Unraveling Links between Chronic Inflammation and Long COVID: Workshop Report.

As COVID-19 continues, an increasing number of patients develop long COVID symptoms varying in severity that last for weeks, months, or longer. Symptoms commonly include lingering loss of smell and taste, hearing loss, extreme fatigue, and "brain fog." Still, persistent cardiovascular and respiratory problems, muscle weakness, and neurologic issues have also been documented. A major problem is the lack of clear guidelines for diagnosing long COVID. Although some studies suggest that long COVID is due to prolonged inflammation after SARS-CoV-2 infection, the underlying mechanisms remain unclear. The broad range of COVID-19's bodily effects and responses after initial viral infection are also poorly understood. This workshop brought together multidisciplinary experts to showcase and discuss the latest research on long COVID and chronic inflammation that might be associated with the persistent sequelae following COVID-19 infection.

Metabolic Regulation of Inflammation and Its Resolution: Current Status, Clinical Needs, Challenges, and Opportunities.

Metabolism and inflammation have been viewed as two separate processes with distinct but critical functions for our survival: metabolism regulates the utilization of nutrients, and inflammation is responsible for defense and repair. Both respond to an organism's stressors to restore homeostasis. The interplay between metabolic status and immune response (immunometabolism) plays an important role in maintaining health or promoting disease development. Understanding these interactions is critical in developing tools for facilitating novel preventative and therapeutic approaches for diseases, including cancer. This trans-National Institutes of Health workshop brought together basic scientists, technology developers, and clinicians to discuss state-of-the-art, innovative approaches, challenges, and opportunities to understand and harness immunometabolism in modulating inflammation and its resolution.

Decrease in CD8+ lymphocyte number and altered cytokine profile in human prostate cancer.

The tumor microenvironment is comprised of multiple cell types arranged in a three-dimensional structure. Interactions amongst the various cell components play an important role in neoplasia, including the inflammatory reaction that occurs as part of the host response. In this study, the regional lymphocyte subpopulations and cytokine profiles associated with prostate cancer were examined using a quantitative imaging approach and expression microarray analysis. Lymphocytes were measured in four different epithelial phenotypes in prostate cancer specimens: carcinoma; prostatic intraepithelial neoplasia (PIN); benign prostate hyperplasia (BPH); and normal epithelium. The data indicate that CD8 positive, cytotoxic T lymphocytes are significantly decreased in regions adjacent to hyperplasia and carcinoma as compared to normal epithelium and PIN. In contrast the relative number of CD4 positive and CD20 positive lymphocytes did not change markedly. Parallel mRNA expression array analysis of the normal and tumor microenvironments identified a distinct cytokine profile in cancer, with 24 dysregulated genes in tumor epithelium and nine altered in tumor-associated stroma. Overall, these data indicate that the spatial distribution of CD8 positive, cytotoxic T lymphocytes is dysregulated in human prostate glands that contain cancer, and cytokine profiles are altered at the mRNA level.

Three dimension optical imaging in a high throughput layered expression system.

Layered peptide array (LPA) system enables multiplex screening of biomarkers [1-3]. One of the main problems of the LPA system is the screening of the layered-membranes stack. Nowadays, each membrane is imaged separately using conventional fluorescent imaging. This process is time consuming and requires extensive manual interaction. This paper describes a general solution for optical imaging of a layered grid medium using photogrammetric methods. The suggested method enables visualization of the LPA membranes stack by using only two images of the stack. This study is a proof of concept of the suggested solution using MATLAB simulation and phantom experiments.

High throughput screening of normal and neoplastic tissue samples.

The capacity to rapidly and efficiently elucidate a reliable set of disease specific biomarkers is paramount to enable a future of personalized medicine. High throughput screening methods applied to human clinical samples for the discovery of diagnostic, prognostic, and therapeutic targets address this need. Although the ability to analyze either thousands of markers from one sample or one marker from thousands of samples is the current state of high throughput screening, it would be ideal to have the ability to analyze thousands of markers from thousands of samples to expedite the early discovery phase of biomarkers and their validation. This review summarizes the current state of high throughput screening of tissue specimens and discusses its applications. In addition, the rationale, difficulties, strategies, and development of new technologies to address the need for improved high throughput capabilities are discussed.

Quantitation of HER2 and telomerase biomarkers in solid tumors with IgY antibodies and nanocrystal detection.

In an effort to improve affinity biomarker validation in fixed patient tissue specimens, we have developed a novel quantum dot-based bioimaging system that utilizes chicken IgY antibody for high sensitivity and specificity relative quantitation of cancer proteins. Monospecific, polyclonal IgYs were generated against human HER2 and telomerase, and analytically validated for specificity by western blot and immunohistochemistry on tumor and normal cells and for relative affinity by layered peptide array (LPA). IgYs bound desired targets in cell lines and fixed tissues and showed greater affinity than commercial mammalian antibodies for both HER2 and telomerase proteins. In tissue microarray experiments, HER2 quantitation with IgY antibody and quantum dot imaging correlated well with chromogenic in situ hybridization (CISH), whereas telomerase quantitation suggested a trend toward correlation with prostate cancer Gleason Grade and differentiation. Although patient numbers were small, these findings demonstrate the feasibility of relative quantitation of cancer biomarkers with IgY and quantum dot fluorophores, and show promise for rigorous clinical validation in large patient cohorts.

