extfieldsofstudy: Biology Medicine

text:
Classic Chromophobe Renal Cell Carcinoma Incur a Larger Number of Chromosomal Losses Than Seen in the Eosinophilic Subtype

Chromophobe renal cell carcinoma (chRCC) is a renal tumor subtype with a good prognosis, characterized by multiple chromosomal copy number variations (CNV). The World Health Organization (WHO) chRCC classification guidelines define a classic and an eosinophilic variant. Large cells with reticular cytoplasm and prominent cell membranes (pale cells) are characteristic for classic chRCC. Classic and eosinophilic variants were defined in 42 Swiss chRCCs, 119 Japanese chRCCs and in whole-slide digital images of 66 chRCCs from the Cancer Genome Atlas (TCGA) kidney chromophobe (KICH) dataset. 32 of 42 (76.2%) Swiss chRCCs, 90 of 119 (75.6%) Japanese chRCCs and 53 of 66 (80.3%) TCGA-KICH were classic chRCCs. There was no survival difference between eosinophilic and classic chRCC in all three cohorts. To identify a genotype/phenotype correlation, we performed a genome-wide CNV analysis using Affymetrix OncoScan® CNV Assay (Affymetrix/Thermo Fisher Scientific, Waltham, MA, USA) in 33 Swiss chRCCs. TCGA-KICH subtypes were compared with TCGA CNV data. In the combined Swiss and TCGA-KICH cohorts, losses of chromosome 1, 2, 6, 10, 13, and 17 were significantly more frequent in classic chRCC (p < 0.05, each), suggesting that classic chRCC are characterized by higher chromosomal instability. This molecular difference justifies the definition of two chRCC variants. Absence of pale cells could be used as main histological criterion to define the eosinophilic variant of chRCC.

Introduction
Chromophobe renal cell carcinoma (chRCC) is a distinct histological entity of renal cell carcinoma (RCC) described by Thoenes et al. [1] in 1985. chRCC accounts for approximately 5-7% of RCC [2][3][4]. Thoenes et al. used the term chromophobe cell for larger cells with reticular, but not clear cytoplasm and prominent cell membranes (plant cell-like) [1,2]. Three years later, these authors described eosinophilic cells with smaller size and with fine oxiphilic granularity as a second cell component of chRCC [3]. Crotty et al. used the term pale cell instead of the formerly used term chromophobe cell and considered pale cell and eosinophilic cell [5] as two main cell types in chRCC. Several ultrastructural studies showed that pale cells are characterized by numerous cytoplasmic microvesicles, a feature probably related to defective mitochondrial development, whereas mitochondria are abundant in eosinophilic cells [2,[6][7][8].
Most chRCCs consist of both cell types, which are typically mixed, with eosinophilic cells usually arranged at the center and pale cells usually arranged at the periphery of the sheets or nests [2]. The 2016 World Health Organization (WHO) renal tumor classification acknowledges an eosinophilic variant of chRCC "that is sometimes difficult to distinguish from renal oncocytoma" [3][4][5]9,10] but there are no exact diagnostic criteria to classify an eosinophilic chRCC.
In this study, we analyzed the histopathological variants of chRCCs in 42 Swiss, 119 Japanese and in whole-slide digital images of 66 chRCCs from The Cancer Genome Atlas (TCGA) Kidney Chromophobe (KICH) dataset. Further, we utilized single-nucleotide polymorphism (SNP) arrays to assess genome-wide copy number variation (CNV) and correlated CNV to the histological variants in the Swiss and the TCGA-KICH data [13].

Chromophobe Renal Cancer Subtype and Survival
Higher pT stage (pT3-4 vs pT1-2) and higher pN stage (pN1-2 vs pN0) were significantly associated with worse survival by log-rank test (Figure 4a,b) and univariate Cox regression analysis (Table 3) in the Swiss-TCGA-Japanese cohort. There was no overall survival (OS) difference between classic and eosinophilic chRCC subtypes in the three independent cohorts of the Swiss (42 cases), TCGA-KICH (64 cases) and the Japanese (119 cases) nor in the Swiss-TCGA-Japanese combined cohort ( Figure 4c). Multivariate Cox regression analysis, including pT stage (pT3-4 vs pT1-2), pN stage (pN1-2 vs pN0), WHO/ISUP grade (Grade 3/4 vs Grade 2), and chRCC subtype showed that pT stage and pN stage were independent prognostic factors for OS, whereas no prognostic impact of the chRCC subtype or WHO/International Society of Urological Pathology (ISUP) grade was observed (Table 3).

Chromophobe Renal Cancer Subtype and Survival
Higher pT stage (pT3-4 vs pT1-2) and higher pN stage (pN1-2 vs pN0) were significantly associated with worse survival by log-rank test (Figure 4a,b) and univariate Cox regression analysis (Table 3) in the Swiss-TCGA-Japanese cohort. There was no overall survival (OS) difference between classic and eosinophilic chRCC subtypes in the three independent cohorts of the Swiss (42 cases), TCGA-KICH (64 cases) and the Japanese (119 cases) nor in the Swiss-TCGA-Japanese combined cohort (Figure 4c). Multivariate Cox regression analysis, including pT stage (pT3-4 vs pT1-2), pN stage (pN1-2 vs pN0), WHO/ISUP grade (Grade 3/4 vs Grade 2), and chRCC subtype showed that pT stage and pN stage were independent prognostic factors for OS, whereas no prognostic impact of the chRCC subtype or WHO/International Society of Urological Pathology (ISUP) grade was observed (Table 3).

Chromosomal Copy Number Variation and Survival
Both, CN data and survival data were available from 30 Swiss chRCCs and 64 chRCCs from TCGA-KICH cohort. In the Swiss cohort, neither CN losses of each chromosome 1, 2, 6, 10, 13, 17, 21 in single analysis nor CN loss of any chromosome among chromosome 1, 2, 6, 10, 13, 17, 21 were associated with worse survival by log-rank test and univariate Cox regression analysis (Table 4). In the combined Swiss-TCGA cohort, only chromosome 21 CN loss was associated with shorter overall survival, whereas all other chromosomes were not associated with survival ( Figure 5a and Table 4). Multivariate analysis showed that pT stage was the only independent prognostic factor for OS whereas no association was found between OS and CN loss of chromosome 21 or CN loss of any other chromosome among chromosome 1, 2, 6, 10, 13, 17, 21 (Table 5). Importantly, chRCCs without any CN loss of chromosome 1, 2, 6, 10, 13, 17, 21 groups revealed 100% survival in the combined Swiss/TCGA-KICH cohorts (Figure 5b and Table 4). Multivariate analysis showed that pT stage was the only independent prognostic factor for OS whereas no association was found between OS and CN loss of chromosome 21 or CN loss of any other chromosome among chromosome 1, 2, 6, 10, 13, 17, 21 (Table 5). Importantly, chRCCs without any CN loss of chromosome 1, 2, 6, 10, 13, 17, 21 groups revealed 100% survival in the combined Swiss/TCGA-KICH cohorts (Figure 5b and Table 4).

