We performed semen analysis including Male infertility Oxidative System (MyOxSIS) analysis, physical examination, and evaluation of endocrine and erectile function. Results: OS, erectile function and spermiogram categories did not differ significantly between the groups. The interval from the onset of symptoms to surgery differed significantly between groups (p < 0.001). Preservation of the testes was associated with a higher round cell count (p = 0.002) and follicle stimulating hormone . OS showed a significant positive correlation with the spermiogram category . A negative correlation was observed between OS and age (p = 0.033), sperm concentration (p < 0.001) and total sperm count (p = 0.006). Conclusions: Endocrine, exocrine and erectile function are not significantly affected by TT in the long term. Orchiectomy results in elevated FSH and a lower round cell count compared to preservation of the testis.Background: This study investigates endocrine and exocrine testicular function, oxidative stress (OS) in semen, and erectile function in patients who underwent surgery for suspected testicular torsion (TT). Methods: We evaluated 49 patients over a mean follow-up of 101 months:  Testicular torsion (TT), usually a spontaneous twisting of the testis around the spermatic cord causing subsequent ischemia, affects 1/4000 men younger than 25 years of age . In ordeExperimental animal studies showed that unilateral TT is capable of reducing fertility by damaging the blood–testis barrier (BTB). However, this only occurs when the twisted testicle is left in place . FurtherPermanent impairment of testicular function is thought to be caused by ischemia at the time of TT and by oxidative stress (OS) following detorsion . ReperfuThe primary endocrine function of the testis, which is the production of testosterone, appears to remain unaffected by TT ,16. HoweImpairment of exocrine testicular function has been described in various studies, which also report altered sperm morphology and decreased sperm motility and sperm count ,16,17,18We lack a sufficient body of long-term data on the impact of testicular function after TT on erectile function ,21. MoreThe present study includes a long-term follow-up of endocrine and exocrine testicular function, OS in semen, and erectile function in patients who underwent surgery for suspected TT.The data of patients who underwent surgery for suspected testicular torsion between 1 January 1999 and 1 September 2020 at the University Clinic of Urology and Andrology, Salzburg, were analyzed. Three hundred and nineteen men were identified and contacted between January and May 2021. Forty-nine patients agreed to participate in the study. All participants gave their written informed consent and were >18 years of age at the follow-up examinations.n = 25; patients with no intraoperative sign of torsion = suspicion of spontaneous detorsion) had been treated with surgical exploration of the testis and bilateral orchidopexy to avoid a recurrent torsion of the testis; Group 2  had undergone surgical exploration of the testis, unilateral detorsion, and bilateral orchidopexy; Group 3  had undergone surgical exploration of the testis, unilateral orchiectomy, and contralateral orchidopexy.We divided the patients into three groups based on surgical procedure: Group 1 . The International Index of Erectile Function (IIEF-5) questionnaire was used to determine sexual function. The physical examination included a scrotal ultrasound investigation, a blood test, and a spermiogram with an assessment of routine seminal parameters according to the World Health Organization (WHO), determination of OS, and anti-sperm antibodies (ASA).The patients’ physical status was assessed on a scale from 1 to 5 . According to their COVID-19 status, patients were divided into two groups: the first had had a COVID-19 infection proven by a polymerase chain reaction test, and the second had not. Clinical data, if available, were obtained from the hospital records.A blood sample was taken between 7 a.m. and 9 a.m. to determine hormonal status. FSH, LH, testosterone, free-testosterone (fT), sexual hormone binding globulin (SHBG), albumin, prolactin, and thyroid-stimulating hormone (TSH) were measured in the hospital laboratory. The normal ranges for the blood test were as follows: FSH 1.5–12.4 mU/mL; LH 1.7–8.6 mU/mL; testosterone 2.49–8.36 ng/mL; fT 6.76–22.76 pg/mL; SHBG 18.30–54.10 nmol/L; albumin 3.4–5 g/dL; prolactin 86–324 µU/mL; TSH 0.5–4.20 mU/L.6 mL.Semen was collected in the hospital by masturbation after two to five days of sexual abstinence. All samples were analyzed within one hour after collection. Semen analysis was performed according to the WHO 2010 criteria. ASA was analyzed by the mixed antigen reaction test (MAR test). OS was determined with the Male infertility Oxidative System  . The norThe consent of the local ethics committee was obtained (EK: 1152/2020).t-test was used for group comparisons. In the case of normality but no variance homogeneity, Welch’s t-test was applied. For variables without normally distributed data and for variables measured on ordinal scales, the Mann–Whitney U test was used. Dichotomous variables were compared by Fisher’s exact test, and the remaining categorical variables by the chi-squared test.All data of continuous variables were checked for normal distribution  and, in the case of normal distribution, also for variance heteroscedasticity . In the case of normality and variance homogeneity, the independent two-sample As no continuous variables showed both normally distributed data and variance homogeneity, all continuous variables, as well as variables measured on ordinal scales, were compared by a non-parametric analysis of variance . Data of categorical variables were analyzed by the chi-squared test, with the provision of adjusted residuals.Correlations were reviewed by Bravais–Pearson correlation coefficients, Spearman’s rank correlation coefficients, and point biserial Spearman’s rank coefficients. Associations of continuous variables and variables measured on ordinal scales with categorical variables were investigated by eta coefficients . Associations of dichotomous variables with categorical variables were investigated by Cramer’s V (combined with the chi-squared test and Fisher’s exact test). The influence of various parameters on oxidative stress was investigated by multiple regression analysis. The type I error was not adjusted for multiple testing. Therefore, the results of inferential statistics are only descriptive. Statistical analyses were performed using the open-source R statistical software package, version 4.0.5 . The detailed statistical analysis can be obtained from the authors on request.n = 7), hematospermia (n = 1), hypersensitivity (n = 3), and a high-riding testis (n = 2); 57.1% of the patients (n = 28/49) were physically active and 96% of the patients (n = 47/49) felt fit.Demographic data are shown in Ten patients (20.4%) had fathered a child after TT in ten cases. Assisted reproduction was not needed. One patient who was normozoospermic after TT reported a spontaneous abortion.One patient reported moderate (Group 1), one patient mild to moderate (Group 1), and six patients mild erectile dysfunction , according to the IIEF-5 questionnaire. Three patients reported not having sexual intercourse.On physical examination, six patients had a hydrocele, six a spermatocele, one relative phimosis, and seven a varicocele . Testicular microlithiasis was seen in two cases.Laboratory parameters are summarized in 6/mL sperm count). Forty-two patients (87.6%) had normozoospermia, five (10.4%) oligozoospermia, and one (2.1%) asthenozoospermia. Three patients (6.3%) with normozoospermia had reduced vitality (one in Group 1 and two in Group 2); morphology was normal in all cases. MAR positivity was seen in four cases, all in Group 1. Agglutinate was detected in 48% of Group 1 , 36.9% of Group 2 , and 25% of Group 3 (+1/4). Five patients in Group 1, three in Group 2, and one in Group 3 had elevated OS. OS could not be evaluated in two cases (Group 2). The three groups did not differ significantly in regard to spermiogram categories .Semen analysis was feasible in 48 cases . One patp < 0.001), FSH (p = 0.003), round cell count (p = 0.002), and leukocytes in the spermiogram (p = 0.040) (p = 0.754). Patients with orchiectomy had elevated FSH and lower testosterone and free-testosterone levels compared to patients with preserved testes .Significant differences were seen between the three subgroups in the time from the onset of symptoms to surgery (= 0.040) . The grad testes . OS did 6 mL) was feasible in 46 cases. One patient could not give a semen sample and the analysis of OS was not applicable in two cases . There was no significant difference between the three groups .The analysis of OS (mV/10p = 0.010 – Spearman’s test). A multivariate analysis of oxidative stress showed a significant correlation for oligozoospermia and normozoospermia . Vitality revealed a non-significant negative correlation with OS . A history of COVID-19 infection showed no significant association with OS and no relevant association with any investigated parameter.The results of the analysis are shown in TT primarily affects young men who desire to father children ,2. Infern = 4). Furthermore, we assumed that patients treated with exploration of the testis and orchidopexy had an intermittent torsion. The patient cohort may have had other reasons for an acute scrotum, such as epididymitis. However, these patients could serve as controls for comparison.The time from the onset of symptoms to surgery, as well as the grade of torsion, are major factors affecting testicular viability . AssuminA possible genetic predisposition to TT has been reported in up to 10% of cases . Of our Barsch et al.  analyzedLower testicular volume is associated with decreased sperm parameters in patients ,31. Furtp = 0.073) or orchidopexy  compared to controls (14.5%). We noted MAR positivity in four cases; all patients had only undergone exploration of the testes. According to experimental animal studies, bilateral testicular damage and reduced fertility may occur after unilateral TT due to damage to the BTB, occurring only if the previously torqued testicle is left in place [Diverse ASA data have been registered after TT . Arap etin place . In our in place , so a toLate hormonal function may be affected after TT. A study comprising 12 patients with detorsion and orchidopexy and eight patients with orchiectomy revealed normal FSH, LH, and testosterone levels for all patients. However, inhibin B levels were significantly reduced in patients with torsion compared to controls . Arap etAccording to Zhan et al. , patientp = 0.025), spermiogram category , and medication history , as well as a significant negative correlation between OS and age at control , sperm concentration , and total sperm count . We noticed a high variability in OS within the groups, which can be explained due to the dependence of OS from various clinical factors. Medication with antioxidants can improve semen quality through reducing OS [OS is considered one of the major factors in male infertility ,11,12,13ucing OS . Howeverucing OS . Increasucing OS . BacteriDespite the fact that TT is an absolute emergency in urological care and surgery is the gold standard of therapy, counselling patients prior to surgery is of great importance. However, due to the low incidence of TT as cause of infertility , the patThe interval from the onset of symptoms to surgery and the degree of torsion are major factors influencing the viability of the testes. Erectile function, testosterone, and OS were not significantly affected by TT in the long-term follow-up. Orchiectomy results in elevated FSH and lower round cell counts compared to preservation of the testis. OS is significantly associated with sperm concentration, total sperm count, spermiogram category, medication history, age at follow-up, and the height of patients.