Layered peptide array for multiplex immunohistochemistry.

Layered peptide array is a new methodology for multiplex molecular measurements from two-dimensional life science platforms. The technology can be used in several different configurations depending on the needs of the investigator. Described here is an indirect layered peptide array (iLPA) that is capable of measuring proteins on a solid surface, such as target antigens on a tissue section. A prototype iLPA system was developed and subsequently examined for reproducibility and specificity and then compared with standard immunohistochemistry. Semiquantitative, multiplex proteomic analysis of histological sections was achieved with up to 20 membranes. The experimental variability was 18%. Overall, the data suggest that iLPA technology will be a relatively simple and inexpensive method for molecular measurements from tissue sections.

Layered peptide arrays: a diverse technique for antibody screening of clinical samples.

The layered peptide array (LPA) is a recently developed technique designed to measure antibody levels in a multiplex, high-throughput manner. LPAs can assess antibody presence either in fluid samples or from tissues while maintaining the two-dimensional orientation of the life science platform. In this manuscript, we evaluated and assessed the performance of the LPA platform, focusing on throughput capability, sensitivity, and specificity of the assay in several different systems.

Layered expression scanning: multiplex molecular analysis of diverse life science platforms.

With the advent of the genomic era, there is an increasing use of high-throughput techniques to generate transcriptome- and proteome-based profiles of biological specimens. Each of these methodologies offers a unique window into the inner workings of cell and tissue samples. Often, these studies generate large data sets and provide investigators with a substantial number of candidate dysregulated genes and pathways. Follow-up studies are then undertaken to independently validate the original findings and to extend the study to additional samples or more quantitative measurements. Although there are several methods available for these validation efforts, they are often tedious and laborious to perform; thus, additional tools that enable this task are needed. One such approach is layered expression scanning (LES), a new technique developed via a cooperative research and development agreement (CRADA) between the National Cancer Institute and 20/20 GeneSystems, Inc. The technique is based on the movement of biomolecules from a two-dimensional life science platform (histological tissue section, electrophoresis gel, multi-well plate, etc.) through a set of analysis membranes while maintaining the original distribution pattern of the molecules. Each membrane measures one analyte and the data are then mapped back to the original specimen, permitting each component of the life science platform to be studied in detail. LES can be configured in several different ways depending on the goals of the study. In this review, we summarize the use of the LES technique for a variety of biological applications.

Transfer and multiplex immunoblotting of a paraffin embedded tissue.

As we transition from genomics to the challenges of the functional proteome, new tools to explore the expression of proteins within tissue are essential. We have developed a method of transferring proteins from a formalin fixed, paraffin embedded tissues section to a stack of membranes which is then probed with antibodies for detection of individual epitopes. This method converts a traditional tissue section into a multiplex platform for expression profiling. A single tissue section can be transferred to up to ten membranes, each of which is probed with different antibodies, and detected with fluorescent secondary antibodies, and quantified by a microarray scanner. Total protein can be determined on each membrane, hence each antibody has its own normalization. This method works with phospho-specific antibodies as well as antibodies that do not readily work well with paraffin embedded tissue. This novel technique enables archival paraffin embedded tissue to be molecularly profiled in a rapid and quantifiable manner, and reduces the tissue microarray to a form of protein array. This method is a new tool for exploration of the vast archive of formalin fixed, paraffin embedded tissue, as well as a tool for translational medicine.

Layered peptide arrays: high-throughput antibody screening of clinical samples.

High-throughput methods to detect and quantify antibodies in sera and other patient specimens have use for many clinical and laboratory studies, including those associated with cancer detection, microbial exposures, and autoimmune diseases. We developed a new technique, termed layered peptide array (LPA), to serve as a screening tool to detect antibodies in a highly multiplexed format. We demonstrate here that a prototype LPA was capable of producing approximately 5000 measurements per experiment and appeared to be scalable to higher throughput levels. Sera and saliva from Sjögren's syndrome patients served as a test set to examine antibody titers in clinical samples. The LPA platform exhibited both a high sensitivity (100%) and high specificity (94%) for correctly identifying SSB antigen-positive samples. The multiplex capability of the platform was also confirmed when serum and saliva samples were analyzed for antibody reactivity to several peptides, including Sjögren's syndrome antigens A and B. The data indicate that LPA analysis will be a useful method for a number of screening applications.

Histomathematical analysis of clinical specimens: challenges and progress.

Proteomic analysis of clinical tissue specimens is a difficult undertaking. Described here is a multiplex study of protein expression levels in histological sections of human prostate that addresses many of the associated challenges. Whole-mount sections from 10 prostatectomy specimens were studied using 15 antibodies, immunohistochemical staining, digital imaging, and mathematical analysis of the data sets. The approach was successful in stratifying cell lineages present in the samples based on proteomic patterns, including differentiating normal epithelium from cancer. This strategy likely will be a useful method for extending the number of proteins that can be analyzed in clinical cancer specimens using currently available laboratory techniques.

