Virus-Shaped Mesoporous Silica Nanostars to Improve the Transport of Drugs across the Blood-Brain Barrier.

Conditions affecting the brain are the second leading cause of death globally. One of the main challenges for drugs targeting brain diseases is passing the blood-brain barrier (BBB). Here, the effectiveness of mesoporous silica nanostars (MSiNSs) with two different spike lengths to cross an in vitro BBB multicellular model was evaluated and compared to spherical nanoparticles (MSiNP). A modified sol-gel single-micelle epitaxial growth was used to produce MSiNS, which showed no cytotoxicity or immunogenicity at concentrations of up to 1 µg mL-1 in peripheral blood mononuclear and neuronal cells. The nanostar MSiNS effectively penetrated the BBB model after 24 h, and MSiNS-1 with a shorter spike length (9 ± 2 nm) crossed the in vitro BBB model more rapidly than the MSiNS-2 with longer spikes (18 ± 4 nm) or spherical MSiNP at 96 h, which accumulated in the apical and basolateral sides, respectively. Molecular dynamic simulations illustrated an increase in configurational flexibility of the lipid bilayer during contact with the MSiNS, resulting in wrapping, whereas the MSiNP suppressed membrane fluctuations. This work advances an effective brain drug delivery system based on virus-like shaped MSiNS for the treatment of different brain diseases and a mechanism for their interaction with lipid bilayers.

Nine treatments of 1000 mL therapeutic phlebotomy in a subject with polycythemia: A case report.

Large-volume therapeutic phlebotomy is the mainstay of hemochromatosis treatment and offers an opportunity to investigate the hemodynamic changes during acute hypovolemia. An otherwise healthy 64-year-old male with hemochromatosis participated. On nine separate visits, 1000 mL therapeutic phlebotomy was performed. On one occasion, pre- and post-phlebotomy orthostatic challenge with 27° reverse Trendelenburg position was administered. Mean arterial pressure, heart rate, and stroke volume were measured continuously during the procedures. The patient's tolerance to the interventions was continuously evaluated. The procedures were well tolerated by the patient. Mean arterial pressure was maintained during hemorrhage and following phlebotomy in both supine and reverse Trendelenburg positions, primarily through an increase in heart rate and systemic vascular resistance. The present study found that 1000 mL therapeutic phlebotomy in a patient with hemochromatosis may be acceptably and safely used to model hemorrhage. The approach demonstrates high clinical applicability and ethically robustness in comparison with volunteer studies.

Reconstitution of the alternative pathway of the complement system enables rapid delineation of the mechanism of action of novel inhibitors.

The complement system plays a critical role in the innate immune response, acting as a first line of defense against invading pathogens. However, dysregulation of the complement system is implicated in the pathogenesis of numerous diseases, ranging from Alzheimer's to age-related macular degeneration and rare blood disorders. As such, complement inhibitors have enormous potential to alleviate disease burden. While a few complement inhibitors are in clinical use, there is still a significant unmet medical need for the discovery and development of novel inhibitors to treat patients suffering from disorders of the complement system. A key hurdle in the development of complement inhibitors has been the determination of their mechanism of action. Progression along the complement cascade involves the formation of numerous multimeric protein complexes, creating the potential for inhibitors to act at multiple nodes in the pathway. This is especially true for molecules that target the central component C3 and its fragment C3b, which serve a dual role as a substrate for the C3 convertases and as a scaffolding protein in both the C3 and C5 convertases. Here, we report a step-by-step in vitro reconstitution of the complement alternative pathway using bio-layer interferometry. By physically uncoupling each step in the pathway, we were able to determine the kinetic signature of inhibitors that act at single steps in the pathway and delineate the full mechanism of action of known and novel C3 inhibitors. The method could have utility in drug discovery and further elucidating the biochemistry of the complement system.

Valency of Ligand-Receptor Binding from Pair Potentials.

