Key Challenges and Recommendations for Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol.

The urine microRNA profile may help monitor post-transplant renal graft function.

Noninvasive, cost-effective biomarkers that allow accurate monitoring of graft function are needed in kidney transplantation. Since microRNAs (miRNAs) have emerged as promising disease biomarkers, we sought to establish an miRNA signature in urinary cell pellets comparing kidney transplant patients diagnosed with chronic allograft dysfunction (CAD) with interstitial fibrosis and tubular atrophy and those recipients with normal graft function. Overall, we evaluated 191 samples from 125 deceased donor primary kidney transplant recipients in the discovery, initial validation, and the longitudinal validation studies for noninvasive monitoring of graft function.

MiRNAs in kidney transplantation: potential role as new biomarkers.

MiRNAs (miRNAs) are a class of small endogenous, noncoding RNAs with important roles in regulating gene expression known to play a role in many cellular functions including cellular differentiation, cell proliferation, cell development and functional regulation of the immune system, among others. As such, miRNAs are emerging not only as potential biomarkers but also as potential therapeutic targets. Here, we review the currently published work on miRNAs and renal transplantation as it pertains to ischemia-reperfusion injury, acute kidney injury, delayed graft function, calcineurin inhibitor toxicity, acute rejection, chronic allograft dysfunction and kidney fibrosis.

Identification of biomarkers to assess organ quality and predict posttransplantation outcomes.

Unlabelled: The increased disparity between organ supply and need has led to the use of extended criteria donors and donation after cardiac death donors with other comorbidities.

Methods: We have examined the preimplantation transcriptome of 112 kidney transplant recipient samples from 100 deceased-donor kidneys by microarray profiling. Subject groups were segregated based on estimated glomerular filtration rate (eGFR) at 1 month after transplantation: the GFR-high group (n=74) included patients with eGFR 45 mL/min per 1.

Reduced expression of inflammatory genes in deceased donor kidneys undergoing pulsatile pump preservation.

Background: The use of expanded criteria donor kidneys (ECD) had been associated with worse outcomes. Whole gene expression of pre-implantation allograft biopsies from deceased donor kidneys (DDKs) was evaluated to compare the effect of pulsatile pump preservation (PPP) vs. cold storage preservation (CSP) on standard and ECD kidneys.

Pretransplant transcriptome profiles identify among kidneys with delayed graft function those with poorer quality and outcome.

Robust biomarkers are needed to identify donor kidneys with poor quality associated with inferior early and longer-term outcome. The occurrence of delayed graft function (DGF) is most often used as a clinical outcome marker to capture poor kidney quality. Gene expression profiles of 92 preimplantation biopsies were evaluated in relation to DGF and estimated glomerular filtration rate (eGFR) to identify preoperative gene transcript changes associated with short-term function.

Gene expression changes are associated with loss of kidney graft function and interstitial fibrosis and tubular atrophy: diagnosis versus prediction.

Background: Loss of kidney graft function due to interstitial fibrosis (IF) and tubular atrophy (TA) is the most common cause of kidney allograft loss.

Methods: One hundred one allograft tissues (26 samples with IF/TA, 17 normal allografts, and an independent biopsy group collected at 3 month [n=34] posttransplantation) underwent microarray analysis to identify early detection/diagnostic biomarkers of IF/TA. Profiling of 24 allograft biopsies collected at or after 9-month posttransplantation (range 9-18 months) was used for validation.

Distinctive gene expression profiles characterize donor biopsies from HCV-positive kidney donors.

Background: Because of the shortage of organs for transplantation, procurement of kidneys from extended criteria donors is inevitable. Frequently, donors infected with hepatitis C virus (HCV) are used. To elucidate an initial compromise of molecular pathways in HCV graft, gene expression profiles were evaluated.

Gene expression profiling of peripheral blood leukocytes identifies potential novel biomarkers of chronic obstructive pulmonary disease in current and former smokers.

Background: Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease with associated systemic effects.

Objective: To use gene expression microarrays in peripheral blood leukocytes of current and former cigarette smokers to identify differences associated with COPD.