We included 319 cognitively unimpaired participants from the ALFA+ cohort with available structural MRI, fluorodeoxyglucose PET and CSF biomarkers of amyloid-β and tau pathology , synaptic dysfunction (neurogranin), neuronal and axonal injury (neurofilament light), glial activation  and α-synuclein using the Roche NeuroToolKit. We first used the amyloid-β/tau framework to investigate differences in the neuroimaging biomarkers between preclinical Alzheimer’s disease stages. Then, we looked for associations between the neuroimaging markers and all the CSF markers. Given the non-negative nature of the concentrations of CSF biomarkers and their high collinearity, we clustered them using non-negative matrix factorization approach (components) and sought associations with the imaging markers. By groups, higher grey matter volumes were found in the amyloid-β-positive tau-negative participants with respect to the reference amyloid-β-negative tau-negative group. Both amyloid-β and tau-positive participants showed higher fluorodeoxyglucose uptake than tau-negative individuals. Using the obtained components, we observed that tau pathology accompanied by YKL-40 (astrocytic marker) was associated with higher grey matter volumes and fluorodeoxyglucose uptake in extensive brain areas. Higher grey matter volumes in key Alzheimer-related regions were also found in association with two other components characterized by a higher expression of amyloid-β in combination with different glial markers: one with higher GFAP and S100b levels (astrocytic markers) and the other one with interleukin-6 (pro-inflammatory). Notably, these components’ expression had different behaviours across amyloid-β/tau stages. Taken together, our results show that CSF amyloid-β and phosphorylated tau, in combination with different aspects of glial response, have distinctive associations with higher grey matter volumes and increased glucose metabolism in key Alzheimer-related regions. These mechanisms combine to produce transient higher grey matter volumes and fluorodeoxyglucose uptake at the earliest stages of the Alzheimer’s continuum, which may revert later on the course of the disease when neurodegeneration drives structural and metabolic cerebral changes.Higher grey matter volumes/cortical thickness and fluorodeoxyglucose uptake have been consistently found in cognitively unimpaired individuals with abnormal Alzheimer’s disease biomarkers compared with those with normal biomarkers. It has been hypothesized that such transient increases may be associated with neuroinflammatory mechanisms triggered in response to early Alzheimer’s pathology. Here, we evaluated, in the earliest stages of the Alzheimer’s  et al. show that amyloid-β and tau pathologies, in combination with different aspects of glial response, have distinctive associations with brain’s structure and function in key Alzheimer-related regions. These mechanisms combine to produce transient higher grey matter volumes and glucose metabolism at the earliest stages of the Alzheimer’s continuum.Salvadó   Well-established biomarkers for Aβ and tau include CSF and PET measurements. Measures of neurodegeneration or neuronal injury are MRI, fluorodeoxyglucose ([18F]FDG) PET, as well as CSF neurofilament light (NfL). However, neurodegeneration markers are not considered to be specific for Alzheimer’s disease as they may be altered due to a variety of underlying aetiologies. Therefore, under this research framework, markers of neurodegeneration, as well as cognition, are used only to stage severity but not to define Alzheimer’s disease.From a research perspective, Alzheimer’s disease is defined as the presence of abnormal biomarkers of amyloid-β (Aβ) and tau, which constitute the biological hallmarks of the disease.continuum. A better understanding of these alterations, their interrelationships and when they emerge in the Alzheimer’s continuum would provide additional information on the pathophysiology of the disease and inform the rational development of disease modifying interventions. It is of particular interest to inform preventive interventions, to identify biological pathways that may eventually lead to neurodegeneration and that are already altered in asymptomatic Alzheimer’s disease stages.Even though alterations in Aβ and tau define Alzheimer’s disease, other pathophysiological pathways are also affected early in the Alzheimer’s 2 In a follow-up work, we reported that these markers were also associated with cerebral Aβ deposition as measured by PET.3 However, the relationship between these CSF biomarkers and structural and metabolic alterations in the brain are not well understood, especially in these early phases of the disease.In a previous study in cognitively unimpaired individuals, we found that just after CSF Aβ42/40 becomes abnormal, there is a steep increase in CSF biomarkers of tau-related [phosphorylated tau (p-tau) and total tau (t-tau)] and synaptic dysfunction (neurogranin) and, to a lesser extent, in neuronal and axonal injury (NfL) and glial markers .continuum; however, brain alterations in the opposite direction have been described in earlier phases of the disease. In particular, increased grey matter (GM) volume and/or cortical thickness has been found with the earliest abnormalities of Aβ in cross-sectional,4–8 as well as in longitudinal studies.9 In addition, increments in glucose metabolism, as measured with [18F]FDG PET, have also been shown in association with Aβ burden in cognitively unimpaired participants.10 Although consistently reported, the biological reasons for these unexpected increments are still not known. Some studies suggest that incipient neuroinflammation to the early Aβ deposition may be responsible for them.11 But other hypotheses point at other mechanisms such as brain swelling, increased neural activity or neural hypertrophy.12–14Neurodegeneration is present in the late stages of the Alzheimer’s 18F]FDG uptake) at the earliest stages of the Alzheimer’s continuum. To this aim, we first compared the GM volume and the [18F]FDG uptake between Aβ and tau (AT)15 groups in a cohort of cognitively unimpaired participants. Then, we analyzed associations between the two imaging modalities and a set of CSF biomarkers measured using the Roche NeuroToolKit which are known to be altered in Alzheimer’s disease and other neurodegenerative diseases, namely markers of amyloid-β (CSF Aβ42/40) and tau pathology (p-tau and t-tau), synaptic dysfunction (neurogranin), neuronal and axonal injury (NfL), glial activation  and α-synuclein. As some of these biomarkers showed a high degree of collinearity, we clustered them into components to decompose the independent effects of these biomarkers on brain structure and function to better understand the biological underpinnings of cerebral changes in preclinical Alzheimer’s disease stages.In this work, we tried to describe and understand the behaviour of these two well-established imaging biomarkers of neurodegeneration: GM volume and brain metabolism  parent cohort.The ALFA+ study (ALFA-FPM-0311) was approved by the Independent Ethics Committee ‘Parc de Salut Mar’, Barcelona, and registered at Clinicaltrials.gov (Identifier: NCT02485730). All participants signed the study’s informed consent form that had also been approved by the Independent Ethics Committee ‘Parc de Salut Mar’, Barcelona. The experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.17 as well as Total-Tau and Phospho-Tau(181P)18 were measured using Elecsys® electrochemiluminescence immunoassays on a fully automated cobas e 601 instrument . The other CSF biomarkers  were measured with robust prototype assays as part of the Roche NeuroToolKit on cobas e 411 and e 601 instruments . All available participants with CSF biomarkers were used in this study. All measurements were performed at the Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden, by board-certified laboratory technicians who were blinded to diagnostic and other clinical data.CSF levels of Aβ42,2 A − T+ participants (n = 12) were excluded from further analyses as they are thought to reflect non-Alzheimer’s disease pathological changes.1 The extreme values of each CSF biomarker, defined as those that fell outside three times the interquartile range above the third quartile or below the first quartile, were also removed.Individuals were then classified by AT groups using very sensitive cut-offs of CSF Aβ42/40 ratio (A+: <0.071) and p-tau (T+: >24 pg/mL), previously validated for research purposes.CSF biomarkers were then grouped into three main categories: core Alzheimer’s disease biomarkers, glial biomarkers and markers of neurodegeneration. Core Alzheimer’s disease biomarkers were related to Aβ and tau pathophysiology and included Aβ42/40 ratio and p-tau and t-tau, respectively. Glial biomarkers were those related to micro- or astroglial-related pathways and included: GFAP, YKL-40, sTREM2, s100b and IL-6. Finally, NfL and α-synuclein were considered neurodegeneration markers and neurogranin a synaptic dysfunction marker.18F]FDG PET scan and a [18F]flutemetamol PET scan acquired within 1 year of the CSF determinations (82 ± 181 days). A high-resolution 3D T1-weighted MRI sequence was acquired in a 3 T Philips Ingenia CX scanner . PET scans were acquired in a Siemens Biograph mCT scanner. [18F]FDG PET scans were acquired 45 min after the administration of 185 MBq of [18F]FDG and images were reconstructed using a OSEM3D algorithm  with PSF + TOF corrections. Centiloid (CL) values 19 were obtained from [18F]flutemetamol PET scans to characterize cerebral Aβ deposition in the participants, as described in a previous study.20All participants had a T1-weighted MRI scan, a [Modulated GM volume maps were obtained using the DARTEL implementation for voxel-based morphometry in SPM12. These images were finally smoothed with a three-dimensional Gaussian kernel of 12 mm full width at half maximum (FWHM).18F]FDG PET scans were co-registered to the corresponding T1-weighted MRI scans at the T1 space and normalized to the standard MNI space using the transformation calculated for the MRI scan using the DARTEL implementation. [18F]FDG uptake in the brain was normalized to the cerebellar vermis.21 Spatially normalized [18F]FDG scans were finally smoothed by a three-dimensional Gaussian kernel of 12 mm FWHM.