Trojan Technologies (Canada)

Incidents of contamination in biopharmaceutical production have highlighted the need to apply alternative or supplementary disinfection techniques. Ultraviolet (UV) irradiation is a well-established method for inactivating a broad range of microorganisms, and is therefore a good candidate as an orthogonal technique for disinfection. To apply UV as a safeguard against adventitious agents, the UV sensitivity of these target agents must be known so that the appropriate dose of UV may be applied to achieve the desired level of inactivation. This document compiles and reviews experimentally derived 254 nm sensitivities of organisms relevant to biopharmaceutical production. In general, different researchers have found similar sensitivity values despite a lack of uniformity in experimental design or standardized quantification techniques. Still, the lack of consistent methodologies has led to suspicious UV susceptibilities in certain instances, justifying the need to create a robust collection of sensitivity values that can be used in the design and sizing of UV systems for the inactivation of adventitious agents.

Introduction: Enhanced TGF-β signaling drives aortic root aneurysm development in Marfan syndrome (MFS) via the non-canonical (ERK) pathway in murine models, but ERK not been studied in human tissue. Conversely, blockade of canonical (Smad) TGF-β signaling is detrimental in MFS mice. Recently, TGF-β-induced increases in smooth muscle cell (SMC) contractile genes and collagen I (COL1) expression have been proposed to lead to aortic stiffness and dilatation in MFS. Hypothesis: We hypothesized that ERK signaling is enhanced in human MFS tissue and dictates the predilection of the aortic root to develop aneurysm by altering SMC gene expression. Methods and Results: Western blot of aortic root tissue from MFS patients with root aneurysms (4.67±0.09 cm) revealed 3.1-fold increased ERK activation relative to control aortic root (p=0.03, n=8). When compared to non-dilated MFS ascending (ASC) aorta (2.94±0.14 cm), there was no difference in ERK activation between the two regions. While RT-PCR of MFS root showed increased expression of the contractile gene regulator myocardin (2.4±0.07 fold, p=0.01), and of MYH11 (3.05±0.13 fold, p=0.03) ACTA2 (2.93±0.10 fold, p<0.01), and SM22-α (1.88±0.13 fold, p=0.02) versus controls, these genes were not differentially expressed between MFS root and ASC. COL1 expression was increased in MFS root relative to controls (5.57±0.06 fold, p<0.01) and MFS ASC (2.25±0.06 p<0.01). In vitro treatment of cultured MFS aortic root SMCs (n=5 patients) with TGF-β and Smad-specific inhibitor (SB431542) failed to increase expression of MYOCD, MYH11, ACTA2, SM22-α or COL1, while co-treatment with TGF-β and ERK inhibitor (PD98059) induced significant increases in expression of these genes (5.4-, 2.6-, 2.7-, 1.4-, and 1.4- fold, respectively p<0.05). Conclusions: ERK signaling is uniformly enhanced in MFS aortic root and ASC tissue, indicating that further study into aortic region-specific responses to cellular signals is needed to assess the development of focal aortic root aneurysm in MFS. Increased contractile gene expression is Smad-dependent and non-focal and thus does not clarify focal root aneurysm. COL1, while focally enhanced in the MFS root, is Smad-driven and suppressed by ERK, and may represent a compensatory response.