Challenges in the Development of Therapy for Dry Age-Related Macular Degeneration.

Dry age-related macular degeneration (AMD), a multifactorial progressive degenerative disease of the retinal photoreceptors, pigmented epithelium and Bruch's membrane/choroid in central retina, causes visual impairment in millions of elderly people worldwide. The only available therapy for this disease is the over-the-counter (OTC) multi-vitamins plus macular xanthophyll (lutein/zeaxanthin) which attempts to block the damages of oxidative stress and ionizing blue light. Therefore development of dry AMD prescribed treatment is a pressing unmet medical need. However, this effort is currently hindered by many challenges, including an incomplete understanding of the mechanism of pathogenesis that leads to uncertain targets, confounded by not yet validated preclinical models and the difficulty to deliver the drugs to the posterior segment of the eye. Additionally, with slow disease progression and a less than ideal endpoint measurement method, clinical trials are necessarily large, lengthy and expensive. Increased commitment to research and development is an essential foundation for dealing with these problems. Innovations in clinical trials with novel endpoints, nontraditional study designs and the use of surrogate diseases might shorten the study time, reduce the patient sample size and consequently lower the budget for the development of the new therapies for the dry AMD.

Bmp indicator mice reveal dynamic regulation of transcriptional response.

Cellular responses to Bmp ligands are regulated at multiple levels, both extracellularly and intracellularly. Therefore, the presence of these growth factors is not an accurate indicator of Bmp signaling activity. While a common approach to detect Bmp signaling activity is to determine the presence of phosphorylated forms of Smad1, 5 and 8 by immunostaining, this approach is time consuming and not quantitative. In order to provide a simpler readout system to examine the presence of Bmp signaling in developing animals, we developed BRE-gal mouse embryonic stem cells and a transgenic mouse line that specifically respond to Bmp ligand stimulation. Our reporter identifies specific transcriptional responses that are mediated by Smad1 and Smad4 with the Schnurri transcription factor complex binding to a conserved Bmp-Responsive Element (BRE), originally identified among Drosophila, Xenopus and human Bmp targets. Our BRE-gal mES cells specifically respond to Bmp ligands at concentrations as low as 5 ng/ml; and BRE-gal reporter mice, derived from the BRE-gal mES cells, show dynamic activity in many cellular sites, including extraembryonic structures and mammary glands, thereby making this a useful scientific tool.

Hyperforin, the active component of St. John's wort, induces IL-8 expression in human intestinal epithelial cells via a MAPK-dependent, NF-kappaB-independent pathway.

St. John's wort is widely used as an herbal antidepressant and is among the top-selling botanical products in the United States. Although St. John's wort has been reported to have minimal side effects compared with other antidepressants, here we show that hyperforin, the active component of St. John's wort, can stimulate interleukin-8 (IL-8) expression in human intestinal epithelia cells (IEC) and primary hepatocytes. Hyperforin is also able to induce expression of mRNA, encoding another major inflammatory mediator--intercellular adhesion molecule-1 (ICAM-1). IEC participate in the intestinal inflammatory process and serve as a first line of defense through bidirectional communication between host and infectious pathogens. Although hyperforin is a potent ligand for the steroid and xenobiotic receptor (SXR), we found that hyperforin induced IL-8 mRNA through an SXR-independent transcriptional activation pathway. IL-8 induction by hyperforin required the activation of AP-1 but not the NF-kappaB transcription factor, thereby distinguishing it from the NF-kappaB-dependent IL-8 induction mediated by tumor necrosis factor alpha (TNFalpha). Further study revealed that extracellular signal-regulated kinase 1 and 2 (ERK1/2) were required for the hyperforin-induced expression of IL-8. Our results suggest a previously unsuspected effect of St. John's wort in modulating the immune and inflammatory responses.

The TATGARAT box of the HSV-1 ICP27 gene is essential for immediate early expression but not critical for efficient replication in vitro or in vivo.

We constructed a recombinant virus containing a promoter mutation altering the immediate-early expression of the HSV-1 ICP27 transcript, ICP27DeltaSma, which contains a deletion of the "TATGARAT" and surrounding sequences, but retains the rest of the ICP27 promoter. This mutant does not exhibit immediate-early expression of ICP27 using criteria of expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. While transcript abundance at 1h after infection at 0.1 PFU/cell in mouse embryo fibroblasts was significantly altered compared to infections with wt -rescues, by 4 h after infection these differences were diminished or absent. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue infections at the earliest times tested, but were equivalent by 8-12 h pi. Further, both single and multi-step virus replication was equivalent with both mutants and rescues. Thus, altering the immediate early kinetics of ICP27 leads to a sub-optimal quantitative lag-phase in gene expression but without consequence to replication fitness in vitro . Infections in vivo also revealed the ability of mutant and rescue virus to invade the CNS of mice following footpad injections was equivalent. The nature of the role of immediate-early ICP27 expression is discussed in light of these observations.

