Hell's Itch: A Unique Reaction to UV Exposure.

We present a survey-based exploration of Hell's Itch, a severe dermatologic reaction often mistaken for sunburn, that reveals distinct symptoms including intense pain, unrelenting itching, paresthesia, and even suicidal ideation, differentiating it from a typical sunburn.

The Correlation of Sleep Disturbance and Location of Glioma Tumors: A Narrative Review.

Sleep disturbance can occur when sleep centers of the brain, regions that are responsible for coordinating and generating healthy amounts of sleep, are disrupted by glioma growth or surgical resection. Several disorders cause disruptions to the average duration, quality, or patterns of sleep, resulting in sleep disturbance. It is unknown whether specific sleep disorders can be reliably correlated with glioma growth, but there are sufficient numbers of case reports to suggest that a connection is possible. In this manuscript, these case reports and retrospective chart reviews are considered in the context of the current primary literature on sleep disturbance and glioma diagnosis to identify a new and useful connection which warrants further systematic and scientific examination in preclinical animal models. Confirmation of the relationship between disruption of the sleep centers in the brain and glioma location could have significant implications for diagnostics, treatment, monitoring of metastasis/recurrence, and end-of-life considerations.

Moving beyond Titers.

Vaccination to prevent and even eliminate disease is amongst the greatest achievements of modern medicine. Opportunities remain in vaccine development to improve protection across the whole population. A next step in vaccine development is the detailed molecular characterization of individual humoral immune responses against a pathogen, especially the rapidly evolving pathogens. New technologies such as sequencing the immune repertoire in response to disease, immunogenomics/vaccinomics, particularly the individual HLA variants, and high-throughput epitope characterization offer new insights into disease protection. Here, we highlight the emerging technologies that could be used to identify variation within the human population, facilitate vaccine discovery, improve vaccine safety and efficacy, and identify mechanisms of generating immunological memory. In today's vaccine-hesitant climate, these techniques used individually or especially together have the potential to improve vaccine effectiveness and safety and thus vaccine uptake rates. We highlight the importance of using these techniques in combination to understand the humoral immune response as a whole after vaccination to move beyond neutralizing titers as the standard for immunogenicity and vaccine efficacy, especially in clinical trials.

Potential Therapeutic Targets for Combination Antibody Therapy against Pseudomonas aeruginosa Infections.

Despite advances in antimicrobial therapy and even the advent of some effective vaccines, Pseudomonas aeruginosa (P. aeruginosa) remains a significant cause of infectious disease, primarily due to antibiotic resistance. Although P. aeruginosa is commonly treatable with readily available therapeutics, these therapies are not always efficacious, particularly for certain classes of patients (e.g., cystic fibrosis (CF)) and for drug-resistant strains. Multi-drug resistant P. aeruginosa infections are listed on both the CDC's and WHO's list of serious worldwide threats. This increasing emergence of drug resistance and prevalence of P. aeruginosa highlights the need to identify new therapeutic strategies. Combinations of monoclonal antibodies against different targets and epitopes have demonstrated synergistic efficacy with each other as well as in combination with antimicrobial agents typically used to treat these infections. Such a strategy has reduced the ability of infectious agents to develop resistance. This manuscript details the development of potential therapeutic targets for polyclonal antibody therapies to combat the emergence of multidrug-resistant P. aeruginosa infections. In particular, potential drug targets for combinational immunotherapy against P. aeruginosa are identified to combat current and future drug resistance.

The next frontier of oncotherapy: accomplishing clinical translation of oncolytic bacteria through genetic engineering.

The development of a 'smart' drug capable of distinguishing tumor from host cells has been sought for centuries, but the microenvironment of solid tumors continues to confound therapeutics. Solid tumors present several challenges for current oncotherapeutics, including aberrant vascularization, hypoxia, necrosis, abnormally high pH and local immune suppression. While traditional chemotherapeutics are limited by such an environment, oncolytic microbes are drawn to it - having an innate ability to selectively infect, colonize and eradicate solid tumors. Development of an oncolytic species would represent a shift in the cancer therapeutic paradigm, with ramifications reaching from the medical into the socio-economic. Modern genetic engineering techniques could be implemented to customize 'Frankenstein' bacteria with advantageous characteristics from several species.

