Collagenase predigestion on paraffin sections enhances collagen immunohistochemical detection without distorting tissue morphology

Reliable immunohistochemical detection of collagen in formalin fixed, paraffin embedded tissues requires protease digestion. While these pan-proteases (pepsin, trypsin, protease K, etc.) enhance collagen detection, they also digest many other tissue proteins and produce poor cellular morphology and unrecognizable cellular structures. Balancing the conditions (protease type, concentration, incubation time and temperature) to digest some, but not all, proteins in a tissue section while optimizing collagen detection requires one to compromise improved collagen immunolabeling with adequate cellular morphology. Furthermore, optimal conditions for digesting tissue proteins to enhance collagen detection vary among tissue types and their fixation. Although brain is not typically subject to these deleterious consequences, structures such as epithelium, spermatids, stroma etc. and other tissues with complicated histology are profoundly affected. To resolve this technical dilemma, we discovered a novel use for collagenase to enhance collagen immunodetection without affecting the noncollagen proteins, thereby preserving tissue morphology. Collagenase, which is typically used in vitro for disassociation of cells, has never been used reliably on formalin fixed, paraffin embedded tissue sections. This new use of collagenase for immunohistochemistry promotes increased collagen immunolabeling, is easy to use, is versatile, and allows preservation of tissue structure that provides maximal and accurate histological information.     

Granzyme B inhibits vaccinia virus production through proteolytic cleavage of eukaryotic initiation factor 4 gamma 3

Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD(1408)S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery.     

High density array screening to identify the genetic requirements for transition metal tolerance in Saccharomyces cerevisiae

Biological systems have developed with a strong dependence on transition metals for accomplishing a number of biochemical reactions. Iron, copper, manganese and zinc are essential for virtually all forms of life with their unique chemistries contributing to a variety of physiological processes including oxygen transport, generation of cellular energy and protein structure and function. Properties of these metals (and to a lesser extent nickel and cobalt) that make them so essential to life also make them extremely cytotoxic in many cases through the formation of damaging oxygen radicals via Fenton chemistry. While life has evolved to exploit the chemistries of transition metals to drive physiological reactions, systems have concomitantly evolved to protect against the damaging effects of these same metals. Saccharomyces cerevisiae is a valuable tool for studying metal homeostasis with many of the genes identified thus far having homologs in higher eukaryotes including humans. Using high density arrays, we have screened a haploid S. cerevisiae deletion set containing 4786 non-essential gene deletions for strains sensitive to each of Fe, Cu, Mn, Ni, Zn and Co and then integrated the six screens using cluster analysis to identify pathways that are unique to individual metals and others with function shared between metals. Genes with no previous implication in metal homeostasis were found to contribute to sensitivity to each metal. Significant overlap was observed between the strains that were sensitive to Mn, Ni, Zn and Co with many of these strains lacking genes for the high affinity Fe transport pathway and genes involved in vacuolar transport and acidification. The results from six genome-wide metal tolerance screens show that there is some commonality between the cellular defenses against the toxicity of Mn, Ni, Zn and Co with Fe and Cu requiring different systems. Additionally, potential new factors been identified that function in tolerance to each of the six metals.     

The human cathelicidin, LL-37, induces granzyme-mediated apoptosis in cytotoxic T lymphocytes