Coarse grained molecular dynamics simulations have been crucial for investigating the dynamics of nanoparticle uptake by cell membranes via ligand-receptor interactions. These models have enabled researchers to evaluate the effects of nanoparticle size, shape, and ligand distribution on cellular uptake. However, when pair potentials are used to represent ligand-receptor interactions, the number of receptors interacting with one ligand, valency, may vary. We demonstrate that the curvature of a nanoparticle, strength of ligand-receptor interactions, and ligand or receptor concentration change the valency, ranging from 3.4 to 5.1 in this study. Such a change in valency can create inaccurate comparisons between nanoparticles or even result in the uptake of smaller nanoparticles than would be expected. To rectify this inconsistency, we propose the adoption of a model based on bond formation and use it to determine the extent to which previous studies may have been affected. This work recommends avoiding pair potentials for modeling ligand-receptor interactions to ensure methodological consistency in nanoparticle studies.

Modeling Au Nanostar Geometry in Bulk Solutions.

The findings within make it possible to reference gold nanostars based on their geometric properties, similar to how a radius describes a nanosphere, rather than just the LSPR of the structure-the current practice. The average tip approximation presented reduces the complexity of nanostars in discrete dipole approximation simulations. By matching the projected area and LSPR of the modeled nanostars to synthesized nanostars, the volume, surface area, and number of tips can be approximated without a lengthy characterization process. Knowing the nanoparticle geometry can determine drug carrier capacity, an approximate number of hot spots for EM imaging, and how the particle will interact with cells. The geometric data obtained will drive the biological application and increase the usability of this particle class.

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis.

The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

Feasibility of effectiveness trials in pediatric anesthesia.

BACKGROUND: Effectiveness studies aim to investigate whether an intervention does more good than harm in normal clinical practice. DISCUSSION: Historically, research in pediatric anesthesia has appropriately concentrated on efficacy studies which even if of excellent design, tell us little about how generalizable or applicable the findings are to routine clinical practice. Simultaneously effectiveness and cost-effectiveness research has been limited due to systematic and cultural factors. To understand the challenges of effectiveness research in children, it is necessary to examine the overlapping relationship between efficacy, effectiveness, and cost-effectiveness studies. This can be illuminated with Cochrane´s hierarchy of investigation and the disease, illness, and sickness modelfrom the philosophy of science. Effectiveness (illness perspective) studies form the middle rung of Cochrane´s research hierarchy, between efficacy (disease perspective) and cost-effectiveness (sickness perspective) research. Effectiveness studies aim to be generalizable and determine whether interventions work in routine clinical practice. Since outcomes that reflect patient priorities offer the most generalizability these are encouraged in effectiveness studies. Due to developing consciousness and communication in young children, identifying and measuring patient-centered outcomes has challenges. These challenges are not insurmountable and with appropriate communication and research techniques the subjective nature of the experience of illness in the young may be elucidated and should direct research goals where possible. Sickness perspective research, in terms of cost-effectiveness, remains underdeveloped in pediatric anesthesia. SUMMARY: Pediatric anesthesia has a strong base in efficacy research, but the need to expand research to include effectiveness and eventually cost-effectiveness studies should not be ignored.

Colonic perforation following polypectomy of a gastrointestinal follicular lymphoma masquerading as a colon polyp.

INTRODUCTION: The gastrointestinal (GI) tract is the predominant site for primary extranodal non-Hodgkin lymphomas (NHL), accounting for 5-10% of all extranodal disease. CASE: A 74-year-old man underwent colonoscopy for a positive fecal occult blood test. Colonoscopy revealed a 3.5-cm polyp in the descending colon and was removed by snare cautery polypectomy. Post-polypectomy site showed a 0.3-0.5-cm mucosal defect. Five endoclips were applied to close the mucosal defect. The patient remained stable during subsequent intensive monitoring and never required surgical intervention. Pathology of the polyp revealed follicular lymphoma (FL) involving the lamina propria of the mucosa with extension into the submucosa. The patient had no systemic symptoms, and staging for NHL with contrast computerized tomography of the chest, abdomen, and pelvis revealed no evidence suggestive of lymphoma. DISCUSSION: Approximately 6-20% of all primary GI-NHL are in the colon. The frequency of GI-FL accounts for 1-3.6% of all GI-NHL. After a search of the current literature, there have been no cases of a follicular lymphoma presenting solely as an isolated colon polyp. Likewise, bowel perforation due to polypectomy of such polyps has never been cited. Retrospectively, the diagnosis and extent of the polyp could have been established using endoscopic end-to-end forceps biopsy and/or endoscopic ultrasound with a radial scanning catheter probe and fine-needle aspiration of the lesion. Such a diagnosis could have changed the strategy for endoscopic removal of the polyp. Our case is interesting because it is the first report of a follicular lymphoma presenting as a single isolated colon polyp involving all layers of the colonic mucosa.