Expression microdissection: operator-independent retrieval of cells for molecular profiling.

Tissue microdissection is an important method for the study of disease states. However, it is difficult to perform high-throughput molecular analysis with current techniques. We describe here a prototype version of a novel technique (expression microdissection) that allows for the procurement of desired cells via molecular targeting. Expression microdissection (xMD) offers significant advantages over available methods, including an increase in dissection speed of several orders of magnitude. xMD may become a valuable tool for investigators studying cancer or other disease states in patient specimens and animal models.

Molecular profiling of cancer.

The objective of molecular profiling of cancer is to determine the differential expression of genes and proteins from human tissue in the progression from normal precursor tissue to preneoplastic tissue to cancer in order to discover diagnostic, prognostic, and therapeutic markers. With the development of high-throughput analytical techniques such as microarrays and 2-D PAGE as well as the development of tools for cell procurement from histological sections such as laser capture microdissection (LCM), it is now possible to perform molecular analyses on specific cell populations from tissue. Since recognition of specific cell populations is critical, there is a need to optimize fixation and embedding not only to improve preservation of biomolecules, but also to maintain excellent histology. We have shown that 70% ethanol fixation of prostate tissue improves the recovery of DNA, RNA, and proteins over routine formalin fixation and maintains histological quality comparable to formalin. There is also a need to develop new technologies in order to expand the range of tissue types that can be analyzed. The development and applications of Layered Expression Scanning (LES) for the molecular analysis of whole tissue sections are discussed.

Interaction between the immune system and tongue squamous cell carcinoma induced by 4-nitroquinoline N-oxide in mice.

Squamous cell carcinoma of the oral cavity (SCC) accounts for 3% of cancers in the western world and 40% of cancers in India. The overall 5-year survival rate is only 50%. Most of the lesions appear intra-orally on the tongue. Results from a previous study demonstrated a significant increase in T and B-lymphocytes under the transformed epithelium when examining human lesions of hyperkeratosis, dysplasia and carcinoma of the tongue. In order to investigate the interaction between the host immunity and SCC, carcinogen induced SCC of the tongue was studied in mice. The water-soluble carcinogen, 4 nitroquinoline N-oxide (4NQO), was applied to BALB/c mice tongues and produced tongue SCC after a long incubation period of several months. Immunologic properties were examined systemically in the spleens and locally, at the tumor site. Examination of spleen lymphocytes from 4NQO induced mice revealed enlargement of the spleens and a significant decrease in the CD3, CD4, CD8 and CD19 cells. In the tongues, expression of TGF-beta, TNF-alpha, GM-CSF, and IL-1 beta mRNA were detected. TNF-alpha protein was detected in the affected tongues using immunoassays. mRNA expression of TNF-alpha was detected in the cancerous epithelium when extracted from the connective tissue. CD11b and CD3 cells were detected in the connective tissue under the developing carcinoma. CD11b positive cells were more prominent. The infiltrate was very scattered and not prominent as the infiltrate in the human tongue tissues. These results indicate that the growing tumor affected the immune response around the tumor and systemically. Most of the cytokines, which appeared in the affected tongues, originated from the tumor surroundings, but TNF-alpha was found also in the tumor. The interaction between the tumor and immune response components is important for diagnosis and treatment purposes.

Novel proteomic approaches for tissue analysis.

Proteomics, the global study of protein expression and characteristics, has recently emerged as a key component in the field of molecular analysis. The dynamic nature of proteins, from ion channels to chaperones, presents a challenge, yet the understanding of these molecules provides a rich source of information. When applying proteomic analysis directly to human tissue samples, additional difficulties arise. The following article presents an overview of the current proteomic tools used in the analysis of tissues, beginning with conventional methods such as western blot analysis and 2D polyacrylamide gel electrophoresis. The most current high-throughput techniques being used today are also reviewed. These include protein arrays, reverse-phase protein lysate arrays, matrix-assisted laser desorption/ionization, surface-enhanced laser desorption/ionization and layered expression scanning. In addition, bioinformatics as well as issues regarding tissue preservation and microdissection to obtain pure cell populations are included. Finally, future directions of the tissue proteomics field are discussed.

In vivo quantitative three-dimensional localization of tumor labeled with exogenous specific fluorescence markers.

We introduce a diffused optical detection system based on the administration of a fluorophore-antibody conjugate to diseased tissue. The conjugate interacts with the antigens expressed by the diseased tissue, resulting in fluorescent labeling of the antigen. By combining an optical detection system with a reconstruction algorithm developed on the basis of the random-walk model, we were able to determine the position of the fluorophore (and, thus, of the diseased cells) in the tissue. We present three-dimensional reconstructions of the location of a fluorophore (FITC-fluorescein isothiocyanate) in the tongues of mice. Measurements were performed with the fluorophore embedded at various simulated depths. The simulations were performed with agarose-based gel slabs applied to the tongue as tissuelike phantoms. Reconstructed fluorophore locations agree well with the actual values.

Evaluation of non-formalin tissue fixation for molecular profiling studies.

Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).