in children  reporting guideline for cross-sectional studies.In each school, we organized face-to-face appointments for the teachers and the principals to introduce the aims, proposed methods and the procedures of our study. We aimed to incentivize permission for parents by using active communication techniques with the help of the teachers and the principals. We provided standard procedures for the school teachers. School teachers were required to explain the study aim, obtain the informed consent, and distribute the questionnaires in regular parent-teacher conferences. Parents could fill out the questionnaires during the conference or take it home and return it in a sealed envelope. Parents had the right to decline to consent and refuse to join the study.We asked parents to fill out the Sleep Disturbance Scale for Children (SDSC) to measure sleep quality in all children. The SDSC was a 26-item parent-rated scale developed by Oliviero Bruni in 1996 . Each itWe have already conducted a validation study to revise the Chinese version of SDSC in the first wave of the Seven Northeastern Cities study, and it is reliable in screening parent-reported sleep problems in Chinese children (Cronbach’s α = 0.81). The detail of the study is described elsewhere .We collected information on SHS exposure via questionnaires. We defined having exposure to early-life SHS based on an affirmative answer to the two questions: (1) Did anyone who lived with the mother during her pregnancy smoke anywhere inside the house? and (2) Did anyone who lived with the child during his or her first 2 years smoke anywhere inside the house? Therefore, exposure to early-life SHS was a category variable encoded as (1) unexposed; (2) ever exposed during pregnancy or the first 2 years of life; and (3) ever exposed during both pregnancy and the first 2 years of life.We also collected information on the current number of cigarettes smoked inside the house per day during weekdays and weekends by all family members who lived with the child, and therefore we defined having current SHS exposure if any family member who lived with the child smoked cigarettes.We conducted data analyses from April 32,021 to May 3, 2021. We calculated means and standard deviations for continuous variables and percentages for categorical variables. The differences across different SHS exposure groups were determined using ANOVA test for continuous variables and chi-square tests for categorical variables.We analyzed the associations of exposure to early-life SHS with sleep problems in children by fitting generalized linear mixed models with an identity link (continuous outcomes with gaussian distribution) or logit link  function. We fitted crude models with school as random intercept. We fitted adjusted models for each outcome, adjusting for covariates collected from questionnaires . We conducted stratified analyses by sex and by child age, and whether the associations varied with sex or child age were assessed from the heterogeneity of effect across strata and the significance of interaction terms.n = 2257, 4.95%) since previous studies have found strong associations between SHS exposure and sleep problems in asthmatic children [To verify the robustness of the results, sensitivity analyses were performed: (1) we examined the associations by excluding children with parent-reported asthma  we P value < 0.05 for two-sided test was considered statistically significant.Statistical analyses were conducted with the statistical software R 4.0.3 (R Core Team 2020). We presented the results as estimates (β) and odds ratios (OR) with the 95% confidence interval (CI). A As shown in Table In the adjusted model Table , compareWhen using the cut-offs to analyze the associations between early-life SHS exposure and the proxies for increased problems in the total scores of SDSC and six domains within the clinical range, we found similar results compared with those using the continuous T-scores Table . For exaPinteraction = 0.0298), while the association between SHS exposure and T-score of SHY was stronger in males (Pinteraction = 0.0253). When considering child age, the association between SHS and T-score of DOES was stronger in older children (Pinteraction < 0.001).The results were similar in stratified analyses by sex or by child age, and we only found significant modification of sex or child age on the associations of early-life SHS exposure with the T-score of DOES and/or SHY Table . The assWhen grouping the early-life SHS exposure into two detailed categories (pregnancy and the first 2 years of life), the results remained similar eTables. and 5. WIn the present study, we found that early-life SHS exposure was associated with global sleep problems in children aged 6–18 years. Specifically, we found that the associations were stronger in children with symptoms related to SWTD. In addition, we found significant sex and age differences in the associations of early-life SHS exposure with the DOES and SHY T-score.Most of the previous studies focused on studying the associations between current SHS exposure and one specific sleep-related symptom without considering the global sleep problems. We only found one cross-sectional study was conducted in Taiwan, China using a comprehensive measure of Pittsburg Sleep Quality Index (PSQI), and indicated a positive but null association between current SHS exposure and poor sleep quality in 213 adolescents (10–15 years) . In our Previous observational studies focused on studying sleep-related symptoms of DIMS, SBD, DA and DOES with inconsistent results. We have extended the findings by considering two more symptoms of SWTD and SHY, which were prevalent but seldom discussed in children. One randomized controlled study of 498 Italian children aged 8–11 years suggested that interventions on parental smoking reduced the prevalence of sleep bruxism (one symptom of SWTD) . One comIn this study, we have identified significant sex differences in the associations between early-life SHS exposure and the T-score of DOES and SHY, but the potential mechanism remained unclear. Our results might be supported by previous animal data that lung function alterations in mice were sex-specific with males being more susceptible to early-life SHS exposure . MoreoveSeveral limitations should be well noted. First, due to the cross-sectional nature of the current data, we were unable to assess the causality of our findings, and longitudinal studies are needed to demonstrate the causal inference. Second, we used self-reported questionnaires to obtain information of SHS exposure, resulting in potential recall bias and exposure misclassification. This bias would likely to have been non-differential by the outcomes, and assuming the sensitivity plus the specificity for the exposure measurement was > 1, the results would have been biased towards the null. However, we observed mostly consistent dose-dependent associations across all SDSC domains, and the method we used remained to be the most cost-effective to assess SHS exposure in observational studies with large sample of children . Third, In conclusion, we found that children aged 6–18 years exposed to higher early-life SHS had higher odds of global sleep problems, and the associations were the strongest in children with symptom related to SWTD . Our findings highlight the importance to strengthen public health efforts and support the critical importance of maintaining a smoke-free environment especially in early life.Additional file 1: eTable.1. Basic information of previous observational studies regarding the associations of exposure to SHS with sleep-related problems in general children populationα. eTable.2. Summaries of the results of previous observational studies regarding the associations of exposure to SHS with sleep-related problems in general children population. eTable.3. Distribution of the SDSC score in the participants. eTable.4. Associations of SHS exposure during early life using prenatal and early postnatal periods with the scores of the SDSC in children aged 6–18 yearsa. eTable.5. Associations of SHS exposure during early life using prenatal and early postnatal periods with the increased problems within the clinical range using the SDSC in children aged 6–18 yearsa. eTable.6. Associations of SHS exposure during early life with sleep problems in asthmatic childrena. eTable.7. Associations of current SHS exposure with simultaneous sleep problems in children aged 6–18 yearsa.


