Joseph Paulauskis

Neutrophil C5a receptor and the outcome in a rat model of sepsis.

Complement fragment 5a (C5a)–C5a receptor (C5aR) signaling plays an essential role in neutrophil innate immunity. Blockade of either the ligand or the receptor improves survival rates in experimental sepsis. In the current study, sepsis was induced in rats by cecal ligation/puncture. Early in sepsis C5aR content on neutrophils significantly dropped, reached the nadir at 24 h after onset of sepsis, and progressively elevated thereafter. Western‐blot, RT‐PCR, and confocal microscopy analyses revealed that the loss and re‐expression of C5aR during sepsis might be due, at least in part, to the receptor internalization and reconstitution. The reduction and reconstitution of C5aR correlate with the loss and restoration of innate immune functions of blood neutrophils (chemotaxis and reactive oxygen species production), respectively. Quantitative measurements of C5aR on blood neutrophils are highly predictive of survival or death during sepsis. These data suggest that neutrophil C5aR content represents an essential component of an efficient defense system in sepsis and may serve as a prognostic marker for the outcome.

Altered Neutrophil Trafficking During Sepsis

In sepsis, dysregulation of the inflammatory system is well known, as reflected in excessive inflammatory mediator production, complement activation, and appearance of defects in phagocytic cells. In the current study sepsis was induced in rats by cecal ligation/puncture. Early in sepsis the β1 and β2 integrin content on blood neutrophils increased in a nontranscriptional manner, and the increase in β2, but not β1, integrin content was C5a dependent. Similar changes could be induced in vitro on blood neutrophils following contact with phorbol ester or C5a. Direct injury of lungs of normal rats induced by deposition of IgG immune complexes (IgG-IC) caused 5-fold increases in the myeloperoxidase content that was β2, but not β1, dependent. In contrast, in cecal ligation/puncture lungs myeloperoxidase increased 10-fold after IgG immune complex deposition and was both β1 and β2 integrin dependent. These data suggest that sepsis causes enhanced neutrophil trafficking into the lung via mechanisms that are not engaged in the nonseptic state.

Investigating fixative-induced changes in RNA quality and utility by microarray analysis.

Described herein is a detailed analysis of the impact of three fixatives (10% neutral buffered formalin, modified methacarn and 70% ethanol) on RNA quality and utility using microarray analysis compared to OCT-embedded and flash frozen tissue. From rat livers fixed and stored in paraffin blocks for 1 month or 1 year, RNA was isolated and applied to rat whole genome microarrays. At both time points, RNA isolated from OCT-embedded tissue lost up to 5% of the information contained in snap frozen control liver. Of the fixatives used, modified methacarn was associated with the smallest loss of RNA information content (approximately 10%), while liver fixed in 70% ethanol and 10% neutral buffered formalin lost roughly 25% and 80%, respectively. We conclude that when optimum morphology is required for techniques such as laser microdissection, modified methacarn is the fixative least harmful to nucleic acids of the three tested in this study. In contrast, using traditional isolation techniques, RNA derived from tissue fixed in 10% NBF will not give reliable results on microarray studies, and should be reserved for techniques less affected by the fragmentation and modification of the template RNA, such as quantitative RT-PCR.

So Many Studies, Too Few Subjects: Establishing Functional Relevance of Genetic Polymorphisms on Pharmacokinetics

Based on current literature, greater clarity in defining the magnitude of polymorphism effects on pharmacokinetics can be achieved by addressing key components of study design, including adequate subject numbers per study group. Convincing evidence of functional relevance exists for polymorphisms in genes such as CYP2D6 and UGT1A1, whereas the published evidence for similar effects for CYP3A5, OATP1B1, and ABCB1 is still emerging or equivocal. Polymorphism‐associated differences in pharmacokinetic parameters were simulated to incorporate (1) the ratio of group mean parameter values for homozygous wild‐type subjects versus homozygous variants, (2) pharmacokinetic variability, and (3) sample size needed to achieve 80% power, assuming 69% coefficient of variation. Subject selection by genotype and choice of probe substrate are also considered. Simulation results and literature examples are incorporated to define key recommendations for future investigations. This will allow for more definitive statements in publications regarding genotype influence on pharmacokinetics.

Differential expression of pancreatitis-associated protein and thrombospondins in arterial versus venous tissues.

Background/Aims: Arteries and veins modulate cardiovascular homeostasis and contribute to hypertension pathogenesis. Functional differences between arteries and veins are based upon differences in gene expression. To better characterize these expression patterns, and to identify candidate genes that could be manipulated selectively in the venous system, we performed whole genome expression profiling of arteries and veins. Methods: We used the CodeLink platform and the major artery (thoracic aorta) and vein (caudal vena cava) of the rat. Results: The most prominent difference was pancreatitis-associated protein (PAP1), expressed 64-fold higher in vena cava versus aorta. Expression of mRNA for thrombospondins (TSP-1, TSP-4) was greater than 5-fold higher in veins versus arteries. Higher mRNA expression of TSP-1, TSP-2, TSP-4 and PAP1 in vena cava versus aorta was confirmed by PCR. Immunohistochemical analysis of tissue sections qualitatively confirmed a higher expression of these proteins in vena cava versus aorta. Conclusion: This is the first gene array study of adult rat arterial and venous tissues, and also the first study to report differences in inflammatory genes between arteries and veins. Data from these studies may provide novel insights into the genetic basis for functional differences between arteries and veins in health and disease.