[2 test depending on the nature of the variable.Differences in demographics by AT groups were assessed using one-way ANOVA or χ18F]FDG PET scans, in two independent models, set as the dependent variable and AT grouping  as the independent variable coded with dummy regressors. Age, sex and APOE-ɛ4 status (i.e. carriers/non-carriers) were used as covariates in both sets of analyses and total intracranial volume (TIV) was also included as covariate for the T1-weighted analysis. Contrasts were designed to test all pairwise comparisons in both directions.The first objective of our study was to investigate differences in two different imaging markers of neurodegeneration by AT groups in a cohort of cognitively unimpaired individuals. To this aim, we performed independent one-way ANCOVA for the two sets of images. A voxel-wise analysis was performed with modulated T1-weighted images and  with no differences across groups and 173 (54.2%) APOE-ɛ4 carriers with A-T- group having a significant lower proportion of carriers than the other AT groups.The group of participants with valid CSF biomarkers and available imaging biomarkers of neurodegeneration were 319 . Participants’ characteristics are described in 18F]FDG uptake between these two groups. In the next groups’ comparison, A + T+ individuals had smaller GM volumes in bilateral frontal gyrus than A + T- individuals. On the other hand, higher [18F]FDG uptake was observed in the bilateral transverse temporal gyrus, right calcarine and bilateral occipital pole in A + T+ compared with A*T- group (i.e. Aβ-negative tau-negative and Aβ-positive tau-negative).Differences in the imaging markers of neurodegeneration by AT groups are depicted in Structural and metabolic associations with raw CSF biomarkers are shown in 18F]FDG uptake associations in bilateral transverse temporal gyrus and striatum, but negative associations in medial frontal/parietal regions of the brain. The other two main CSF biomarker’s levels showing significant positive associations with [18F]FDG uptake were sTREM2, mainly in anterior and posterior cingulate gyri and thalamus and α-synuclein in the thalamus.A similar pattern was observed for p-tau, t-tau and neurogranin with positive association with FDG uptake). Using an unbiased approach, we showed distinct patterns of pathophysiological markers associated with increased GM volume and [18F]FDG uptake in preclinical Alzheimer’s disease stages.In this work, we assessed, in cognitively unimpaired individuals, the associations between CSF biomarkers of several pathophysiological mechanisms of neurodegenerative diseases and GM volume and cerebral glucose consumption  groups. Albeit such these structural and metabolic increments might seem paradoxical since decrements have been typically described in association with Alzheimer’s disease progression and neurodegeneration in symptomatic stages, they are in line with numerous previous studies which have observed increased GM volumes/cortical thickness and/or [18F]FDG uptake in preclinical Alzheimer’s disease stages.24 The fact that we are observing these increments on structure and metabolism in different AT stages may be related to the several biological mechanisms other than neurodegeneration that might be independently driving the observed effects on brain structure and function (see below). We hypothesize that these behaviours may be decoupled at these early preclinical stages and, synchronize in later stages of the disease when neurodegeneration is driving the changes. However, this needs further validation in independent cohorts.We first focused our attention on specific biomarkers of Alzheimer’s disease pathology and found differences in brain structure and metabolism across AT stages,24 temporo-parietal areas,24 putamen6 and occipital lobe.6 Regarding metabolism increases, we observed fewer areas with increments, involving mainly bilateral transverse temporal gyrus, right calcarine and bilateral occipital pole. Studies comparing cerebral glucose metabolism in these early stages are scarce, however, the increments found in our study in the temporal lobe are in agreement with a previous study.4 In this study from Johnson et al., the authors showed that from a group of cognitively unimpaired participants, those with higher levels of Aβ pathology showed higher glucose metabolism in similar temporal areas and bilateral thalamus compared with Aβ negative participants. Suggesting that, although paradoxical, our results concur with those found in previous studies.The pattern of increased GM volume found in this study was scattered across the whole brain. Some of the more prominent areas with larger GM volumes in A+T- participants were temporal and temporo-parietal areas, bilateral cuneus, bilateral putamen, and bilateral angular gyrus, but we also found some significant clusters in the occipital and frontal lobe. Some of these areas have been described before as having larger volumes in relationship to Aβ pathology, such as the temporal lobe,APOE-ɛ4 carriership and family history of Alzheimer’s disease prevalence. Thus, our participants may be at earlier stages of the disease, if so, although having a higher risk for Alzheimer’s disease due to their genetic risk factors. It is also important to note, that our sample size is significantly larger than most of those in the previous studies, which may have allowed us to be more sensitive to detect significant differences. Finally, we used a group comparison approach as some of previous studies,24 based on the extensively used AT staging.15 This allowed us to consider the two main pathological hallmarks of Alzheimer’s disease, given that previous studies have suggested synergistic effects on brain structure.25It must be noted, that despite the resemblance of some of the results, our study has significant differences with respect to many of the aforementioned studies. Given that our cohort comes from a research perspective, our participants differ in important characteristics from previous studies, which mainly come from clinical cohorts. In particular, our subjects are significantly younger (mean age = 61.1 years old) and have higher 25 Similarly, another longitudinal study showed a decrease in the rate of atrophy, likely age-related, in those subjects that were only Aβ positive compared to biomarker negative subjects. As expected, though, atrophy rates significantly increased in subjects more advanced in the disease stage, when both Aβ and tau were positive.9 Altogether, these results support an inverted U-shape for structural changes in Alzheimer’s disease, as previously suggested,24 with a slight GM thickening in early stages, mainly related to Aβ positivity, that will be followed by a pathology-related atrophy process when subjects become tau-positive.We hypothesize that the increments in GM volumes and glucose metabolism are transient changes before the expected neurodegeneration in more advanced stages of the disease. Supporting this idea, previous longitudinal studies have also found significant pathology-related atrophy only in later stages. For instance, a longitudinal study by Desikan and colleagues showed that only subjects that had both Aβ and tau pathology, but not those with only Aβ, presented atrophy in the entorhinal cortex.i.e. components) that are expressed together.Once we had established the differences in the imaging variables by AT stages, we used the set of CSF biomarkers to better understand the biological mechanisms underlying these structural and metabolic alterations. CSF markers were strongly correlated among them and resulted in similar patterns of associations with the imaging biomarkers. In particular, many of the glial and neurodegeneration markers showed similar associations than those of the tau pathology markers (p-tau and t-tau). Therefore, a direct correlation analysis did not allow us to disentangle the specific effects associated with different pathophysiological mechanisms. To address this, we followed a clustering approach using non-matrix factorization (NNMF), which has allowed us to discover several combinations of CSF biomarkers , glial markers , neurogranin and α-synuclein (neurodegeneration-related markers). Higher values of this component were associated with increased [18F]FDG uptake with other pathophysiological mechanisms using different CSF biomarkers. In this regard, this pattern was observed in association with increased YKL-40 levels and, to a lower extent, sTREM2. YKL-40 seemed more specifically expressed in association with p-tau in this first component as sTREM2 was similarly expressed in this and the rest of the components. In the Alzheimer’s disease continuum, higher levels of CSF YKL-40, a marker of reactive astrogliosis, have been frequently described with increased levels of p-tau27 and in associations with increased GM volume and cortical thickness in preclinical Alzheimer’s disease stages.29 Microglial activation, here represented by higher sTREM2 levels, has also been related to higher GM volumes in early stages of Alzheimer’s disease.11 Here, we suggest that all these pathophysiological mechanisms may be more tightly associated than previously thought. Overall, results with this component suggest that astrocytic and microglial reactivity related with early tau pathology underlies the observed pattern of increased GM volume and hypermetabolism. However, our experimental approach cannot decipher the underlying inflammatory mechanisms underlying these associations. Previous reports observing increments in these early phases of Alzheimer’s disease have suggested multiple non-exclusive biological explanations such as cellular hypertrophy,24 oedema or brain swallowing5 and the buildup of Aβ plaques.30 On this regard, neuropathological studies have provided some proofs of neuronal hypertrophy in close relationship with Aβ pathology.13 Future studies trying to further investigate these effects may benefit from including other imaging techniques such as T1/T2 relaxometry for looking at oedema or other cortical fluid/myelin changes,31 or neurite orientation dispersion and density imaging to investigate microstructural aspects of axons and dendrites32 using MRI. PET tracers targeting microglial and astroglial activation, like those binding to translocator protein 18 kDa (TSPO) or monoamine oxidase B (MAO-B), may also help us to decipher these underlying mechanisms in the near future.33 Finally another hypothesis that cannot be disregarded is that these differences might be related to brain reserve,34 which may have helped these subjects to maintain their cognitive abilities although their pathology.5The novelty in our study is that we can link this pattern of higher GM volume and FDG uptake in preclinical Alzheimer’s disease, which are mainly associated with increased levels of CSF glial biomarkers in association with altered Alzheimer’s disease pathology biomarkers. Our results suggest that distinct mechanisms trigger glial activation at different preclinical Alzheimer’s disease stages in response to Aβ or tau pathology, which may lead to transient increments in imaging markers considered to reflect neurodegeneration. Such increments appear to revert at later stages of disease in association with higher levels of CSF NfL, even in preclinical stages. Our results may help to better understand the interrelationship among biomarkers of core Alzheimer’s disease pathology and those of associated pathophysiological mechanisms that are already altered in the earliest stages of Alzheimer’s disease, which can inform the design of clinical trials of preventive therapies targeting these mechanisms.To conclude, we showed higher GM volumes and [fcac134_Supplementary_DataClick here for additional data file.