Tumor-associated macrophages (TAMs) play numerous roles in cancer biology and are an important component of the relationship between immune system response and tumor progression. Several new immunotherapy techniques have been developed that target TAMs and are under investigation in both clinical and preclinical settings. Despite this surge of new immunotherapy techniques, a means to specifically and quantifiably measure the presence of TAMs to ensure the viability of these therapies, has yet to be widely investigated. The development of molecular imaging agents that target TAMs provides a path to noninvasively gain valuable insight into the molecular and functional characteristics of the tumor microenvironment and how the immune response facilitates the progression of cancer or therapy response. This article reviews published preclinical and clinical research in the imaging of TAMs through Positron Emission Tomography (PET).Macrophages are large phagocytic cells that can be classified as a type of white blood cell and may be either mobile or stationary in tissues. The presence of macrophages in essentially every major disease makes them attractive candidates to serve as therapeutic targets and diagnostic biomarkers. Macrophages that are found in the microenvironment of solid tumors are referred to as tumor-associated macrophages (TAMs) and have been shown to influence chemoresistance, immune regulation, tumor initiation and tumor growth. The imaging of TAMs through Positron Emission Tomography (PET) has the potential to provide valuable information on cancer biology, tumor progression, and response to therapy. This review will highlight the versatility of macrophage imaging in cancer through the use of PET. The year 1883 marked the beginning of an exciting new era for immunology. Ellie Metchnikoff introduced the concept of macrophages by determining phagocytes functions as a critical host-defense mechanism. This eventually led Metchnikoff and Paul Ehrlich to receive a Nobel Prize for their work on immunity in 1908. This study provided the earliest description of macrophages and opened up a new field of research as numerous scientists propelled the field forward by working to advance current knowledge and understanding of the role of macrophages in many disease states .Today, macrophages are described as large phagocytic cells that can be classified as a type of white blood cell that may be either mobile or stationary in tissues . They arMacrophages represent a diverse set of cells which are associated with a distinct set of disease states such as tuberculosis , sepsis Macrophages can be broadly classified into two main groups with differing roles in immune defense and immune surveillance: classically activated macrophages M1) and alternatively activated macrophages (M2)  and alte. M1 and Macrophage polarization can be influenced by antitumoral treatments such as chemotherapy and radiotherapy and thus can facilitate resistance to such therapies ,18. ImmuIt has previously been demonstrated that the presence of TAMs in a variety of human cancers can result in a poor clinical prognosis. Such examples include gastric cancer , colorecTAMs have become increasingly more relevant in cancer biology as they represent a biomarker that could lead to improved diagnostic and prognostic outcomes . The undDespite this need, clinical imaging of tumor-associated macrophages has yet to be widely investigated. The development of molecular imaging agents that specifically target TAMs is an area that is growing in order to address this need. The imaging of macrophages can provide a noninvasive approach to gain valuable insight into the molecular and functional characteristics of the tumor microenvironment and how the immune response facilitates the progression of cancer or therapy response.Positron emission tomography (PET) is a form of molecular imaging that allows for a noninvasive assessment of physiologic information by utilizing radioactive contrast agents. It is widely used in both clinical and preclinical settings due to its wide array of translational possibilities, sensitivity, and quantitative accuracy. The use of PET has proved to be an instrumental tool in the diagnosis and monitoring of a variety of disease states such as cancer . This reIn the field of oncology, 2-deoxy-2-[fluorine-18]fluoro-D-glucose (FDG) is a widely used PET imaging probe for the detection and staging of numerous types of cancer. FDG is an analog of glucose which allows for the ability to provide functional information of the glucose metabolism of cancer cells .18F]FDG PET has also been explored in relation to TAMs. In 2015, Cencini and colleagues investigated the relationship between [18F]FDG-PET assessment of classical Hodgkin lymphoma (cHL) and TAMs in their roles as potential prognostic predictors. Their study involved the retrospective analysis of 200 cHL patients from 2005–2012. They hypothesized that a positive early [18F]FDG-PET assessment could be synonymous with a high TAM score. The patients’ disease progression was staged using a bone marrow biopsy and [18F]FDG-PET/CT. Therapy consisted of 2-6 cycles of ABVD  [18F]FDG-PET which exhibited a positive correlation for progression free survival in that it was significantly higher in [18F]FDG-PET negative patients. CD68 expression was classified as low, intermediate, or high and demonstrated no significant difference between the groups when compared with progression free survival. The authors concluded that TAMs did not show a correlation with [18F]FDG-PET in classical Hodgkin lymphoma possibly due to the selected TAM markers, antibodies, or cut-off values for TAM scores [[rbazine) . TAMs weM scores .18F]FDG-PET was utilized by Jeong and colleagues to explore the relationship between TAMs with hypoxia and aerobic glycolysis as these have been linked to poor response to chemotherapy and radiotherapy [18F]FDG-PET and immunohistochemistry was conducted for TAMs by using CD68 antibodies to differentiate between tumors with low CD68 and high CD68 levels. Contrary to the previous study, significant results showed that there was a strong correlation between [18F]FDG uptake and TAMs in tumors. The authors also observed that high CD68 expression was associated with a higher [18F]FDG Standardized Uptake Value (SUV). However, when considering the subtype of non-small cell lung cancer (adenocarcinomas or squamous cell carcinomas), [18F]FDG uptake and TAM levels only showed a significant correlation in adenocarcinomas. In addition, a strong correlation was found between CD68 and glycolysis related molecules such as GLUT1 and HK2 through the utilization of The Cancer Genome Atlas [18F]FDG uptake by PET/MRI. Clodronate liposome was