Hepatic expression of tumor necrosis factor-α and evaluation of MAPK-p38 and NFκB signaling pathways in autoimmune hepatitis.

OBJECTIVES: Autoimmune hepatitis (AIH) is a necroinflammatory disease that occurs when genetically susceptible individuals are exposed to an environmental trigger. It is a rare disease, and its epidemiological aspects are nearly unknown in Northeast Brazil. In the literature, the activation of components of the inflammatory cascade pathways, including interleukins such as TNF-α and signaling factors like MAPK-p38 and NFκB, in the pathogenesis of AIH is well described in animal models. This study evaluated, for the first time, the immunostaining of TNF-α, MAPK-p38, and NFκB in immunohistochemical analysis of liver biopsies from AIH patients. The activation of the MAPK-p38 pathway was also studied through immunoassay analysis in the peripheral blood of AIH patients. METHODS AND RESULTS: Data from medical records of 25 AIH patients were analyzed. Histological and immunohistochemical analysis of liver tissue obtained from biopsies was performed to detect NFκB, MAPK-p38, and TNF-α. Immunoassay analysis of the MAPK-p38 pathway was performed in peripheral blood from 18 AIH patients and 8 healthy volunteers. Medical record analysis showed an average age of 33.3 years, with a female predominance in a ratio of 7.3:1. Concomitance with other autoimmune diseases was observed in 36 % of patients, with thyroid disorders being the most prominent among them, and an 8 % indication for liver transplantation. In the evaluation of autoantibodies, ANA was detected in 52 %, followed by SMA at 20 %, and Anti-LKM-1 at 16 %. Liver biopsy findings were like the global literature, with interface hepatitis and lymphoplasmacytic infiltration observed. Immunohistochemical analysis showed immunostaining for NFκB, MAPK-p38, and TNF-α, corroborating the inflammatory and immunological characteristics of the disease. Immunoassay analysis in peripheral blood confirmed the activation of the MAPK-p38 signaling pathway, with a statistically significant difference between AIH patients and healthy controls. CONCLUSIONS: The epidemiological and histological findings of AIH in this study in Northeast Brazil were like global population data. Immunohistochemical analysis of liver tissue and immunoassay analysis in peripheral blood confirmed the activation of TNF-α and the NFκB and MAPK-p38 signaling pathways in AIH patients.

Effect of combined red and infrared wavelengths on inflammation, hemorrhage, and muscle damage caused by Bothrops leucurus snake venom.

To evaluate the effects of red and infrared wavelengths, separately and combined, on the inflammatory process and collagen deposition in muscle damage caused by B. leucurus venom. 112 mice were inoculated with diluted venom (0.6mg/kg) in the gastrocnemius muscle. The animals were divided into four groups: one control (CG) and three treatments, namely: 1) red laser (λ=660 nm) (RG), 2) infrared laser (λ=808 nm) (IG) and 3) red laser (λ=660 nm) + infrared (λ=808 nm) (RIG). Each group was subdivided into four subgroups, according to the duration of treatment application (applications every 24 hours over evaluation times of up to 144 hours). A diode laser was used (0.1 W, CW, 1J/point, ED: 10 J/cm2). Both wavelengths reduced the intensity of inflammation and the combination between them significantly intensified the anti-inflammatory response. Photobiomodulation also changed the type of inflammatory infiltrate observed and RIG had the highest percentage of mononuclear cells in relation to the other groups. Hemorrhage intensity was significantly lower in treated animals and RIG had the highest number of individuals in which this variable was classified as mild. As for collagen deposition, there was a significant increase in RG in relation to CG, in RIG in relation to CG and in RIG in relation to IG. Photobiomodulation proved to be effective in the treatment of inflammation and hemorrhage caused by B. leucurus venom and stimulated collagen deposition. Better results were obtained with the combined wavelengths.

Marrubiin Inhibits Peritoneal Inflammatory Response Induced by Carrageenan Application in C57 Mice.