Giant gastric hyperplastic polyp: not always a benign lesion.

Giant gastric hyperplastic polyps constitute of around 76% of all gastric polyps found. They are often found incidentally on upper GI endoscopy. They often present with occult GI bleeding causing iron deficiency anemia or partial gastric outlet obstruction. Although mostly benign, they do have potential for malignant transformation and hence must be excised endoscopically or surgically, whichever may be feasible. We hereby present a couple of cases of gastric hyperplastic polyps in an attempt to add to the current literature on this rather rare entity.

Specific pathogen-free macaques: definition, history, and current production.

Specific pathogen-free (SPF) macaque colonies are now requested frequently as a resource for research. Such colonies were originally conceived as a means to cull diseased animals from research-dedicated colonies, with the goal of eliminating debilitating or fatal infectious agents from the colony to improve the reproductive capacity of captive research animals. The initial pathogen of concern was Mycobacterium tuberculosis (M.tb.), recognized for many years as a pathogen of nonhuman primates as well as a human health target. More recently attention has focused on four viral pathogens as the basis for an SPF colony: simian type D retrovirus (SRV), simian immunodeficiency virus (SIV), simian T cell lymphotropic/leukemia virus (STLV), and Cercopithecine herpesvirus 1 (CHV-1). New technologies, breeding, and maintenance schemes have emerged to develop and provide SPF primates for research. In this review we focus on the nonhuman primates (NHPs) most common to North American NHP research facilities, Asian macaques, and the most common current research application of these animals, modeling of human AIDS.

Optimization of lipid-indinavir complexes for localization in lymphoid tissues of HIV-infected macaques.

In HIV-infected persons on highly active antiretroviral therapy, residual virus is found in lymphoid tissues. Indinavir concentrations in lymph node mononuclear cells of patients on highly active antiretroviral therapy were approximately 25% to 35% of those in blood mononuclear cells, suggesting that drug insufficiency contributes to residual virus in lymphoid tissues. Therefore, we developed novel lipid-indinavir nanoparticles targeted to lymphoid tissues. Given subcutaneously, these nanoparticles provided indinavir concentrations 250% to 2270% higher than plasma indinavir concentrations in both peripheral and visceral lymph nodes. Improved indinavir delivery was reflected in reduced viral RNA and CD4(+) T-cell rebound. This study optimized lipid nanoparticle formulation with respect to indinavir in lymphoid tissues of HIV-infected macaques. Regardless of lipid characteristic tested (charge, fluidity, and steric modification), indinavir binds completely to lipid at pH 7.4 but is reversed at pH 5.5 or lower. Compared with previous formulations, nanoparticles composed of disteroyl phosphatidylcholine and methyl polyethylene glycol-disteroyl phosphatidylethanolamine (DSPC:mPEG-DSPE) provided 6-fold higher indinavir levels in lymph nodes and enhanced drug exposure in blood. Enhanced anti-HIV activity paralleled improved intracellular drug accumulation. Collectively, these data suggest that indinavir nanoparticles composed of DSPC:mPEG-DSPE provided the most effective lymphoid delivery and could maximally suppress the virus in lymphoid tissues.

Serendipitous insights involving nonhuman primates.