LL-37 is a human cationic host defense peptide (antimicrobial peptide) belonging to the cathelicidin family of peptides. In this study, LL-37 was shown to kill stimulated CD8⁺ T cells (Cytotoxic T lymphocytes; CTLs) via apoptosis, while having no cytotoxic effect on non-stimulated CD8⁺ or CD4⁺ T cells or stimulated CD4⁺ T cells. Of interest, the CD8⁺ cells were much more sensitive to LL-37 than many other cell types. LL-37 exposure resulted in DNA fragmentation, chromatin condensation, and the release of both granzyme A and granzyme B from intracellular granules. The importance of granzyme family members in the apoptosis of CTLs following LL-37 treatment was analyzed by using C57BL/6 lymphocytes obtained from mice that were homozygous for null mutations in the granzyme B gene, the granzyme A gene, or both granzymes A and B. Granzymes A and B were both shown to play an important role in LL-37-induced apoptosis of CTLs. Further analysis revealed that apoptosis occurred primarily through granzyme A-mediated caspase-independent apoptosis. However, caspase-dependent cell death was also observed. This suggests that LL-37 induces apoptosis in CTLs via multiple different mechanisms, initiated by the LL-37-induced leakage of granzymes from cytolytic granules. Our results imply the existence of a novel mechanism of crosstalk between the inflammatory and adaptive immune systems. Cells such as neutrophils, at the site of a tumor for example, could influence the effector, activity of CTL through the secretion of LL-37.     

Bovine pancreatic trypsin inhibitor is a new antifungal peptide that inhibits cellular magnesium uptake

Antimicrobial peptides (AMPs) are promising agents for control of bacterial and fungal infections. Traditionally, AMPs were thought to act through membrane disruption but recent experiments have revealed a diversity of mechanisms. Here we describe a novel antifungal activity for bovine pancreatic trypsin inhibitor (BPTI). BPTI has several features in common with a subset of antimicrobial proteins in that it is small, cationic and stabilized by disulphide bonds. BPTI inhibits growth of Saccharomyces cerevisiae and the human pathogen Candida albicans. Screening of the yeast heterozygous essential deletion collection identified the magnesium transporter Alr1p as a potential BPTI target. BPTI treatment of wild type cells resulted in a lowering of cellular Mg²⁺ levels. Populations treated with BPTI had fewer cells in S‐phase of the cell cycle and a corresponding increase of cells in G₀/G₁ and G₂ phases. The same patterns of cell cycle arrest obtained with BPTI were also obtained with the magnesium channel inhibitor hexamine(III)cobalt chloride. Analysis of the growth inhibition of C. albicans revealed that BPTI is inhibiting growth via the same mechanism in the two yeast species. Inhibition of magnesium uptake by BPTI represents a novel mechanism of action for AMPs.     

A quarter century of granzymes

Granzymes (Grs) were discovered just over a quarter century ago. They are produced by cytotoxic T cells and natural killer cells and are released upon interaction with target cells. Intensive biochemical, genetic, and biological studies have been performed in order to study their roles in immunity and inflammation. This review summarizes research on the family of Grs.     

Modulation of the tumor cell phenotype by IFN-gamma results in resistance of uveal melanoma cells to granule-mediated lysis by cytotoxic lymphocytes

IFN-gamma, a pleiotropic immune regulator, is implicated in both tumor immune surveillance and selection of tumor variants resistant to immune control, i.e., immunoediting. In uveal melanoma patients, elevated serum levels of IFN-gamma correlate with the spread of metastasis and represent a negative prognostic marker. Treatment with IFN-gamma boosted the MHC class I presentation machinery in uveal melanoma cells but suppressed their MHC class I-restricted CTL lysis. Tumor cells exposed to IFN-gamma efficiently activated specific CTL but were less susceptible to permeabilization by perforin and exhibited a decreased capacity to bind and incorporate granzyme B. These results define a novel mechanism of resistance to granule-mediated CTL lysis in human tumors. Furthermore, the data suggest that immunoediting is not limited to genetic or epigenetic changes resulting in stable cellular phenotypes but also involves an inducible modulation of tumor cells in response to a microenvironment associated with immune activation.     

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Stent implantation through a self-expanding stent

Jailing of a side-branch is a known complication of stent implantation, and makes access to the side-branch difficult, especially if the stent is of the self-expanding type. Although plain balloon angioplasty is feasible for the jailed side-branches, the use of newer devices (a stent, Rotablation or atherectomy) has not been described. We describe a novel way of treating a side-branch jailed by a self-expanding stent by using stent implantation through the strut of a self-expanding stent.