Discussion
In our study, we used the absence of voluminous pale cells to define eosinophilic chRCC. Using this definition, classic chRCC is associated with significantly more frequent losses of chromosomes 1, 2, 6, 10, 13, and 17.
Various cytogenetic, comparative genomic hybridization, and recent molecular studies have confirmed the very unique and characteristic genotype with multiple chromosomal losses in chRCC [4,9,[11][12][13][14][15]. However, previous attempts to correlate histological variants of chRCCs with a specific genotype have failed. More than 10 years ago, Brunelli et al. analyzed classic and eosinophilic chRCCs by fluorescence in situ hybridization, but they have not observed different frequencies of chromosomal 2, 6, 10, and 17 losses [11]. This is in contrast to our OncoScan results with more chromosomal CNV in classic than in eosinophilic chRCC. Our results are in line with a TCGA-KICH study by Davis et al., demonstrating in almost all classic chRCC there are characteristic chromosomal copy-number losses, whilst approximately 50% of all eosinophilic chRCC (9 of 19) experienced no chromosomal copy-number alterations [13]. Recently, Trpkov et al. proposed low-grade oncocytic tumors (LOT) as an emerging renal tumor entity [22]. They argue that LOT lacks multiple chromosomal losses and gains, and exhibits indolent clinical behavior. This tumor does not fit completely into either oncocytoma or eosinophilic chRCC, despite showing some similarities with both entities. Further studies are warranted to proof that LOT potentially represents a distinct type of tumor or if they should be regarded as variant of eosinophilic chRCC.
During our study design and the re-evaluation of histological slides for this study, we realized that there are no stringent diagnostic criteria to classify eosinophilic chRCC. The current 2016 WHO classification states that eosinophilic chRCC is almost purely composed of eosinophilic cells and that the majority of cells should be eosinophilic cells [2]. Given this lack of exact criteria, we decided to use the complete absence of pale cells as definition for eosinophilic chRCC, because pale cells are easily identifiable and can be clearly separated from eosinophilic cells.
As a consequence of this lack of stringent criteria for subtyping chRCC, distribution of chRCC variants varies extremely between different studies [2,3,10,11,23,24]. Davis et al. recently classified the TCGA-KICH tumors as classic and eosinophilic variants [13]. Our evaluation of TCGA-KICH digital whole slide images for chRCC only partially matched his classification of classical and eosinophilic variants, which can be explained by our more conservative cut-off to define eosinophilic chRCC (complete absence of pale cells).
Interestingly, there were no survival differences between eosinophilic and classic chRCC in 3 cohorts from TCGA, Japan and Europe. Given the morphological overlap between eosinophilic chRCC and benign oncocytoma, one could assume that eosinophilic chRCC have a better prognosis than classic chRCC. The prognostic similarity between eosinophilic and classic chRCC further underlines the importance to clearly separate eosinophilic chRCC from oncocytoma. Most importantly, classic chRCC had significantly more losses of chromosome 2 and 6 in the Swiss tumors and more losses of chromosome 1, 2, 6, 10, 13, and 17 in the TCGA dataset. Swiss classic chRCC showed only a trend to more chromosome 1, 10, 13, or 17 losses, probably due to the lower number of cases (Table 2). Almost all eosinophilic and all classic chRCC (91-100%) revealed chromosome 1 loss, suggesting that chromosome 1 loss may be an early event in chRCC tumorigenesis. Chromosome 1 losses have even been identified in oncocytoma [9,11,12,15]. This could be due to the misclassification of eosinophilic chRCCs as oncocytoma, but it is also tempting to speculate that there is a stepwise progression from oncocytoma to eosinophilic or classic chRCC with chromosome 1 loss as a genetic driver.
Treatment outcomes are poorly characterized in patients with metastatic chRCC. This is a consequence of rare metastasis of this subtype. Patients with metastatic chRCC can be treated with tyrosine kinase inhibitors. It has been recently shown that outcomes between metastatic chRCC and clear cell renal cell carcinoma (ccRCC) are similar when treated with conventional targeted therapies [25]. In addition, chRCC has to activate mutations in phosphatase and tensin homolog (PTEN)-phosphatidylinositol 3-kinase (PI3K)/Protein Kinase B (Akt)/mammalian target of the rapamycin (mTOR) pathway [13], which would result in an appropriate target for an mTOR inhibitor. Genomic instability, including whole-chromosome aneuploidy, is a hallmark of human cancer, but the level of chromosomal losses in chRCC is unique. We have recently identified SF3B1 on chromosome 2 as a Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS (CYCLOPS) gene with a highly significant positive correlation to hypoxia-inducible factor-1α (HIF1α) [26]. It is therefore tempting to speculate that an Splicing factor 3B subunit 1 (SF3B1)/HIF1α pathway with potential therapeutical relevance exists in chRCC.
Due to the unique genomic background, chRCC should be enrolled in separate clinical trials to measure outcomes. However, chRCC is mostly included in clinical trials together with other non-clear cell RCCs. Accurate classification of metastatic lesions is therefore important as chRCC should be managed with different treatment algorithms. Kouba et al. have recently demonstrated that cytogenetics, showing multiple genetic losses is an additional tool in a metastatic RCC lesion for differential diagnosis of the primary [27]. Our own data show that chRCC without chromosomal losses have an indolent behavior. Therefore, analysis of chromosomal losses by fluorescence in situ hybridization (FISH) or other technologies could be used to assess the behavior of chRCC in organ-confined tumors or to better characterize metastatic lesions of RCC.

