Collagen mutation and age contribute to differential craniofacial phenotypes in mouse models of osteogenesis imperfecta.

Craniofacial and dentoalveolar abnormalities are present in all types of osteogenesis imperfecta (OI). Mouse models of the disorder are critical to understand these abnormalities and underlying OI pathogenesis. Previous studies on severely affected OI mice report a broad spectrum of craniofacial phenotypes, exhibiting some similarities to the human disorder. The Brtl/+ and G610c/+ are moderately severe and mild-type IV OI, respectively. Little is known about the aging effects on the craniofacial bones of these models and their homology to human OI. This study aimed to analyze the Brtl/+ and G610c/+ craniofacial morphometries during aging to establish suitability for further OI craniofacial bone intervention studies. We performed morphological measurements on the micro-CT-scanned heads of 3-wk-old, 3-mo-old, and 6-mo-old female Brtl/+ and G610c/+ mice. We observed that Brtl/+ skulls are shorter in length than WT (P < .05), whereas G610c/+ skulls are similar in length to their WT counterparts. The Brtl/+ mice exhibit alveolar bone with a porotic-like appearance that is not observed in G610c/+. As they age, Brtl/+ mice show severe bone resorption in both the maxilla and mandible (P < .05). By contrast, G610c/+ mice experience mandibular resorption consistently across all ages, but maxillary resorption is only evident at 6 mo (P < .05). Western blot shows high osteoclastic activities in the Brtl/+ maxilla. Both models exhibit delayed pre-functional eruptions of the third molars (P < .05), which are similar to those observed in some bisphosphonate-treated OI subjects. Our study shows that the Brtl/+ and G610c/+ mice display clear features found in type IV OI patients; both show age-related changes in the craniofacial growth phenotype. Therefore, understanding the craniofacial features of these models and how they age will allow us to select the most accurate mouse model, mouse age, and bone structure for the specific craniofacial bone treatment of differing OI groups.

Vaccine-elicited IL-1R signaling results in Th17 TRM-mediated immunity.

Lung tissue resident memory (TRM) cells are thought to play crucial roles in lung host defense. We have recently shown that immunization with the adjuvant LTA1 (derived from the A1 domain of E. coli heat labile toxin) admixed with OmpX from K. pneumoniae can elicit antigen specific lung Th17 TRM cells that provide serotype independent immunity to members of the Enterobacteriaceae family. However, the upstream requirements to generate these cells are unclear. Single-cell RNA-seq showed that vaccine-elicited Th17 TRM cells expressed high levels of IL-1R1, suggesting that IL-1 family members may be critical to generate these cells. Using a combination of genetic and antibody neutralization approaches, we show that Th17 TRM cells can be generated independent of caspase-1 but are compromised when IL-1α is neutralized. Moreover IL-1α could serve as a molecular adjuvant to generate lung Th17 TRM cells independent of LTA1. Taken together, these data suggest that IL-1α plays a major role in vaccine-mediated lung Th17 TRM generation.

Sex hormone signaling and regulation of immune function.

Immune responses to antigens, including innocuous, self, tumor, microbial, and vaccine antigens, differ between males and females. The quest to uncover the mechanisms for biological sex differences in the immune system has intensified, with considerable literature pointing toward sex hormonal influences on immune cell function. Sex steroids, including estrogens, androgens, and progestins, have profound effects on immune function. As such, drastic changes in sex steroid concentrations that occur with aging (e.g., after puberty or during the menopause transition) or pregnancy impact immune responses and the pathogenesis of immune-related diseases. The effect of sex steroids on immunity involves both the concentration of the ligand and the density and distribution of genomic and nongenomic receptors that serve as transcriptional regulators of immune cellular responses to affect autoimmunity, allergy, infectious diseases, cancers, and responses to vaccines. The next frontier will be harnessing these effects of sex steroids to improve therapeutic outcomes.