Lay abstract Side effects of chemotherapeutics are thought to often be a reflection of our inability to target these toxic substances to only cancer cells; hence, scientists have spent centuries searching for alternative treatments that would confine their actions to tumor cells, sparing healthy tissue. Unfortunately, the dense nature of tumor tissue along with altered blood vessels, that lead to diminished tumor tissue oxygenation, altered tissue pH and cellular metabolic inactivity or even cell death have proven challenging. Importantly, these barriers have contributed to local and even sometimes systemic suppression of the patient's immune system that can allow the tumor to grow and progress unchecked. While most non-cancer cells are inhibited by the local tumor environment, certain microbes, including some bacteria and viruses, are drawn to it, possessing a natural ability to selectively infect, colonize and eradicate solid tumors. These microbes may also restore the patient's immune balance. However, use of these microbes is not without its own problems; nevertheless, modern genetic engineering techniques could be implemented to develop customized, safe, effective bacteria with advantageous characteristics. The development and clinical translation of cancer-fighting bacteria would represent a shift in cancer therapeutics and would have ramifications that reach beyond medical efficacy into the realm of socioeconomics. This review seeks to marry the current field of oncolytic bacteria with the expanding field of modern bacterial genetic engineering techniques in prospect of such a therapeutic.

Epitope characterization of anti-drug antibodies-a tool for discovery and health: an overview of the necessity of early epitope characterization to avoid anti-drug antibodies and promote patient health.

Introduction: The market for monoclonal antibody (mAb) therapies is growing rapidly as the pharmaceutical industry expands its development across a broad spectrum of diseases. Unfortunately, as shown in the recent failure of bococizumab by Pfizer, these treatments often stimulate the formation of problematic anti-drug antibodies (ADAs). ADAs can cause side effects and limit efficacy for many patients. To increase efficacy and decrease safety concerns from ADAs, immunogenicity characterization is needed early in the drug development process. Here, we present emerging techniques that hold promise to improve ADA assays and their potential applications to pharmaceutical development and personalized medicine.Areas covered: This manuscript outlines the importance of epitope characterization to better understand immunogenicity and describes a strategy for using this information in treating patients taking mAb therapies.Expert opinion: We propose using high-information assays to characterize epitopes to help mAb therapy engineering and potentially improve individual patient outcomes. To understand this, we will discuss three different aspects of ADAs: (1) the problem of ADAs and what is currently being done about them, (2) the current state of epitope characterization and how it is being utilized, and (3) how early epitope characterization can advance drug discovery and improve outcomes for patients taking mAb therapies.

Antibody Printing Technologies.

Antibody microarrays are routinely employed in the lab and in the clinic for studying protein expression, protein-protein, and protein-drug interactions. The microarray format reduces the size scale at which biological and biochemical interactions occur, leading to large reductions in reagent consumption and handling times while increasing overall experimental throughput. Specifically, antibody microarrays, as a platform, offer a number of different advantages over traditional techniques in the areas of drug discovery and diagnostics. While a number of different techniques and approaches have been developed for creating micro and nanoscale antibody arrays, issues relating to sensitivity, cost, and reproducibility persist. The aim of this review is to highlight current state-of the-art techniques and approaches for creating antibody arrays by providing latest accounts of the field while discussing potential future directions.

Localization of the Interaction Site of Herpes Simplex Virus Glycoprotein D (gD) on the Membrane Fusion Regulator, gH/gL.

A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, and gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL-blocking MAbs were located within flexible regions of the gH N terminus and the gL C terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, "stabilizing" MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV.IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with a receptor to activate the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we have identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibodies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion, which identifies targets for vaccine development.

Characterizing Epitope Binding Regions of Entire Antibody Panels by Combining Experimental and Computational Analysis of Antibody: Antigen Binding Competition.

Vaccines and immunotherapies depend on the ability of antibodies to sensitively and specifically recognize particular antigens and specific epitopes on those antigens. As such, detailed characterization of antibody-antigen binding provides important information to guide development. Due to the time and expense required, high-resolution structural characterization techniques are typically used sparingly and late in a development process. Here, we show that antibody-antigen binding can be characterized early in a process for whole panels of antibodies by combining experimental and computational analyses of competition between monoclonal antibodies for binding to an antigen. Experimental "epitope binning" of monoclonal antibodies uses high-throughput surface plasmon resonance to reveal which antibodies compete, while a new complementary computational analysis that we call "dock binning" evaluates antibody-antigen docking models to identify why and where they might compete, in terms of possible binding sites on the antigen. Experimental and computational characterization of the identified antigenic hotspots then enables the refinement of the competitors and their associated epitope binding regions on the antigen. While not performed at atomic resolution, this approach allows for the group-level identification of functionally related monoclonal antibodies (i.e., communities) and identification of their general binding regions on the antigen. By leveraging extensive epitope characterization data that can be readily generated both experimentally and computationally, researchers can gain broad insights into the basis for antibody-antigen recognition in wide-ranging vaccine and immunotherapy discovery and development programs.