Swiss Patients
chRCC patients were identified from the files of the Department of Pathology and Molecular Pathology of the University Hospital Zurich between 1993 and 2013. Our retrospective study fulfilled the legal conditions according to Article 34 of the Swiss Law "Humanforschungsgesetz (HFG)", which, in exceptional cases, allows the use of biomaterial and patient data for research purposes without informed consent, if i) it is impossible or disproportionately difficult to obtain patient consent; ii) there is no documented refusal; iii) research interests prevail the individual interest of a patient. Law abidance of this study was reviewed and approved by the ethics commission of the Canton Zurich (KEK-ZH-Nr. 2014-0604 on 1st April 2015; PB_2016-00811 on 22nd February 2016). This study was conducted in accordance with the Declaration of Helsinki. The demographic and clinicopathological characteristics for 42 chRCCs with clinical data are summarized in Table 1.
All tumors were reviewed by two pathologists (R.O. and H.M.). At least two sections were observed for determination of the existence of pale cells in tumor tissue according to the standard international protocol for pathological examination of RCCs [28,29]. ChRCCs were defined according to the 2016 WHO classification as tumors composed of large polygonal cells with reticular, clear or eosinophilic cytoplasm showing distinct cell borders, sometimes perinuclear halo and irregular (raisinoid) nuclei.
Pale cells were described as being larger than eosinophilic cells, with voluminous pale, finely reticular, but not clear cytoplasm and with distinct cell borders. We used hematoxylin and eosin-stained sections and paid particular attention to the periphery of tumor cell sheet or nest, i.e., around the vascular septa and fibrous stroma in the tumor.

The Cancer Genome Atlas (TCGA) Dataset
Clinical information of TCGA-KICH was obtained from the National Cancer Institute Genomic Data Commons Data Portal [30]. In TCGA-KICH dataset, there were 66 primary chRCCs with matched copy number variation data [13]. The demographic and clinical characteristics for the selected 66 patients are summarized in Table 1. Detailed clinical data can be found in Table S2. For survival analysis, the patients with missing or with too short a follow-up (i.e., less than 30 days) were excluded from this study.
Digital whole slide images of TCGA cases were reviewed by using the Cancer Digital Slide Archive [21]. Publically available Level 3 TCGA data were downloaded from the FIREHOSE database [31], including GISTIC CN data. The study did not include consultation cases. The study protocol was approved by the institutional review boards in all participating institutions. This study was a retrospective observational study, and an opt-out approach was used with the disclosure of this study on the website of each institution. The patients with missing or too short a follow-up (i.e., less than 30 days) were excluded from this study. All chRCCs were negative for vimentin except for focal sarcomatoid areas. The demographic and clinicopathological characteristics for the 119 chRCCs are summarized in Table 1.

OncoScan ® CNV Assay of chRCCs
Tumor areas displaying >80% cancer cells without hemorrhage or necrosis were marked on the hematoxylin and eosin slides. DNA from FFPE tumor tissue samples was obtained by punching 4 to 6 tissue cylinders (diameter 0.6 mm) from each sample. DNA extraction from FFPE tissue was done as described [32]. The double-strand DNA concentration (dsDNA) was determined using the fluorescence-based Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Tumors with poor DNA quality were excluded from the study. Genome-wide DNA copy-number alterations and allelic imbalances of 33 chRCC were determined using the Affymetrix OncoScan ® CNV Assay (Affymetrix/Thermo Fisher Scientific, Waltham, MA, USA) as previously described [33]. The demographic and clinicopathological characteristics for 33 Swiss chRCCs with clinical data are summarized in Table 1. Samples were further processed by IMGM Laboratories GmbH (Martinsried, Germany) for CNV (copy number variation) determination according to the Affymetrix OncoScan CNV Assay recommended protocol. The data were analyzed by the Nexus Copy Number 10.0 (Biodiscovery, Inc., El Segundo, CA, USA) software using Affymetrix TuScan algorithm. All array data were also manually reviewed for subtle alterations not automatically called by the software.

Statistical Analysis
All statistical analysis was done using R version 3.4.1 (R Foundation for Statistical Computing, Vienna, Austria) and EZR, Version 1.37 (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R [34]. Fisher's exact test was used to assess the association between two categorical variables. A Kaplan-Meier analysis and the log-rank test were used to derive and compare survival curves. Univariate and multivariate regression analyses with the Cox proportional hazards model were used to identify prognostic factors. The significance threshold was set at a p-value of 0.05.

Conclusions
In conclusion, the molecular difference between classic and eosinophilic chRCCs justifies the definition of 2 chRCC variants. Using the absence of pale cells as a diagnostic criterion for the eosinophilic variant may improve the reproducibility of histopathological subtyping.


text:
Host Determinants of MERS-CoV Transmission and Pathogenesis

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic pathogen that causes respiratory infection in humans, ranging from asymptomatic to severe pneumonia. In dromedary camels, the virus only causes a mild infection but it spreads efficiently between animals. Differences in the behavior of the virus observed between individuals, as well as between humans and dromedary camels, highlight the role of host factors in MERS-CoV pathogenesis and transmission. One of these host factors, the MERS-CoV receptor dipeptidyl peptidase-4 (DPP4), may be a critical determinant because it is variably expressed in MERS-CoV-susceptible species as well as in humans. This could partially explain inter- and intraspecies differences in the tropism, pathogenesis, and transmissibility of MERS-CoV. In this review, we explore the role of DPP4 and other host factors in MERS-CoV transmission and pathogenesis—such as sialic acids, host proteases, and interferons. Further characterization of these host determinants may potentially offer novel insights to develop intervention strategies to tackle ongoing outbreaks.