Marrubiin is a diterpene with a long history of a wide range of biological activities. In this study, the anti-inflammatory effects of marrubiin were investigated using several in vitro and in vivo assays. Marrubiin inhibited carrageenan-induced peritoneal inflammation by preventing inflammatory cell infiltration and peritoneal mast cell degranulation. The anti-inflammatory activity was further demonstrated by monitoring a set of biochemical parameters, showing that the peritoneal fluid of animals treated with marrubiin had lower levels of proteins and lower myeloperoxidase activity compared with the fluid of animals that were not treated. Marrubiin exerted the most pronounced cytotoxic activity towards peripheral mononuclear cells, being the main contributors to peritoneal inflammation. Additionally, a moderate lipoxygenase inhibition activity of marrubiin was observed.

Peri-tumoral infiltrate associates with postoperative prognosis of patients with OSCC: Stronger association in HPV negative patients.

PURPOSE: The current data on the relationship between local inflammatory infiltration and prognosis in oral squamous cell carcinoma (OSCC) are limited and controversial, especially in different HPV status. In this study, we analyzed the relationship between peri-tumoral inflammatory infiltrate (PTI) and HPV status and prognosis of patients with OSCC after surgery. METHODS: A retrospective cohort of 99 primary OSCC patients who underwent surgery was constructed. P16 immunohistochemistry was used to determine HPV status. PTI was determined by hematoxylin-eosin staining and quantified into four levels: none (Score 0), weak (Score 1), moderate (Score 2) and strong (Score 3). The associations of PTI with clinico-pathological characteristics, HPV status and survival were examined. RESULTS: Most OSCC patients had weak to moderate PTI. PTI was significantly associated with lymph node metastasis (P = 0.041), and patients with moderate PTI had significantly better OS (P = 0.009) than those with no PTI. In HPV negative OSCC, patients with moderate PTI also had significantly better OS (P = 0.019) than those with no PTI. However, PTI was not significantly associated with survival in HPV positive OSCC. CONCLUSIONS: In HPV negative OSCC, moderate PTI may suggest a better postoperative prognosis than no PTI.

Local delivery of a CXCR3 antagonist decreases the progression of bone resorption induced by LPS injection in a murine model.

OBJECTIVES: This experimental study was carried out to investigate the effects of locally delivered nanoparticles (AMG-487 NP) containing a CXCR3 antagonist in inhibiting the progression of LPS-induced inflammation, osteoclastic activity, and bone resorption on a murine model. MATERIALS AND METHODS: Thirty, 7-week-old C57BL/6 J male mice were used. Inflammatory bone loss was induced by Porphyromonas gingivalis-lipopolysaccharide (P.g.-LPS) injections between the first and second maxillary molars, bilaterally, twice a week for 6 weeks (n = 20). AMG-487 NP were incorporated into a liposome carrier and locally delivered on sites where P.g.-LPS was injected. Control mice (n = 10) were injected with vehicle only. Experimental groups included (1) control, (2) LPS, and (3) LPS + NP. At the end of 1 and 6 weeks, mice were euthanized, maxillae harvested, fixed, and stored for further analysis. RESULTS: Volumetric bone loss analysis revealed, at 1 week, an increase in bone loss in the LPS group (47.9%) compared to control (27.4%) and LPS + NP (27.8%) groups. H&E staining demonstrated reduced inflammatory infiltrate in the LPS + NP group compared to LPS group. At 6 weeks, volumetric bone loss increased in all groups; however, treatment with the CXCR3 antagonist (LPS + NP) significantly reduced bone loss compared to the LPS group. CXCR3 antagonist treatment significantly reduced osteoclast numbers when compared to LPS group at 1 and 6 weeks. CONCLUSIONS: This study showed that local delivery of a CXCR antagonist, via nanoparticles, in a bone resorption model, induced by LPS injection, was effective in reducing inflammation, osteoclast numbers, and bone loss. CLINICAL RELEVANCE: CXCR3 blockade can be regarded as a novel target for therapeutic intervention of bone loss. It can be a safe and convenient method for periodontitis treatment or prevention applicable in clinical practice.

