Novel toll-like receptor 4 ligands: synthesis, biological studies and applications in molecular vaccines

Lipid A, a unique disaccharide glycolipid, is the active principle of Gram-negative bacterial lipopolysaccharide in activating the innate immune response via Toll-like receptor 4 (TLR4). Given the important role that TLR4 plays in innate immunity, and ultimately, the development of an adaptive immune response, ligands that can modulate TLR4-mediated signalling have great therapeutic potential as both vaccine adjuvants, and anti-sepsis agents. In attempting to develop novel ligands which can successfully modulate TLR4-mediated signalling in a well defined fashion, simplified structures which aim to mimic the natural lipid A structure have shown great promise. The notion of cancer immunotherapy, in which the vast power of the immune system is tapped to prevent and/or eradicate the disease has begun to garner considerable attention. Tumour associated carbohydrate antigens, carbohydrate containing epitopes which are either unique of over-expressed by cancer cells, are viable targets of said immunotherapy. A major limitation, however, is the low antigenicity displayed by these carbohydrate epitopes. Studies have shown that the inclusion of adjuvant structures, especially when directly chemically conjugated to the antigen, improve the success of anti-cancer vaccination efforts. The primary goal of this study has been aimed at the development of novel vaccine adjuvants, specifically the design of novel molecular frameworks to mimic the structure of lipid A in the activation of TLR4. A secondary goal of this study has aimed at the application of successful novel lipid A mimics as the immunostimulatory component of self-adjuvanting carbohydrate antigens for use in therapeutic cancer vaccines. One novel molecular framework that has been designed and synthesized employs a flexible, acyclic diethanolamine-based scaffold to mimic one of the sugar moieties natural to the lipid A disaccharide. Several structural variations of this framework were generated for structure-activity relationship studies in an effort to maximize immunostimulatory potency. The mimics were evaluated in vitro for their ability to induce TLR4-mediated cytokines. All variations showed confirmed TLR4 stimulatory activity, the potency of which was dependent on the functionalization of the terminal ethanol moiety of the diethanolamine-based acyclic scaffold. In vivo studies evaluating the adjuvant potential of this novel family of lipid A mimics are currently underway. As part of an industrial partnership aimed at the development of novel vaccine adjuvants, a second lipid A mimic framework was designed and synthesized, in which an aromatic residue has been incorporated into the structural backbone. Two structural variations of the framework were generated which vary in the functionalization of the phenolic hydroxyl of the aromatic-based backbone. Several in vivo studies have shown that both mimics exhibit potent TLR4 immunostimulatory activity, and successful adjuvant properties. In an effort to construct a fully synthetic, self-adjuvanting tumour associated carbohydrate antigen for eventual use in therapeutic cancer vaccines, the immunostimulatory activity of the diethanolamine-based lipid A mimic framework designed herein, was tapped. As such, a conjugate structure in which the lipid A mimic framework and the Thomsen-Friedenreich carbohydrate antigen are directly linked via a flexible chemical linker was designed and synthesized. Future studies will determine the ability of the conjugate to induce an effective antibody response towards the carbohydrate epitope

Synthesis of novel Lipid A analogs as potential ligands for toll-like receptor 4

Lipid A, the glycolipid component of Gram-negative bacterial lipopolysaccharide has been shown to engage the Toll-like receptor 4 (TLR4) signalling pathway. While this signalling cascade results in cellular mediators which serve to activate the immune system and eliminate the invading organism, the associated toxicity is potentially harmful to the host. With an ever increasing molecular understanding of TLR4 mediated signalling, synthetic analogs of the natural Lipid A structure and the structure-activity relationship information they provide have emerged as a potential method of harnessing the desired immunomodulating effects from the undesired toxicity. As such, the therapeutic potential of TLR4 signalling is becoming an increasing possibility. Two novel synthetic frameworks have been designed and synthesized to potentially mimic the natural Lipid A structure. A dimeric monosaccharide framework was obtained, however, attempts at functionalization (R = Lipid) to generate a series of analogs were generally unsuccessful due to unforeseen circumstances. A revised synthetic strategy has thereby been undertaken to allow more facile functionalization of this framework. A monosaccharide framework in which diethanolamine is employed as an acyclic scaffold replacing one of the sugar groups common to the natural disaccharide Lipid A structure has also been obtained. Functionalization (R = Lipid) has been successfully achieved, with further functionalization of this framework through the acyclic hydroxyl moiety intended. Ultimately, biological evaluation will determine the potential of these frameworks as being ligands for TLR4

Vaginal support as determined by levator ani defect status 6 weeks after primary surgery for pelvic organ prolapse

ObjectiveTo evaluate whether major levator ani muscle defects were associated with differences in postoperative vaginal support after primary surgery for pelvic organ prolapse (POP).MethodsA retrospective chart review of a subgroup of patients in the Organ Prolapse and Levator (OPAL) study. Of the 247 women recruited into OPAL, 107 underwent surgery for prolapse and were the cohort for the present analysis. Major levator ani defects were diagnosed when more than 50% of the pubovisceral muscle was missing on MRI. Postoperative vaginal support was assessed via POPâ quantification system. Postoperative anatomic outcome was analyzed according to levator ani defect status, as determined by MRI.ResultsSupport of the anterior vaginal wall 2 cm above the hymen occurred among 62% of women with normal levator ani muscles/minor defects and 35% of those with major defects. Support of the anterior wall 1 cm above the hymen occurred among 32% women with normal muscles /minor defects and 59% of those with major defects. Levator ani defects were not associated with differences in postoperative apical/posterior vaginal support.ConclusionSix weeks after primary surgery for prolapse, women with normal levator ani muscles/minor defects had better anterior vaginal support than those with major levator defects.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135557/1/ijgo141.pd

Why do women have stress urinary incontinence?