P < 0.01) but did not reduce recurrent neutropenia later. Prophylactic G-CSF was associated with increased grade ≥2 CRS , but not ICANS. In multiple myeloma, prophylactic G-CSF was not used; patients were stratified by early G-CSF exposure (≤2 days vs. ≥3 days after CAR T or no exposure), with no significant difference in toxicities. Future trials should clarify the optimal G-CSF strategy to improve outcomes after CAR T.Chimeric antigen receptor T cells (CAR T) are groundbreaking therapies but may cause significant toxicities including cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and cytopenias. Granulocyte colony-stimulating factor (G-CSF) is often used to mitigate neutropenia after CAR T, but there is no consensus recommended strategy due to hypothesized, but largely unknown risks of exacerbating toxicities. To investigate the impact of G-CSF, we retrospectively analyzed 197 patients treated with anti-CD19 CAR T for lymphoma and 47 patients treated with anti-BCMA CAR T for multiple myeloma. In lymphoma, 140 patients (71%) received prophylactic G-CSF before CAR T (mostly pegylated G-CSF) and were compared with 57 patients (29%) treated with G-CSF after CAR T or not exposed. Prophylactic G-CSF was associated with faster neutrophil recovery (3 vs. 4 days,  Chimeric antigen receptor T cell therapy (CAR T) has made a transformative impact on the treatment of blood cancers including lymphoma and multiple myeloma –5. CAR TGranulocyte colony-stimulating factor (G-CSF) is often used prophylactically to expedite neutrophil recovery in patients treated with chemotherapy, but there is no consensus on the optimal use of G-CSF for patients receiving CAR T –16. PartNotably, the package insert for tisagenlecleucel recommends avoiding growth factors until three weeks after CAR T or until CRS has resolved; the package inserts for axicabtagene ciloleucel and idecabtagene vicleucel do not specifically comment –30. Most9/L, < 1.0 × 109/L, and < 0.5 × 109/L, respectively. Neutrophil recovery was defined as ANC > 0.5 × 109/L for three consecutive days. Severe thrombocytopenia was defined as platelets < 20 × 109/L; engraftment was defined as platelets > 20 × 109/L for three consecutive days, with absence of transfusions for seven days.This study was approved by our Institutional Review Board and conducted in accordance with the Declaration of Helsinki. The primary cohort included adults ≥18 years old treated with commercial anti-CD19 CAR T cells (tisagenlecleucel or axicabtagene ciloleucel) for lymphoma at Massachusetts General Hospital and Brigham and Women’s Hospital/Dana-Farber Cancer Institute after their respective United States Food and Drug Administration approval dates, from October 18, 2017 to December 31, 2019. The following diagnoses were eligible: relapsed/refractory diffuse large B-cell lymphoma (DLBCL), including DLBCL transformed from low-grade lymphoma, high-grade B-cell lymphoma (HGBCL), follicular lymphoma (FL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma, and marginal zone lymphoma. Patients who received novel adjuvant therapies after CAR T as part of clinical trials, including PD-1 inhibitors and ibrutinib, were excluded. Lymphoma response and disease progression were defined according to the International Working Group criteria . A seconThe Mann–Whitney U test was used to compare continuous variables. Fisher’s exact test was used to compare categorical variables. Progression-free survival (PFS) was defined as the time from CAR T cell infusion to either disease progression or death. Overall survival (OS) was defined as the time from CAR T cell infusion to death. The probability of PFS and OS were estimated using the Kaplan-Meier method. Patients who remained alive were censored at the time of last follow-up. PFS and OS were compared using the log-rank test. The time of last follow-up for both cohorts was January 1, 2022. Median follow-up time was calculated using the reverse Kaplan-Meier method .The cumulative incidence of CRS and ICANS were estimated from the date of CAR T cell infusion and compared using the method of Gray, with patients censored at the time of last follow-up or death . In addiP < 0.10  in univariate analyses were included in multivariate models, in addition to the variable of interest (prophylactic G-CSF) if not already included from univariate analyses. Hazard ratios (HR) and odds ratios (OR) were shown with 95% confidence intervals (CI). For the competing risk model, subdistribution HR were generated using the method of Fine and Gray [P < 0.05. Analyses and figures were generated using R version 3.6.1 and R studio version 2022.02.2 + 485.Cox proportional hazards models were developed for time to grade ≥2 CRS, grade ≥2 ICANS and neutrophil recovery in the lymphoma cohort using a forward selection method. Logistic regression models were developed for severe neutropenia and thrombocytopenia. Univariate models included age, sex, CAR T construct, Eastern Cooperative Oncology Group (ECOG) performance status, lines of therapy prior to CAR T, receipt of bridging chemotherapy, time from diagnosis to CAR T, pre-lymphodepletion ANC, hemoglobin, platelets, lactate dehydrogenase (LDH) and albumin. Variables with and Gray . StatistN = 166, 84%) were treated with axicabtagene ciloleucel; the other 31 (16%) received tisagenlecleucel. The median age at CAR T was 62. The diagnoses included DLBCL , transformed DLBCL , HGBCL , FL , PMBCL  and mantle cell or marginal zone lymphoma .We identified 197 patients who received anti-CD19 CAR T cells for the treatment of relapsed/refractory lymphoma. Most patients  received antibiotic prophylaxis, chiefly with fluoroquinolones ; triazole fungal prophylaxis was less frequent . The median follow-up time was 29.9 months (95% CI 28.3–32.0).Patients received a median of three prior lines of therapy (range 1–9). Bridging therapy was administered in 77 patients (39%) prior to CAR T. Fifty-four patients (27%) underwent prior autologous hematopoietic cell transplantation (HCT). Five patients (3%) received prior allogeneic HCT. All received fludarabine and cyclophosphamide for lymphodepletion per standard protocols. Most patients  were not exposed to G-CSF immediately prior to CAR T, and hereafter referred to as the control group. Forty-two of the 57 control group patients went on to receive G-CSF after CAR T to treat neutropenia, a median of six days post-infusion range, 1–23). The other 15 of 57 did not receive any G-CSF within 30 days after CAR T. Baseline characteristics are shown in Table –23. The N = 187, 95%) developed mild neutropenia (ANC < 1.5 × 109/L) immediately after CAR T. Further, 151 patients (77%) developed severe neutropenia (ANC < 0.5 × 109/L), occurring within a median of three days . A multivariate logistic regression model was generated, whereby CAR T construct (axicabtagene ciloleucel) was associated with an increased relative odds of severe neutropenia . There was a trend toward prophylactic G-CSF being associated with a decreased relative odds of severe neutropenia in a multivariate analysis  was faster in the prophylactic G-CSF group compared to the control group  . In a univariate Cox regression model, predictors of neutrophil recovery (ANC > 0.5 × 109/L) included bridging chemotherapy, hemoglobin, platelets, albumin, LDH, and prophylactic G-CSF (Table P < 0.01).The median duration of severe neutropenia was three days . Time to neutrophil recovery (ANC > 0.5 × 1001) Fig. . Median SF Table . Prophyl9/L) after day ten post-CAR T infusion was 24% and 29% at 30- and 90-days, respectively. The cumulative event is shown stratified by prophylactic G-CSF administration in Fig. P = 0.26).A subset of patients developed recurrent neutropenia after initial neutrophil recovery and many of these were treated with G-CSF. The cumulative incidence of later severe neutropenia and/or treatment with G-CSF (since some patients were treated with G-CSF prior to having an ANC < 0.5 × 109/L) after CAR T, occurring within median of five days . Median duration of thrombocytopenia was 24 days . A multivariate logistic regression model was generated, whereby predictors of severe thrombocytopenia included number of prior lines of therapy , platelets , hemoglobin , and LDH  developed severe thrombocytopenia (< 20 × 105% develoP = 0.87). There was a trend towards a shorter duration of IV antibiotic exposure in the prophylactic G-CSF group . Thirty-seven patients (19%) had documented infections within 30 days after CAR T, including pneumonia (N = 11), bacteremia (N = 5), and candidemia (N = 3). There was no difference in the proportion of infections between the prophylactic G-CSF and control groups . Within 30 days after CAR T, three patients had fatal infections ; two of three occurred in the control group.In total, 126 patients (64%) were treated with intravenous (IV) antibiotics after CAR T, chiefly for febrile neutropenia. There was no difference in the proportion of patients treated with IV antibiotics between the prophylactic G-CSF and control groups , but grade ≥3 CRS was comparable . Moreover, 69 patients (35%) experienced grade ≥2 ICANS, with 37 patients (19%) having grade ≥3 ICANS. The frequency of grade ≥2 ICANS was comparable between the prophylactic G-CSF and control groups , as well as grade ≥3 ICANS . Interrogating the control group alone  and G-CSF non-exposed patients (N = 15), both for grade ≥2 CRS  and grade ≥2 ICANS .Maximum CRS and ICANS after anti-CD19 CAR T for lymphoma are shown in Table P = 0.03) and prophylactic G-CSF exposure retained significance for an increased risk of grade ≥2 CRS . The subgroup of 166 patients treated with axicabtagene ciloleucel were also analyzed separately. Again, prophylactic G-CSF was associated with grade ≥2 CRS in a multivariate analysis  (Table P = 0.9). The findings were similar when the 166 patients treated with axicabtagene ciloleucel were analyzed separately  Table . ProphylP < 0.01). There was a non-significant trend toward increased corticosteroid administration in the prophylactic G-CSF group as well .For CRS and ICANS treatment, 100 patients (51%) received tocilizumab, 91 (46%) received corticosteroids, and seven (4%) received anakinra. In keeping with the documented increased severity of CRS, more patients in the prophylactic G-CSF group received tocilizumab . The duration of hospitalization from CAR T infusion was significantly longer in the prophylactic G-CSF group . However, the rate of re-admission to hospital within 30 days after CAR T infusion was not significantly different between the two groups .Twenty-three patients (12%) were admitted to intensive care units (ICU) after CAR T, mostly in the setting of severe CRS and/or ICANS. There was no significant difference in proportion of patients admitted to ICU between the prophylactic G-CSF and control groups  from the primary analysis was excluded. Of the remaining 57 G-CSF naïve patients, 42 (74%) went on to experience grade ≥1 CRS. We excluded eight of 42 patients who received G-CSF prior to the onset of grade 1 CRS. Of the remaining 34 eligible patients, 17 (50%) were treated