Introduction
Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel pathogen that was isolated in late 2012 [1]. Since then, the virus has caused multiple outbreaks and infected more than 2000 individuals, [2] who then develop a respiratory infection ranging in severity from asymptomatic to fatal [3,4]. Severe-to-fatal MERS-CoV patients have a higher chance of transmitting this virus since they shed a higher amount of virus progeny in comparison to the asymptomatic-to-mild ones [5][6][7][8].
Identifying and quarantining these patients in healthcare facilities where outbreaks have occurred, together with implementing proper infection control, has been effective in reducing transmission and containing these outbreaks [9,10]. However, new MERS-CoV cases are still being reported, especially in the Arabian Peninsula [2,11]. This is partly due to the continuous zoonotic introduction of this virus to the human population in this region by dromedaries [12]. The dromedary camel is the only animal species that has been reported to transmit this virus to humans [13][14][15][16]. MERS-CoV infection in these animals merely causes mild upper respiratory tract infection [17,18], but seroepidemiological studies showed that this virus has been circulating in dromedary camels for decades, suggesting the efficient transmission of MERS-CoV in this species [19][20][21][22].
Although the clinical manifestations, as well as transmission, are remarkably different in MERS-CoV-infected humans and dromedary camels, the viruses isolated from these two species are highly similar, if not indistinguishable [12,16]. This indicates that host factors play a significant role in MERS-CoV pathogenesis and transmission. However, the identity of these host factors and how they affect the pathogenesis and transmission of MERS-CoV are generally not well understood. Dipeptidyl peptidase-4 (DPP4)-the MERS-CoV receptor, sialic acids, proteases, and interferons are ; a cartoon representation of MERS-CoV S1 protein binding to DPP4 (PDB code 4L72) (B). The S protein consists of the S1 and S2 subunits. The α/β hydrolase domain of DPP4 is indicated in red, β-propeller domain in green, while part of the MERS-CoV S1 protein is shown in blue.
Other MERS-CoV-interacting host factors besides DPP4 are less extensively studied and have mostly been investigated in vitro. Glycotopes of α2,3-sialic acids coupled with 5-N-acetylated neuraminic acid are recognized by the S1 protein of MERS-CoV during attachment [24]. In the absence of these glycotopes, MERS-CoV entry is reduced but not abolished, indicating their function as an attachment factor rather than a receptor [24]. Besides α2,3-sialic acids, CEACAM5 and GRP78 have also been suggested to be attachment factors for MERS-CoV, but their roles in vivo during MERS-CoV infection are not clear at this moment [37,38]. Post attachment, MERS-CoV uses the C-terminal part of its S protein-known as S2 ( Figure 1A)-to interact with host proteases, such as furin, TMPRSS2, and cathepsins [39][40][41][42]. These proteases cleave the S protein and induce conformational changes, allowing fusion between viral and host cellular membranes, resulting in the release of viral RNA into the cell cytoplasm [27]. TMPRSS2 and DPP4 are held in one complex at the cell surface by a scaffolding protein, the tetraspanin CD9, leading to a rapid and efficient entry of MERS-CoV into the susceptible cells [43]. Once fusion with host cell membranes has occurred, MERS-CoV subsequently replicates its genetic material and produces viral proteins in the cell cytoplasm to generate new virus progeny. During this stage, MERS-CoV uses its nsp3-4 polyproteins to build its replication organelles as well as its accessory proteins such as the 4a and 4b proteins to inhibit host anti-viral defense mechanisms [44][45][46][47][48][49][50][51][52][53][54]. However, the capacity of MERS-CoV accessory proteins to impede several pathways of host immune response in the lungs may be limited. MERS-CoV inoculation of macaques and genetically modified mice generally results in limited clinical manifestations; thus, adapting this virus through serial passaging or defecting the type I interferon pathway may be needed to enhance viral replication and pathogenesis in these animals [32, [55][56][57][58]. These observations, together with studies showing type I interferon capacity to inhibit MERS-CoV infection in vitro [25,59], highlight the importance of the innate immune response, especially type I interferon, as an inhibiting factor for MERS-CoV.

Host Factors in MERS-CoV Transmission
So far MERS-CoV has been isolated from dromedary camels and humans [1,60]. Both species are not only susceptible to MERS-CoV infection, but also capable of transmitting this virus [7,[12][13][14][15][16][17][18]22]. However, current data indicate that virus spread is more efficient in dromedary camels than in humans [5,7,[19][20][21]61]. This difference in transmissibility could be partially due to the different tropism of MERS-CoV in these two species. In dromedaries, MERS-CoV has been shown to replicate in the nasal epithelium upon experimental in vivo infection [17], while in humans, MERS-CoV mainly replicates in the lower respiratory tract, particularly in the bronchiolar and alveolar epithelia [23,[62][63][64][65]. Higher viral RNA levels in the sputum and lavage samples of MERS-CoV patients compared to nasal and throat swabs are consistent with the tropism of MERS-CoV in humans [66][67][68]. This different MERS-CoV tropism in dromedary camels and humans is in line with the localization of DPP4 in the respiratory tract tissues of these two species. In humans, DPP4 is absent in the nasal epithelium but present in the lower respiratory tract epithelium, mainly in type II pneumocytes [69,70]. In contrast, DPP4 is expressed in the nasal epithelium of dromedary camels [69]. This difference in DPP4 localization between humans and dromedary camels therefore explains MERS-CoV tropism in these two species and highlights DPP4 as an essential determinant of MERS-CoV tropism.
DPP4 localization has also been investigated in many other MERS-CoV-susceptible species. In Gambian and Egyptian fruit bats, DPP4 is expressed in the respiratory tract and intestinal epithelium, suggesting that MERS-CoV can target both tissues [71]. In line with this finding, MERS-CoV inoculation via intranasal and intraperitoneal routes in the Jamaican fruit bat led to viral RNA shedding both in the respiratory tract and the intestinal tract [72]. In contrast to frugivorous bats, DPP4 is limitedly expressed in the respiratory tract epithelium of two insectivorous bats, i.e., common pipistrelle and common serotine bats, but abundant in their intestinal epithelium [71]. Accordingly, sequences of MERS-like-CoVs were mainly obtained from rectal swabs and fecal samples of insectivorous bats [73][74][75][76][77][78][79][80]. These findings not only support insectivorous bats as the origin host of MERS-CoV [73][74][75][76][77][78][79][80], but also indicate the importance of intestinal tropism and fecal-oral transmission of MERS-like-CoV in these insectivorous bats.
Besides bats, humans, and dromedary camels, other animal species have also been proposed as potential hosts of MERS-CoV. Remarkably, DPP4 of horses, llamas, alpacas, pigs, bovines, goats, sheep, and rabbits has been demonstrated to recognize the S protein of MERS-CoV [81,82]. In most of these species, there is a preferential upper respiratory tract expression of DPP4 observed. Rabbits express DPP4 in the upper and lower respiratory tract epithelium, and thus may allow MERS-CoV to replicate in both compartments [33,83]. Horses, llamas, and pigs mainly express DPP4 in the upper respiratory tract-particularly the nasal epithelium [84]. Upon intranasal MERS-CoV inoculation, llamas, alpacas, and pigs developed upper respiratory tract infection, while horses did not seroconvert and only shed infectious virus in a limited amount [84][85][86][87][88]. The reason why horses seem to be less permissive to MERS-CoV remains to be investigated, but a chronic co-infection in the guttural pouch, a common disease among horses, might be one of the explanations. This guttural pouch infection results in excessive mucus production that might hinder MERS-CoV from attaching and entering the nasal epithelium [84,89,90]. Sheep, on the other hand, did not seem to express significant levels of DPP4 in their respiratory tract, and thus did not seroconvert nor shed infectious virus upon experimental MERS-CoV inoculation [84,88]. Comparable to sheep, goats limitedly shed infectious virus upon experimental infection and did not transmit this virus to other naïve goats upon direct contact [88]. The results of experimental MERS-CoV infection in livestock animals are in line with data from epidemiological studies. MERS-CoV seropositive llamas and alpacas are present in the field, while horses, goats, and sheep are generally found to be seronegative [22,86,87,[91][92][93][94][95][96][97][98].
Given the fact that experimental in vivo infection studies and DPP4 expression analysis in different animal species revealed that dromedary camels are not the only animals in which MERS-CoV has an upper respiratory tract tropism [17,18,83,84], it is then relevant to question whether other animals can potentially spread MERS-CoV as well. New World camelids, i.e., alpacas and llamas, are able to transmit the virus to respective naïve animals upon contact [86]. Pigs and rabbits, on the other hand, hardly transmit the virus-neither by contact nor airborne routes [83,99]. Most likely, this is caused by the fact that pigs and rabbits, unlike dromedary camels, shed low levels of infectious virus upon MERS-CoV inoculation ( Figure 2). This difference indicates that other host factors besides DPP4 could cause interspecies variation in MERS-CoV infection. Indeed, several glycotopes of α2,3-sialic acids that function as attachment factors of MERS-CoV are present in the nasal epithelium of dromedary camels but absent in that of rabbits and pigs ( Figure 3) [24,100]. The lack of these glycotopes in pigs and rabbits might limit the susceptibility and transmission of MERS-CoV in these animals. Although the role of these glycotopes in MERS-CoV transmission still requires further investigation, it remains plausible that an efficient transmission of this virus might require the presence of both DPP4 and MERS-CoV-recognized glycotopes of α2,3-sialic acids (Figure 3). . It has also been reported that the lysosomal proteases from bat cells support coronavirus spike-mediated virus entry more efficiently than their counterparts from human cells [39]. These observations suggest that host proteases from different host species may determine the species and tissue tropism of MERS-CoV.
Because MERS-CoV has been circulating in dromedary camels for decades before emerging in the human population [19][20][21][22], it is plausible that this virus inhibits the immune response of dromedary camels more efficiently than that of other species, including pigs and rabbits. The difference in immune response among MERS-CoV-susceptible species is therefore another factor that might yield interspecies variation in permissiveness to MERS-CoV. Characterizing the difference in host proteases and immune responses among MERS-CoV-susceptible species, as performed for DPP4 and MERS-CoV-recognized α2,3-sialic acid glycotopes (Figure 3), has not yet been investigated. These data, however, may further explain interspecies variation in MERS-CoV infection and transmission.