Exploring metformin as a candidate drug for rosacea through network pharmacology and experimental validation.

Rosacea is a common chronic inflammatory disease that affects the middle of the face. Due to the unclear pathogenesis, the effective treatment options for rosacea remain limited. In this study, weighted gene co-expression network analyses (WGCNA) identified three rosacea-related hub modules, which were involved in immune-, metabolic- and development- related signaling pathways. Next, the key genes from green and brown modules were submitted to CMap database for drug prediction and metformin was identified as a candidate drug for rosacea. Moreover, network pharmacology analysis identified pharmacological targets of metformin and demonstrated that metformin could help in treating rosacea partly by modulating inflammatory and angiogenesis signaling pathways. Finally, we verified the therapeutic role and mechanism of metformin on rosacea in vivo and vitro. We found that metformin treatment significantly improved rosacea-like skin lesions including immune cells infiltration, cytokines/chemokines expression and angiogenesis. Moreover, metformin suppressed LL37- and TNF-α-induced the ROS production and MAPK-NF-κB signal activation in keratinocytes cells. In conclusion, our findings identified and verified metformin as a novel therapeutic candidate for rosacea, and it alleviates the pathological symptoms, possibly by suppressing inflammatory responses, angiogenesis in rosacea.

Comparison of Bio-C Pulpo and MTA Repair HP with White MTA: effect on liver parameters and evaluation of biocompatibility and bioactivity in rats.

AIM: To evaluate the tissue response promoted by Bio-C Pulpo (Bio), MTA Repair HP (MTA-HP) and White MTA (WMTA) and whether these materials cause liver changes in a rat experimental model. METHODOLOGY: Polyethylene tubes filled with Bio, MTA-HP and WMTA, and empty tubes (control group, CG) were implanted into the subcutaneous tissues of rats for 7, 15, 30 and 60 days. Inflammatory reaction score (IRS), capsule thickness, number of inflammatory cells (IC), von Kossa reaction, interleukin-6 (IL-6) and alkaline phosphatase (ALP) immunohistochemistry reactions were performed. Combined methods, von Kossa followed by immunohistochemistry for detection of ALP, were performed. At 60 days, the serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels were measured and liver fragments were collected for histological analysis; the data were assessed by one-way ANOVA analysis followed by Sidak's post-test. The biocompatibility and bioactivity data were subjected to the two-way ANOVA analysis followed by Tukey post hoc test, except the IRS. The IRS data were subjected to the Kruskal-Wallis ANOVA non-parametric test followed by Dunn's test (p ≤ .05). RESULTS: No significant difference was detected in serum GOT and GPT concentrations and in the number of hepatocytes among the experimental and CG samples. Although Bio-C Pulpo had the highest IC and IL-6-immunolabelled cells (p < 0.0001) at all periods, no significant difference was observed in the IRS among the materials, except at 60 days. In this period, the WMTA had lower IRS. All groups had a significant reduction in the capsule thickness and in the number of IC and IL-6-immunolabelled cells over time. Bio-C Pulpo, MTA-HP and WMTA specimens had greater immunoexpression of ALP than CG (p < .0001). At all periods, von Kossa-positive and birefringent structures were observed in the capsules around the materials. ALP-immunolabelled cells were also seen near von Kossa-positive structures. CONCLUSIONS: Bio-C Pulpo, MTA-HP and WMTA materials did not cause morphological changes in the liver and no significant alteration in the serum GOT and GPT levels. Moreover, these bioceramic materials were biocompatible and exhibited bioactive potential. However, Bio-C Pulpo induced greater inflammatory infiltrate than MTA-HP and WMTA at all periods.

Chronic hepatitis B: The interplay between intrahepatic lymphocyte population and viral antigens in relation to liver damage.