Materials And Methods: Random forest modelling and a split-sample case-control approach were used to identify candidate predictors.

Molecular pathways involved in loss of graft function in kidney transplant recipients.

Interstitial fibrosis (IF) and tubular atrophy (TA) are integral parts of chronic allograft dysfunction and represent in the new classification a separate entity with or without the identification of a specific etiology. Loss of kidney graft function with IF/TA is one of the causes of most kidney allograft losses. Despite progress in immunosuppression, chronic allograft dysfunction remains the main clinical challenge for improving long-term graft survival.

Chemical analysis of cigarette smoke particulate generated in the MSB-01 in vitro whole smoke exposure system.

Cigarette mainstream smoke (MS) is a dynamic aerosol consisting of a gas-vapor phase and a particulate phase. In recent years, novel in vitro whole smoke exposure systems have been developed to expose cells directly to whole MS. One such system is the Burghart Mimic Smoker-01 (MSB-01).

Cigarette smoke extract induced protein phosphorylation changes in human microvascular endothelial cells in vitro.

Phosphorylation is the most widely studied posttranslational modification (PTM) and is an important regulatory mechanism used during cellular responses to external stimuli. The kinases and phosphatases that regulate protein phosphorylation are known to be affected in many human diseases. Cigarette smoking causes cardiovascular disease (CVD).

Characterization of a whole smoke in vitro exposure system (Burghart Mimic Smoker-01).

In vitro systems are frequently used to study mechanisms of mainstream cigarette smoke (MS)-induced lung injury. Traditional methods of exposure involve the capture of MS particulate phase with filter pads or bubbling MS through phosphate buffered saline (PBS) or cell culture medium. Although useful for in vitro experiments, these exposure methods may fail to capture potential interactions between the gas and particulate phases.

Tumor-derived p53 mutants induce NF-kappaB2 gene expression.

Overexpression of mutant p53 is a common theme in tumors, suggesting a selective pressure for p53 mutation in cancer development and progression. To determine how mutant p53 expression may lead to survival advantage in human cancer cells, we generated stable cell lines expressing p53 mutants p53-R175H, -R273H, and -D281G by use of p53-null human H1299 (lung carcinoma) cells. Compared to vector-transfected cells, H1299 cells expressing mutant p53 showed a survival advantage when treated with etoposide, a common chemotherapeutic agent; however, cells expressing the transactivation-deficient triple mutant p53-D281G (L22Q/W23S) had significantly lower resistance to etoposide.

Modulation of gene expression by tumor-derived p53 mutants.

p53 mutants with a single amino acid substitution are overexpressed in a majority of human cancers containing a p53 mutation. Overexpression of the mutant protein suggests that there is a selection pressure on the cell indicative of an active functional role for mutant p53. Indeed, H1299 cells expressing mutant p53-R175H, p53-R273H or p53-D281G grow at a faster rate compared with a control cell line.

Tumor-derived p53 mutants induce oncogenesis by transactivating growth-promoting genes.

We have studied the mechanism of mutant p53-mediated oncogenesis using several tumor-derived mutants. Using a colony formation assay, we found that the majority of the mutants increased the number of colonies formed compared to the vector. Expression of tumor-derived p53 mutants increases the rate of cell growth, suggesting that the p53 mutants have 'gain of function' properties.

Transactivation and transrepression studies with p53.

The methods outlined in this chapter are designed to facilitate the study of the transactivation and transrepression properties of p53 (as well as p63 and p73). Once a gene of interest is identified, its presumptive promoter region can be cloned upstream of a luciferase gene in a plasmid. The most common reason for transfection experiments is to study gene expression patterns in the presence or absence of a particular gene product (e.

Hetero-oligomerization does not compromise 'gain of function' of tumor-derived p53 mutants.

Tumor-derived p53 mutants activate transcription from promoters of various growth-related genes. We tested whether this transactivation function of the mutant protein is sufficient to induce tumorigenesis ('gain of function'). Tumor-derived mutant p53-281G transactivates the promoters of human epidermal growth factor receptor (EGFR) and human multiple drug resistance gene (MDR-1).