Host Factors in MERS-CoV Pathogenesis
MERS-CoV causes respiratory infection in humans ranging from asymptomatic to severe pneumonia [3,4]. However, it is currently unclear what causes this intraspecies variation. Epidemiology data indicate that individuals with certain risk factors are at higher risk of developing severe MERS-CoV infection [4,102]. This implies that some host factors may dictate the outcome of MERS-CoV infection, thus rendering intraspecies variation. Two of the risk factors, i.e., smoking and chronic obstructive pulmonary disease (COPD), have been shown to upregulate DPP4 expression in the lungs [70,[102][103][104], suggesting DPP4 as a possible reason for intraspecies variation observed among MERS-CoV patients. In healthy human lungs, DPP4 is almost exclusively expressed in type II pneumocytes [69,70]. Type II pneumocytes are small cuboidal cells that can regenerate alveolar epithelium upon injury, and roughly cover 2% of the alveolar surface area. Meanwhile, around 95% of the surface area of the alveolus is occupied by type I pneumocytes that are morphologically flat and responsible for gas exchange [105,106]. In the lungs of smokers and COPD patients, unlike in healthy human lungs, DPP4 is prominently expressed in both type I and II pneumocytes, indicating upregulated expression on type I pneumocytes [104]. Autopsy reports from fatal MERS-CoV patients showed that both type I and II pneumocytes expressed DPP4 and became infected by MERS-CoV, proposing a role of DPP4-expressing type I pneumocytes in MERS-CoV pathogenesis [64,107]. Damage to type I cells in the lung alveoli during viral infection may lead to diffuse alveolar damage [108]. In line with observations made in human MERS cases, common marmosets that express DPP4 in both type I and II pneumocytes have been reported to produce more infectious virus upon experimental MERS-CoV infection, compared to rhesus and cynomolgus macaques that merely expressed DPP4 in type II pneumocytes [58,[109][110][111][112]. Accordingly, these common marmosets developed moderate-to-severe infection, while macaques generally developed mild transient pneumonia [32,58,[109][110][111][112]. Similarly, in genetically modified mice that displayed MERS-CoV tropism for type II pneumocytes, only mild clinical manifestations were observed upon MERS-CoV infection [56,113]. Adapting MERS-CoV through serial passaging or upregulating DPP4 expression throughout the airway epithelium in mice, however, will induce severe clinical disease [55,56]. These data altogether support the role of DPP4-expressing type I pneumocytes in the pathogenesis of severe MERS-CoV infection.
The differential expression of host factors that limits the infection should also be taken into account. DPP4 in soluble form has been demonstrated to protect against MERS-CoV infection in vitro and in a mouse model [23,114]; however, its presence in the lungs and role in MERS-CoV pathogenesis remain to be investigated. The host immune response also has the capacity to inhibit MERS-CoV infection. MERS-CoV has been shown to replicate to higher levels in immunocompromised rhesus macaques [115], consistent with the observation that immunocompromised individuals have difficulties clearing MERS-CoV upon infection [68,107,116]. The survivors of MERS-CoV infection have been shown to develop virus-specific CD4+ and CD8+ T cell responses, implying the role of T cells in virus clearance [117]. However, the depletion of T cells in mice can either lead to failure in MERS-CoV clearance or improvement in clinical outcome, depending on the type of mouse model used [57,118]. Therefore, the role of adaptive immune response in MERS-CoV pathogenesis is currently unclear. On the other hand, one of the main components of the host innate immune response, type I interferon, inhibits MERS-CoV replication in susceptible cells, partly by inhibiting double membrane vesicles (DMV) formation [25,57,59,119,120]. The absence of type I interferon signaling in mice also resulted in more severe clinical manifestations and histopathological lesions upon MERS-CoV infection [57]. Advance age, which can cause delayed type I interferon response upon viral infection, is a well-known risk factor for fatal MERS-CoV infection [4,102,[121][122][123]. Collectively, these data highlight the role of host innate immune response as a potent inhibitor for MERS-CoV infection.
It is indubitable that severe MERS-CoV infection is not solely driven by the pathogen. Additional underlying conditions increase MERS-CoV replication and induce severe-to-fatal clinical manifestations [4,11,103,124,125]. It is plausible that more than one underlying condition is needed to yield a fatal outcome. DPP4 upregulation in type I pneumocytes and insufficient type I interferon response might be crucial determinants for severe MERS-CoV infection (Figure 4). Further investigation of the host determinants of MERS-CoV pathogenesis may offer insights for developing novel therapeutic measures.