Serendipity is discussed as a form of controlled chaos, a phenomenon in a class with synchronicity and other actions affecting research in terms of theory versus observation (e.g., "optional stopping"). Serendipity is a fundamental aspect of basic research, a profitable and normal outcome in the context of "informed observation." The serendipitous finding fits into the following pattern: it is unanticipated, anomalous, and strategic. All observations that have meaning must fit into some context in the observer's mind or suggest a revolutionary new context. It is critically important to maintain access to the resources provided by established primate centers and similar laboratories to capitalize in a timely way on serendipitous findings and to benefit from valuable discoveries made in more directly targeted development investments. Examples are given of serendipitous insights gained in experimentation and observation relative to nonhuman primate research, including both broad and narrow topics. Genomics, which uses comparison-based strategies and capitalizes on the DNA sequences of genetic information, presents what might seem the basis for endless serendipity because nonhuman primates are likely to share most genes present in the human genome. Other topics discussed include infant behavior, birth periodicity, leprosy, cystic fibrosis, environmental enrichment, endocrinology, drug development, and the rapidly expanding study of infectious diseases and pathogen-based bioterrorism.

SPL7013 gel as a topical microbicide for prevention of vaginal transmission of SHIV89.6P in macaques.

SPL7013 is a dendrimer with a polyanionic outer surface that allows multiple interactions with target sites. It potently binds and blocks HIV-1 and chimeric simian/HIV-1 viruses (SHIVs) replication in vitro. Gels containing different concentrations of SPL7013 were used as topical microbicides in female pigtailed macaques (Macaca nemestrina) to study their ability to prevent vaginal transmission of SHIV(89,6P). On virus challenge, all untreated macaques (8/8) and seven of eight macaques treated with placebo gel were infected within 2 weeks postinfection (PI) and showed high plasma viremia and dramatic CD4(+) cell decline within 4 weeks PI. In contrast, 6/6 macaques, 5/6 macaques, and 2/6 macaques treated with 5% w/w (50 mg/ml), 3% w/w (30 mg/ml), and 1% w/w (10 mg/ml) SPL7013 gels, respectively, resisted viral challenge. The results showed that animals treated with SPL7013 showed a dose-dependent resistance to virus challenge. Neither SPL7013 nor placebo gels produced any adverse effects following the single application in the study. These results showed that 3-5% w/w SPL7013 gels were effective in blocking vaginal transmission of SHIV in macaques after single gel application followed by single virus challenge. These results suggest that SPL7013 gel may be a promising anti-HIV microbicide formulation for further evaluation.

HIV in central nervous system and behavioral development: an HIV-2287 macaque model of AIDS.

OBJECTIVE: To determine which route of inoculation produced consistent and frequent HIV infection in the central nervous system (CNS) and alterations in cognitive and motor development in infant macaques. METHODS: Infant macaques (Macaca nemestrina) were inoculated with the highly pathogenic strain HIV-2287 intravenously (n = 3) or intrathecally (n = 3). Uninfected infants were evaluated as controls. Disease progression was evaluated by virological assessment of blood and cerebral spinal fluid (CSF), CD4 T cell count in blood, and quinolinic acid levels in CSF (a surrogate marker of neuronal cell damage). The effect of HIV infection on cognitive and motor development in infants was monitored during the 6-month study. RESULTS: Either route of HIV-2287 inoculation produced detectable viral RNA in CSF and productive infection in blood. Detection of virus in CSF paralleled a rise in quinolinic acid levels. All HIV-infected infants experienced a severe and rapid decline in CD4 T cell counts by 10 weeks after viral infection. HIV-infected infants, particularly those infected by the intravenous route, exhibited delays in reaching cognitive and motor milestones, which paralleled neuropathological changes. CONCLUSIONS: The HIV-2287 infant model produced a high incidence of viral infection in the CNS regardless of the route of inoculation. Significant alteration in neurobehavioral development was observed in HIV-infected infants, and this measure was significantly impaired particularly in infants infected by the intravenous route. These data, coupled with the ability to detect viral RNA and changes in quinolinic acid levels in CSF, may allow quantitative evaluation of drug and immune candidates for treating neurological effects of AIDS.