Immediate-early expression of the herpes simplex virus type 1 ICP27 transcript is not critical for efficient replication in vitro or in vivo.

We constructed a promoter mutation altering the immediate-early expression of the herpes simplex virus type 1 (HSV-1) ICP27 transcript and its cognate wild-type rescue viruses in order to assess the role of the ICP27 protein in the earliest stages of viral infection by global transcriptional analysis with a DNA microarray. This mutant, ICP27/VP16, replaces the whole ICP27 promoter/enhancer with the VP16 promoter. It demonstrates loss of immediate-early expression of ICP27 according to the criteria expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. Significant differences in relative transcript abundances between the mutant and wild-type rescue viruses were limited at the earliest times measured and not evident at all by 4 h after infection. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue virus infections at the earliest times tested, but were equivalent by 8 h postinfection. Further, both single and multistep levels of virus replication were equivalent with both mutant and rescue viruses. Thus, altering the immediate-early kinetics of ICP27 leads to a suboptimal quantitative lag phase in gene expression but without consequence for replication fitness in vitro. Infections in vivo also revealed equivalent ability of mutant and rescue viruses to invade the central nervous system of mice following footpad injections. Limitations to an immediate-early role of ICP27 in the biology of HSV are discussed in light of these observations.

Vitamin K2 regulation of bone homeostasis is mediated by the steroid and xenobiotic receptor SXR.

Vitamin K2 is a critical nutrient required for blood clotting that also plays an important role in bone formation. Vitamin K2 supplementation up-regulates the expression of bone markers, increases bone density in vivo, and is used clinically in the management of osteoporosis. The mechanism of vitamin K2 action in bone formation was thought to involve its normal role as an essential cofactor for gamma-carboxylation of bone matrix proteins. However, there is evidence that suggests vitamin K2 also has a transcriptional regulatory function. Vitamin K2 bound to and activated the orphan nuclear receptor SXR and induced expression of the SXR target gene, CYP3A4, identifying it as a bona fide SXR ligand. Vitamin K2 treatment of osteosarcoma cells increased mRNA levels for the osteoblast markers bone alkaline phosphatase, osteoprotegerin, osteopontin, and matrix Gla protein. The known SXR activators rifampicin and hyperforin induced this panel of bone markers to an extent similar to vitamin K2. Vitamin K2 was able to induce bone markers in primary osteocytes isolated from wild-type murine calvaria but not in cells isolated from mice deficient in the SXR ortholog PXR. We infer that vitamin K2 is a transcriptional regulator of bone-specific genes that acts through SXR to favor the expression of osteoblastic markers. Thus, SXR has a novel role as a mediator of bone homeostasis in addition to its role as a xenobiotic sensor. An important implication of this work is that a subset of SXR activators may function as effective therapeutic agents for the management of osteoporosis.

[Reversal of anti-apoptotic action by tetrandrine in human breast carcinoma multidrug-resistant MCF-7 cells].

OBJECTIVE: To study whether the anti-apoptotic action is reversed by tetrandrine in a combination with vincristine in human breast carcinoma MCF-7 multidrug-resistant cells. METHOD: Chromatin condensation was observed by co-staining of fluorescent dyes Hoechst 33342 and propidium iodide; and G1 sub-peak was detected by flow cytometry. Apoptotic cells were detected with TUNEL method. Cellular free ca2+ was determined with Fluo-3 staining method. RESULT: Two types of chromatin condensation were observed after the sensitive and drug-resistant MCF-7 cells were treated with an antitumor drug vincristine 5 mumol.L-1 for 24 h. The number of cell with chromatin condensation was obviously reduced in the drug-resistant cells treated with the same concentration of vincristine, as compared with the sensitive MCF-7 cells. The number of the apoptotic cells was increased by a combination of non-cytotoxic tetrandrine 20 mumol.L-1 and vincristine in both the sensitive and drug-resistant cells, which was confirmed with fluorescent indication and TUNEL method. The increment of introcellular free Ca2+ level in the cells treated with tetrandrine in a combination of vincristine was detected with Fluo-3 staining method. CONCLUSION: The anti-apoptotic action of human breast carcinoma MCF-7 cells can be effectively reversed by tetrandrine.