Optimization of Host Cell-Compatible, Antimicrobial Peptides Effective against Biofilms and Clinical Isolates of Drug-Resistant Bacteria.

Here, we describe the continued synthetic molecular evolution of a lineage of host-compatible antimicrobial peptides (AMP) intended for the treatment of wounds infected with drug-resistant, biofilm-forming bacteria. The peptides tested are variants of an evolved AMP called d-amino acid CONsensus with Glycine Absent (d-CONGA), which has excellent antimicrobial activities in vitro and in vivo. In this newest generation of rational d-CONGA variants, we tested multiple sequence-structure-function hypotheses that had not been tested in previous generations. Many of the peptide variants have lower antibacterial activity against Gram-positive or Gram-negative pathogens, especially variants that have altered hydrophobicity, secondary structure potential, or spatial distribution of charged and hydrophobic residues. Thus, d-CONGA is generally well tuned for antimicrobial activity. However, we identified a variant, d-CONGA-Q7, with a polar glutamine inserted into the middle of the sequence, that has higher activity against both planktonic and biofilm-forming bacteria as well as lower cytotoxicity against human fibroblasts. Against clinical isolates of Klebsiella pneumoniae, innate resistance to d-CONGA was surprisingly common despite a lack of inducible resistance in Pseudomonas aeruginosa reported previously. Yet, these same isolates were susceptible to d-CONGA-Q7. d-CONGA-Q7 is much less vulnerable to AMP resistance in Gram-negative bacteria than its predecessor. Consistent with the spirit of synthetic molecular evolution, d-CONGA-Q7 achieved a critical gain-of-function and has a significantly better activity profile.

Candida albicans-specific Th17 cell-mediated response contributes to alcohol-associated liver disease.

Alcohol-associated liver disease is accompanied by intestinal mycobiome dysbiosis, yet the impacts on liver disease are unclear. We demonstrate that Candida albicans-specific T helper 17 (Th17) cells are increased in circulation and present in the liver of patients with alcohol-associated liver disease. Chronic ethanol administration in mice causes migration of Candida albicans (C. albicans)-reactive Th17 cells from the intestine to the liver. The antifungal agent nystatin decreased C. albicans-specific Th17 cells in the liver and reduced ethanol-induced liver disease in mice. Transgenic mice expressing T cell receptors (TCRs) reactive to Candida antigens developed more severe ethanol-induced liver disease than transgene-negative littermates. Adoptively transferring Candida-specific TCR transgenic T cells or polyclonal C. albicans-primed T cells exacerbated ethanol-induced liver disease in wild-type mice. Interleukin-17 (IL-17) receptor A signaling in Kupffer cells was required for the effects of polyclonal C. albicans-primed T cells. Our findings indicate that ethanol increases C. albicans-specific Th17 cells, which contribute to alcohol-associated liver disease.

A Bioactive Synthetic Outer-Core Oligosaccharide Derived from a Klebsiella pneumonia Lipopolysaccharide for Bacteria Recognition.

There is an urgent need for new treatment options for carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae), which is a common cause of life-threatening hospital- and community-acquired infections. Prophylactic or therapeutic vaccination may offer an approach to control these infections, however, none has yet been approved for human use. Here, we report the chemical synthesis of an outer core tetra- and pentasaccharide derived from the lipopolysaccharide of K. pneumoniae. The oligosaccharides were equipped with an aminopentyl linker, which facilitated conjugation to the carrier proteins CRM197 and BSA. Mice immunized with the glycoconjugate vaccine candidates elicited antibodies that recognized isolated LPS as well as various strains of K. pneumoniae. The successful preparation of the oligosaccharides relied on the selection of monosaccharide building blocks equipped with orthogonal hydroxyl and amino protecting groups. It allowed the differentiation of three types of amines of the target compounds and the installation of a crowded 4,5-branched Kdo moiety.

Innovative vaccine approaches-a Keystone Symposia report.

