Faculty Bibliography

The neurovirulent retroviruses FrCasE and Moloney MLV-ts1 cause noninflammatory spongiform neurodegeneration in mice, manifested clinically by progressive spasticity and paralysis. Neurons have been thought to be the primary target of toxicity of these viruses. However the neurons themselves appear not to be infected, and the possible indirect mechanisms driving the neuronal toxicity have remained enigmatic. Here we have re-examined the cells that are damaged by these viruses, using lineage-specific markers. Surprisingly, these cells expressed the basic helix-loop-helix transcription factor Olig2, placing them in the oligodendrocyte lineage. Olig2+ cells were found to be infected, and many of these cells exhibited focal cytoplasmic vacuolation, suggesting that infection by spongiogenic retroviruses is directly toxic to these cells. As cytoplasmic vacuolation progressed, however, signs of viral protein expression appeared to wane, although residual viral RNA was detectable by in situ hybridization. Cells with the most advanced cytoplasmic effacement expressed the C/EBP-homologous protein (CHOP). This protein is up-regulated as a late event in a cellular response termed the integrated stress response. This observation may link the cellular pathology observed in the brain with cellular stress responses known to be induced by these viruses. The relevance of these observations to oligodendropathy in humans is discussed

Interferons (IFNs) are potent antiviral cytokines that inhibit infection by a wide spectrum of viruses by activating the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. Several IFN-induced antiviral proteins including 2',5'-oligoadenylate synthetase, dsRNA-activated protein kinase and Mx play a critical role in conferring the antiviral properties of IFN. However, studies have shown that additional antiviral factors are involved in addition to these proteins during IFN-mediated antiviral action. In an effort to characterize these novel antiviral factors, the antiviral mechanism of alpha IFN (IFN-alpha) against vesicular stomatitis virus (VSV) was investigated in human lung epithelial A549 cells. These studies demonstrated that soluble secreted antiviral proteins as the constituents of conditioned medium prepared from IFN-alpha-treated cells reduced VSV infectivity by more than 2 logs, compared with a 4 log inhibition observed following treatment of cells with IFN-alpha. The antiviral mechanism of these secreted proteins appeared to act at the level of cellular entry of VSV. Interestingly, the IFN-alpha-induced antiviral proteins were secreted independently of STAT1 (an essential component of the JAK/STAT pathway), demonstrating that the release of such extracellular soluble antiviral proteins from cells may represent an alternative mechanism of the antiviral defence strategy of IFN towards VSV infection.

Objective: Five double-blind, randomized, saline-controlled trials (RCTs) were included in the United States marketing application for an intra-articular hyaluronan (IA-HA) product for the treatment of osteoarthritis (OA) of the knee. We report an integrated analysis of the primary Case Report Form (CRF) data from these trials. Method. Trials were similar in design, patient population and outcome measures - all included the Lequesne Algofunctional Index (LI), a validated composite index of pain and function, evaluating treatment over 3 months. Individual patient data were pooled; a repeated measures analysis of covariance was performed in the intent-to-treat (ITT) population. Analyses utilized both fixed and random effects models. Safety data from the five RCTs were summarized. Results: A total of 1155 patients with radiologically confirmed knee OA were enrolled: 619 received three or five IA-HA injections; 536 received. 'placebo' saline injections. In the active and control groups, mean ages were 61.8 and 61.4 years; 62.4% and 58.8% were women; baseline total Lequesne scores 11.03 and 11.30, respectively. Integrated analysis of the pooled data set found a statistically significant reduction (P < 0.001) in total Lequesne score with hyaluronan (HA) (-2.68) vs placebo (-2.00); estimated difference -0.68 (95%