Dynamics of a Viscous Droplet in Return Bends of Microfluidic Channels.

Return bends are frequently encountered in microfluidic systems. In this study, a three-dimensional spectral boundary element method for interfacial dynamics in Stokes flow has been adopted to investigate the dynamics of viscous droplets in rectangular return bends. The droplet trajectory, deformation, and migration velocity are investigated under the influence of various fluid properties and operational conditions, which are depicted by the Capillary number, viscosity ratio, and droplet size, as well as the dimensions of the return bend. While the computational results provide information for the design of return bends in microfluidic systems in general, the computational framework shows potential to guide the design and operation of a droplet-based microfluidic delivery system for cell seeding.

Surface Plasmon Resonance Reveals Direct Binding of Herpes Simplex Virus Glycoproteins gH/gL to gD and Locates a gH/gL Binding Site on gD.

Herpes simplex virus (HSV) requires fusion between the viral envelope and host membrane. Four glycoproteins, gD, gH/gL, and gB, are essential for this process. To initiate fusion, gD binds its receptor and undergoes a conformational change that hypothetically leads to activation of gH/gL, which in turn triggers the fusion protein gB to undergo rearrangements leading to membrane fusion. Our model predicts that gD must interact with both its receptor and gH/gL to promote fusion. In support of this, we have shown that gD is structurally divided into two "faces": one for the binding receptor and the other for its presumed interaction with gH/gL. However, until now, we have been unable to demonstrate a direct interaction between gD and gH/gL. Here, we used surface plasmon resonance to show that the ectodomain of gH/gL binds directly to the ectodomain of gD when (i) gD is captured by certain anti-gD monoclonal antibodies (MAbs) that are bound to a biosensor chip, (ii) gD is bound to either one of its receptors on a chip, and (iii) gD is covalently bound to the chip surface. To localize the gH/gL binding site on gD, we used multiple anti-gD MAbs from six antigenic communities and determined which ones interfered with this interaction. MAbs from three separate communities block gD-gH/gL binding, and their epitopes encircle a geographical area on gD that we propose comprises the gH/gL binding domain. Together, our results show that gH/gL interacts directly with gD, supporting a role for this step in HSV entry.IMPORTANCE HSV entry is a multistep process that requires the actions of four glycoproteins, gD, gH/gL, and gB. Our current model predicts that gD must interact with both its receptor and gH/gL to promote viral entry. Although we know a great deal about how gD binds its receptors, until now we have been unable to demonstrate a direct interaction between gD and gH/gL. Here, we used a highly sensitive surface plasmon resonance technique to clearly demonstrate that gD and gH/gL interact. Furthermore, using multiple MAbs with defined epitopes, we have delineated a domain on gD that is independent of that used for receptor binding and which likely represents the gH/gL interaction domain. Targeting this interaction to prevent fusion may enhance both therapeutic and vaccine strategies.

Vaccine-induced antibodies to herpes simplex virus glycoprotein D epitopes involved in virus entry and cell-to-cell spread correlate with protection against genital disease in guinea pigs.

Herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) subunit antigen is included in many preclinical candidate vaccines. The rationale for including gD2 is to produce antibodies that block crucial gD2 epitopes involved in virus entry and cell-to-cell spread. HSV-2 gD2 was the only antigen in the Herpevac Trial for Women that protected against HSV-1 genital infection but not HSV-2. In that trial, a correlation was detected between gD2 ELISA titers and protection against HSV-1, supporting the importance of antibodies. A possible explanation for the lack of protection against HSV-2 was that HSV-2 neutralization titers were low, four-fold lower than to HSV-1. Here, we evaluated neutralization titers and epitope-specific antibody responses to crucial gD2 epitopes involved in virus entry and cell-to-cell spread as correlates of immune protection against genital lesions in immunized guinea pigs. We detected a strong correlation between neutralizing antibodies and protection against genital disease. We used a high throughput biosensor competition assay to measure epitope-specific responses to seven crucial gD2 linear and conformational epitopes involved in virus entry and spread. Some animals produced antibodies to most crucial epitopes while others produced antibodies to few. The number of epitopes recognized by guinea pig immune serum correlated with protection against genital lesions. We confirmed the importance of antibodies to each crucial epitope using monoclonal antibody passive transfer that improved survival and reduced genital disease in mice after HSV-2 genital challenge. We re-evaluated our prior study of epitope-specific antibody responses in women in the Herpevac Trial. Humans produced antibodies that blocked significantly fewer crucial gD2 epitopes than guinea pigs, and antibody responses in humans to some linear epitopes were virtually absent. Neutralizing antibody titers and epitope-specific antibody responses are important immune parameters to evaluate in future Phase I/II prophylactic human vaccine trials that contain gD2 antigen.