In Chronic hepatitis B (CHB) infection, virus and immune response interplay is thought to be responsible for pathogenesis. Yet, the impact of each immune cell population and viral protein expression in liver damage is still unknown. Our aim was to study the interplay between intrahepatic immune response and viral activity in relation to CHB liver damage. Immunostaining was performed in 29 liver biopsies from untreated CHB patients to characterize liver infiltrate [Th (CD4+), CTL (CD8+), Treg (FoxP3+), Th17 (IL-17A+) and Th1 (T-bet+)] and viral antigen expression (HBsAg and HBcAg). Inflammatory activity and fibrosis were assessed using the HAI and METAVIR scoring system. All studied populations were identified in the portal-periportal (P-P) areas with a CD4+ lymphocyte predominance, while only CD8+ and FoxP3+ cells were observed in the intralobular area. Both P-P CD4+ and intralobular CD8+ cell frequencies were increased among severe hepatitis cases. Concerning HBsAg and HBcAg expression, a mutually exclusive pattern was observed. HBcAg was mainly detected among HBeAg-positive patients and was associated with hepatitis severity and higher frequency of P-P FoxP3+, intralobular CD8+ and FoxP3+ cells. HBsAg was identified among HBeAg-negative cases with less severe hepatitis grade and lower frequency of P-P CD4+ and intralobular FoxP3+ lymphocytes. In conclusion, the HBV antigen profile expression seen during CHB infection may be reflecting different stages of viral replication which impacts the host immune response and liver damage process. While HBcAg might be an inducer of a regulatory microenvironment, the intralobular CTL population seemed to have a key role in hepatitis severity.

A novel prognostic model for tongue squamous cell carcinoma based on the characteristics of tumour and its microenvironment: iBD score.

AIMS: Tumour budding and invasive depth can predict survival of patients with tongue squamous cell carcinoma (TSCC), while the prognostic value of tumour microenvironment (TME) remains unknown. Here, both characteristics of the tumour and its microenvironment were examined and a novel prognostic model has been proposed. METHODS AND RESULTS: A total of 246 patients with TSCC were included. Using H&E-stained sections, pathological parameters of tumour and the TME were assessed. Inflammatory response (i), tumour budding (B) and invasive depth (D) were combined as iBD score. The association between these variables and the patient survival was determined. Both tumour budding and inflammatory status were independent variables for predicting overall survival (OS) and disease-free survival (DFS) of TSCC patients. Invasive depth was correlated with differentiation, T classification, lymph node metastasis, clinical stage and recurrence (P < 0.05). The novel iBD model was strongly correlated with T classification, lymph node metastasis, clinical stage and recurrence, and showed clear distinction of scores 0, 1 and 2. High iBD score had a strong association with reduced OS and DFS (P < 0.01). CONCLUSIONS: The iBD scoring model is strongly associated with lymph node metastasis and recurrence in TSCC and could be a promising survival predictor for TSCC patients.

Vascular density and inflammatory infiltrate in primary oral squamous cell carcinoma and after allogeneic hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) recipients have been reported to have an increased risk of chronic graft versus host disease (cGVHD) and hematological and solid cancers. Oral manifestations are the first signs of cGVHD observed in the majority of patients, and oropharyngeal cancer is the most frequent secondary malignancy occurred after HSCT. In this study, we have evaluated the inflammatory infiltrate cell content and correlated with the vascular density in patients affected by primary oral squamous cell carcinoma (OSCC) from previous healthy controls and OSCC after cGVHD. Results have demonstrated that patients with OSCC after GVHD show a more consistent inflammatory infiltrate as compared with the OSCC ones. In detail, the inflammatory background composed of CD3-positive T cells, tryptase-positive mast cells, CD31-positive endothelial cells, and CD68-positive macrophages may be more pronounced in the setting of GVHD + OSCC than in the control group. By contrast, CD20-positive B cells and CD1a-positive dendritic cells were more abundant in the latter population. Finally, a positive correlation was found as between vascular density and inflammatory cell infiltration in both GVHD + OSCC and OSCC groups. Overall, these results confirm the role played by immune cells in enhancing tumor progression and angiogenesis and suggest a potential therapeutic strategy involving inhibition of recruitment of immune cells to the tumor microenvironment and blockade of pro-tumoral effects and pro-angiogenic functions.