also administered intravenously in order to deplete TAMs. Small animal imaging results demonstrated that [18F]FDG signals were significantly lowered by the administration of Clodronate liposome FDG-PET in head and neck squamous cell carcinoma (HNSCC). Their study involved 73 advanced HNSCC patients in which four parameters were studied using [18F]FDG PET/CT: the standardized uptake value, mean standardized uptake value metabolic tumor volume, and total lesion glycolysis. Key results demonstrated that increased glucose uptake, SUVmax, and SUVmean were correlated with higher M2-macrophage polarization Ga-NOTA-anti-MMR-sdAb in organs such as the liver, spleen, and 3LL-R Lewis lung carcinoma tumors which are known to be areas of higher amounts of CD206 TAMs. CD206 deficient mice served as controls and verified specificity as there was no uptake in healthy organs or the 3LL-R tumors. Additionally, it was shown in imaging and biodistribution studies that the probe was rapidly excreted through the kidneys and urine  phenyl)-5, 7-dimethylpyrazolo  pyrimidin-3-yl) acetamide ([18F]-DPA-714), a tracer targeting TSPO, to differentiate inflammation from tumor [18F]-DPA-714 showed significantly lower values for each tumor model which suggested that this tracer could have the potential to separate tumors from other inflammatory diseases using PET PBR28, a PET tracer that targets TSPO, in a mouse model of pancreatic ductal adenocarcinoma(PDAC). PDAC is an aggressive type of cancer with a five-year survival rate of less than 10 percent fluoro)phenyl)-5,7-dimethylpyrazolopyrimidin-3-yl)acetamide ([18F]F-DPA), another TSPO tracer, in head and neck squamous cell carcinoma (HNSCC) [18F]F-DPA is similar to the tracer discussed previously ([18F]-DPA-714) but differs in the placement of the fluorine atom. Nude female mice were injected with either FaDu or Cal33 cells and separated into non-irradiated and irradiated groups. Two experiments were conducted with the FaDu xenografts in which [18F]F-DPA and [18F]FDG imaging occurred either one or two weeks after radiotherapy. The third experiment was conducted with the Cal33 xenografts in which solely [18F]F-DPA imaging was performed one week after radiotherapy. TAM analysis, immunohistochemistry, ex vivo biodistribution, and Western Blot experiments were also executed. Key results demonstrated that [18F]F-DPA had a significantly higher uptake in FaDu irradiated tumors when compared to non-irradiated tumors in both one week and two weeks after treatment. [18F]FDG uptake in irradiated tumors one week after treatment remained constant whereas uptake was significantly lower in irradiated tumors two weeks after treatment. TSPO expression in tumors showed varied expression with no statistical difference between irradiated and non-irradiated tumors one and two weeks after treatment. Ex vivo biodistribution and autoradiography results also portrayed significantly higher uptake of [18F]F-DPA in irradiated tumors versus non-irradiated tumors. Results also demonstrated that radiotherapy decreased the proportion of macrophages in M1 and M2 when compared to non-irradiated tumors but increased non-polarized migratory/macrophages. In addition, TSPO blocking studies done with pre-treatment with PK1195 reduced uptake by 88 percent in non-irradiated tumors and 78 percent in irradiated tumors compared with non-treated cells F-ed cells .18F]-DPA-714 in numerous tumor models  compared to in a muscular inflammation model. Targeting TSPO has also shown promise in tracking the accumulation and depletion of macrophages with [11C]PBR28 in a mouse model of pancreatic ductal adenocarcinoma (PDAC). In addition, the effect of radiotherapy on TAMs was investigated with a TSPO targeting agent, [18F]F-DPA.Overall, TSPO imaging studies have shown variation in terms of experimental design and aim of the study. TSPO can be useful in differentiating inflammation from tumor which was illustrated through the use of FDG in relation to TAMs has shown variable results. In 2015, Cencini and colleagues concluded that TAMs did not show a correlation with [18F]FDG-PET in classical Hodgkin lymphoma despite a 2010 study by Steidl and colleagues that was able to identify a correlation between a high TAM score and results of intervention, also in classical Hodgkin lymphoma [18F]FDG-PET in classical Hodgkin lymphoma, in 2015, showed conflicting results in that one observed a correlation between the two, while the other did not FDG uptake and TAMs in non-small cell lung cancer tumors. However, when considering the subtype of non-small cell lung cancer (adenocarcinomas or squamous cell carcinomas), [18F]FDG uptake and TAMs only showed a significant correlation in adenocarcinomas [18F]FDG uptake and TAMs in the two predominant subtypes of non-small lung cell cancer [Jeong and colleagues were able to show that there was a strong correlation between [rcinomas . Goodwinrcinomas . The difl cancer .18F]FDG- PET assessment in correlation with TAMs can hint at a possible limitation when considering the use of [18F]FDG as means to draw conclusions or observations on TAMs. In addition, [18F]FDG-PET also has the limitation of differentiating tumors and non-tumor inflammatory diseases due to its lack of specificity [The variability of [cificity . The uticificity . CD206 acificity .18F]fluoro-PEG-folate (polyethylene glycol folate) which was used to visualize rheumatoid arthritis in a first in man study in early 2020 [Given the importance of TAMs in cancer and advances in macrophage imaging in other disease states, there is an opportunity to employ tracers that have been developed for PET imaging in inflammatory and cardiovascular diseases for imaging in cancer models. For example, the β-isoform of the folate receptor, a glycosylphosphatidylinositol-anchored cell membrane protein, has been shown to be an emerging marker for M2 polarized macrophages, but has not been extensively explored with TAMs. This target has been successfully imaged with [rly 2020 . Future TAMs have proven their relevance in cancer biology as valuable tools to gain insight on the relationship between immune system response and tumor progression which has resulted in an exciting new era of the development of therapies that target TAMs. Despite an emergence of new immunotherapy techniques, clinical imaging of TAMs has yet to be widely investigated. The imaging of macrophages can provide a noninvasive approach to gain valuable insight into the molecular and functional characteristics of the tumor microenvironment and how the immune response facilitates the progression of cancer or therapy response. The promising data included in this review emphasizes the importance of continuing to expand the research in this area.