Using hydrodynamic focusing to predictably alter the diameter of synthetic silk fibers.

Spiders and silkworms provide a model of superior processing for multifunctional and highly versatile high-performance fibers. Mimicking the spider's complex control system for chemical and mechanical gradients has remained an ongoing obstacle for synthetic silk production. In this study, the use of hydrodynamic fluid focusing within a 3D printed biomimetic spinning system to recapitulate the biological spinneret is explored and shown to produce predictable, small diameter fibers. Mirroring in silico fluid flow simulations using a hydrodynamic microfluidic spinning technique, we have developed a model correlating spinning rates, solution viscosity and fiber diameter outputs that will significantly advance the field of synthetic silk fiber production. The use of hydrodynamic focusing to produce controlled output fiber diameter simulates the natural silk spinning process and continues to build upon a 3D printed biomimetic spinning platform.

Global sensing of the antigenic structure of herpes simplex virus gD using high-throughput array-based SPR imaging.

While HSV-2 typically causes genital lesions, HSV-1 is increasingly the cause of genital herpes. In addition, neonatal HSV infections are associated with a high rate of mortality and HSV-2 may increase the risk for HIV or Zika infections, reinforcing the need to develop an effective vaccine. In the GSK Herpevac trial, doubly sero-negative women were vaccinated with a truncated form of gD2 [gD2(284t)], then examined for anti-gD serum titers and clinical manifestations of disease. Surprisingly, few vaccinees were protected against genital HSV-2 but 86% were protected from genital HSV-1. These observations suggest that subtle differences in gD structure might influence a protective response. To better understand the antigenic structure of gD and how it impacts a protective response, we previously utilized several key anti-gD monoclonal antibodies (mAbs) to dissect epitopes in vaccinee sera. Several correlations were observed but the methodology limited the number of sera and mAbs that could be tested. Here, we used array-based surface plasmon imaging (SPRi) to simultaneously measure a larger number of protein-protein interactions. We carried out cross-competition or "epitope binning" studies with 39 anti-gD mAbs and four soluble forms of gD, including a form [gD2(285t)] that resembles the Herpevac antigen. The results from these experiments allowed us to organize the mAbs into four epitope communities. Notably, relationships within and between communities differed depending on the form of gD, and off-rate analysis suggested differences in mAb-gD avidity depending on the gD serotype and length. Together, these results show that gD1 and gD2 differ in their structural topography. Consistent with the Herpevac results, several mAbs that bind both gD1 and gD2 neutralize only HSV-1. Thus, this technology provides new insights into the antigenic structure of gD and provides a rationale as to how vaccination with a gD2 subunit may lead to protection from HSV-1 infection.

The Complex Relationship between Virulence and Antibiotic Resistance.

Antibiotic resistance, prompted by the overuse of antimicrobial agents, may arise from a variety of mechanisms, particularly horizontal gene transfer of virulence and antibiotic resistance genes, which is often facilitated by biofilm formation. The importance of phenotypic changes seen in a biofilm, which lead to genotypic alterations, cannot be overstated. Irrespective of if the biofilm is single microbe or polymicrobial, bacteria, protected within a biofilm from the external environment, communicate through signal transduction pathways (e.g., quorum sensing or two-component systems), leading to global changes in gene expression, enhancing virulence, and expediting the acquisition of antibiotic resistance. Thus, one must examine a genetic change in virulence and resistance not only in the context of the biofilm but also as inextricably linked pathologies. Observationally, it is clear that increased virulence and the advent of antibiotic resistance often arise almost simultaneously; however, their genetic connection has been relatively ignored. Although the complexities of genetic regulation in a multispecies community may obscure a causative relationship, uncovering key genetic interactions between virulence and resistance in biofilm bacteria is essential to identifying new druggable targets, ultimately providing a drug discovery and development pathway to improve treatment options for chronic and recurring infection.

Energy-triggered drug release from polymer nanoparticles for orthopedic applications.

Sequestra, present in many cancers and orthopedic infections, provide a safe harbor for the development of drug resistance. In the face of burgeoning drug resistance, the importance of nanoscale, microenvironment-triggered drug delivery cannot be overestimated. Such strategies may preserve pharmaceutical efficacy and significantly alter the etiology of many orthopedic diseases. Although temperature-, pH- and redox-responsive nanoparticle-based systems have been extensively studied, local drug delivery from polymeric nanoparticles can be triggered by a variety of energy forms. This review offers an overview of the state of the field as well as a perspective on the safety and efficacy of ultrasound, hyperthermia and radio frequency-triggered internal delivery systems in a variety of applications.

