Optimized multiplex immunofluorescence for the characterization of tumor immune microenvironment in neoplastic paraffin-preserved tissues.

The study of neoplastic cells enabled the discovery of important tumor-related biomarkers which resulted in new forms of early diagnosis, therapeutic options, and prognostic markers. Thus, immunofluorescence (IF), a high throughput imaging technology, represents a valuable method that enables the virtual characterization and localization of diverse cell types and targets, preserving tissue architecture and spatial surroundings. IF staining and analysis of formalin-fixed paraffin-embedded (FFPE) tissues are considered a challenge due to several difficulties, such as tissue autofluorescence, non-specific antibody binding, and image acquisition and quality. This study aimed to develop a multiplex-fluorescence staining technique with high-contrast and high-quality multiple-color images to enrich the investigation of important biomarkers. We present a robust optimized multiple-immunofluorescence procedure that reduced sample autofluorescence, enabled the use of simultaneous antibodies on the same sample, and showed super-resolution imaging through precise antigen localization. We illustrated the utility of this powerful method in FFPE neoplastic appendix, lymph node and bone marrow biopsies, and a 3D-coculture system, in which cells are enabled to grow and interact with their surroundings in all 3D dimensions. Our optimized multiple-immunofluorescence method represents a powerful tool for better understanding the complexity of tumor cells, characterizing cell populations and spatial localization, revealing predictive and prognostic biomarkers, and identifying immunologic phenotypes in a single and limited sample. This valuable IF protocol successfully enables tumor microenvironment profiling that could contribute to the study of cellular crosstalk and the niche, and to the identification of predictive biomarkers for neoplasms.

Exposure to soluble platinum salts in precious metal refinery workers over a 17-year period.

BACKGROUND: Occupational exposure to soluble chlorinated platinum (Pt) salts, commonly called chloroplatinates, is a known cause of Pt salt sensitisation (PSS) and occupational asthma. We aimed to model inhalable soluble Pt salts exposure levels based on measurements in precious metal refineries for use in a retrospective cohort study on PSS. METHODS: Five platinum refineries located in the United Kingdom (3 sites), United States, and South Africa provided time weighted average inhalable soluble Pt salts exposure data, measured in 2,982 personal air samples over a 17-year period (2000-2016). We used a Bayesian hierarchical model to estimate geometric mean (GM) exposure levels for each refinery and job title over time. RESULTS: The GM of measured exposure levels over all facilities was 92 ng/m3 with a geometric standard deviation (GSD) of 9.07. Facility-specific GMs ranged from 48 ng/m3 (GSD 15.3) to 242 ng/m3 (GSD 5.99). Exposure modelling showed that soluble Pt salts exposure levels declined approximately 10% per year in two of the five facilities, but there were no clear time trends in the other facilities. A priori specified exposure groups captured most of the between-jobs differences, which helps to accurately predict exposures for jobs with no measurement data available. CONCLUSIONS: We applied exposure modelling to estimate time, refinery, and job-specific soluble Pt salts exposures. A significant annual decline in exposure levels was observed in two of the five participating facilities. Modelled exposure levels can be linked to individual workers' job history for exposure-response analysis of PSS in an epidemiological study.