In vivo imaging of blood-oxygen-level-dependent (BOLD) signals associated with functional activity in the somatosensory cortex was used to assess the sensory functions during vibrotactile hind paw stimulation. The signal displays an exaggerated response in animals with lateral hemisection that recovers to the level seen in the sham-operated mice by injection of HB-GAM to the trauma site. The effect of HB-GAM treatment on sensory-motor functions was assessed by performance in demanding behavioral tests requiring integration of afferent and efferent signaling with central coordination. Administration of HB-GAM either by direct injection into the trauma site or by intrathecal injection improves the climbing abilities in animals with cervical hemisection and in addition enhances the grip strength in animals with lateral hemicontusion without affecting the spontaneous locomotor activity. Recovery of sensory signaling in the sensorimotor cortex by HB-GAM to the level of sham-operated mice may contribute to the improvement of skilled locomotion requiring integration of spatiotemporal signals in the somatosensory cortex.Heparin-binding growth-associated molecule (pleiotrophin) is a neurite outgrowth-promoting secretory protein that lines developing fiber tracts in juvenile CNS . Previously, we have shown that heparin-binding growth-associated molecule (HB-GAM) reverses the CSPG (chondroitin sulfate proteoglycan) inhibition on neurite outgrowth in the culture medium of primary CNS neurons and enhances axon growth through the injured spinal cord in mice demonstrated by two-photon imaging. In this study, we have started studies on the possible role of HB-GAM in enhancing functional recovery after incomplete spinal cord injury (SCI) using cervical lateral hemisection and hemicontusion mouse models.  The adult mammalian central nervous system displays a low capability to regenerate its connections after trauma. This has led to a plethora of experimental studies to enhance the regenerative potential in the central nervous system (CNS). A prominent reason for the regeneration failure of the CNS traumas such as spinal cord injury (SCI) is the hostile environment preventing the regrowth of neuronal connections , 2. UntiIn mature CNS, several types of chondroitin sulfate proteoglycans (CSPGs) with overlapping acute to chronic expression patterns provide an inhibitory environment for axon regeneration within several months after SCI . CSPGs aThe concept of CSPG inhibition and its role in the regulation of CNS plasticity and regeneration has been largely based on the use of chondroitinase ABC that cleaves chondroitin sulfate chains of CSPGs or on inhibition of CSPG synthesis , 8–15. IHeparin-binding growth-associated molecule  was initially isolated as a neurite outgrowth-promoting protein that binds strongly to heparin . In addiin vitro  of the somatosensory cortex during hind paw vibrotactile stimulation was used to monitor the effect of HB-GAM local administration on sensory functions after lateral spinal cord hemisection at the cervical level in mice. An additional cohort of animals was used to study the effect of HB-GAM treatment on the recovery of motor and sensory-motor functions in the same SCI model.Lateral spinal cord hemisection is used as an animal model of the Brown-Séquard syndrome. Motor dysfunction and loss of light tactile sensations and proprioreception on the ipsilateral side of the trauma and loss of pain and temperature sensations contralaterally are the clinical symptoms of the Brown-Séquard syndrome. The symptom description in animal models is much more controversial ranging from loss of sensory functions to hyperalgesia and allodynia development –35. MoreTo find a route for protein administration that is better compatible with human therapy and allows easily repeated dosing of the protein, we assessed the distribution of radioactively labeled HB-GAM to the thoracic and cervical areas after a single L5-L6 intrathecal injection. Repeated intrathecal administration of HB-GAM was tested in the mouse models of C5 hemisection and hemicontusion. Local injection to the trauma site and intrathecal injections into the cerebrospinal fluid give similar results in behavioral assays. Our studies suggest that HB-GAM induces recovery of sensory signaling in the sensorimotor cortex and promotes partial recovery of motor functions after SCI and might be valuable for human therapy.Adult (8–10 weeks at the beginning of the study) female C57BL/6NHsd and C57BL/6JRcc mice were used for functional and imaging studies respectively. Mice of C57BL6 strain are prone to develop ulcerative dermatitis that in severe cases could significantly impact animal behavior and general welfare . We founad libitum.The mice were housed in groups of four to five animals in individually ventilated cages before surgery. Animals used for BOLD signal imaging were housed individually for a few days after optical window installation to secure tight frame fixation. Ventilated cages were set at a stable temperature (21°C) in a 12-h light/dark cycle and provided with food and water Before surgery, all animals were equally distributed based on the baseline vertical screen performance and body weight to the sham and the SCI groups. Animals received an antibiotic shot 30 min prior to the surgery and 3 days after the surgery . Painkillers were administered 15 min before the surgery and after the surgery during 2–3 days  depending on the animal recovery. In the first studies  we chose an injectable anesthesia cocktail (Ketamine + Rompun 8 ml/kg) to induce the trauma in order to avoid the use of inhalational masks and get better access to surgery area and more freedom to manipulate with an animal in technically challenging surgery followed by compound administration with microinjector. Inhaled isoflurane anesthesia (4%) was used for later functional studies , which improved animal survival, especially in the cervical hemicontusion model. Ketamine/xylazine injectable anesthesia results in cardiac  and respSingle injections of the recombinant HB-GAM characterized previously  (1 mg/mlThe mouse was anesthetized with isoflurane (2%) and placed in the prone position. The skin was shaved and disinfected in the lumbar area. The injection was done by a 31 G needle connected to Hamilton 10 μl syringe by polyethylene tubing. The L5 vertebra was localized between the iliac crests of the hip bones, and the needle was inserted through the skin between the L5 and L6 vertebras. After positioning of the needle in the intrathecal space verified by tail-flick reflex, 7 μl of solution (HB-GAM 1 mg/ml or 1xPBS) was slowly injected. The needle was kept at the place for an additional 15 s to prevent solution backflow. Intrathecal injections were repeated for 4 weeks once per week starting from day 7 after the spinal cord trauma. Sham animals were injected with PBS.123]HB-GAM  were injected through the skin by an intrathecal puncture to the lumbar area between the L5 and L6 vertebrae. Radioactivity in the syringe was measured prior to and post-injection. Single-photon emission computerized tomography-CT (SPECT-CT) imaging was carried out with NanoSPECT/CT , a four-headed small animal scanner featuring a 1 mm multi-pinhole mouse apertures . SPECT images were acquired dynamically at around 30, 60, 90, and 120 min post-injection taking 24 projections at 90 s for two animals. Additionally, one mouse was imaged at 5 h and another at 15 h post-injection increasing the exposure to 120 s. CT imaging was acquired at 45 kVp in 180 projections, 500 mc/projection. SPECT images were reconstructed with HiSPECT NG software  and fused with CT datasets with VivoQuant software . To quantify tracer uptake in spinal cord areas, reconstructed SPECT images were reoriented and analyzed with VivoQuant  software by using CT data as a reference. Three-dimensional (3D) volumes of interest (VOIs) were defined for three main spinal cord regions  by using the 3D ROI tool. The radioactivity in each VOI was calculated and results were presented as standard uptake value (SUV) calculated as the ratio of the activity in the VOI divided by the total activity injected per gram of mouse.The HB-GAM protein was labeled with iodine-123 and purified from a free label at the Laboratory of Radiochemistry, University of Helsinki, using solid-state iodination of a single tyrosine in the protein sequence