with G-CSF within seven days after the onset of grade 1 CRS, whereas 17 (50%) were not exposed to G-CSF. From the onset of grade 1 CRS, there was no signal of significantly worsened CRS after G-CSF initiation (P = 0.24)  Fig. . SimilarP = 0.40). For the entire cohort of 197 patients, 2-year PFS and OS were 43.9% (95% CI 37.1–51.9%) and 63.5% (95% CI 56.6–71.2%), respectively ; 2-year OS 65.0% vs. 59.6%, respectively, (P = 0.18). Overall, seven patients (4%) developed therapy-related myelodysplastic syndrome or acute myeloid leukemia after CAR T, within a median of 18.5 months . These patients received significantly more lines of therapy prior to CAR T ; six received prophylactic G-CSF prior to CAR T.For the lymphoma cohort, the overall response rate was 82%, with 134 (68%) complete responses (CR) and 28 (14%) partial responses. Thirty patients (15%) had either stable disease or progression, and five patients (3%) died prior to response assessment. The CR rate was similar between the prophylactic G-CSF and control groups   were treated with G-CSF after CAR T per investigator discretion, starting a median of two days after CAR T cell infusion , for a median of eight doses . Since we were unable to stratify based on prophylactic G-CSF exposure, we separated patients at the median, i.e. whether they received early G-CSF within two days after CAR T (hereafter referred to as the ‘early G-CSF group’) or not . There were no major differences in baseline characteristics between the two groups except that the pre-lymphodepletion platelet count was lower in the early G-CSF group (91 vs. 169 ×10% were trP = 0.32). The overall median duration of neutropenia was 5 days , and was longer in the early G-CSF group compared to the control group . Thirty-nine of 47 patients (83%) were treated with IV antibiotics. The overall median duration of IV antibiotic treatment was 8 days , but was significantly shorter in the early G-CSF group . One severe infection (bacteremia) occurred in each group.Thirty-six of 47 (77%) patients developed severe neutropenia, which was similar between the early G-CSF and control groups  . Thirteen patients (28%) were treated with corticosteroids, which also did not differ . One patient in each group was admitted to an ICU for CRS/ICANS.Overall, grade ≥2 CRS occurred in 17 (36%) of patients, with only one case of grade 3 CRS (Table ly) Fig. . OverallP = 0.76). The median PFS was 9.9 months (95% CI 7.4–21.2), and median OS was 23.2 months (95% CI 16.3-not reached). The overall response rate after CAR T was 89%, including 32 (68%) CR. The CR rate was similar between the early G-CSF and control groups  prior to anti-CD19 CAR T was associated with a significantly increased risk of grade ≥2 CRS. However, we did not identify an association between prophylactic G-CSF and severe ICANS. In spite of these findings, prophylactic G-CSF exposure was associated with a small but significantly faster time to neutrophil recovery (3 vs. 4 days); however, the clinical significance of this finding is uncertain as the rate of infections was not significantly different between the prophylactic G-CSF and control groups. There was a trend toward a shorter duration of IV antibiotic exposure with prophylactic G-CSF, but also a longer length of stay in the hospital. Of note, prophylactic G-CSF did not translate to a decreased risk of recurrent severe neutropenia later, reflecting the recognized risk of prolonged cytopenias after CAR T in a substantial proportion of patients , 11, 13.We also interrogated a smaller cohort of 47 multiple myeloma patients treated with anti-BCMA CAR T cells. None of these patients received prophylactic G-CSF, but the majority were treated with G-CSF shortly after CAR T. As such, we analyzed the effect of early G-CSF (within 2 days after CAR T) vs. control (G-CSF ≥ 3 days after CAR T or no exposure). We did not identify a difference in CRS or ICANS between the two groups. There was a longer duration of neutropenia in the early G-CSF group, which is counterintuitive, but may reflect confounding by indication, i.e., patients with more severe hematologic toxicity necessitated earlier G-CSF treatment. While this is a limitation of our anti-BCMA CAR T analysis, it highlights a strength of our anti-CD19 CAR T analysis, given the latter was based on prophylactic G-CSF exposure prior to CAR T.Several retrospective studies have explored the effects of G-CSF on outcomes after CAR T in lymphoma. A group from Mayo Clinic compared 35 patients who received G-CSF after CAR T to 35 patients who did not—with no signal of increased severity of CRS or ICANS, and shorter duration of neutropenia with post-CAR T G-CSF . AnotherIndeed, a strength of our analysis is that we stratified based on G-CSF exposure prior to CAR T, which, to the best of our knowledge, is the first study to address this question. Another strength is that we re-graded toxicities based on ASTCT consensus recommendations . We alsoFuture clinical trials should explore the optimal safe use of G-CSF in patients treated with CAR T. Until then, our results suggest a strategy where G-CSF is used after CAR T as a treatment for severe neutropenia as opposed to prophylaxis prior to CAR T could be associated with less severe CRS. However, this strategy must be balanced by potential risks associated with a longer period of early neutropenia. Though limited by small numbers, we did not find that later initiation of G-CSF in patients who already had low-grade CRS significantly exacerbated toxicity—further evidence that a neutropenia-driven G-CSF treatment approach could be feasible. Perhaps by identifying patients with risk factors for prolonged severe neutropenia (i.e. with multivariate models described herein or elsewhere) , methodsMechanistically, we hypothesize that G-CSF given prior to CAR T primes myeloid cells at the critical juncture before CAR T in vivo expansion, resulting in intensified cytokine cross-talk and thus increased severity of CRS, but this theory requires elucidation with translational studies , 19. TheSupplemental Material


Escherichia coli (E. coli) expression system; subsequently, a gB + gD subunit vaccine adjuvanted by GM-CSF was constructed. A rabbit model infected with a PRV SD-2017 strain was established, the TCID50 and LD50 were measured, and the typical clinical symptoms were observed. After a lethal challenge of 5 LD50 with a PRV SD-2017 strain, the rabbits exhibited typical clinical symptoms, including itching and high temperature, and histopathology revealed severe inflammation in the brain, which is the dominant target organ of PRV. Rabbits immunized with the gB + gD + GM-CSF subunit vaccines produced higher levels of antibodies than those immunized with gB + gD + ISA 201, which was adjuvanted with a frequently used oil adjuvant. The survival rate of rabbits vaccinated with gB + gD + GM-CSF was 100%, which was superior to that of rabbits vaccinated with gB + gD + ISA 201 (80%), inactivated PRV + GM-CSF (60%) and commercial inactivated vaccine (60%) after challenge with PRV SD-2017. These data suggested that the gB + gD + GM-CSF-based subunit vaccine had good protective efficacy against the PRV SD-2017 strain in rabbits and that GM-CSF could be developed as a candidate adjuvant for use in a vaccine regimen to prevent and even eradicate PR.Pseudorabies (PR), which is caused by the pseudorabies virus (PRV), is a severe infectious disease that causes abortions in adult sows and fatal encephalitis in piglets; the disease can occur in pigs of all ages and other mammals, which can lead to significant economic loss around the worldwide. The new PRV variant invalidated the available commercial attenuated and inactivated vaccines. Consequently, subunit vaccines have been suggested as novel strategies for PR control, while they are usually formulated with adjuvants due to their lower immunogenicity. We aimed to select a safe and efficient adjuvant for subunit vaccines for PR. In our study, glycoprotein B (gB) and glycoprotein D (gD) were expressed based on a baculovirus expression system, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was expressed using an  Pseudorabies (PR), caused by pseudorabies virus (PRV), is a severe infectious disease characterized by abortions in pregnant pigs, fatal nervous system disorders in piglets, and respiratory illness in growing-fattening pigs, leading to significant economic loss to the swine industry worldwide . In receThe causative agent, PRV, which belongs to subfamily alpha-Herpesvirinae, is a double-stranded DNA virus containing at least 72 genes encoding 11 different glycoproteins in the viral envelope  . gB and Due to their well-defined components with excellent safety, subunit vaccines are considered advantageous for the control of PR over common inactivated and attenuated vaccines, thereby attracting researchers’ focus to protect animal welfare and reduce public health costs . The difAccumulating evidence shows that gB and gD are crucial proteins that induce humoral and cellular immune responses and are regarded as attractive targets for the exploration of novel types of vaccines . It has in vivo experiment-based information about GM-CSF is proving that it is a usable adjuvant for human or animal vaccines (Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), a proinflammatory cytokine, helps elicit robust antiviral and antitumor efficacy in preclinical trials, thereby regulating the development and functions of dendritic cell (DC) subsets, as well as the activation of T lymphocytes . Similarvaccines . Thus, GTo develop more effective subunit vaccines against the PRV SD-2017 strain, gB and gD were expressed based on the baculovirus expression system; furthermore, an efficient adjuvant was selected to enhance the immune response to the subunit vaccines. In our study, rabbits were employed to explore the immunoprotective capacity of the gB + gD subunit vaccines and GM-CSF adjuvant. The clinical symptoms, mortality, neutralizing antibody (NA) titers, gB- and gD- specific antibody levels, and pathology were assessed. The results indicated that gB + gD + GM-CSF subunit vaccines induced a robust immune response and provided efficient protection for rabbits, and that GM-CSF may be a potential candidate adjuvant for subunit vaccines.E. coli DH10Bac™  competent cells to harvest the recombinant baculovirus shuttle plasmids according to the Bac-To-Bac Baculovirus Expression System (BEVS) directions. The recombinant baculovirus shuttle plasmids were transinfected into Sf9 cells with Cell Infection II Reagent (Gibco) to obtain soluble gB and gD proteins. Soluble gB and gD proteins were purified using a Ni column, and the concentrations of gB and gD were measured using a BCA Protein Assay Kit. Proteins were identified by SDS-PAGE and Western blotting.The extracellular domain sequence of the gD gene and the N-terminal sequence of the gB gene of the PRV strain (GenBank: MT949536.1) were retrieved from the GenBank database and amplified by PCR utilizing primers designed by Primer 5. The PCR products were cloned into the pFastBac HT-B