Concluding Remarks and Future Perspectives
Although MERS-CoV has been reported to undergo some genotypic changes since it emerged in the human population [12,[126][127][128][129], this has not resulted in distinct phenotypic changes so far [63,126]. Therefore, host factors remain the most significant determinant in explaining inter-and intraspecies variations observed in MERS-CoV pathogenesis and transmission. DPP4 and MERS-CoV-recognized α2,3-sialic acids might partially explain these variations, since their localization has been demonstrated to be variable between MERS-CoV-susceptible species [69,71,84,100]. DPP4 expression in human lungs has also been shown to vary due to certain comorbidities [70,96,104]. Nevertheless, it is undoubtable that the inter-and intraspecies variation in MERS-CoV pathogenesis and transmission is a complex phenomenon influenced by more than one host factor. Current data suggest proteases and interferons as other critical host factors, but how they instigate inter-and intraspecies variations, as well as their role in MERS-CoV pathogenesis and transmission, still remain to be further elucidated. Characterization of the host determinants of MERS-CoV pathogenesis and transmission could potentially offer insight into this virus epidemiology and guide novel therapeutic development. It may also help to identify the most vulnerable individuals to protect against MERS-CoV infection-for example, by using vaccination.
Author Contributions: All authors contributed to the writing of the manuscript and carefully evaluated the manuscript before submission.


Protective effect of Platymiscium floribundum Vog. in tree extract on periodontitis inflammation in rats

Periodontitis is an immuno-inflammatory disease, which can lead to tooth loss. This study aimed to investigate the efficacy of Platymiscium floribundum Vog., a Brazilian tree which has been used in folk medicine as an anti-inflammatory agent, in a pre-clinical trial of periodontitis in rats. Periodontitis was induced by placing a sterilized nylon (3.0) thread ligature around the cervix of the second left upper molar of the rats, which received (per os) P. floribundum extract (0.1, 1 or 10 mg/kg) or vehicle 1h before periodontitis-challenge and once daily during 11 days. Treatment with P. floribundum (10mg/kg) decreased alveolar bone loss, MPO activity nitrite/nitrate levels, oxidative stress, TNF-α, IL1-β, IL-8/CINC-1, and PGE2 gingival levels, and transcription of TNF-α, IL1-β, COX-2, iNOS, RANK, and RANKL genes, while elevated both BALP serum levels and IL-10 gingival levels. The animals did not show signs of toxicity throughout the experimental course. These findings show that P. floribundum has anti-inflammatory and anti-resorptive properties in a pre-clinical trial of periodontitis, representing an interesting biotechnological tool.

Introduction
Periodontitis is a complex immuno-inflammatory disease being characterized by periodontal ligament damage and alveolar bone loss, which can lead ultimately to tooth loss. Its complexity arises from the interplay between tooth-associated microbial biofilm and the host's immune-a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 inflammatory response [1]. Due its prevalence, periodontitis has negative effects on the quality of life of the subjects.
The conventional treatment for periodontitis comprises the non-surgical (scaling and root planning) and surgical approaches. Furthemore, adjunct therapy with systemic nonsteroidal anti-inflammatory drugs and antibiotics is sometimes required [4]. However, this strategy has attracted a lot of criticisms because the side effects such as emerging of bacterial resistance, gastrointestinal bleeding as well as cardiovascular and renal effects [5]. In an attempt to overcome these limitations the search for safer therapeutic agents still continues.
Natural products isolated from plants are considered good alternatives to synthetic chemicals. Platymiscium floribundum Vog. (genus Platymiscium and family Fabaceae-Papilionoideae) is a medium sized tree which belongs to the Fabaceae-Papilionoideae family. In the Brazilian Northeast it is popularly known as "sacambu" and "jacaranda-do-litoral" and it has been used in folk medicine as an anti-inflammatory agent. Phytochemical investigation of this genus highlighted flavonoids, isoflavones and coumarins as main constituents [6][7].
The use of natural products in the management of periodontitis still lacks preclinical and clinical studies that can prove the efficacy and safety. Thus, the present study was aimed at investigating the unexplored efficacy of P. floribundum in a rat model of periodontitis. Also, we investigated the putative role of cytokines/chemokines PGE 2 , NO, and oxidative stress in P. floribundum efficacy. Additional, a systemic evaluation of the sub-chronic toxicity of P. floribundum was carried out.

Animals
144 female Wistar rats (200-220g) were obtained from the Federal University of Ceara animal research facility. Rats were housed in polypropylene boxes with controlled temperature and 12 h-12 h light dark cycles with free acess to chow diet and water. All animal procedures followed a protocol in compliance with the guidelines from the Brazilian Society of Laboratory Animal Science (SBCAL) and which was approved by a standing Institutional Animal Care and Use Committee at School of Medicine, Federal University of Ceará, Sobral, Ceará, Brazil (Permit number: 05/2015).

Determination of bone remodeling
After 11 days of treatment, the maxillae were harvested and fixed in buffered formalin (10%). After 24 hour, the maxillas were defleshed and stained with methylene blue (1%), fixed in a piece of wax, and photographed. The morphometric analysis of bone resorption was performed using the ImageJ1 Software (National Institute of Health, Bethesda, MD, USA), as described previously [10]. Further, serum levels of Bone Alkaline Phosphatase (BALP) were quantified as previously described [11] for analysis of bone formation.

Histopathological analysis of alveolar bone
The H&E-stained maxillae were semiquantitatively evaluated for the presence of cell infiltrate and osteoclasts, and state of preservation of cement and alveolar process. giving a (0-3) score grade for each of these parameters [9].

Scanning Electron Microscopic (SEM) of alveolar bone
The maxillae were fixed in Karnovisky for at least 6 hours, and then transferred to Cacodylate buffer. They were then cut into a diamond-shaped blade in a medial-distal plane to obtain the maxillary fragment (0.5 × 0.2 cm and 0.5 mm thick). The fragments were left in desiccator drying for 24 h and were assembled into gold dust plating stubs (Quorum Metallizer QT150ES, Quorum Technologies, Laughton, England) for Scanning Electron Microscopy (SEM inspect50, FEI, Hillsboro, Oregon, USA). The analysed region was that between the first and second molar.