The Hedgehog pathway is vital for the development of many epidermal appendages, but its role in mammary development has been unclear. Here, we show that although Gli2 and Gli3 are expressed during embryonic mammary development, transcriptional reporters of positive Hedgehog signaling are absent. Nevertheless, Gli3(xt/xt) embryos show aberrant early mammary marker expression and lack two pairs of mammary buds, demonstrating that Gli3 is essential for mammary bud formation and preceding patterning events. Misactivation of the Hedgehog pathway by targeted expression of the constitutive activator Gli1, from the Gli2 promoter in Gli3(xt/+) mice, also induces mammary bud loss. Moreover, loss of Gli3 expression induces Gli1 misexpression in mammary mesenchyme. These results establish that the essential function of Gli3 during embryonic mammary development is to repress Hedgehog/Gli1-inducible targets. During postnatal mammary development, Gli2 and Gli3 are expressed in stromal and myoepithelial cells, and Gli3 is also found within the lumenal epithelium. Again, transcriptional reporters of positive Hedgehog signaling are absent from these cell types, yet are expressed robustly within mammary lymphatics. Thus, positive Hedgehog signaling is absent throughout mammary development, distinguishing the mammary gland from other epidermal appendages, such as hair follicles, which require Hedgehog pathway activity

KvLm, a novel bacterial depolarization-activated K(+) (Kv) channel isolated from the genome of Listeria monocytogenes, contains a voltage sensor module whose sequence deviates considerably from the consensus sequence of a Kv channel sensor in that only three out of eight conserved charged positions are present. Surprisingly, KvLm exhibits the steep dependence of the open channel probability on membrane potential that is characteristic of eukaryotic Kv channels whose sensor sequence approximates the consensus. Here we asked if the KvLm sensor shared a similar fold to that of Shaker, the archetypal eukaryotic Kv channel, by examining if interactions between conserved residues in Shaker known to mediate sensor biogenesis and function were conserved in KvLm. To this end, each of the five non-conserved residues in the KvLm sensor were mutated to their Shaker-like charged residues, and the impact of these mutations on the voltage dependence of activation was assayed by current recordings from excised membrane patches of Escherichia coli spheroplasts expressing the KvLm mutants. Conservation of pairwise interactions was investigated by comparison of the effect of single mutations to the impact of double mutations presumed to restore wild-type fold and voltage sensitivity. We observed significant functional coupling between sites known to interact in Shaker Kv channels, supporting the notion that the KvLm sensor largely retains the fold of its eukaryotic homologue.

The ability to specifically image macrophages may enable improved detection and characterization of atherosclerosis. In this study we evaluated the in vitro uptake of gadolinium (Gd)-containing immunomicelles (micelles linked to macrophage-specific antibody), micelles, and standard contrast agents by murine macrophages, and sought to determine whether immunomicelles and micelles improve ex vivo imaging of apolipoprotein E knockout (ApoE KO) murine atherosclerosis. Murine RAW 264.7 macrophages were incubated with Gd-DTPA, micelles, and immunomicelles. Cell pellets were prepared and imaged using a 1.5 T MR system with an inversion recovery spin-echo sequence to determine the in vitro T1 values. Ex vivo analysis of mouse aortas was performed using a 9.4T MR system with a high-spatial-resolution sequence (78x39x78 microm3). The T1 value was significantly decreased in cells treated with micelles compared to Gd-DTPA (P<0.0001), and in cells incubated at 4 degrees C with immunomicelles compared to micelles (P<0.05). Ex vivo MRI signal intensity (SI) was significantly increased by 81% and 20% in aortas incubated with immunomicelles and micelles, respectively. Confocal microscopy demonstrated in vitro and ex vivo uptake of fluorescent immunomicelles by macrophages. Immunomicelles and micelles improve in vitro and ex vivo MR detection of macrophages, and may prove useful in the detection of macrophage-rich plaques.