vector, named pFastBac-gB and pFastBac-gD, which were synthesized by Shanghai Sangn Biotech (China). All recombinant plasmids were transformed into The pseudorabies virus (PRV) SD-2017 strain was isolated by Shandong Key Lab of Preventive Veterinary Medicine, Qingdao Agricultural University (QAU), China, and was identified as a PRV mutant (data unpublished). PRV was propagated in porcine kidney cells (PK-15 cells) and stored at –80°C. The PK-15 cells were propagated in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum .Three-month-old rabbits were purchased from Qingdao Kangda Biological Co., Ltd. . All rabbit experiments were performed at Shandong SINDER Technology Co., Ltd. , and in accordance with the protocols approved by the Animal Care and Ethics Committee of the SINDER, under the number 20180024.E. coli expression system. Briefly, sequences of porcine-origin GM-CSF genes (GenBank: U67175.1) were retrieved from the GenBank database, and whole gene synthesis was performed by Shanghai Sangon Biotech, with the gene cloned into the pET-32a vector . The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells (Takara), and IPTG (Takara) was used to induce the generation of soluble proteins. GM-CSF was purified using a His-tagged protein purification kit  and the concentrations of gB and gD were measured using a BCA Protein Assay Kit. The expressed product was identified by SDS-PAGE.ISA 201 oil adjuvant  and GM-CSF were used as candidate adjuvants of subunit vaccine, of which GM-CSF was expressed by an 5) of cells were seeded in 96-well plates and incubated at 37°C in 5% CO2. Then, 100 μL/well of tenfold-diluted (from 10–1 to 10–10) PRV in DMEM was added to single-layer PK-15 cells in 96-well plates for eight replicates of each dilution, and 100 μL/well DMEM was added as a control group and incubated at 37°C in 5% CO2. After incubation for 2 h, the supernatant was discarded, and the 96-well plate was washed twice with PBS. Subsequently, DMEM supplemented with 2% FBS was added to the 96-well plate, and the plate was placed at 37°C in 5% CO2 to produce cytopathic effects (CPEs). CPEs were observed and recorded for approximately 5 days, and TCID50 was calculated based on the Reed-Muench method  and inoculated intranasally with different doses of PRV . The rectal temperature and survival rate were recorded for 10 days. The LD50 was determined based on the Reed-Muench method (The PRV SD-2017 strain was diluted with DMEM to 1000 TCIDh method .The compound of gB and gD proteins (each 100 μg) were blended with ISA 201 adjuvant as a water-in-oil vaccine (gB + gD + ISA 201) and with GM-CSF as a water-in-water vaccine (gB + gD + GM-CSF) at a ratio of 1:1 (m/m).7.29 TCID50) was mixed in GM-CSF adjuvant at a ratio of 1:1 (v/v) as a water-in-water inactivated PRV vaccine (inactivated PRV + GM-CSF).The PRV antigen was inactivated by beta-propiolactone at a final concentration of 0.1% for 24 h at 4°C and then placed in a water bath for 2 h at 37°C for complete inactivation. The inactivated PRV (10n = 25) were randomly divided into five groups. After acclimation, three groups were vaccinated by different types of vaccines as described above. One group was immunized with commercial inactivated vaccine  and last group received no immunization as the mock group. After the first immunization for 14 days, a second boost immunization was administered. Blood near the ears was collected for serum separation to assess the levels of gB and gD specific antibodies and neutralizing antibodies at 14 and 28 days post-immunization (dpi).Three-month-old rabbits , all surviving rabbits were euthanized, and brain, liver, and lung samples were collected for histopathological analysis.To observe the positive effect of GM-CSF on the survival rate of rabbits, all vaccinated rabbits were challenged intranasally with 5 LDThe serum samples described above were separated and tested for the production of gB-specific antibodies with commercial enzyme-linked immunosorbent assay (ELISA) kits (Wuhan Keqian Biology) according to the manufacturer’s instructions.nm630  value of fluorescence was measured by a microplate reader within 10 min. When the difference between the average value of the negative control, named “N,” and the positive control was equal to or greater than 0.4, the trial was set up. If the quotient between the value of the serum sample, named “S,” and the value of the negative control was less than or equal to 0.6 (S/N ≤ 0.6), the serum sample was judged as “positive”; if the value of S/N was lower, the antibody titers of the serum sample were higher.Recombinant gD protein (200 ng) was diluted in PBS and coated into 96-well ELISA plates overnight at 4°C. Then, 1% BSA was used to block the binding sites for 2 h at 37°C. Both the serum sample and controls were added to the well to combine with coated gD protein for 1 h at 37°C, and an HRP-labeled monoclonal antibody was added for 30 min at room temperature. Finally, the OD–1 to 2–8) with DMEM. Then, 500 μL of each dilution of inactivated serum was mixed with an equal volume of PRV SD-2017 strain at a concentration of 100 TCID50. 100 μL of the mixture was added to a 96-well plate filled with a single-layer of PK-15 cells, and each dilution was repeated eight times. The virus-positive control well received an equal volume of PRV without serum, and the cell negative control well received an equal volume of DMEM containing 2% FBS. The plate was incubated at 37°C in a 5% CO2 atmosphere, and CPEs were observed for approximately 5 days. The neutralizing antibody titer was determined based on the Reed-Muench method.Neutralizing antibodies against the PRV SD-2017 strain were measured by cell tests. Serum samples were heat inactivated for 30 min at 56°C and twofold-diluted .In total, 100 mg of brain, liver, and lung tissues were cut into tiny pieces and fully ground in sterilized lysis buffer under aseptic conditions. Following complete grinding, the suspension of tissues in PBS was collected and filtered through a 0.22 μm filter after centrifugation at 8,000 rpm at 4°C for 10 min. Genomic DNA from these samples was extracted using a BioFlux Plasmid DNA Mini Extraction Kit  and stored at –20°C. Real-time fluorescent quantitative PCR (qPCR) was performed with a probe qPCR mix (with UNG) (Takara) on a qPCR system .The representative rabbits from each group were dissected, and the brains, lungs, and livers were collected randomly and fixed in 10% neutral formalin at room temperature for 24 h. The fixed tissues were embedded in paraffin wax, stained with hematoxylin and eosin (H&E), and examined by light microscopy.SD) of three replicates. Data were analyzed by one-way ANOVA and two-way ANOVA using GraphPad Prism software. Significance is presented as *p < 0.05 and **p < 0.01.Data are presented as the mean ± standard deviation , while two, one, and two dead rabbits were found in the gB + gD + ISA 201 group , inactivated PRV + GM-CSF group , and CIV group , respectively .The collected sera in all rabbits immunized with vaccines were positive by ELISA for gB-specific antibodies, as S/N value was lower than 0.6. The S/N values after the second immunization were generally lower than those after the first immunization, which was similar to the results for gD-specific antibodies, reflecting that the serum antibody titers continued to increase steadily. In contrast, the mock group was negative at 14 days post-immunization, as S/N value was higher than 0.7 .For the gB + gD + GM-CSF and gB + gD + ISA 201 groups, as expected, the antibody levels of both groups of rabbits were higher than those of the inactivated PRV + GM-CSF group and CIV group, which indicated the better immunogenicity of the recombinant proteins. The gB + gD-GM-CSF group exhibited higher antibody titers than the other vaccinated groups .in vitro assay with PK-15 cells. PRV-specific neutralizing antibodies were detected in all immunized rabbit sera, but neutralizing antibodies in the mock group remained negative during the whole study period. On the 14th day after initial immunization, neutralizing antibodies were detected in all vaccinated groups. Subsequently, 14 days after the second boost immunization, the titers of neutralizing antibodies displayed a sharp increase in all vaccinated groups. NAs titers of the gB + gD + GM-CSF group (27.28) were remarkably higher than the gB + gD + ISA 201 group (26.56), inactivated PRV + GM-CSF group (25.89), and CIV group (26.1)  .To further investigate the cellular responses of the vaccinated group, ELISA kits were used to quantify the IL-4 and IFN-γ cytokines, which are involved in Th2-biased and Th1-biased cellular responses, respectively. The results showed that IL-4 and IFN-γ levels were increased in the vaccinated groups compared with those in mock group at 28 dpi. Moreover, IL-4 in gB + gD + GM-CSF group was higher than those in other vaccinated groups. IL-4 in gB + gD + GM-CSF group was higher than those in gB + gD + ISA 201 group, which suggested efficacy of GM-CSF for Th2-biased cellular responses .7.39 copies/mg), livers (105.54 copies/mg), and lungs (107.29 copies/mg)  .The rabbits immunized with gB + gD + GM-CSF subunit vaccines did not exhibit any pathological lesions, including bleeding points or necrosis .PR is an acute and high-mortality infectious disease characterized by fever, respiratory disorders, and neurological symptoms caused by PRV, which can infect a wide range of mammals and cause nearly 100% mortality . In rece50 of the PRV SD-2017 strain in rabbits was 3.55 × 102 TCID50, indicating the strong virulence of the PRV SD-2017 strain and the high sensitivity of rabbits to the PRV SD-2017 strain. During the initial period, the rectal temperature of all challenged rabbits increased until 4 dpc , which can offer multifunctional proteins, laying the favorable foundations for the production of high-titer antibodies. In this study, gB and gD expression values were calculated for masses of 63 and 55 kDa, respectively , and theIn our previous study, gB or gD individually was used to formulate subunit vaccines for immunization. Nonetheless, the single-protein subunit vaccine did not stimulate sufficient production of specific antibodies to protect rabbits from lethal PRV SD-2017 strain infection. Hence, we redesigned a protocol in which gB and gD in combination as subunit vaccines were used for immunization and achieved the expected outcomes in comparison to inactivated vaccines. Given the lower survival rate of rabbits treated with a dose of 50 μg of gD or gB in previous trials, we increased the immunization dose to 100 μg for rabbit immunization.in vivo studies have clearly indicated that GM-CSF could be developed into a novel class of immune enhancers  than the rabbits immunized with gB + gD + ISA 201 (26.56), inactivated PRV + GM-CSF (25.89) and CIV (26.1). The survival rate of the gB + gD + GM-CSF group (100%) was higher than that of the gB + gD + ISA 201 (80%), inactivated PRV + GM-CSF (60%) groups and CIV (60%)  , which iBecause of the lack of a commercially available ELISA kit for quantification of anti-PRV gD