followed by high performance liquid chromatography (HPLC purification. The mice were anesthetized with isoflurane (4%); 7 μl of , and an additional emission filter 590LP filter  was placed in front of the camera to reduce possible short-wavelength noise. During the imaging session data, frames were captured with the acquisition frequency of 10 Hz and combined into 10 block files (300 data frames/block) resulting in 3,000 data frames per 5 min session. The sensory stimulation pattern (0.1 Hz on-off period) was synchronized with camera frame capturing via a custom MatLab script  and MCC USB-1209FS  controller and resulted in a three-phased stimulus-evoked hemodynamic signal (https://imagej.net/plugins/maximum-entropy-threshold). The outcome illustrates a 2D representation of the initial dip phase to perform surface area calculation. The magnitude of the initial dip phase was analyzed using Open-source FIJI software (available at https://imagej.net/software/fiji/).Intrinsic signal optical imaging (ISOI) was used to monitor local blood oxygenation changes associated with functional activity in the cortex. Animals were slightly anesthetized with isoflurane (0.9–1.2%), placed on a heating plate (36°C), and heads were fixed by implanted metal frames. Through the imaging experiment, the anesthesia level was monitored with a PhysioSuite physiological monitor  and adjusted to keep the breath rate at around 120+/−10 breaths per minute. Custom vibrators (1 cm diameter) were fixed to the plantar surface of both hind paws for tactile stimulation. To obtain images of light reflectance, a 12-bit MV1-D1312-160 CL CMOS-camera  equipped with paired objective lenses  was used resulting in a 4 mm x 4 mm field of view. Image capture was done c signal . Raw datThe mouse was placed on the edge of the horizontal wire screen  faced to the edge and then immediately turned with the screen vertically to the upside-down position. The latency to climb to the upper edge was measured with a 1 min cutoff time. The test was repeated in three subsequent trials with at least 1 min interval and the averaged value was used for data analysis. To understand better the role of ipsilateral front paw motor functions in vertical screen test performance we evaluated the usage of front paws in wire grasping before each testing session to detect possible paralysis or spasticity of paw/digits: the mouse was lifted by the tail and slowly move down to the horizontal wire screen allowing to extend the front paws and grasp the wire. The grasping abilities were scored between 0 and 2, where 0 is the normal functioning of ipsilateral paw, 1 is weak grasping with occasional paw displacement but relaxed paw and extended fingers after the wire release, and 2 is keeping paw flexed unable to grasp the wire.The mouse was placed into the glass cylinder  and videotaped for the following analysis for the first 20 exploratory vertical rearings with 5 min cutoff time. Trials with <5 rearings were excluded from the analysis. An experimenter blinded to the animal group quantified the number of ipsilateral front paw use  and the total number of rearings with front paws used for bodyweight support against the wall.In the hemisection model, the grip strength was measured separately for ipsilateral and contralateral front paws keeping animals from the scruff and allowing them to grasp the bar with one paw only. Waiting until complete wound healing after the trauma surgery was required to start the grip strength testing (4 weeks). To be able to measure grip strength at an earlier time point (2 weeks) in the hemicontusion model, cumulative grip strength for both front paws was measured by an automated grip-strength meter . Animals handled by tail were allowed to grasp the horizontal metal bar. Mice were then gently pulled horizontally away from the bar until the grasp was released. Grip strength was measured as the maximum produced force averaged across five trials performed with at least 1 min intertrial interval.The animal was released to the corner of the open field chamber  faced to the wall and allowed to explore the chamber for 15 min. Spontaneous locomotor activity was measured automatically by infrared sensors and analyzed by the Activity Monitor v5.1 software  as the traveled distance and the rearing time (time spent standing on rear paws).Mice were placed in individual restraining chambers (10 x 10 cm) with the wire-mesh floor for adaptation at least 30 min before the test. An electronic aesthesiometer  was used for the assessment of mechanical sensitivity and allodynia. A metal probe was placed under the central area of the hind paw palm, and pressure  was applied until paw withdrawal. Latency to withdraw was analyzed for each hind paw as an average of four trials repeated with at least 2 min intertrial interval.Statistical analysis was performed using GraphPad Prism 9, San Diego, CA. For normally distributed data two-way ANOVA followed by pairwise comparison with Tukey or Dunnett test was used for assessing the group difference at individual timepoints or within-group difference  respectively. Non-normally distributed data were analyzed by Friedman test with Dunn's correction to compare individual time points (vs. 1 week) within the same group and Kruskal-Wallis test with uncorrected Dunn's pairwise comparison to test the group difference at individual time points. Data in the figures are presented as the mean +/– SEM.in vivo imaging and functional regeneration in repeated behavioral testing during 7–9 weeks after the SCI  is similar to functional MRI (fMRI) in that it reveals activity-dependent changes in the nervous system using BOLD imaging . In ISOIp = 0.0087, week 4 p = 0.0244, week 5 p = 0.0217, week 6 p = 0.0203, week 7 p = 0.0406, Kruskal-Wallis test with uncorrected Dunn's pairwise comparison). In contrast, the HB-GAM-treated animals performed like the sham-operated animals during the whole testing period , week 3 (p = 0.0163), and the week 7 , while the HB-GAM treatment partially restored the vertical activity demonstrating the absence of a significant difference vs. the sham animals  which could be interpreted as a sign of allodynia, whereas an increased latency to withdrawal for ipsilateral paw in the PBS group in comparison with the HB-GAM group was observed at the week 6 . Overall, the HB-GAM-treated animals were not different from the sham mice in the von Frey test , 7 (p = 0.0408), and 9  vs. the pre-treatment value at the week 1, and there was no group difference vs. the sham animals at the weeks 5 and 9. In contrast to the HB-GAM-treated animals, no recovery was observed in the PBS-treated mice: starting from week 1 the PBS animals constantly demonstrated a significantly longer climbing time than the sham animals. Moreover, the climbing time for the PBS group vs. the HB-GAM group was significantly longer at weeks 5 (p = 0.0136), 7 (p = 0.0041), and 9   . Similarp = 0.0282) and 9  compared to the level at the week 1 . as was also found in the grip strength tests .p = 0.0025, Friedman test with Dunn's pairwise comparison) in comparison with the climbing speed at week 1 after the SCI . The severity of trauma was controlled by the automatically assessed impact force that was similar in both groups with spinal cord contusion . As in the hemisection model, hemicontusion resulted in a significant increase in the climbing time that was spontaneously reduced both in the PBS-treated and in the HB-GAM-treated groups, while the climbing time reduction reached statistical significance only in the HB-GAM group at the week 7    in compap = 0.005, 2-way ANOVA with Turkey post-hoc pairwise comparison)  . As in tparison) , the oveHeparin-binding growth-associated molecule (HB-GAM) was initially isolated by screening for factors that enhance neurite outgrowth in central neurons . MoleculD values  signaling in the somatosensory cortex induced by vibrotactile stimulation of the hind paw. While the cervical lateral SCI results in a change of motor and sensory function both in front and hind paws, stimulations of hind paws allow assessing of the functioning of ascending pathways and their representation in the somatosensory cortex with minimum interactions with possible pathological processes in the peripheral or central nervous system on the level of trauma. We have applied the technique designated ISOI that is in principle similar to BOLD