KRAS mutant rectal cancer cells interact with surrounding fibroblasts to deplete the extracellular matrix.

Somatic mutations in the KRAS oncogene are associated with poor outcomes in locally advanced rectal cancer but the underlying biologic mechanisms are not fully understood. We profiled mRNA in 76 locally advanced rectal adenocarcinomas from patients that were enrolled in a prospective clinical trial and investigated differences in gene expression between KRAS mutant (KRAS-mt) and KRAS-wild-type (KRAS-wt) patients. We found that KRAS-mt tumors display lower expression of genes related to the tumor stroma and remodeling of the extracellular matrix. We validated our findings using samples from The Cancer Genome Atlas (TCGA) and also by performing immunohistochemistry (IHC) and immunofluorescence (IF) in orthogonal cohorts. Using in vitro and in vivo models, we show that oncogenic KRAS signaling within the epithelial cancer cells modulates the activity of the surrounding fibroblasts in the tumor microenvironment.

Validation of Anti-Mouse PDL-1 Goat Polyclonal Antibody Staining with Mouse PDL-1 In Situ Hybridization on Adjacent Sections of Cell Pellets and Mouse Tumors.

Finding a valid antibody to detect mouse programmed death ligand 1 (PDL-1) by immunohistochemistry or immunofluorescence staining has been notoriously difficult. Successful validation of an antibody requires the use of multiple detection methods with the ability to compare appropriate positive and negative controls. Here, we describe in detail the protocols used to validate a mouse-specific PDL-1 antibody used in immunohistochemistry staining with an mRNA in situ hybridization on adjacent sections of mouse B16 tumor. This validation is supported by immunohistochemistry staining of PDL-1 on B16 cell pellets either treated or not treated with IFN-gamma.

Automated Double In Situ Detection of Mouse Lgr5 mRNA and Lysozyme Protein in Examining the Neighboring Cell Types of the Mouse Intestinal Crypt.

Automated detection of mRNAs and proteins in the same tissue sections is not a routine procedure. Successful experiment depends on the preparation of the tissue, the detection procedure, as well as the quality of the probes and antibodies. The multiplexed detections require experimental conditions, preserving the state of the molecular targets of interest and providing expression pattern of each target the same as in a single detection. Here we describe in detail the automated protocols used to detect mouse Lgr5 mRNA by in situ hybridization and immunofluorescence detection of lysozyme in the same mouse intestinal sections. Both the in situ hybridization and the protein detection were performed with an automated staining processor and provided strong and reproducible results.

Deconvoluting hepatic processing of carbon nanotubes.

Single-wall carbon nanotubes present unique opportunities for drug delivery, but have not advanced into the clinic. Differential nanotube accretion and clearance from critical organs have been observed, but the mechanism not fully elucidated. The liver has a complex cellular composition that regulates a range of metabolic functions and coincidently accumulates most particulate drugs. Here we provide the unexpected details of hepatic processing of covalently functionalized nanotubes including receptor-mediated endocytosis, cellular trafficking and biliary elimination. Ammonium-functionalized fibrillar nanocarbon is found to preferentially localize in the fenestrated sinusoidal endothelium of the liver but not resident macrophages. Stabilin receptors mediate the endocytic clearance of nanotubes. Biocompatibility is evidenced by the absence of cell death and no immune cell infiltration. Towards clinical application of this platform, nanotubes were evaluated for the first time in non-human primates. The pharmacologic profile in cynomolgus monkeys is equivalent to what was reported in mice and suggests that nanotubes should behave similarly in humans.

Understanding the three-dimensional world from two-dimensional immunofluorescent adjacent sections.