antibodies, we used the recombinant gD protein that was expressed to coat the ELISA plate. After exploring the optimal conditions of the components, we constructed an indirect ELISA for anti-PRV gD antibodies to detect the anti-gD antibodies in rabbit serum (data unpublished). The results of gB and gD ELISAs showed that the gB- and gD-specific antibodies of all immunized groups were positive, and the antibody titer after the secondary immunization was higher than that after the first immunization . AccordiTo further evaluate the immune efficacy of gB + gD + GM-CSF, IL-4, and IFN-γ were quantified to evaluate the cellular immune responses. IL-4 is vital for Th2 immunity, and IFN-γ is vital for Th1 immunity. Approximately 10.4 pg/mL of IL-4 was produced in response to gB + gD + GM-CSF immunization compared with gB + gD + ISA 201 immunization (9.2 pg/mL), indicating that GM-CSF promoted Th2-biased cellular immune responses, which suggested GM-CSF was mainly involved in promoting antibody-related immune responses. In addition, better Th1-biased cellular immune responses were produced by gB + gD + ISA 201 (137.6 pg/mL) compared with those induced by gB + gD + GM-CSF (127.2 pg/mL), which exhibited the better cellular and humoral immune efficacy of ISA 201 .Additionally, the results of the gB + gD + GM-CSF group and inactivated PRV + GM-CSF group suggested that GM-CSF may be more suitable for subunit vaccines, which is related to the spatial structure of recombinant proteins, while the mechanism is unknown. Furthermore, the protective effect of gB + gD + GM-CSF was superior to that of gB + gD + ISA 201, and we inferred that GM-CSF promoted the maturation and differentiation of antigen presenting cells (APCs), strengthening the immune response. The better protection afforded by gB + gD + ISA 201 may be related to the prolonged duration of the antigen and slow release compared with those of inactivated PRV + GM-CSF. Additionally, the vaccine adjuvant GM-CSF is a water solution and easy to prepare, which are superior attributes compared to those of ISA 201.PRV infects a wide range of animal species including swine as the natural host as well as ruminants, carnivores, rodents, and lagomorphs . RabbitsIn conclusion, gB + gD + GM-CSF subunit vaccines can provide comprehensive protection for rabbits compared with the inactivated PRV vaccines, and the adjuvant GM-CSF can remarkably enhance the immune effect compared with that for ISA 201, which implies that GM-CSF may be a preferred adjuvant candidate for subunit vaccines for the control and eradication of PR.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by Shandong SINDER Technology Co., Ltd. , and accordance with the protocols approved by the Animal Care and Ethics Committee of the SINDER, under number 20180024.ZC, KZ, and HLZ: data curation. HZ and YY: formal analysis. ZC, KZ, HZ, and DY: methodology. ZQ and HS: project administration. ZC and ZQ: supervision. ZC and KZ: writing – original draft. ZC, HS, and ZQ: writing – review and editing. All authors contributed to the article and approved the submitted version.


Atherosclerosis is known as one of the chronic diseases with high prevalence in the human species. Many studies have elucidated the relationship between this disease and chronic periodontitis caused by Porphyromonas gingivalis (P.g). The aim of this study was to investigate the prevalence of P.g fimbriae A (fimA) genotypes II and IV in patients with periodontitis and atherosclerosis.  This cross-sectional study investigated the frequency of P.g II and IV genotypes in the subgingival plaque specimens of 42 subjects in three experimental groups: periodontitis (A), atherosclerosis (B), periodontitis + atherosclerosis (C) and aortic wall specimens obtained from 30 patients (groups B and C) by the PCR technique.  P.g bacterium was seen in 46.6% of patients with chronic periodontitis. The same bacterium was not found in aortic wall specimens of patients with chronic periodontitis (group C) and there was only one P.g-positive aortic wall specimen (7.7%) among the patients with healthy periodontium (group B). Genotypes II and IV were not observed in any specimen.  The results of statistical analysis showed no significant correlation between the prevalence of P.g and genotypes II and IV in the subgingival plaques and the incidence and severity of atherosclerosis. Atherosclerosis accounts for one out of three deaths occurring in the United States. Many studies have shown the effects of periodontal diseases on elevated atherosclerotic risk in individuals. This can be attributed to different reasons. For example, the initial origins of these diseases might be the same . Alternatively, inflammation and periodontal infection might release endotoxins or bacteria in the circulatory system, which might in turn lead to the chronic systemic inflammation or destruction of the endothelial wall by the bacterium itself. This can be attributed to findings from previous studies which have shown the presence of some large periopathogens in the carotid artery atheromas. Bacteria that are closely related to the onset and progression of periodontal diseases such as A. actinomycetemcomitans(A.a), Porphyromonas gingivalis (P.g), T. denticula(T.d) are called periodontopathic bacteria.Atherosclerosis, a major cause of mortality in industrial societies, is a pathological condition in which lipid plaques are deposited in the inner wall of arteries.Porphyromonas gingivalisis a gram-negative, anaerobic bacterium which is observed in many periodontal diseases such as periodontal abscess, adult periodontitis and therapy-resistant periodontitis. It is often detected in deep dental pockets and is less prevalent in people with healthy periodontal conditions. This bacterium has filament protrusions on its surface called fimbria, which is a strong virulence factor of this bacterium in the onset and progression of the disease. The fimbria protein components facilitate the binding and accumulation of bacteria to the surface of the host's tissues, just as studies have shown its effect on bacterial adhesion to the salivary gland walls. According to nucleotide sequences, the genes of this bacterium (fimA gene) consist of 6 fimA genotypes, which can affect its pathogenesis. Evidence from previous studies showed that genotypes II and IV are more commonly seen in subjects with periodontal problems. Therefore, coupled with inadequate research among the Iranian population, the present study investigated the prevalence of Porphyromonas gingivalisfimA genotypes II and IV in the sub-gingival and atheromatous plaques in patients with chronic periodontitis and atherosclerosis. (15 subjects in each group) and the frequency considered in this formula was based on previous studies. A sample size of 15 each was collected for patients with periodontitis, atherosclerosis and patients with periodontitis + atherosclerosis. Samples were selected from the volunteers referred to Estakhr Clinic and Tehran Heart Center, Tehran, Iran and Sina Heart Center, Isfahan, Iran. Subjects with chronic moderate-to-severe periodontitis (as defined by Armitage) and pockets with a probing depth of at least 4 mm and CAL of at least 3 mm were assigned to groups A and C. Patients in group B had a healthy periodontal condition, i.e., the maximum probing depth of 3 mm and maximum CAL of 2 mm. Coronary artery bypass graft (CABG) patients were diagnosed by a cardiac surgeon. People who had a history of antibiotic use for the last 6 months, long-term use of immunosuppressive drugs, uncontrolled systemic diseases, immune deficiency, use of any tobacco products, periodontal treatment during the past 12 months, pregnant and lactating women, individuals with <12 teeth in their mouth and those with any contagious viral diseases such as HBV, HCV, HIV, etc. were excluded from the study.The present study was a cross-sectional observational study and approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran (Thesis number: 310131). The sample size was obtained based on the Cochran's sample size formulaIn group A, the tooth sample was first isolated using cotton rolls and the supragingival plaque was removed from the tooth surface using sterile gauze. After determining the probing depth and identifying the deepest pockets in the mouth, the subgingival plaque specimen was removed from the 3 areas of the deepest pockets using a Gracey curette  (PD≥3 mm). While working with the curette, attempts were made to avoid damaging the soft tissue and gums and not to stain the plaque specimen with blood. The specimens were immediately placed in 1.5-mL microtubes containing lysis buffer (NaCl = 0.145 g/EDTA 0.5 M = 12.5 mL/Tris 0.5 M = 5 mL/SDS = 0.5 g) and the solution frozen in a freezer (-40°C). The gingival sulcus plaque and the aortic wall sampling were performed in group B. The gingival sulcus plaque sampling was performed similar to that in group A. To provide blood supply to the grafted vein during CABG surgery, the ascending aortic wall was pierced using a punch and one end of the vein was connected to the resulting hole. The surgeon later determined the location of the atheromatous plaque at the aortic wall during the surgery by a tactile sensation. In this study, the tissue isolated from the ascending aortic wall in the aortic punching stage was collected as the aortic wall specimen. This tissue was immediately immersed in 1.5-mL microtubes containing the lysis buffer, and the solution was frozen in a freezer at -40°C. In group C, the sampling of the aortic wall and the gingival sulcus plaque sampling were similar to that in previous groups.Microtubes were transferred to a laboratory in the Molecular Cell Research Center, Faculty of Medicine, Shahid Beheshti University of Medical Sciences at 0°C. Using PCR technique and 16S rDNA primer, the presence or absence of P.g bacterium in the specimens was investigated in the laboratory. After retrieval from the lysis buffer, the bacterial gingival sulcus plaques underwent the extraction process without any extra process. However, the aortic wall specimens were divided into finely divided pieces after being removed from the lysis buffer with a surgical blade and then were subjected to the extraction process. DNA extraction was performed using tissue kit . 