The isolated human umbilical vein as a bioassay for kinin-generating proteases: An in vitro model for therapeutic angioedema agents.

Aims: The isolated human umbilical vein is a robust contractile bioassay for ligands of the bradykinin (BK) B2 receptor (B2R), also extendable to B1 receptor (B1R) pharmacology. We hypothesized that, as a freshly isolated vessel, it also contains traces of plasma proteins that may confer responses to exogenous proteases via the formation of kinins.

Main Methods: Rings of human umbilical veins were mounted in organ baths containing Krebs buffer maintained at 37°C and purified proteases were introduced in the bathing fluid along with additional drugs/proteins that permit mechanistic analysis of effects.

Pharmacological effects of recombinant human tissue kallikrein on bradykinin B2 receptors.

Tissue kallikrein (KLK-1), a serine protease, initiates the release of bradykinin (BK)-related peptides from low-molecular weight kininogen. KLK-1 and the BK B2 receptor (B2R) mediate beneficial effects on the progression of type 2 diabetes and renal disease, but the precise role of KLK-1 independent of its kinin-forming activity remains unclear. We used DM199, a recombinant form of human KLK-1, along with the isolated human umbilical vein, a robust bioassay of the B2R, to address the previous claims that KLK-1 directly binds to and activates the human B2R, with possible receptor cleavage.

Autoimmune diabetes is suppressed by treatment with recombinant human tissue Kallikrein-1.

The kallikrein-kinin system (KKS) comprises a cascade of proteolytic enzymes and biogenic peptides that regulate several physiological processes. Over-expression of tissue kallikrein-1 and modulation of the KKS shows beneficial effects on insulin sensitivity and other parameters relevant to type 2 diabetes mellitus. However, much less is known about the role of kallikreins, in particular tissue kallikrein-1, in type 1 diabetes mellitus (T1D).

Preclinical characterization of recombinant human tissue kallikrein-1 as a novel treatment for type 2 diabetes mellitus.

Modulation of the kallikrein-kinin system (KKS) has been shown to have beneficial effects on glucose homeostasis and several other physiological responses relevant to the progression of type 2 diabetes mellitus (T2D). The importance of bradykinin and its receptors in mediating these responses is well documented, but the role of tissue kallikrein-1, the protease that generates bradykinin in situ, is much less understood. We developed and tested DM199, recombinant human tissue kallikrein-1 protein (rhKLK-1), as a potential novel therapeutic for T2D.

Gene regulation and chromatin remodeling by IL-12 and type I IFN in programming for CD8 T cell effector function and memory.

A third signal that can be provided by IL-12 or type I IFN is required for differentiation of naive CD8 T cells responding to Ag and costimulation. The cytokines program development of function and memory within 3 days of initial stimulation, and we show here that programming involves regulation of a common set of approximately 355 genes including T-bet and eomesodermin. Much of the gene regulation program is initiated in response to Ag and costimulation within 24 h but is then extinguished unless a cytokine signal is available.

Akt-mediated liver growth promotes induction of cyclin E through a novel translational mechanism and a p21-mediated cell cycle arrest.

The control of hepatocyte growth is relevant to the processes of liver regeneration, development, metabolic homeostasis, and cancer. A key component of growth control is the protein kinase Akt, which acts downstream of mitogens and nutrients to affect protein translation and cell cycle progression. In this study, we found that transient transfection of activated Akt triggered a 3-4-fold increase in liver size within days but only minimal hepatocyte proliferation.

Global assessment of cross-hybridization for oligonucleotide arrays.

Cross-hybridization is the tendency for chains of nucleic acids to bind to other chains of nucleic acids that have similar but not identical sequences. This has the potential to make the interpretation of microarray experiments difficult since intensity at a spot on the array does not simply depend on the quantity of target in the sample. We propose a method for evaluating the extent of cross-hybridization in oligonucleotide arrays for data arising from a typical microarray experiment.

Reovirus induces and benefits from an integrated cellular stress response.

Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response.

Coordinate stabilization of growth-regulatory transcripts in T cell malignancies.

We used microarray technology to compare mRNA decay rates of approximately 7000 transcripts in normal purified human T lymphocytes or the malignant T cell lines Jurkat and H9 following transcriptional arrest with actinomycin D. We found that over 2000 transcripts were expressed at abnormal levels in malignant T cells, including approximately 100 transcripts that were overexpressed and exhibited abnormally stable mRNA. Seventeen transcripts that encoded components of the ubiquitin-proteasome system were coordinately overexpressed and stabilized in both malignant cell lines.

Patterns of coordinate down-regulation of ARE-containing transcripts following immune cell activation.

We evaluated the expression of over 900 AU-rich element (ARE)-containing transcripts in primary human T lymphocytes following stimulation with anti-CD3 and anti-CD28 antibodies and found that approximately 48% of these transcripts were regulated following T cell activation. We identified approximately 145 ARE-containing transcripts that were rapidly induced and then rapidly disappeared within 1 h after activation. Another 250 ARE-containing transcripts expressed in resting T cells were rapidly turned off within 30 min after activation.

Microarray-based analyses of mRNA decay in the regulation of mammalian gene expression.

Mammalian cells coordinately regulate their gene expression programmes to ensure appropriate responses to stimuli. While transcriptional events provide an important level of gene expression regulation, modulation of messenger RNA (mRNA) decay provides an additional critical regulatory step. Much of the current knowledge of regulated mRNA decay comes from investigations of cytokine and other early response genes involved in inflammation and immunity.

Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes.

We used microarray technology to measure mRNA decay rates in resting and activated T lymphocytes in order to better understand the role of mRNA decay in regulating gene expression. Purified human T lymphocytes were stimulated for 3 h with medium alone, with an anti-CD3 antibody, or with a combination of anti-CD3 and anti-CD28 antibodies. Actinomycin D was added to arrest transcription, and total cellular RNA was collected at discrete time points over a 2 h period.