Cyanovirin-N inhibits AIDS virus infections in vaginal transmission models.

The cyanobacterial protein cyanovirin-N (CV-N) potently inactivates diverse strains of HIV-1 and other lentiviruses due to irreversible binding of CV-N to the viral envelope glycoprotein gp120. In this study, we show that recombinant CV-N effectively blocks HIV-1(Ba-L) infection of human ectocervical explants. Furthermore, we demonstrate the in vivo efficacy of CV-N gel in a vaginal challenge model by exposing CV-N-treated female macaques (Macaca fascicularis) to a pathogenic chimeric SIV/HIV-1 virus, SHIV89.6P. All of the placebo-treated and untreated control macaques (8 of 8) became infected. In contrast, 15 of 18 CV-N-treated macaques showed no evidence of SHIV infection. Further, CV-N produced no cytotoxic or clinical adverse effects in either the in vitro or in vivo model systems. Together these studies suggest that CV-N is a good candidate for testing in humans as an anti-HIV topical microbicide.

Lipid-drug association enhanced HIV-1 protease inhibitor indinavir localization in lymphoid tissues and viral load reduction: a proof of concept study in HIV-2287-infected macaques.

Analysis of indinavir levels in HIV-positive patients indicated that drug concentrations in lymph node mononuclear cells (LNMCs) were about 25-35% of mononuclear cells in blood. To enhance lymphatic delivery of anti-HIV drugs, a novel drug delivery strategy was designed consisting of lipid-associated indinavir (50-80 nm in diameter) complexes in suspension for subcutaneous (SC) injection. Due to the pH-dependent lipophilicity of indinavir, practically all the drug molecules are incorporated into lipid phase when formulated at pH 7.4 and 5:1 lipid-to-drug (m/m) ratio. At pH 5.5, about 20% of drugs were found in lipid-drug complexes. Effects of lipid association on the time course of plasma indinavir concentrations were determined in macaques (Macaca nemestrina) administered with either soluble or lipid-associated formulation of indinavir (10 mg/kg, SC). Results yielded about a 10-fold reduction in peak plasma concentration and a 6-fold enhancement in terminal half-life (t1/2beta = 12 vs. 2 hours). In addition, indinavir concentrations in both peripheral and visceral lymph nodes were 250-2270% higher than plasma (compared with <35% with soluble lipid-free drug administration in humans). Administration of lipid-associated indinavir (20 mg/kg daily) to HIV-2287-infected macaques (at 30-33 weeks after infection) resulted in significantly reduced viral RNA load and increased CD4 T cell number concentrations. Collectively, these data indicate that lipid association greatly enhances delivery of the anti-HIV drug indinavir to lymph nodes at levels that cannot be achieved with soluble drug, provides significant virus load reduction, and could potentially reverse CD4 T cell depletion due to HIV infection.

Cyanovirin-N gel as a topical microbicide prevents rectal transmission of SHIV89.6P in macaques.

Cyanovirin-N (CV-N), an 11-kDa cyanobacterial protein, potently inactivates diverse strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and also prevents virus-to-cell fusion, virus entry, and infection of cells in vitro. These properties make CV-N an attractive candidate for use as a topical microbicide to prevent the sexual transmission of HIV. We evaluated the efficacy of gel-formulated, recombinant CV-N gel asa topical microbicide in male macaques (Macaca fascicularis) that were rectally challenged with a chimeric SIV/HIV-1 virus known as SHIV89.6P. All of the untreated macaques were infected and experienced CD4+T cell depletion. In contrast, none of the macaques that received either 1% or 2% CV-N gel showed evidence of SHIV89.6P infection. Neither CV-N nor placebo gels produced any adverse effects in any macaque following the rectal application. These results indicate that CV-N gel as a topical microbicide can prevent rectal transmission of SHIV in macaques. These studies encourage clinical evaluation of CV-N as a topical microbicide to prevent sexual transmission of HIV in humans.