Clinical Management Protocol for Dental Implants Inserted in Patients with Active Lichen Planus. Part II 4-Year Follow-Up.

PURPOSE: To evaluate marginal bone loss (MBL) of dental implants inserted in active lichen planus patients. MATERIALS AND METHODS: The study included 59 subjects divided into 3 groups depending on their lichen planus diagnosis and administration of a low dose of corticosteroids: 17 healthy individuals, 20 controlled lichen planus patients controlled using low doses of systemic corticosteroids, and 22 noncontrolled lichen planus patients. During 4-year follow-up sessions MBL was evaluated, and biopsies were collected from lichen planus patients and examined. Two-way ANOVA was used to analyze the data (α = 0.05). RESULTS: There was no statistically significant difference in MBL between healthy and controlled patients; however, noncontrolled patients exhibited increased MBL (F = 1309, p < 0.001) which reached 2.53 mm after 4 years. There were significant interactions between state of the disease (F = 1309, p < 0.001), evaluation time (F = 317, p < 0.001), and interaction between state of the disease and observation time (F = 159, p < 0.001). Histopathologic examination of collected biopsies revealed healthy tissue architecture of the controlled patients, while inflammatory cellular infiltration and signs of classical destructive tissue were observed for noncontrolled patients. CONCLUSIONS: Lichen planus patients receiving dental implants should be controlled on a low-dose of corticosteroids to prevent accelerated MBL and to reduce remission of clinical manifestations.

Decreased RARß expression induces abundant inflammation and cervical precancerous lesions.

It is well known that vitamin A and its receptors protect against cancer development and that Retinoid Acid Receptor ß (RARß) is epigenetically silenced during tumoral progression. Cervical Cancer (CC) has been causally linked to high risk human papillomavirus (HR-HPV) infection. However, host factors are important in determining the outcome of persistent HR-HPV infection as most cervical precancerous lesions containing HR-HPVs do not progress to invasive carcinomas. Increasing evidence suggests that low diet in vitamin A and their receptors participate in the development of CC. The aim of this study has been to investigate the effects of abated RARß expression in the development of cervical premalignant lesions in 4 month-old conditional mice (RARß(L-/L-)). Results demonstrated the development of spontaneous squamous metaplasia, inflammatory infiltrate, enhanced mitotic activity, loss of cell differentiation, as well as decreased apoptosis and p16(INK4a) protein levels in RARß(L-/L-) mice cervix. All these changes are hallmarks of moderate dysplasia. Importantly, our results suggest that the low expression of RARß, may induce the down regulation of p16(INK4a), chronic inflammation and decreased apoptosis and may be involved in vulnerability to HR-HPV and early stage cervical carcinogenesis.

Parasitism and inflammation in ear skin and in genital tissues of symptomatic and asymptomatic male dogs with visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is transmitted through vector, although venereal transmission has been suggested. This study aimed to compare the parasitic loads and inflammatory processes in genital tissues with ear skin from seropositive male dogs. Forty-five seropositive dogs were separated into groups containing symptomatic (n = 23) and asymptomatic (n = 22) animals. The control group (n = 2) healthy animals with seronegative and negative results in direct parasitological test. Samples of ear tip skin, prepuce, glans penis, testis, epididymis, and prostate were collected for evaluation of parasitic load and inflammatory infiltrate. Although ear tip skin was the most intensely parasitized, prepuce and epididymis revealed no difference in parasitism when compared with ear tip skin (P > 0.05). Parasitic loads in testis and prostate were lower than other tissues (P < 0.05). Parasitism in glans penis was high, similar to prepuce and epididymis, but lower than ear tip skin. High parasitism was more frequent in symptomatic dogs than asymptomatic animals. Severe inflammatory processes were more frequent within the symptomatic animals compared with asymptomatic and more predominant in prepuce and epididymis. Ear tip skin and genital tissues presented signs of chronic inflammation. There were weak and moderate positive correlations between parasitic loads and inflammatory processes. Our results demonstrate that, likewise with the ear tip skin, the genital of seropositive dogs can carry a large number of Leishmania infantum amastigotes and this process are more intense in symptomatic animals. These data have important implications for understanding the possibility of venereal transmission of CVL.