MPO activity
MPO activity was analyzed in gingival tissues collected at 6 th hour of periodontitis-challenge, as described by Bradley et al. [12]. The results were expressed as MPO activity/mg tissue.

NO production
The total nitrite/nitrate dosage was performed as an indicator of NO production in gingival samples by Griess reaction [13]. The values obtained were compared with those obtained for standard curve and expressed in NOx (μM).

SOD and CAT activity assessment
SOD was measured using a protocol previously described [14]. The results are expressed in grams of SOD/mL. CAT activity was performed using a protocol described by Maehly and Chance [15] Cytokines and PGE2 dosage TNF-α, IL-1β, IL-8/CINC-1, and IL-10 levels were evaluated in gingival samples using commercial kits DuoSet ELISA (R&D Systems Inc., MN, USA). In the same way, the determination of PGE2 was performed by ELISA, using R&D Systems1, Kit Parameter TMPGE2 Assay (catalog PKGE004B, USA kit). Both kits were used according to manufacturer's instructions. The results were arranged as pg/mL.

RNA isolation and quantitative real-time PCR
Total RNA was extracted from gingival samples using the TRIzol reagent (Invitrogen, São Paulo, Brazil). The reverse transcription was performed using SuperScript IV Reverse Transcriptase (Invitrogen, São Paulo, Brazil) following the manufacturer's instructions.
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in StepOne Real-Time PCR thermocycler (Applied Biosystems,Warrington, UK) using SYBRR Green Master Mix (Applied Biosystems, Warrington, UK), as indicated by the manufacturer. The relative gene expression was determined using the 2 -ΔΔCt method [16], with GAPDH (S-GGACC AGGTTGTCTCCTGTG/A-CATTGAGAGCAATGCCAGCC) as the housekeeping gene. The primers pairs used in this study were:

Toxicity assessment
After 11 days of treatment, rats were anesthetized and blood samples were collected from the right ventricle and centrifuged for dosing of total alkaline phosphatase (TALP), creatinine, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Dosages were done following the manufacturer's instructions (Labtest1, Lagoa Santa, MG, Brazil). Additionally, after euthanasia, heart, stomach, liver and kidneys were removed for histopathological analysis (H&E).

Statistical analysis
All data were normalized using the Shapiro-Wilk normality test. Results were showed as mean ±standard error (SEM) or as median, when appropriate. ANOVA followed by the Tukey test or Games-Howell test were used to compare the means and the Kruskal-Wallis and Dunn tests were used to compare the medians. P <0.05 was considered significant. Analyzes were performed using IBM SPSS Statistics for Windows, Version 20.0. Software Armonk, NY or GraphPad Prism 6, San Diego, CA, USA.

Effect of P. floribundum on alveolar bone loss
Administration of P. floribundum (10 mg/kg) 1 hour before the placement of the ligature and for 11 days resulted in a significant inhibition (p<0.001) of alveolar bone loss (Fig 1A), compared to the groups that received only the vehicle (non-treated group). Periodontitis-challenge was associated with alveolar bone resorption, root exposition and loss of interdental contact (Fig 1B [b]), when compared to unchallenged group (Fig 1B [a]). These findings were confirmed by the histopathological analysis (H&E), which showed an intense inflammatory cell influx, reabsorption of the alveolar bone with partial destruction of the cementum and presence of osteoclasts ( Fig 1B [f]), receiving median scores of 2 (2-3) ( Table 1). P. floribundum (10 mg/kg) decreased root exposition and loss of interdental contact (Fig 1B [i]), when compared to non-treated group (Fig 1B [b]). Also, the histopathological analysis (H&E) depicted a discrete inflammatory cellular infiltrate, partial preservation of the alveolar process, preserved cement, and reduction of osteoclasts number (Fig 1B [j]), receiving scores from 0-1 (Table 1). These values were statistically different (P < 0.001), when compared with the non-treated group. When analyzing the region between the first and second molar by using Scanning Electron Microscopy (SEM) in both 65x and 350x magnification, respectively, it can be seen that periodontitis-challenge was associated with an irregular topography (Fig 1B [g-h]), when compared to the bone tissue from unchallenged group (Fig 1B [c-d]). The rats treated with P. floribundum (10 mg/kg) (Fig 1B [k-1]) showed regular tissue topography, when compared to nontreated group (Fig 1B [g-h]).
Also, periodontitis-challenge was associated with a decrease in BALP levels, which was prevented by the P. floribundum (10 mg/kg) injection, when compared with the non-treated group (Fig 1C).  Table 1. Effect of P. floribundum on histopathology (H&E) in the maxilla from rats subjected to periodontitis.

Effect of P. floribundum on MPO activity
A subset of rats was euthanized at the 6 th hour for analysis of MPO activity, where it was found a significant increase in MPO activity, when compared to naive group (Fig 2). P. floribundum (10 mg/kg) decreased MPO activity, when compared with to non-treated group (Fig 3). Periodontitis-challenged group displayed a significant increase in nitrite/nitrate levels (P < 0.05) (Fig 4A). Also, it was found a significant reduction in SOD (P<0.0003) and CAT (P < 0.007) levels, when compared to naive group (Fig 4B and 4C). P. floribundum (10 mg/kg) decreased nitrite/nitrate levels (P < 0.0317) (Fig 4A), and it increased both markers for oxidative stress (SOD and CAT) (P < 0.013 and P < 0.002, respectively) (Fig 4B and 4C), when compared with to non-treated group.

Evaluation of P. floribundum toxicity
During the 11 days of treatment with P. floribundum the animals do not showed any signs of toxicity or mortality. The serum levels of Total Alkaline Phosphatase (TALP), creatinine, as well as serum levels of liver enzymes (AST and ALT) were unchanged, when compared to non-treated animals ( Table 2).
The histopathological analysis of the liver and kidney from animals treated with P. floribundum (10 mg/kg) depicted reversible and harmless changes, including: mild cellular swelling, few haemorrhagic areas, and areas of discrete congestion (Fig 7D and 7F), when compared to the vehicle treated group. However, all these histological changes are considered reversible. The heart and gastric mucosa of the animals did not show any histopathological changes ( Fig  7B-7H).