Calreticulin (CRT), an intracellular chaperone protein crucial for the proper folding and transport of proteins through the endoplasmic reticulum, has more recent acclaim as a critical regulator of extracellular functions, particularly in mediating cellular migration and as a requirement for phagocytosis of apoptotic cells. Consistent with these functions, we show that the topical application of CRT has profound effects on the process of wound healing by causing a dose-dependent increase in epithelial migration and granulation tissue formation in both murine and porcine normal and impaired animal models of skin injury. These effects of CRTare substantiated, in vitro, as we show that CRT strongly induces cell migration/wound closure of human keratinocytes and fibroblasts, using a wound/scratch plate assay, and stimulates cellular proliferation of human keratinocytes, fibroblasts, and vascular endothelial cells, providing mechanistic insight into how CRT functions in repair. Similarly, in both animal models, the histology of the wounds show marked proliferation of basal keratinocytes and dermal fibroblasts, dense cellularity of the dermis with notably increased numbers of macrophages and well-organized collagen fibril deposition. Thus, CRT profoundly affects the wound healing process by recruiting cells essential for repair into the wound, stimulating cell growth, and increasing extracellular matrix production

PURPOSE: This study investigated the presence of the 27-kd heat shock protein (HSP27) and its responses to ultraviolet B (UVB) irradiation in human corneal epithelium and in cultured corneal epithelial cells. METHODS: Human corneal epithelial cells including presumed corneal epithelial stem cells were cultured in vitro. HSP27 expression and intracellular localization in normal corneas or cultured corneal cells were examined using immunofluorescence staining. The expression of HSP27 in cultured corneal cells was also detected using western blotting, and the phosphorylated isoforms of HSP27 were identified using isoelectric focusing. RESULTS: In normal corneal tissue, HSP27 was present in limbal basal and suprabasilar epithelial cells. In cultured epithelial corneal cells, HSP27 expression was heterogeneous: Some cells expressed virtually no HSP27 and others showed relatively strong expression. HSP27 was localized to the cytoplasm in nonstressed cells and translocated to the perinuclear and nuclear areas after UVB irradiation. UVB irradiation also induced the phosphorylation of HSP27, resulting in the increase in monophosphorylated isoform and formation of biphosphorylated isoform. UV induced the phosphorylation of HSP27 apparently through activation of p38 mitogen-activated protein kinase. CONCLUSION: HSP27 is present mainly as a nonphosphorylated isoform in corneal epithelium and cultured corneal epithelial cells under nonstressed conditions. The constitutional expression of HSP27 suggests that it plays a physiologic role in the cornea. After UVB irradiation, HSP27 undergoes rapid phosphorylation and translocation. This stress response may be related to a protective role of HSP27 for survival of UVB-exposed corneal cells

AIMS: Pre-clinical data suggest that the racemic phyto-oestrogen 8-prenylnaringenin (8-PN) may have beneficial effects in postmenopausal women and may become an alternative to classical hormone replacement therapy (HRT) treatment regimes. The aim of this study was to investigate the pharmacokinetics, endocrine effects and tolerability of chemically synthesized 8-PN in postmenopausal women. METHODS: The study was performed using a randomized, double-blind, placebo-controlled, dose-escalation design with three groups of eight healthy postmenopausal women. In each group six subjects received 8-PN and two subjects placebo. 8-PN was given orally in doses of 50, 250 or 750 mg. Drug concentrations in serum, urine and faeces were measured up to 48 h and follicle-stimulating hormone/luteinizing hormone (LH) concentrations up to 24 h. RESULTS: All treatments were well tolerated and associated with a low incidence of (drug unrelated) adverse events. Serum concentrations of free 8-PN showed rapid drug absorption and secondary peaks suggestive of marked enterohepatic recirculation. Independent of the treatment group, approximately 30% of the dose was recovered in excreta as free compound or conjugates over the 48-h observation period. The first C(max) and AUC(0-48 h) showed dose linearity with ratios of 1 : 4.5 : 13.6 (C(max)) and 1 : 5.2 : 17.1 (AUC). The750- mg dose decreased LH concentrations by 16.7% (95% confidence interval 0.5, 30.2). CONCLUSION: Single oral doses of up to 750 mg 8-PN were well tolerated by postmenopausal women. The pharmacokinetic profile of 8-PN was characterized by rapid and probably complete enteral absorption, high metabolic stability, pronounced enterohepatic recirculation and tight dose linearity. The decrease in LH serum concentrations found after the highest dose demonstrates the ability of 8-PN to exert systemic endocrine effects in postmenopausal women.