16s rDNA primers as well as primers of genotypes II and IV were designed and developed by the NCBI Bank Gene and made by Sinaclon Co . the solution was initially placed in a cycle for 5 minutes at 95°C, followed by 30 cycles of 30 seconds at 94, 60 and 72°C, respectively. Finally, the last cycle was performed for 7 minutes at 72°C. After completing the PCR process, the resultant solution was mixed with dye solution (0.6% SDS + 60 Mm EDTA + 12% Glycerol + Syber + Chromophenol blue) and electrophoresed on 1.5% agarose gel. After electrophoresis, the gel was placed on a UV duct  and the results were determined after observing the bands. Chi-squared and Fisher’s exact tests were used to determine the relationship between the prevalence of P.g and its genotypes with severity of chronic periodontitis and atherosclerosis. One-way ANOVA and independent t-test were also used to study the relationship between the prevalence of P.g and its genotypes with severity of atherosclerosis.The PCR reaction was performed using an Eppendorf gradient  on a solution , and 9 μL of deionized water). Based on previous studies,P. gingivalis and its fimA II and IV genotypes, by using the PCR technique. Fisher’s exact test results showed a significant difference between patients in groups B and C in terms of P.gingivalisprevalence rate in the gingival sulcus specimens at P=0.001 (odds ratio=0.03). However, there was no significant difference between patients in groups A and C in terms of the prevalence of moderate periodontitis (MP) and severe periodontitis (SP)  .P.gingivalisamong patients at P=0.462  in patients with healthy periodontium, moderate chronic periodontitis and severe chronic periodontitis were 46.19±19.93, 61.05±30.79 and 63.38±26.21, respectively. These results were not statistically significant at P=0.30. Mean Gensini scores of 22.82±48.78 and 28.31±62.18 were obtained for patients without and with P.gingivalisgenotype in their gingival sulcus plaques, respectively. These results were not statistically significant at P=0.19  reported a P.g prevalence rate of 90% in their study in the gingival sulcus plaques in patients with chronic periodontitis. Also, Aimeti et al reported a P.g prevalence rate of 63.3% in a study on the gingival sulcus plaques in patients with chronic periodontitis, while Morita et al (2014) reported a P.g prevalence rate of 62.5% in a study on subgingival plaques in patients with chronic periodontitis. These researchers used paper points for sampling gingival sulcus plaques, whereas curettes were used for sampling in the present study. Different methods of sampling and racial differences are among the possible causes of differences in the results. In this study, P.g II and IV genotypes were not detected in the gingival sulcus specimens in any groups. Additionally, there are various studies on the prevalence of different P.g genotypes in the gingival sulcus plaques of patients with chronic periodontitis in other countries but there are no consistent results as a whole. Previous studies carried out in Southeast Asia by Zhao et al (China) and Moon et al (South Korea) showed that genotypes II and IV of this bacterium recorded the highest prevalence in the subgingival plaques of patients with chronic periodontitis. However, previous studies performed on the gingival sulcus plaques in patients with chronic periodontitis in Spain by Puig-silla et al and in Brazil by Missailidis et al showed that II and IV were the most prevalent genotypes. In their independent studies on the gingival sulcus plaques in patients with chronic periodontitis, Nakano et al in Japan and Beikler et al in Germany showed that I and II were the most prevalent genotypes. Interestingly, Zao et al reported Type I genotype as the most common genotype in the gingival sulcus plaque in individuals with healthy periodontium.Infections with oral origin have been considered as a cause of infections of heart tissues since 5 centuries ago. showed a statistically significant difference among the studies in terms of the amount of bacterial specimen taken by these two methods. Furthermore, the racial similarity between the Spanish and the Brazilian population seems to play a very important role in the prevalent genotype of this bacterium in the gingival sulcus plaques of patients with chronic periodontitis.Sterile curettes were used in Brazil and South Korea , while sterile paper points were used for sampling in Spain, Germany and China. Hence, the sampling method can be considered as a factor responsible for this difference in the results. However, Al Ahdab et al (2014), using real time-PCR technique, reported a P.g prevalence rate of 0%. Aimeti et al (2007) investigated the carotid artery and the subgingival atheromatous plaques of 33 patients with chronic periodontitis using the PCR technique. Although these researchers found bacterial DNA in 93.94% of the atheromatous plaques, they reported a prevalence rate of 0% for DNA associated with oral pathogens, including P.g. In another study, Cairo et al (2004) divided patients with atherosclerosis into two groups of (N=26) edentulous and dentate individuals. Oral specimens and carotid artery atheromatous plaques were taken from each group and these researchers did not find any P.g genotype DNA in the atheromatous plaques.Results from different studies show that genotype II of this bacterium seems to be the most common genotype in the gingival sulcus specimens of patients with chronic periodontitis. Nevertheless, this genotype was not seen in any of the specimens in this study. It seems that both race and sampling method are factors involved in this difference. Our results showed that the P.g-16s rDNA primer was seen only in one aortic wall specimen (7.7%) in group B, while the P.g-16s rDNA primer was not observed in any of the specimens obtained from group C. The results of the present study are similar to those of three previous studies. In a study on the carotid arteries and aortic atheromatous plaques of 13 patients with different dental conditions, Fernandes et al (2011) performed a study on 40 Iranian patients with atherosclerosis, and divided them into two groups of patients with and without chronic periodontitis. They later reported a P.g prevalence rate of 80% in the coronary artery atheromatous plaques. Mahendra et al (2014) investigated the coronary artery atheromatous plaques and gingival sulcus plaques of 51 patients with chronic periodontitis, who were candidates for CABG surgery. These researchers reported a P.g prevalence rate of 45.1% and 64.71% in the coronary artery atheromatous plaques and the gingival sulcus plaques, respectively. In a study on carotid artery atheromatous plaques of 42 Austrian patients, Figuero et al reported a P.g prevalence of 78.57% in these plaques. Morita et al (2013) investigated 16 cases of carotid artery atheromatous plaques of Japanese patients and reported a P.g prevalence of 62.5%. Pucar et al (2007) examined the coronary artery atheromatous plaques of 15 Serbian patients who were candidates for CABG surgery. These researchers reported a P.g prevalence rate of 53.33% in the specimens.The results of the study of aortic wall atheromatous plaques were inconsistent with the results of most studies on different vessels. For example, Talebi et alWhile reviewing the above studies, it is important to note the following points: and any manipulation or removal of atheromatous plaques in the coronary artery is contraindicated in the new protocols for CABG, in this study, aortic wall atheromatous plaques were taken. Also, according to a review article entitled “Aortic atherosclerotic disease and stroke”, which was published by Kronzon et al (2006), there is a correlation between the aortic calcification observed in chest radiography and the progression of coronary heart disease. In addition, it has been shown that the aortic plaque found in transesophageal echocardiography (TEE) is associated with a higher incidence of CAD and also a significant presence of stenosis in coronary artery angiography. Research has also shown that the lack of aortic plaques in the TEE can act as a predictor for the absence of CAD. In this study, a part of the aortic wall was removed by a punch during CABG surgery. Thus, use of a part of the atheromatous plaque as a specimen provides a chance to examine a larger segment of the plaque while using the part removed by the aortic punch as a specimen restricts this examination  despite the homogeneous nature of the atheroma plaque. This limitation of access to plaque mass in this study as compared to the above-mentioned studies can be considered as one of the possible reasons for the difference in the results obtained.In these studies, atheromatous plaques were taken from coronary arteries or carotid arteries. Given the fact that atherosclerosis is a generalized disease in all blood vessels,Studies have been conducted in different countries and on people of different races. As was earlier mentioned, race plays a decisive role in the spread of various bacterial species. This racial difference can be considered as one of the possible causes of the difference in results.  and Morita et al, as these patients were included in the studies of these researchers. Although in these studies, attempts were made to adjust these individuals in groups, these conditions still have a strong effect on the diversity of microorganisms with normal flora and pathogens. In the present study, interventional factors were considered in the exclusion criteria and those with these factors were excluded from the study. The effects of cigarette and diabetes on the variation of microorganisms can be considered as one of the possible causes of the difference in results.Exclusion criteria  were not considered in the studies of Pucar et al entitled “Periodontal pathogens in the atheromatous plaques controlled clinical and laboratory trial A”, methodological differences were mentioned as one of the possible reasons for the difference in the results obtained .In the article of Cairo, used systemic antibiotics during sampling.The groups in a study by Figuero reported that another possible cause for the differences observed in the results might be the fact that the prevalence of periopathogenic bacteria in atheromatous lesions is affected by various epidemiological factors such as stage of disease, nutrition, and geographical and nationality factors. In this study, there was no significant relationship between Gensini score (severity of atherosclerosis) and the prevalence of P.g in the atheromatous plaques.Finally, CairoThis finding can be explained by considering the following important points:1.To detect P.g in the atheromatous plaques, their count must reach a certain level.2.As previously mentioned, the sampling method used in this study evaluated a small amount of atheromatous plaque.3.Both P.g II and IV genotypes were studied in the present study. It is likely that there is a relationship between Gensini score and other genotypes of this bacterium. There was a significant difference between patients with atherosclerosis in chronic periodontitis and normal periodontium cases (P<0.0001). However, there was no relationship between the frequency of this bacterium in the subgingival plaques of patients with normal and affected periodontium at OR=0.03.The results of this study showed no significant relationship between the prevalence of P.g II and IV genotypes in the subgingival plaques in the case of healthy periodontium, chronic periodontitis and the incidence and severity of atherosclerosis.The study was planned by MRTA and AEN and MP. Data collection statistical analyses and interpretation of data was carried out by MRTA and AEN and BK and SKFN and MP and HM and MRHK. The manuscript was prepared by MRTA and AEN and MP and MRHK.No conflict of interest exists in relation to this study.Not applicable.