T cells drive negative feedback mechanisms in cancer associated fibroblasts, promoting expression of co-inhibitory ligands, CD73 and IL-27 in non-small cell lung cancer.

The success of immune checkpoint therapy shows tumor-reactive T cells can eliminate cancer cells but are restrained by immunosuppression within the tumor micro-environment (TME). Cancer associated fibroblasts (CAFs) are the dominant stromal cell in the TME and co-localize with T cells in non-small cell lung cancer. We demonstrate the bidirectional nature of CAF/T cell interactions; T cells promote expression of co-inhibitory ligands, MHC molecules and CD73 on CAFs, increasing their production of IL-6 and eliciting production of IL-27.

Tissue-Specific Immunopathology in Fatal COVID-19.

In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown. To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury.

Monocyte-derived macrophages promote breast cancer bone metastasis outgrowth.

Bone metastasis is the major cause of death in breast cancer. The lack of effective treatment suggests that disease mechanisms are still largely unknown. As a key component of the tumor microenvironment, macrophages promote tumor progression and metastasis.

Detection of viral hepatitis E in clinical liver biopsies.

Aims: To determine the relative utility of in-situ testing for hepatitis E virus (HEV) RNA and paraffin-section polymerase chain reaction (PCR) to diagnose HEV infection in paraffin-embedded clinical liver biopsies, and to correlate with clinicopathological characteristics.

Methods And Results: We evaluated in-situ and quantitative PCR (qPCR)-based approaches to identifying HEV in clinical liver biopsies from infected patients from multiple centres, correlating with clinical setting (immunocompetent, allograft or immunosuppressed native liver) and histological findings. Thirty-six biopsies from 29 patients had histological data, 27 and 23 of which had satisfactory material for in-situ RNA testing and tissue qPCR, respectively.

Working with Commercially Available Quantum Dots for Immunofluorescence on Tissue Sections.

Quantum dots are semiconductor fluorescent nanocrystals that exhibit excellent characteristics compared with more commonly used organic fluorescent dyes. For many years quantum dot conjugated products have been available in multiple forms for fluorescence imaging of tissue sections under the trademark name Qdot®. They have much increased brightness, narrow emission spectrum, large Stokes shift and photostability compared with conventional organic fluorescent dyes, which together make them the fluorophores of choice for demanding requirements.

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections.

The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength.

The value of immunophenotyping hepatocellular adenomas: consecutive resections at one UK centre.

Aims:   To determine the utility of immunophenotyping for classification of hepatocellular adenomas resected at one Scottish centre.

Methods And Results:   This study comprised a retrospective review and immunophenotyping of consecutive resected benign hepatocellular tumours. Fifty-five patients (seven men) had 64 adenomas and 26 focal nodular hyperplasias (FNHs) resected.

Independent regulation of P53 stabilisation and activation after Rb deletion in primary epithelial cells.

We have previously reported that deletion of the retinoblastoma gene Rb leads to rapid but transient p53 stabilisation. We investigated here the pathways involved. We show that upon Rb-deletion dysregulated E2F activates p19ARF expression that localises in the nucleoli.

TGFbeta induces apoptosis and EMT in primary mouse hepatocytes independently of p53, p21Cip1 or Rb status.

Background: TGFbeta has pleiotropic effects that range from regulation of proliferation and apoptosis to morphological changes and epithelial-mesenchymal transition (EMT). Some evidence suggests that these effects may be interconnected. We have recently reported that P53, P21Cip1 and pRB, three critical regulators of the G1/S transition are variably involved in TGFbeta-induced cell cycle arrest in hepatocytes.

Deficiency of G1 regulators P53, P21Cip1 and/or pRb decreases hepatocyte sensitivity to TGFbeta cell cycle arrest.

Background: TGFbeta is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRb is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGFbeta in disease.

Microarray analysis of gene expression of mouse hepatocytes of different ploidy.

Polyploidisation in hepatocytes has been associated with many physiologic and pathologic processes such as proliferation, metabolism, regeneration, aging, and cancer. We studied gene expression patterns in hepatocytes of different ploidy. Primary hepatocytes were obtained from mice of different ages: young (4-6 weeks old), adult (8-10 weeks old), and older (22-24 weeks old).

Characterisation of Ercc1 deficiency in the liver and in conditional Ercc1-deficient primary hepatocytes in vitro.

The ERCC1/XPF complex is responsible for incision at the 5' side of the lesion during nucleotide excision repair and is also involved in homologous recombination and interstrand cross-link repair. The aim of the current study was to set up a better model for examination of Ercc1 deficiency in the murine liver and to determine the DNA lesions responsible for the premature polyploidy observed. We used the Cre/lox system with an adenovirus carrying Cre recombinase to conditionally induce Ercc1 deficiency in murine hepatocytes in vitro.

Additive effect of p53, p21 and Rb deletion in triple knockout primary hepatocytes.

Using Cre-Lox technology to inducibly delete Rb from wild-type, p21- and/or p53-deficient primary hepatocytes, we investigated the role of p53, p21 and pRb in the regulation of liver cell proliferation, polyploidization and death. These cellular decisions are critical to maintaining liver cell replacement in disease, and in determining the likelihood of carcinogenesis in chronic liver injury. Clearly, the present study shows a complex interplay between p53, p21 and pRb, which regulates the likelihood of hepatocytes stimulated from quiescence, to proliferate, undergo polyploidy or die.

TNF-alpha induced DNA damage in primary murine hepatocytes.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, usually arising from a background of chronic inflammatory disease. Tumor necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine produced in response to tissue injury, endotoxin exposure or infection and TNF-alpha signalling in hepatocytes is associated with an increase in oxidative stress. DNA is vulnerable to reactive oxygen species (ROS)-induced damage, which is highly mutagenic.