The rapid development of COVID-19 vaccines was the result of decades of research to establish flexible vaccine platforms and understand pathogens with pandemic potential, as well as several novel changes to the vaccine discovery and development processes that partnered industry and governments. And while vaccines offer the potential to drastically improve global health, low-and-middle-income countries around the world often experience reduced access to vaccines and reduced vaccine efficacy. Addressing these issues will require novel vaccine approaches and platforms, deeper insight how vaccines mediate protection, and innovative trial designs and models. On June 28-30, 2021, experts in vaccine research, development, manufacturing, and deployment met virtually for the Keystone eSymposium "Innovative Vaccine Approaches" to discuss advances in vaccine research and development.

Vaccine-driven lung TRM cells provide immunity against Klebsiella via fibroblast IL-17R signaling.

Tissue-resident memory (TRM) cells are thought to play a role in lung mucosal immunity to pathogens, but strategies to elicit TRM by mucosal vaccines have not yet been fully realized. Here, we formulated a vaccine composed of outer membrane protein (Omp) X from Klebsiella pneumoniae and LTA1 adjuvant that was administered by the intrapulmonary route. This vaccine elicited both TH1 and TH17 cells that shared transcriptional features with cells elicited by heat-killed K. pneumoniae. Antibody responses were required to prevent bacterial dissemination but dispensable for lung-specific immunity. In contrast, lung immunity required CD4+ T cells, STAT3 expression, and IL-17R signaling in fibroblasts. Lung-specific CD4+ T cells from OmpX+LTA1­immunized mice were observed homing to the lung and could mediate protection against infection in an adoptive transfer model. Vaccine-elicited TH17 cells showed reduced plasticity and were resistant to the immunosuppressant FK506 compared with TH1 cells, and TH17 cells conferred protection under conditions of transplant immunosuppression. These data demonstrate a promising vaccine strategy that elicits lung TRM cells and promotes serotype-independent immunity to K. pneumoniae.

Inhibition of Streptococcus mutans biofilms with bacterial-derived outer membrane vesicles.

BACKGROUND: Biofilms are microbial communities surrounded by a self-produced extracellular matrix which protects them from environmental stress. Bacteria within biofilms are 10- to 1000-fold more resistant to antibiotics, making it challenging but imperative to develop new therapeutics that can disperse biofilms and eradicate infection. Gram-negative bacteria produce outer membrane vesicles (OMV) that play critical roles in communication, genetic exchange, cargo delivery, and pathogenesis. We have previously shown that OMVs derived from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. RESULTS: Here, we examine the antibiofilm activity of Burkholderia thailandensis OMVs against the oral biofilm-forming pathogen Streptococcus mutans. We demonstrate that OMV treatment reduces biofilm biomass, biofilm integrity, and bacterial cell viability. Both heat-labile and heat-stable components, including 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline and long-chain rhamnolipid, contribute to the antibiofilm activity of OMVs. When OMVs are co-administered with gentamicin, the efficacy of the antibiotic against S. mutans biofilms is enhanced. CONCLUSION: These studies indicate that bacterial-derived OMVs are highly effective biological nanoparticles that can inhibit and potentially eradicate biofilms.

Regulation and Function of ILC3s in Pulmonary Infections.

Lower respiratory infections are among the leading causes of morbidity and mortality worldwide. These potentially deadly infections are further exacerbated due to the growing incidence of antimicrobial resistance. To combat these infections there is a need to better understand immune mechanisms that promote microbial clearance. This need in the context of lung infections has been further heightened with the emergence of SARS-CoV-2. Group 3 innate lymphoid cells (ILC3s) are a recently discovered tissue resident innate immune cell found at mucosal sites that respond rapidly in the event of an infection. ILC3s have clear roles in regulating mucosal immunity and tissue homeostasis in the intestine, though the immunological functions in lungs remain unclear. It has been demonstrated in both viral and bacterial pneumonia that stimulated ILC3s secrete the cytokines IL-17 and IL-22 to promote both microbial clearance as well as tissue repair. In this review, we will evaluate regulation of ILC3s during inflammation and discuss recent studies that examine ILC3 function in the context of both bacterial and viral pulmonary infections.