fMRI. Interestingly, the ISOI signals due to vibrotactile hind paw stimulation are clearly enhanced after the spinal cord hemisection on the area of the hind paw representation in the somatosensory cortex . ExaggerA-mediated inhibition (A receptor has been shown to enhance neurite outgrowth (Heparin-binding growth-associated molecule (HB-GAM) injected into the trauma site strongly reduces the ISOI signals in the somatosensory cortex. At all time points measured (from week 1 until week 7) the ISOI signals in the HB-GAM-treated mice are at the level seen in the sham-operated mice . Loss ofhibition . Furtherutgrowth , which mutgrowth .Unilateral lesions of the spinal cord cause problems in skilled locomotion . This waAs a difference from the climbing test, HB-GAM administration did not affect spontaneous locomotor activity that depends on both physical and mental status of the animals and was somewhat reduced in both the PBS-treated and the HB-GAM-treated SCI mice compared to the sham-operated animals. The HB-GAM effect in demanding tasks such as the climbing test or the cylinder test cannot be therefore simply explained by enhanced general activity, motivation, or arousal of the mice but rather suggests a specific effect on neuronal circuits required in coordinated motor functions.Locomotion in a complex environment requires integrated spatiotemporal information that is processed in the somatosensory cortex . InteresHyperalgesia is often observed in spinal cord lesions , 35 and It is also noteworthy that factors inducing sprouting of neurites are known to lead to the development of hyperalgesia. For example, overexpression of nerve growth factors is known to cause hyperalgesia . HB-GAM A decrease in the grip strength of the forepaws is one parameter to follow healing after cervical injury of the spinal cord and was found in the hemicontusion model but not in the hemisection model in the current study. The comparison of the hemisection and hemicontusion results in the grip strength test is not very accurate due to a technical difference in the method: in the hemisection model the grip strength was assessed separately for ipsi- and contralateral paws, while cumulative grip strength measurement for both paws simultaneously was used in the hemicontusion study. To address this question, we tested a separate batch of animals with cervical hemisection exactly in the same test of cumulative grip strength assessment as in hemicontusion (data not shown). These data confirmed the lack of effect for HB-GAM in the grip strength in the hemisection model and the beneficial effect of HB-GAM treatment in the vertical screen test.The loss of grasping abilities of the front paws after cervical spinal cord injury is driven mostly by disruption of descending pathways and partially by damage of segmental circuity including motoneurons loss on the level of the trauma , 59. ConRecently discovered the copy-number variant in the Dock2 gene and altered immunological phenotype of C57Bl6/NHsd  raises qAergic activity .NK designed and carried out the animal surgeries and the behavioral experiments and performed the data analysis. DM developed the protocol for BOLD signaling assessment. SS and DM performed optical windows installations, BOLD imaging, and BOLD data analysis. EM performed behavioral experiments and assisted in animal surgeries. AG designed and performed SPECT-CT study of the compound distribution, imaging analysis, and data interpretation. NK, MP, and HR participated in the study design and data interpretation. HR provided funding and study administration. All authors contributed to manuscript preparation.The study was funded by the Wings for Life foundation, the Academy of Finland, and the Sigrid Jusélius foundation. SPECT/CT imaging was performed at the Real-Time Imaging lab, supported by Biocenter Finland and Helsinki Institute of Life Science. Animal experiments were done with the use of facilities and equipment of Mouse Behavioral Phenotype Facility (MBPF), supported by Biocenter Finland and Helsinki Institute of Life Science.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.


The 5-campus UMass system received designation as an Age-Friendly University (AFU) in 2019. AFU Principle 1 highlights the importance of involving older adults in University activities. UMass Lowell’s Center for Gerontology Research and Partnerships collaborated with the Learning in Retirement Association (LIRA) in Spring 2020 to offer aging-related courses around healthy aging. However, due to COVID-19, these were canceled and are re-scheduled for Spring 2021 via Zoom. The paper will describe the process of selecting course offerings with LIRA and the subsequent cancellation/rescheduling process and adaptation needed. A course will focus on AFU initiative and the opportunities and challenges at UMass Lowell. Likewise, the other course will offer a session on technology and aging where age-based digital divide and strategies for reducing it will be discussed. This paper will reflect on how the collaboration with LIRA and course selection process relates to the AFU principles 1, 5 and 9.


The results of these studies showed a good agreement of EF50 with the invasive assessment of lung resistance and dynamic compliance with a somewhat lower sensitivity of EF50. The measurement of EF50 together with basic respiratory parameters is particularly appropriate for simple and repeatable screening of pulmonary function in large numbers of mice or if noninvasive measurement without use of anesthesia is required. Beyond known applications, head-out body plethysmography also provides a much-needed high-throughput screening tool to gain insights into the impact and kinetics of respiratory infections such as SARS-COV-2 on lung physiology in laboratory mice.Mouse models have become an indispensable tool in translational research of human airway disease and have provided much of our understanding of the pathogenesis of airway disease such as asthma. In these models the ability to assess pulmonary function and particularly airway responsiveness is critically important. Existing methods for testing pulmonary function in mice in vivo include noninvasive and invasive technologies. Noninvasive head-out body plethysmography is a well-established and widely accepted technique which has been proven as a reliable method to measure lung function on repeated occasions in intact, conscious mice. We have performed several validation studies in allergic mice to compare the parameter midexpiratory flow (EF The ability to measure pulmonary function in individual mice on repeated occasions is of great interest because of the prominent role played by these animals into the causes and mechanisms of respiratory and allergic diseases such as asthma. This is largely due to relatively low maintenance costs, a well characterized genome and immune system, high breeding efficiency, the large availability of inbred and transgenic strains, and the technologies for genetic manipulation not yet available in other animals. Mouse models of experimental asthma have contributed substantially to our understanding of pathomechanisms underlying allergic airway inflammation and airway hyperresponsiveness (AHR) –3. HowevAHR defined as the predisposition of the airways to react excessively to bronchoconstrictor agents is an important component of the asthma phenotype. Pulmonary function tests are important to assess lung function and airway responsiveness (AR) in intact organisms, but the development of these tests has been a great challenge due to the small size of murine airways. In recent years, considerable progress has been made in developing valid and suitable pulmonary function measurements –8. CurreL) or dynamic compliance (Cdyn) is the classical method for accurate and specific determination of pulmonary mechanics. The low-frequency forced oscillation technique (LFOT) is currently considered to be the most precise technology to determine both central airway resistance and peripheral tissue compliance. ) , breathing frequency (f) and an increase in time of expiration (TE). This noninvasive approach is particularly useful to measure several animals at a time, to monitor pulmonary function repeatedly, and to capture the kinetics of a response over time. Furthermore, the method is straightforward and can be learned in a relatively short time.Yves Alarie and coworkers