The Morphology of Nasal Polyps in Different Age Groups: Histopathological Features.

BACKGROUND: Nasal polyps (NPs) represent the end-stage manifestation of chronic rhinosinusitis (CRS), a relatively common pathological condition encountered in all ages of life. METHODOLOGY: The aim of our study was to evaluate the histological features and inflammatory cellular components of NPs in a retrospective cohort (143 cases) of pediatric, adult and elderly populations in order to discuss the possible morphological age-related differences statistically documented. RESULTS: In the pediatric group, the inflammatory infiltrate presented many eosinophils mixed with lymphocytes, while in the adult population, lymphocytes and plasma cells were mainly evident, frequently with a perivascular distribution or with the formation of subepithelial lymphoid nodules. In the elderly population, inflammation was less evident and was associated with cavernous-like angecthatic structures with thrombotic stratification. Nearly all morphological findings exhibited statistically significant values among differently aged subgroups. CONCLUSIONS: Our results support the presence of histological specificities of NPs at different ages of life, providing new insight into the etiopathogenesis of NPs. The future role of biological therapies, mainly in cases refractory to already available standard medical and surgical treatments, may be analyzed by a prospective study using a larger cohort with a long-term evaluation also in relation to a possible relapse.

Lymphomonocytic inflammatory infiltrate with numerous eosinophilic granulocytes in the interstitium in a surviving heart transplant recipient: a case report.

Findings of eosinophilic and lymphomonocytic inflammatory infiltrates in endomyocardial biopsies (EMBs) may help in myocardial disease diagnosis identification. Eosinophilic myocarditis (EM), a rare condition, is fatal if left untreated and has rarely been described in heart transplant recipients. An extensive work up is necessary to achieve an early etiological diagnosis; however, the underlying cause remains unexplained in nearly one-third of the patients. The cornerstone of treatment is corticosteroids, comprehensive therapy and heart failure management (including advanced mechanical support for fulminant myocarditis). We have described the case of a 17-year-old heart transplant recipient who presented with a cardiogenic shock. He was admitted to our intensive care unit and treated with inotropic drugs, such as milrinone, adrenaline, vasopressin, and levosimendan; the doses of these drugs were in accordance with our internal protocol. The patient underwent cardiac catheterization, coronarography, and right ventricular EMB. EMB revealed inflammatory lymphomonocytic and eosinophil granulocyte infiltrates; thus, steroid therapy was initiated, with complete recovery achieved after 15 days. Performing an early differential diagnosis among eosinophilic infiltration, acute cellular rejection (ACR), and possible chemotherapeutic damage is emerging as an important challenge. To our knowledge, this is the first reported case of a lymphomonocytic inflammatory infiltration with numerous eosinophilic granulocytes in the interstitium in a surviving heart transplant recipient.

Changes in the extracellular matrix at microvascular obstruction area after reperfused myocardial infarction: A morphometric study.