Prostate-Specific Membrane Antigen Targeted Polymersomes for Delivering Mocetinostat and Docetaxel to Prostate Cancer Cell Spheroids.

Prostate cancer cells overexpress the prostate-specific membrane antigen (PSMA) receptors on the surface. Targeting the PSMA receptor creates a unique opportunity for drug delivery. Docetaxel is a Food and Drug Administration-approved drug for treating metastatic and androgen-independent prostate cancer, and mocetinostat is a potent inhibitor of class I histone deacetylases. In this study, we prepared reduction-sensitive polymersomes presenting folic acid on the surface and encapsulating either docetaxel or mocetinostat. The presence of folic acid allowed efficient targeting of the PSMA receptor and subsequent internalization of the polymeric vesicles in cultured LNCaP prostate cancer cell spheroids. The intracellular reducing agents efficiently released docetaxel and mocetinostat from the polymersomes. The combination of the two drug-encapsulated polymersome formulations significantly (p < 0.05) decreased the viability of the LNCaP cells (compared to free drugs or control) in three-dimensional spheroid cultures. The calculated combination index value indicated a synergistic effect for the combination of mocetinostat and docetaxel. Thus, our PSMA-targeted drug-encapsulated polymersomes has the potential to lead to a new direction in prostate cancer therapy that decreases the toxicity and increases the efficacy of the drug delivery systems.

An Osteoconductive Antibiotic Bone Eluting Putty with a Custom Polymer Matrix.

With the rising tide of antibiotic resistant bacteria, extending the longevity of the current antibiotic arsenal is becoming a necessity. Developing local, controlled release antibiotic strategies, particularly for difficult to penetrate tissues such as bone, may prove to be a better alternative. Previous efforts to develop an osteoconductive local antibiotic release device for bone were created as solid molded composites; however, intimate contact with host bone was found to be critical to support host bone regrowth; thus, an osteocondconductive antibiotic releasing bone void filling putty was developed. Furthermore, a controlled releasing polymer matrix was refined using pendant-functionalized diols to provide tailorable pharmacokinetics. In vitro pharmacokinetic and bioactivity profiles were compared for a putty formulation with an analogous composition as its molded counterpart as well as four new pendant-functionalized polymers. A best-fit analysis of polymer composition in either small cylindrical disks or larger spheres revealed that the new pendant-functionalized polymers appear to release vancomycin via both diffusion and erosion regardless of the geometry of the putty. In silico simulations, a valuable technique for diffusion mediated controlled release models, will be used to confirm and optimize this property.

Optimal tube length for the submerged printing of ovarian cancer cells.

Ovarian cancer is the fifth most common cancer affecting US women, killing more women each year than all other gynecologic cancers combined. Treatment of ovarian cancer is challenging with an overall 5-year survival rates of only 28-46% based on the metastatic state of the disease. While overall survival has improved with modern chemotherapy, poor outcomes have persisted. One of the greatest challenges in cancer therapeutic research remains that late-stage drug development trials for drug candidates have high attrition rates, up to 70% in Phase II and 59% in Phase III trials. The development of in vitro, high-throughput, cell based assays could provide a tool to overcome the challenges associated with high attrition rates by allowing for controlled cell deposition with a defined, controlled phenotype. Submerged, three-dimensional (3D) microfluidic printing technology is uniquely capable of controlling cell deposition without sacrificing the viability of cells for cell-based assays. Here, we investigate the phenotypic effects of tube length during printing on the cells. We observe that the length of the tube has minimal effects on the viability and density of A2780 ovarian cancer cells different cell lines. This study details foundational information for developing a high-throughput cell-based assays (CBA) for screening effective cancer drug candidates.

Comparison of submerged and unsubmerged printing of ovarian cancer cells.

A high-throughput cell based assay would greatly aid in the development and screening of ovarian cancer drug candidates. Previously, a three-dimensional microfluidic printer that is not only capable of controlling the location of cell deposition, but also of maintaining a liquid, nutrient rich environment to preserve cellular phenotype has been developed (Wasatch Microfluidics). In this study, we investigated the impact (i.e., viability, density, and phenotype) of depositing cells on a surface submerged in cell culture media. It was determined that submersion of the microfluidic print head in cell media did not alter the cell density, viability, or phenotype.. This article describes an in depth study detailing the impact of one of the fundamental components of a 3D microfluidic cell printer designed to mimic the in vivo cell environment. Development of such a tool holds promise as a high-throughput drug-screening platform for new cancer therapeutics.