Visualizing tissue structures in three-dimensions (3D) is crucial to understanding normal and pathological phenomena. However, staining and imaging of thick sections and whole mount samples can be challenging. For decades, researchers have serially sectioned large tissues and painstakingly reconstructed the 3D volume. Advances in automation, from sectioning to alignment, now greatly accelerate the process. In addition, immunofluorescent staining methods allow multiple antigens to be simultaneously detected and analyzed volumetrically. The objective was to incorporate multi-channel immunofluorescent staining and automation in 3D reconstruction of serial sections for volumetric analysis. Paraffin-embedded samples were sectioned manually but were processed, stained, imaged and aligned in an automated fashion. Reconstructed stacks were quantitatively analyzed in 3D. By combining automated immunofluorescent staining and tried-and-true methods of reconstructing adjacent sections, we were able to visualize, in detail, not only the geometric structures of the sample but also the presence and interactions of multiple proteins and molecules of interest within their 3D environment. Advances in technology and software algorithms have significantly expedited the 3D reconstruction of serial sections. Automated, multi-antigen immunofluorescent staining will significantly broaden the range and complexity of scientific questions that can be answered with this methodology.

Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection.

Immunofluorescent staining is an informative tool that is widely used in basic research. Automation of immunostaining improves reproducibility and quality of the results. Up to now, use of automation in immunofluorescent staining was mostly limited to one marker. Here we present tyramide signal amplification based method of multiple marker immunofluorescent detection, including detection of antibodies, raised in the same species, in tissue sections and cultured cells. This method can be beneficial for both basic and clinical research.

A sensitive and specific histopathologic prognostic marker for H3F3A K27M mutant pediatric glioblastomas.

Pediatric glioblastomas (GBM) are highly aggressive and lethal tumors. Recent sequencing studies have shown that ~30 % of pediatric GBM and ~80 % of diffuse intrinsic pontine gliomas show K27M mutations in the H3F3A gene, a variant encoding histone H3.3. H3F3A K27M mutations lead to global reduction in H3K27me3. Our goal was to develop biomarkers for the histopathologic detection of these tumors. Therefore, we evaluated the utility of measuring H3K27me3 global reduction as a histopathologic and prognostic biomarker and tested an antibody directed specifically against the H3.3 K27M mutation in 290 samples. The study cohort included 203 pediatric (including 38 pediatric high-grade astrocytomas) and 38 adult brain tumors of various subtypes and grades and 49 non-neoplastic reactive brain tissues. Detection of H3.3 K27M by immunohistochemistry showed 100 % sensitivity and specificity and was superior to global reduction in H3K27me3 as a biomarker in diagnosing H3F3A K27M mutations. Moreover, cases that stained positive for H3.3 K27M showed a significantly poor prognosis compared to corresponding negative tumors. These results suggest that immunohistochemical detection of H3.3 K27M is a sensitive and specific surrogate for the H3F3A K27M mutation and defines a prognostically poor subset of pediatric GBM.

Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction.

Immune mechanisms have been implicated in placental dysfunction in patients with recurrent miscarriages and intrauterine growth restriction (IUGR), but the mediators are undefined. Here we show that complement activation, particularly C5a, is a required intermediary event in the pathogenesis of placental and fetal injury in an antibody-independent mouse model of spontaneous miscarriage and IUGR, and that complement activation causes dysregulation of the angiogenic factors required for normal placental development. Pregnancies complicated by miscarriage or growth restriction were characterized by inflammatory infiltrates in placentas, functional deficiency of free vascular endothelial growth factor (VEGF), elevated levels of soluble VEGF receptor 1 (sVEGFR-1, also known as sFlt-1; a potent anti-angiogenic molecule), and defective placental development. Inhibition of complement activation in vivo blocked the increase in sVEGFR-1 and rescued pregnancies. In vitro stimulation of monocytes with products of the complement cascade directly triggered release of sVEGFR-1, which sequesters VEGF. These studies provide the first evidence linking the complement system to angiogenic factor imbalance associated with placental dysfunction, and identify a new effector of immune-triggered pregnancy complications.

Amplification of autoimmune disease by infection.

Reports of infection with certain chronic persistent microbes (herpesviruses or Chlamydiae) in human autoimmune diseases are consistent with the hypothesis that these microbes are reactivated in the setting of immunodeficiency and often target the site of autoimmune inflammation. New experimental animal models demonstrate the principle. A herpesvirus or Chlamydia species can be used to infect mice with induced transient autoimmune diseases. This results in increased disease severity and even relapse. The evidence suggests that the organisms are specifically imported to the inflammatory sites and cause further tissue destruction, especially when the host is immunosuppressed. We review the evidence for the amplification of autoimmune inflammatory disease by microbial infection, which may be a general mechanism applicable to many human diseases. We suggest that patients with autoimmune disorders receiving immunosuppressing drugs should benefit from preventive antiviral therapy.