Burkholderia pseudomallei OMVs derived from infection mimicking conditions elicit similar protection to a live-attenuated vaccine.

Burkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus that causes the disease melioidosis. B. pseudomallei expresses a number of proteins that contribute to its intracellular survival in the mammalian host. We previously demonstrated that immunization with OMVs derived from B. pseudomallei grown in nutrient-rich media protects mice against lethal disease. Here, we evaluated if OMVs derived from B. pseudomallei grown under macrophage-mimicking growth conditions could be enriched with intracellular-stage proteins in order to improve the vaccine. We show that OMVs produced in this manner (M9 OMVs) contain proteins associated with intracellular survival yet are non-toxic to living cells. Immunization of mice provides significant protection against pulmonary infection similar to that achieved with a live attenuated vaccine and is associated with increased IgG, CD4+, and CD8+ T cells. OMVs possess inherent adjuvanticity and drive DC activation and maturation. These results indicate that M9 OMVs constitute a new promising vaccine against melioidosis.

A novel 3D vascular assay for evaluating angiogenesis across porous membranes.

Microfluidic technology has been extensively applied to model the functional units of human organs and tissues. Since vasculature is a key component of any functional tissue, a variety of techniques to mimic vasculature in vitro have been developed to address complex physiological and pathological processes in 3D tissues. Herein, we developed a novel, in vitro, microfluidic-based model to probe microvasculature growth into and across implanted porous membranes. Using ePTFE and polycarbonate as examples, we characterize the vascularization potential of these thin porous membranes using this device. This tool will allow for the assessment of porous materials early in their development, prior to their use for encapsulating implants or drugs, while minimizing the need for animal studies. Employing quantitative morphometric analysis and measurements of vascular permeability, we demonstrate our model to be an effective platform for evaluation of angiogenic potential of an implanted membrane biomaterial. Results show that endothelial cells can either migrate as single cells or form continuous sprouts across porous membranes, which is a material structure-dependent behavior. Our model is advantageous over conventional Transwell assays as it is amenable to quantitative assessment of vascular sprouting in 3D, and in contrast to animal models it can be employed more efficiently and with real-time assessment capabilities. This new tool could be applied either to test the suitability of a wide range of biomaterials for implantation or to screen different pro-angiogenic factors for therapeutic applications, and will advance the design of new biomaterials.

Burkholderia thailandensis outer membrane vesicles exert antimicrobial activity against drug-resistant and competitor microbial species.

Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current study, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. We show that a number of antimicrobial compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid are present in or tightly associate with B. thailandensis OMVs. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against methicillin-resistant Staphylococcus aureus (MRSA). These findings indicate that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against bio-film-forming and multidrug-resistant organisms.

Synthetic molecular evolution of host cell-compatible, antimicrobial peptides effective against drug-resistant, biofilm-forming bacteria.

Novel classes of antibiotics and new strategies to prevent and treat infections are urgently needed because the rapid rise in drug-resistant bacterial infections in recent decades has been accompanied by a parallel decline in development of new antibiotics. Membrane permeabilizing antimicrobial peptides (AMPs) have long been considered a potentially promising, novel class of antibiotic, especially for wound protection and treatment to prevent the development of serious infections. Yet, despite thousands of known examples, AMPs have only infrequently proceeded as far as clinical trials, especially the chemically simple, linear examples. In part, this is due to impediments that often limit their applications in vivo. These can include low solubility, residual toxicity, susceptibility to proteolysis, and loss of activity due to host cell, tissue, and protein binding. Here we show how synthetic molecular evolution can be used to evolve potentially advantageous antimicrobial peptides that lack these impediments from parent peptides that have at least some of them. As an example of how the antibiotic discovery pipeline can be populated with more promising candidates, we evolved and optimized one family of linear AMPs into a new generation with high solubility, low cytotoxicity, potent broad-spectrum sterilizing activity against a panel of gram-positive and gram-negative ESKAPE pathogens, and antibiofilm activity against gram-positive and gram-negative biofilms. The evolved peptides have these activities in vitro even in the presence of concentrated host cells and also in vivo in the complex, cell- and protein-rich environment of a purulent animal wound model infected with drug-resistant bacteria.