were the first to demonstrate that the noninvasive measurement of midexpiratory flow (EFous mice . Using hs]) Fig. . Changes50 with invasively recorded gold standard parameters in established mouse models of allergic airway inflammation.In general, the majority of experimental asthma models usually determine the following outcomes: immunological , histopathological  and functional . Several validation studies were performed to compare the utility of EF50 as measured by head-out body plethysmography and to detect changes in pulmonary function, we measured the bronchoconstrictive response to aerosolized methacholine (MCh) and differentiated between normal levels of airway reactivity in control animals and airway hyperresponsiveness in Ovalbumin (Ova)-sensitized and -challenged BALB/c mice [To evaluate the sensitivity of EFB/c mice .50 response to aerosolized MCh  in OVA-sensitized and challenged animals was both shifted to the left and amplified compared with that in control animals. The MCh-related decrease in EF50 is presumably mediated by specific muscarinic M3-receptor activation on airway smooth muscles as shown by the rapid onset and resolution of the response to aerosolized MCh.The EF50 values in allergen-sensitized and -challenged mice were associated with the production of allergen-specific IgE and IgG1 and the development of eosinophil infiltration in the lungs. In addition, increased numbers of eosinophils and lymphocytes as well as elevated titers of the Th2 cytokines IL-4 and IL-5 in broncho-alveolar (BAL) fluid were detected showing a Th2-skewed adaptive immune response. Importantly, EF50 closely reflected the enhanced airway response to aerosolized MCh observed with simultaneously measured pulmonary conductance (GL = 1/RL) and dynamic compliance (Cdyn) in allergen-sensitized and -challenged BALB/c mice. We further demonstrated that the decline in EF50 to MCh challenge was partly inhibited by pretreatment with an inhaled beta-2-agonist and that associated changes in f and VT did not directly influence EF50 values. A limitation of the current validation investigation particularly has included pleural catheterization to measure invasive pulmonary mechanics (GL and Cdyn) and the contribution of upper airway resistance. This methodological shortcoming introduced variability including uncertainty of the site of obstruction into the results which made them difficult to compare with other invasive techniques [Decreases in EFchniques .L, Cdyn and EF50 were measured simultaneously in intact mice including local aerosol challenges via an orotracheal tube. The primary objective of this follow-up study in a mouse model of fungal asthma (Aspergillus fumigatus) was to compare the capability of noninvasive EF50 measurements to reflect the allergen-specific and cholinergic AR as observed with invasive determination of pulmonary mechanics [To overcome this limitation we developed and validated a plethysmograph system for invasive lung function measurement combined with an integrated computer-controlled aerosol delivery system .This novechanics . Groups With both methods, allergic mice sensitized and boosted with A. fumigatus revealed allergen-specific early airway responsiveness (EAR) in response to aerosolized allergen, whereas sham-exposed controls were unresponsive Fig. . 48 h la50 can discriminate the degree of AR and reflects the changes in invasively recorded GL and Cdyn during bronchoconstriction. Moreover, the relation of the cholinergic EF50 response between allergic and control animals was similar for invasive and noninvasive measurements. Compared with GL and Cdyn recordings, invasive and noninvasive recordings of EF50 were less sensitive in detecting the maximum degree of bronchoconstriction during challenges with inhaled allergen and MCh [These outcomes suggest that EF and MCh –18.50 method with the classical parameters of lung mechanics, and are thus particularly suitable for simple and repeatable screening of pulmonary function in large animal numbers on multiple occasions. Since EF50 may underestimate the degree of bronchoconstriction it is still unclear how much this limits its use in detecting less pronounced changes of AR. Accordingly, when such circumstances are present, measurements should be validated against invasive standard measures of lung mechanics.The results of these studies showed a good correlation of the EFIn barometric whole-body plethysmography unrestrained mice are placed in a whole-body plethysmograph and the pressure changes that occur in the chamber during the breathing cycle are analyzed. From the box pressure signal during inspiration and expiration, and the timing comparison of early and late expiration, a dimensionless parameter called "enhanced pause" (Penh) has been calculated . Current50 differs substantially in several important ways: EF50 declines with bronchoconstriction and in line with invasive parameters of lung mechanics is associated with a decrease in tidal volume during bronchoconstriction [50 is a physiological variable with a physical meaning (ml/s), enables direct comparison from one animal to another and is closely related to airway resistance. By contrast, Penh is not a valid measure of airway resistance and its usefulness was further weakened by a study showing that changes in Penh were no better than simply measuring time of expiration (TE) in various strains of mice [50 measurements are independent of f, TE, and VT was determined in studies with various airborne stimuli (CBC and n-propranolol) in conscious animals in which EF50 values were unaffected by irritant induced changes in f, VT, and TE, suggesting that EF50 does not correlate simply with changes in breathing patterns [50 more reliably reflects airway resistance than Penh, which is largely a function of respiratory timing.Compared with Penh, EFtriction , 17. In  of mice . That EFpatterns . These dBeyond known applications in asthma research, integrated use of head-out body plethysmography might contribute to a better understanding of other respiratory disorders including, e.g., experimental models of COPD/emphysema, lung fibrosis, tests on airborne irritant/pollutant effects , 19, 28 50 [50 discriminated both virus and dose-specific responses and identified long-term respiratory changes in experimental mouse models of coronavirus and Influenza A virus infection [50 in a mouse model of SARS-CoV-2 infection [In addition, head-out body plethysmography can provide noninvasive and repeatable long-term monitoring of disease responses in the same animals throughout the course of bacterial and viral infections which may propel new ways toward exploring pathogenesis and new drug therapies. As an example, head-out body plethysmography has been reported to be very useful for monitoring infection with Pseudomonas aeruginosa in mice showing a decrease in VT and EF50 . Even monfection . Anothernfection . This apAlthough still underutilized, pulmonary function testing has gained popularity due the recognition of its clear value in the functional assessment of experimental models of various acute and chronic respiratory disorders, including allergic asthma. However, at present, there is no gold standard for measuring pulmonary function in mice, because none of the available invasive and noninvasive methods is optimal in all regards. Some investigations  require the most sensitive and reliable determination of lung mechanics in ventilated, anesthetized animals. The noninvasive assessment of respiratory standard metrics is a complimentary approach for screening pulmonary function and the kinetics of respiratory changes in large numbers of conscious mice. We and others have shown that head-out body plethysmography is a valid, reliable and cost-effective method, that can fulfill an important need in many respiratory research applications. The ability to assess and analyze standard respiratory function parameters concurrently with an accepted index of airflow limitation on multiple occasions in the same animal makes this method particularly useful for acute and longitudinal studies without the use of anesthesia. Beyond known applications, head-out body plethysmography will also provide a much-needed high-throughput screening tool for noninvasively evaluating the impact of SARS-Cov-2 infections on pulmonary function and antiviral drug performance in wild-type and genetically altered mice.