This article reviews progress made in understanding the causes of stress urinary incontinence. Over the last century, several hypotheses have been proposed to explain stress urinary incontinence. These theories are based on clinical observations and focus primarily on the causative role of urethral support loss and an open vesical neck. Recently these hypotheses have been tested by comparing measurements of urethral support and function in women with primary stress urinary incontinence to asymptomatic volunteers who were recruited to be similar in age, race, and parity. Maximal urethral closure pressure is the parameter that differs the most between groups being 43% lower in women with stress incontinence than similar asymptomatic women having as effect size of 1.6. Measures of urethral support effect sizes range from 0.5 to 0.6. Because any one objective measure of support may not capture the full picture of urethrovesical mobility, review of blinded ultrasounds of movements during cough were reviewed by an expert panel. The panel was able to identify women with stress incontinence correctly 57% of the time; just 7% above the 50% that would be expected by chance alone, confirming that urethrovesical mobility is not strongly associated with stress incontinence. Although operations that provide differential support to the urethra are effective, urethral support is not the predominant cause of stress incontinence. Improving our understanding of factors affecting urethral closure may lead to novel treatments targeting the urethra and improved understanding of the small but persistent failure rate of current surgery. Neurourol. Urodynam. 29:S13–S17, 2010. © 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71377/1/20888_ftp.pd

Exploiting the DNA Damaging Activity of Liposomal Low Dose Cytarabine for Cancer Immunotherapy

Perhaps the greatest limitation for the continually advancing developments in cancer immunotherapy remains the immunosuppressive tumor microenvironment (TME). The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis is an emerging immunotherapy target, with the resulting type I interferons and transcription factors acting at several levels in both tumor and immune cells for the generation of adaptive T cell responses. The cGAS-STING axis activation by therapeutic agents that induce DNA damage, such as certain chemotherapies, continues to be reported, highlighting the importance of the interplay of this signaling pathway and the DNA damage response in cancer immunity/immunotherapy. We have developed a multi-targeted mannosylated cationic liposomal immunomodulatory system (DS) which contains low doses of the chemotherapeutic cytarabine (Ara-C). In this work, we show that entrapment of non-cytotoxic doses of Ara-C within the DS improves its ability to induce DNA double strand breaks in human ovarian and colorectal cancer cell lines, as well as in various immune cells. Importantly, for the first time we demonstrate that the DNA damage induced by Ara-C/DS translates into cGAS-STING axis activation. We further demonstrate that Ara-C/DS-mediated DNA damage leads to upregulation of surface expression of immune ligands on cancer cells, coinciding with priming of cytotoxic lymphocytes as assessed using an ex vivo model of peripheral blood mononuclear cells from colorectal cancer patients, as well as an in vitro NK cell model. Overall, the results highlight a broad immunotherapeutic potential for Ara-C/DS by enhancing tumor-directed inflammatory responses. </br

Improving diaper design to address incontinence associated dermatitis

<p>Abstract</p> <p>Background</p> <p>Incontinence associated dermatitis (IAD) is an inflammatory skin disease mainly triggered by prolonged skin contact with urine, feces but also liberal detergent use when cleansing the skin. To minimize the epidermal barrier challenge we optimized the design of adult incontinence briefs. In the fluid absorption area we interposed a special type of acidic, curled-type of cellulose between the top sheet in contact with the skin and the absorption core beneath containing the polyacrylate superabsorber. The intention was to minimize disturbance of the already weak acid mantle of aged skin. We also employed air-permeable side panels to minimize skin occlusion and swelling of the stratum corneum.</p> <p>Methods</p> <p>The surface pH of diapers was measured after repeated wetting with a urine substitute fluid at the level of the top sheet. Occlusive effects and hydration of the stratum corneum were measured after a 4 hour application of different side panel materials by corneometry on human volunteers. Finally, we evaluated skin symptoms in 12 patients with preexisting IAD for 21 days following the institutional switch to the optimized diaper design. Local skin care protocols remained in place unchanged.</p> <p>Results</p> <p>The improved design created a surface pH of 4.6 which was stable even after repeated wetting throughout a 5 hour period. The "standard design" briefs had values of 7.1, which is alkaline compared to the acidic surface of normal skin. Side panels made from non-woven material with an air-permeability of more than 1200 l/m²/s avoided excessive hydration of the stratum corneum when compared to the commonly employed air-impermeable plastic films. Resolution of pre-existing IAD skin lesions was noted in 8 out of 12 patients after the switch to the optimized brief design.</p> <p>Conclusions</p> <p>An improved design of adult-type briefs can create an acidic pH on the surface and breathable side panels avoid over-hydration of the stratum corneum and occlusion. This may support the epidermal barrier function and may help to reduce the occurrence of IAD.</p