BACKGROUND: Extracellular matrix (ECM) suffers substantial alterations after myocardial infarction (MI), including the invasion of leukocyte subtypes. Despite a complete reopening at epicardial level, hypoperfusion within the infarcted myocardium, known as microvascular obstruction (MVO), occurs and exerts a negative impact on ventricular remodeling. In this study, ECM composition at MVO regions was described using a morphometric analysis. METHODS: MI was induced in female swine (n = 10) by transitory 90-minute coronary occlusion followed by seven days of reperfusion. Prior to euthanasia, intracoronary thioflavin-S was infused. Within the infarcted myocardium, regions displaying MVO (thioflavin-S-) or no MVO (thioflavin-S+) were isolated and stained to morphometrically compare ECM composition. RESULTS: As reflected by cell invasion through ECM, areas with MVO displayed an enlarged presence of neutrophils and lymphocytes, whilst no differences in the amount of macrophages and myofibroblasts were detected compared to infarcted myocardium without MVO. Indeed, those regions with macroscopic MVO showed lower capillary density than areas without MVO. Lastly, a significant reduction in the extension of total collagen, type I, but not type III, collagen, laminin, and fibronectin together with an augmentation of polysaccharides were noted in areas showing MVO compared to those without microvascular injury. CONCLUSIONS: ECM composition in infarcted regions with MVO isolated from female swine displays a higher presence of inflammatory infiltrate and polysaccharides as well as reduced number of microvessels and collagen content compared to those areas without microvascular hypoperfusion. These characteristics might underlie the development of adverse ventricular remodeling in MI patients with extensive MVO.

Modulatory influence of melatonin on apical periodontitis in Wistar rats fed a high-fat diet.

OBJECTIVE: This study aimed to evaluate the influence of high-fat diet (HFD) and melatonin (MEL) treatment on the progression of inflammation and alveolar bone resorption (ABR) in rats with AP. DESIGN: Forty male Wistar rats were divided into four groups: apical periodontitis (AP), HFDAP, APMEL and HFDAPMEL. The animals were fed an HFD or standard diet for 107 days. On the 7th day, the rats were subjected to AP, and after 70 days, the rats in the MEL groups were treated with MEL for 30 days. Post treatment, the animals were euthanized, and their jaws were retrieved for evaluation of bone resorption, intensity of the inflammatory response, and immunohistochemical analysis including tartrate-resistant acid phosphatase (TRAP) and interleukin-1ß (IL-1ß) levels and tumor necrosis factor (TNF)-α expression. RESULTS: The APMEL group showed reduction in the inflammatory infiltrate and IL-1ß expression relation to HFDAP, while the TNF-α levels did not differ among the groups. The HFDAP group showed an increase in the ABR. MEL reduced the TRAP levels in the APMEL and HFDAPMEL groups. CONCLUSIONS: while MEL could reduce TRAP levels in the APMEL and HFDAPMEL groups, the reduction in the HFDAPMEL group was smaller than that in the APMEL group, demonstrating that the interaction between AP and HFD decreased the anti-resorptive effects of MEL.

Mapping the Immune Cell Microenvironment with Spatial Profiling in Muscle Tissue Injected with the Venom of Daboia russelii.

Pathological and inflammatory events in muscle after the injection of snake venoms vary in different regions of the affected tissue and at different time intervals. In order to study such heterogeneity in the immune cell microenvironment, a murine model of muscle necrosis based on the injection of the venom of Daboia russelii was used. Histological and immunohistochemical methods were utilized to identify areas in muscle tissue with a different extent of muscle cell damage, based on the presence of hypercontracted muscle cells, a landmark of necrosis, and on the immunostaining for desmin. A gradient of inflammatory cells (neutrophils and macrophages) was observed from heavily necrotic areas to less damaged and non-necrotic areas. GeoMx® Digital Spatial Profiler (NanoString, Seattle, WA, USA) was used for assessing the presence of markers of various immune cells by comparing high-desmin (nondamaged) and low-desmin (damaged) regions of muscle. Markers of monocytes, macrophages, M2 macrophages, dendritic cells, neutrophils, leukocyte adhesion and migration markers, and hematopoietic precursor cells showed higher levels in low-desmin regions, especially in samples collected 24 hr after venom injection, whereas several markers of lymphocytes did not. Moreover, apoptosis (BAD) and extracellular matrix (fibronectin) markers were also increased in low-desmin regions. Our findings reveal a hitherto-unknown picture of immune cell microheterogeneity in venom-injected muscle which greatly depends on the extent of muscle cell damage and the time lapse after venom injection.