M. tuberculosis in the cytoplasm of ciliated bronchiolar epithelium and alveolar pneumocytes in association with elements of the programmed death ligand 1 (PD-L1), cyclo-oxygenase (COX)-2, and fatty acid synthase (FAS) pathways in the early lesion. This suggests that M. tuberculosis uses its secreted antigens to coordinate prolonged subclinical development of the early lesions in preparation for a necrotizing reaction sufficient to produce a cavity, post-primary granulomas, and fibrocaseous disease.The characteristic lesion of primary tuberculosis is the granuloma as is widely studied in human tissues and animal models. Post-primary tuberculosis is different. It develops only in human lungs and begins as a prolonged subclinical obstructive lobular pneumonia that slowly accumulates mycobacterial antigens and host lipids in alveolar macrophages with nearby highly sensitized T cells. After several months, the lesions undergo necrosis to produce a mass of caseous pneumonia large enough to fragment and be coughed out to produce a cavity or be retained as the focus of a post-primary granuloma. Bacteria grow massively on the cavity wall where they can be coughed out to infect new people. Here we extend these findings with the demonstration of secreted mycobacterial antigens, but not acid fast bacilli (AFB) of  Many investigators today believe that cavities form by erosion of caseating granulomas into bronchi. Pathologists in the pre-antibiotic era knew that this is wrong. Cavities form by dissolution of caseous pneumonia, not by erosion of granulomas . EarlierThe early lesions of PPTB do not begin until after establishment of sufficient immunity to prevent disease in all extra pulmonary and most pulmonary sites . PPTB bePost-primary granulomas are easily identified because they form to surround preexisting foci of caseous pneumonia and therefore contain ghosts of alveoli rather than the homogeneous caseum of primary granulomas . PrimaryHere we report finding M. tuberculosis (MTB) antigens, but not acid-fast bacilli (AFB), in alveolar lining cells (pneumocytes) and ciliated bronchial cells together with elements of the programmed death ligand 1 (PD-L1), cyclo-oxygenase (COX)-2, and fatty acid synthase (FAS) pathways in developing lesions of PPTB. This suggests that MTB use secreted antigens to orchestrate an extended sequence of subclinical lesions that prepare for a sudden massive necrotizing pneumonia sufficient to produce cavities from which the organisms can escape to new hosts. Post-primary granulomas arise to surround foci of caseous pneumonia that are not coughed out to form cavities. They persist to become fibrocaseous disease. Most nascent post-primary lesions regress spontaneously leaving apical scars. If we understood why they regress, it might be possible to make them all regress and thereby drive MTB to extinction.Histologic slides from over 50 autopsies of people who died of untreated TB were examined to identify tissue sections with the early lesion of PPTB as previously described ,7. BriefThe early lesion of PPTB is a post-obstructive lobular lipid pneumonia that spreads via bronchi for months prior to caseation ,6,8 Fig. SecreteSince MTB is an obligate human parasite, everything that it does has been selected to ensure survival and transmission among people. MTB secretes many protein antigens that are required for virulence in humans but are not necessary for growth in culture . The funThe early lesion of PPTB is an asymptomatic obstructive lobular pneumonia that develops for 1–2 years before onset of symptoms . It has Bronchial obstruction traps alveolar macrophages to produce post-obstructive lipid pneumonia .Alveolar lining cells use FAS to produce lipids.Alveolar macrophages with M2 phenotype (CD163 Staining) become foamy by accumulating host lipids and secreted mycobacterial antigens.Sensitized tissue resident T cells (TRM) accumulate in alveolar walls.+ T cell activity.PD-L1 expression on alveolar macrophages and alveolar pneumocytes suppress PD-1The finding of MTB antigens in multiple types of cells of the early lesion suggests that MTB use these antigens to coordinate diverse components of the host response to subclinically produce the materials necessary for a necrotizing reaction sufficient to form a cavity large enough to support transmission of infection. The components of the early lesion include:These lesions spread via bronchi as an obstructive lobular pneumonia, recognized by CT scans as the “tree-in-bud” sign, until they either regress or undergo necrosis to become caseous pneumonia that is either coughed out to form cavities or retained to become the focus of post-primary granulomas .Our findings include (1) demonstration of mycobacterial antigen within both the type 1 and type 2 alveolar pneumocytes and ciliated bronchiolar lining cells in addition to alveolar monocytes/macrophages; (2) COX-2 expression in reactive alveolar pneumocytes and ciliated bronchiolar epithelium; (3) expression of FAS that could contribute fatty acids to the foamy alveolar macrophages; (4) expression of programmed death ligand (PD-L1) on the alveolar monocytes/macrophages, pneumocytes, and bronchiolar epithelial cells; and (5) CD163 expression on monocytes/macrophages in alveoli.+ cells were present, but abundant CD8+, PD-1+ cells were in the alveolar walls. Foamy macrophages stained strongly with CD68 and frequently also with the dendritic cell marker DEC-205. T-regulatory cells were also observed in the early lesion [In previous studies, we reported that the early lesion of PPTB contains foamy alveolar macrophages with the M2 phenotype that marked with CD163, PD-L1, phosphorylated mTOR, insulin-like growth factor-1 receptor (IGF-1R), and human cyclooxygenase 2 (COX-2) . Relativy lesion . These fy lesion .The early lesion of PPTB appears to be an attractive target for host directed therapies ,18. MiceSeveral lines of evidence also suggest that the early lesion might also be an attractive target for vaccines. First, many nascent lesions regress spontaneously. If we knew why, it might be possible to induce all to regress. Second, slowly progressive pulmonary tuberculosis in the mouse is a model of the early lesion of PPTB . SeveralFormalin-fixed and paraffin-embedded tissue blocks of human tuberculous lung samples were obtained during regular autopsy practice and after the completion of all medical, legal, and ethical requirements and were deidentified. Information provided included the age, sex, pulmonary TB as the primary cause of death, and negative HIV. Drug sensitivities were not known. Furthermore, this study was conducted according to the principles expressed in the Declaration of Helsinki. Hematoxylin and eosin (H&E) staining was used to identify blocks with the early lesion of PPTB . GrosslyImmunohistochemistry was performed on five micrometer sections that were deparaffinized and stained with monoclonal antibodies conjugated with 3,3′-diaminobenzidine (DAB) for detection by immunohistochemistry. The antibodies used were MTB antigen -2 , programmed death-ligand 1 , programmed death-1 (PD-1), , and fatty acid synthase . Positive and negative controls were run concurrently. The immunohistochemical reactions/expressions were assessed as weak or strong by visual inspection in comparison with positive and negative controls. All procedures were conducted in a fully accredited clinical laboratory with all relevant positive and negative controls. None of the markers studied are present in appreciable concentrations in normal lung tissue G.We previously reported that the early lesion of post-primary TB is a distinct disease entity . The preM. tuberculosis needs both to survive. It must simultaneously protect its host and develop means to escape to new hosts. If protection by granulomas fails, the host dies of disseminated infection and the organisms die with it. M. tuberculosis only survives if post-primary lesions induce a cavity from which it can escape to find new hosts and the host remains healthy enough to circulate in the community.The early lesions produce cavities from which the organisms can escape to find new hosts, while granulomas protect the host from disseminated infection. As an obligate human parasite, The study of post-primary lesions will remain challenging because of the paucity of informative human tissues that can be used both to study the disease and to validate animal models. Fortunately, new multiplex technologies can measure proteins and nucleic acids on slides with a depth and precision undreamed of a decade ago . Such te