Discussion
Herein we have demonstrated the anti-inflammatory and anti-resorptive effect of P. floribundum in a ligature-induced periodontitis in rats. In this study P. floribundum (10 mg/kg) reduced alveolar bone loss and increased the serum levels of BALP, an isoenzyme of alkaline phosphatase considered a marker of bone formation. Furthermore, P. floribundum (10mg/kg) decreased MPO activity, TNF-α, IL1-β, IL-8/CINC-1, and PGE2 gingival levels, oxidative stress, and transcription of TNF-α, IL1-β, COX-2, iNOS, RANK and RANKL genes, while elevated IL-10 gingival levels. Additional, the animals did not show signs of toxicity throughout the experimental course.
Several species of Fabaceae family shows pharmacological activities, thus being a promising source of new bioactive products. Many of these plants are used in popular medicine showing effects such as expectorant, analgesic, treatment of asthma and abdominal pain [17][18]. Although P. floribundum is popularly used as an anti-inflammatory [19], the literature has few studies concerning its mechanism of action. In this regard, phytochemical investigations demonstrated the induction of apoptosis by flavonoids from P. floribundum in HL-60 human leukemia cells [6]. Further, it was reported P. floribundum antifungal, DNA-damaging and anticholinesterasic activities [7]. The use of natural products in the management of periodontitis still lacks preclinical and clinical studies that can prove its efficacy and safety. P. floribundum was chosen to be studied because it has been used in the Brazilian Northeast in folk medicine as an anti-inflammatory agent and no previous pharmacology study was reported yet. Indeeed, the present study is the first demonstration of both efficacy and safety of P. floribundum as anti-inflammatory agent.
BALP has been implicated in bone formation [20]. In the present study, P. floribundum (10 mg/kg) increased serum levels of BALP, which suggests that it may promote bone formation and prevent alveolar bone loss. P. floribundum have some secondary metabolites such as flavonoids, isoflavones, and coumarins [7], and some authors have shown that these metabolites are able to decrease inflammatory mediators during bone resorption [21].
MPO activity was used as a marker of inflammatory cell infiltrates (especially neutrophils) in the inflamed periodontium. According to Palm et al. [22], MPO may be used as a biomarker of periodontitis and high saliva MPO concentrations may suggest periodontal tissue destruction. In the present study, periodontitis-challenge was associated with a significant increase in MPO activity. P. floribundum (10 mg/kg) decreased MPO acivity, suggesting that the efficacy of P. floribundum may be associated with reduction of neutrophil influx.
The periodontitis-associated microbiota can activate resident and recruited inflammatory cells to secrete proinflammatory cytokines/chemokines, which stimulate bone resorption by stimulating the activity of osteoclasts [23]. Indeed, these cytokines increase the recruitment and activity of these cells through improved production of a key osteoclastogenic factor, the Receptor Activator of Nuclear Factor κ B Ligand (RANK-L) and favor bone damage [2]. Also, some authors have suggested the potential involvement of both pro-and anti-inflammatory cytokines in the regulation of the chronic inflammatory periodontitis. In fact, our research group recently showed that ligature-induced periodontitis in rats was related to a significant decrease in gingival levels of IL-10, an anti-inflammatory cytokine [24] In the preset study, P. floribundum (10mg/kg) decreased TNF-α, IL1-β, and IL-8/CINC-1 gingival levels and transcription of TNF-α, IL1-β, RANK and RANKL genes, while elevated IL-10 gingival levels,
The biosynthesis of PGE2 is close related to pro-inflammatory cytokines activity. Some authors showed that the biosynthesis of PGE2 is increased in inflamed gingiva and this synthesis is enhanced by pro-inflammatory cytokines (TNF-α, IL-1β), creating a vicious circle in states of inflammatory osteolysis, promoting alveolar bone loss [2]. Sánchezz et al. [25] suggested the salivary levels of both IL-1β and PGE2 as biomarkers of periodontal status. Our research group recently demonstrated that periodontitis-challenge in rats is associated with both high gingival PGE-2 levels and increased periodontal immunostaining for its processing  enzyme COX-2 [26]. In the present study, we demonstrated that ligature-induced periodontitis rats expressed a significant increase in the mRNA of COX-2. P. floribundum reduced both PGE2 levels and the transcription of COX-2 genes in inflamed gingival tissues, which suggests that its antiresorptive effects are, at least in part, related to inhibition of COX-2.
Both preclinical and clinical trials support the hypothesis that the inhibition of prostaglandins with nonsteroidal anti-inflammatory drugs (NSAIDs) slows periodontitis progression [27]. Once selective COX-2 inhibitors had a better risk-benefit ratio than the standard NSAIDs, we performed the histophatological analysis of the gastric mucosa of P. floribundum treated-rats, which showed no evidence of macroscopic and microscope alterations. Our present data is significant considering that gastric mucosal damage is a common side effect of NSAIDs.
Since NO has been shown to have dual effects during inflammatory processes, it would not be different in the periodontitis. While some authors suggested that NO plays an important role in the destruction of periodontal tissues [28], Martins et al. [29] demonstrated that NO donors decreased inflammation and bone loss in a model of periodontits in rats. Pro-inflammatory cytokines participate in the amplification of the inflammatory response during periodontitis disease by the transcription of the inducible nitric oxide synthase (iNOS) that results in the production of high amounts of NO [30] In the present study, periodontitis-challenged rats displayed a significant increase in nitrite/nitrate levels and expressed a significant increase in the mRNA of iNOS in gingival tissue, which was reduced by the treatment with P. floribundum (10 mg/kg).
In order to keep the ROS levels in a normal range, enzymatic mechanisms such as SOD and CAT are activated continuously. Here we demonstrated that rats with periodontitis showed a significant reduction in both SOD and CAT levels, when compared to naive rats. P. floribundum increased both markers for oxidative stress. These data are in accordance with previous results of our group showing the antioxidant effect of natural extracts from Stemodia maritima L. and Calendula offinalis [24], [31]  Effects of P. floribundum extract on periodontitis in rats Considering the growing interest in natural products, research concerning not only the efficacy but also the toxicity profile of these products is very encouraged [32][33]. Hence, in the present study, we performed biochemical analysis in peripheral blood from rats 11 days after daily injection of P. floribundum. Further, we evaluated the integrity (H&E) of heart, liver and kidneys from rats that received P. floribundum. Neither biochemical analysis nor the histopathological ones revealed any signs of toxicity.
In conclusion, P. floribundum reduces inflammatory markers associated with periodontitis, such as bone loss, pro-inflammatory cytokines, and oxidative stress. Our findings suggest that this natural product-based, may act in different pathways involved in inflammatory process during periodontitis. Thus, P. floribundum may offer a huge of possibilities for planning a supportive periodontal therapy, being promising to the oral health care industry.