Novel inhibitor of hematopoietic cell kinase as a potential therapeutic agent for acute myeloid leukemia.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are characterized by risk of relapses, poor survival, unwanted side effects and high toxicity with the current therapies. In light of these facts, there are efforts to develop new drugs specific for deregulated molecules that participate in leukemia pathogenesis. Hematopoietic cell kinase (HCK), an Src kinase family member, is overexpressed on hematopoietic stem cells of MDS and de novo AML patients and involved in the oncogenic process. Thus, we investigated in vitro, ex vivo and in vivo effects of a novel chemical compound targeting HCK inhibition (iHCK-37), in combination with the most used drugs for the treatment of MDS and de novo AML, 5-Azacytidine and Cytarabine. Herein, the combination treatment with iHCK-37 and 5-Azacytidine or Cytarabine demonstrated additive effects against leukemia cells, compared to either drug alone. iHCK-37 plus 5-Azacytidine or Cytarabine treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to reduction of ERK and AKT phosphorylation, and increased BAX and decreased BCL-XL protein expression. Moreover, treatment with iHCK-37 reduced MDS and AML CD34-positive cell numbers inside a 3D-structure but did not affect normal CD34-positive cell numbers. In vivo analysis showed that leukemic mice treated with iHCK-37 had reduced ERK and AKT proteins phosphorylation levels and leukocyte numbers. In conclusion, the iHCK-37 inhibitor has anti-neoplastic activity in leukemia cells without altering apoptosis and survival rate of normal cells, suggesting on-target malignant cell killing activity as a single agent or in combination with 5-Azacytidine or Cytarabine.

Differential regulation of MMP-13 expression in two models of experimentally induced periodontal disease in rats.

OBJECTIVE: Evaluate expression of MMP-13 during the course of two models experimentally induced periodontal disease in rats. DESIGN: Expression of MMP-13 at mRNA and protein levels was studied, respectively, by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Two experimental models were used: LPS injections and ligature placement. 30mug of LPS from Eschericia coli was injected twice a week into the palatal aspect of upper molars. Ligatures were placed at the gingival margin around lower first molars. Controls received injections of PBS vehicle and no ligatures on lower molars. Samples were collected 5, 15 and 30 days after initiation of periodontal disease and processed for extraction of total RNA, total protein, and routinely processed for histology. RESULTS: Both experimental models produced a significant increase on the inflammatory infiltrate that paralleled elevated levels of MMP-13 mRNA and protein at 5 and 15 days. The LPS model was associated with a sustained level of inflammation and increased MMP-13 mRNA throughout the 30 days, whereas the ligature model showed a decrease on the severity of inflammation and MMP-13 mRNA at the 30-day period. Interestingly, MMP-13 protein levels were diametrically contrary to the mRNA levels. CONCLUSION: MMP-13 expression during LPS- and ligature-induced experimental periodontal disease follows the increase on severity of inflammation at the earliest periods. At 30 days, there is a decrease on the severity of inflammation on the ligature model associated with decreased MMP-13 mRNA. There is a lack of transcription-translation coupling of MMP-13 gene in both experimental models.

Signaling pathways associated with the expression of inflammatory mediators activated during the course of two models of experimental periodontitis.

AIMS: Evaluate the signaling pathways associated with inflammatory mediators activated in two models of experimental periodontitis. MAIN METHODS: Two models were used: lipopolysaccharide (LPS) injections and ligature placement. Wistar rats were used and 30 microg LPS from Escherichia coli was injected twice a week into the palatal aspect of the upper molars. Ligatures were placed around lower first molars. A control group received injections of PBS on the palatal gingivae whereas no ligatures were placed on the lower molars. Samples were collected 5, 15 and 30 days and processed for analysis by Western blotting and stereometry. KEY FINDINGS: The ligature model was associated with rapid and transient activation of extracellular-regulated kinases (ERK) and p38 mitogen-activated protein kinase (MAPK) as well as of nuclear factor kappa B (NF-kappaB). Activation of these signaling pathways on the LPS model was delayed but sustained throughout the 30-day experimental period. Inflammatory changes induced by both models were similar; however there was a significant reduction on inflammation degree on the ligature model, which paralleled the decrease observed on the activation of the signaling pathways. Activation of signal transducer and activator of transcription (STAT)-3 by phosphorylation of Tyrosine residues and of STAT-5 was observed only on the ligature model. SIGNIFICANCE: Regulation of gene expression results from the activation of signaling pathways initiated by receptor-ligand binding of external antigens and also of cytokines produced by the host immune system. Understanding the signaling pathways relevant for a given condition may provide information useful for novel therapeutic approaches.