A mouse herpesvirus induces relapse of experimental autoimmune arthritis by infection of the inflammatory target tissue.

It is not known what is required for successive relapses in autoimmune diseases or evolution to a progressive chronic disease. Autoimmune arthritis caused by passive transfer of autoantibodies against glucose 6-phosphate isomerase is transient and therefore lends itself well to test for what might extend the disease. Herpesviruses have long been suspected of contributing to human autoimmune disease. We infected mice with a murine gamma-herpesvirus (MHV-68). In immunodeficient mice, transient arthritis was followed by a relapse. This was due to lytic viral infection of synovial tissues demonstrated by PCR, immunohistochemistry, and electron microscopy. Latent infection could be reactivated in the synovium of normal mice when treated with Cytoxan and this was associated with increased clinical arthritis. We conclude that herpesviruses may play an ancillary pathogenic role in autoimmune arthritis by infection of the inflammatory target tissue.

Oligoclonal expansions of antigen-specific CD8+ T cells in aged mice.

Oligoclonal T cell expansions (TCE) are common in old humans and mice. It is not known whether an Ag-specific response becomes more oligoclonal with age, and, if so, how this might alter biological responses or compromise the immune response, thus contributing to the immunodeficiency of aging. We used a tumor antigen response to study these questions. Early on, antigen reactive T cell numbers at the site of tumor injection were lower and clonally more restricted in old mice. Subsequently, long-term oligoclonal TCE emerged in the blood and spleen of old mice. IL-15 was not necessary for development of TCE in the blood. Overall, the data pointed to a dysregulated immune response in old mice, perhaps due to lack of optimal IL-2 and CD4 help at the earliest stages and a lack of an efficient local peritoneal CTL response. This was associated with a deficient humoral response and, likely, persistence of tumor cells or tumor antigens. Perhaps the spleen is the site of persistence which explains clonal TCE observed primarily in PBL and spleen. The TCE appear to be inefficient as they are often anergic. As a result an occasional peritoneal or splenic tumor may arise in old mice.

Competitive Specificity Analysis of Natural Antibodies against Epitope of Bacterial Cell Wall Peptidoglycoan: Glucosaminylmuramyl Dipeptide Carrying Adjuvant Activity.

The natural antibodies against glucosaminylmuramyl dipeptide (GMDP), the epitope of peptidoglycan of bacterial cell wall, isolated from human serum by thermal extraction possess a capability to cross-react with determinant of glycan chain - tetrasaccharide consisting of N-acetylglucosamine and N-acetylmuramic acid. The intensity of interaction of natural anti-GMDP-antibodies with specific ligand is significantly higher than with tetrasaccharide. The natural antibodies against tetrasaccharide carry properties of heteroclitic antibodies, i.e. the intensity of their interaction with heterologous ligand, GMDP, is significantly higher than with homologous one, tetrasaccharide GMGM. GMDP is supposed to be specific antigenic peptidoglycan determinant against which the antibodies reacting with various intensity to homologous (GMDP) and relative (tetrasaccharide) hapten are formed in the process of natural immunization.

Affinity of Serum Natural Antibodies against Peptidoglycan Bacterial Cell Component with Adjuvant Activity.

We studied affinity of natural antibodies of human serum against glucosaminylmuramyl dipeptide (GMDP), the epitope peptidoglycan bacterial cell wall component which carries adjuvant activity. Antibodies against GMDP were isolated from the blood sera of healthy donors using thermal extraction of antibodies from specific ligand on plastic. Determination of the dissociation constant (K(d)) showed equal K(d) in the serum and affinity-purified anti-GMDP-antibodies, i.e. extraction by this method led to the isolation of all subpopulations of antibodies in spite of their affinity. K(d) of serum, affinity-purified and monoclonal anti-GMDP-antibodies proved of low value - 10(-6) M, and according to this index anti-GMDP-antibodies may be classified between anti-protein and anti-carbohydrate antibodies.