Molecular Diversity from Arid-Land Plants: Valorization of Terpenes and Biotransformation Products.

Asteraceae plants from arid lands are a source of biomass, resin and latex rich in terpenoids with diverse biological effects. Thirty-six previously isolated terpenes, comprising sesquiterpenes, diterpenes, triterpenes and quassinoids, isolated from arid-land plants and a series of metabolites from the biotransformation of some lead compounds were evaluated against insect pests (Spodoptera littoralis, Leptinotarsa decemlineata, Myzus persicae and Rhopalosiphum padi), cells (insect, hamster, murine and human tumoral cells) and parasites (Trypanosoma cruzi and Leishmania infantum). Among the insecticidal sesquiterpenes, maalian-1α,8α-diol (12) and γ-eudesmol (17) were antifeedant against L. decemlineata, M. persicae and cytotoxic to Sf9 insect cells, and (-)-maali-3-en-8α-ol (10), (+)-maaliane-5α,8α,9α-triol (11), chrysothame (31) and holacanthone (35) were antifeedant against S. littoralis. The parasite L. infantum was slightly more sensitive than T. cruzi to the test compounds (39 % vs. 33 % of active compounds) with compound 17 and the biotransformed diterpene 27 being antiparasitic to L. infantum, with no cytotoxic effects on mammalian cells. Moreover, sesquiterpenes 3 and 17, and grindelane diterpenes 22, 23 and 26 showed selective activity against chemoresistant human colon, cervical and melanoma cancer cells. Thus, considering our results, the best candidates for future studies are compounds 17 and 3, due to their activity on insect pests, parasites (17) and tumoral cells (3, 17, 22, 23 and 26).

In situ Treatment With Novel Microbiocide Inhibits Methicillin Resistant Staphylococcus aureus in a Murine Wound Infection Model.

Increased prevalence of antibiotic resistance in skin and soft tissue infections is a concerning public health challenge currently facing medical science. A combinatory, broad spectrum biocidal antiseptic has been developed ("ASP") as a topically applied solution to potential resistant and polymicrobial infected wounds that may be encountered in this context. The ASP-105 designate was evaluated in vitro by determining the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), against different strains of methicillin-resistant Staphylococcus aureus (MRSA), resulting estimates of which approximated the positive control (bacitracin). To evaluate in vivo microbicide efficacy, we utilized a murine full thickness wound model to study bacterial infection and wound healing kinetics. Mice were experimentally wounded dorsally and infected with bioluminescent MRSA. The infected wound was splinted, dressed and treated topically with either ASP-105, vehicle (-control), or bacitracin. Bacterial burden and wound healing was monitored using an in vivo imaging system and evaluation of biofilm formation using scanning electron microscopy of wound dressing. Treatment with ASP-105 significantly reduced bacterial burdens in the first 3 days of infection and inhibited MRSA biofilm formation on the surgical dressing. Notably, treatment with ASP-105 resulted in a sterilizing effect of any detectable MRSA in nearly all (80%; 4/5) of treatment group. All mice receiving vehicle control developed highly MRSA-luminescent and purulent wound beds as a result of experimental infection. The ASP-105 therapy facilitated natural healing in the absence of MRSA infection. Results of this study suggests that that the novel "ASP" combinatory topical antiseptic can be used directly in wounds as a potent, broad-spectrum microbicide against drug resistant S. aureus without injury to the wound bed and impediment of natural restorative processes associated with wound healing. Further studies are warranted to test the effectiveness of this biocidal formulation against other recalcitrant bacterial and fungal pathogens in the context of serious wound infections, and to assess utility of use in both clinical and self-treat scenarios.

Three-dimensional photolithographic micropatterning: a novel tool to probe the complexities of cell migration.

In order to independently study the numerous variables that influence cell movement, it will be necessary to employ novel tools and materials that allow for exquisite control of the cellular microenvironment. In this work, we have applied advanced 3D micropatterning technology, known as two-photon laser scanning lithography (TP-LSL), to poly(ethylene glycol) (PEG) hydrogels modified with bioactive peptides in order to fabricate precisely designed microenvironments to guide and quantitatively investigate cell migration. Specifically, TP-LSL was used to fabricate cell adhesive PEG-RGDS micropatterns on the surface of non-degradable PEG-based hydrogels (2D) and in the interior of proteolytically degradable PEG-based hydrogels (3D). HT1080 cell migration was guided down these adhesive micropatterns in both 2D and 3D, as observed via time-lapse microscopy. Differences in cell speed, cell persistence, and cell shape were observed based on variation of adhesive ligand, hydrogel composition, and patterned area for both 2D and 3D migration. Results indicated that HT1080s migrate faster and with lower persistence on 2D surfaces, while HT1080s migrating in 3D were smaller and more elongated. Further, cell migration was shown to have a biphasic dependence on PEG-RGDS concentration and cells moving within PEG-RGDS micropatterns were seen to move faster and with more persistence over time. Importantly, the work presented here begins to elucidate the multiple complex factors involved in cell migration, with typical confounding factors being independently controlled. The development of this unique platform will allow researchers to probe how cells behave within increasingly complex 3D microenvironments that begin to mimic specifically chosen aspects of the in vivo landscape.

Three-dimensional biomimetic patterning in hydrogels to guide cellular organization.

An image-guided micropatterning method is demonstrated for generating biomimetic hydrogel scaffolds with two-photon laser scanning photolithography. This process utilizes computational methods to directly translate three-dimensional cytoarchitectural features from labeled tissues into material structures. We use this method to pattern hydrogels that guide cellular organization by structurally and biochemically recapitulating complex vascular niche microenvironments with high pattern fidelity at the microscale.

Discovery and development of anticancer agents from plants.

A novel in vitro assay for the discovery of anticancer agents was used to examine aqueous and organic extracts from 1847 plants collected mainly in the U.S. Southwest and West. The assay results were separated into 5 categories: inactive (62%), equally active (36%), equally active and potent (0.5%), solid tumor selective (1.4%), and human selective (0.8%). Extracts from the latter three categories were fractionated using the in vitro assay to biodirect each step. Psorothamnus emoryi extracts were solid tumor selective and yielded two active compounds upon fractionation: dalrubone and 5-methoxydalrubone. Calocedrus decurrens was equally active and potent and yielded deoxypodophyllotoxin as the active compound. Linanthus floribundus was human selective and yielded strophanthidin as the active compound. The potential of this assay to discover novel anticancer agents from the active extracts is discussed.

Triterpenoids from Parthenium argentatum x P. tomentosa.

Four tetracyclic triterpenoids and lupeol were isolated from the hybrid Parthenium argentatum x P. tomentosa. The new triterpenoids were identified as 16, 24-epoxy-3 alpha-hydroxylanost-8-ene (argentatin E); 16, 24-epoxy-25-hydroxycycloart-1, 11, 22-trien-3-one (argentatin F); 16,24-dihydroxycycloart-20, 25-dien-3-one (argentatin G) and 16, 24-dihydroxycycloart-25-en-3-one (argentatin H). The chemical identities of these compounds were confirmed by the different spectrometric measurements.