Evaluation of Malignant Hyperthermia Features in Patients with Pathogenic or Likely Pathogenic RYR1 Variants Disclosed through a Population Genomic Screening Program.

BACKGROUND: Malignant hyperthermia (MH) susceptibility is a heritable musculoskeletal disorder that can present as a potentially fatal hypermetabolic response to triggering anesthesia agents. Genomic screening for variants in MH-associated genes RYR1 and CACNA1S provides an opportunity to prevent morbidity and mortality. There are limited outcomes data from disclosing variants in RYR1, the most common MH susceptibility gene, in unselected populations. The authors sought to identify the rate of MH features or fulminant episodes after triggering agent exposure in an unselected population undergoing genomic screening including actionable RYR1 variants. METHODS: The MyCode Community Health Initiative by Geisinger (USA) is an electronic health record-linked biobank that discloses pathogenic and likely pathogenic variants in clinically actionable genes to patient-participants. Available electronic anesthesia and ambulatory records for participants with actionable RYR1 results returned through December 2020 were evaluated for pertinent findings via double-coded chart reviews and reconciliation. Descriptive statistics for observed phenotypes were calculated. RESULTS: One hundred fifty-two participants had an actionable RYR1 variant disclosed during the study period. None had previous documented genetic testing for MH susceptibility; one had previous contracture testing diagnosing MH susceptibility. Sixty-eight participants (44.7%) had anesthesia records documenting triggering agent exposure during at least one procedure. None received dantrolene treatment or had documented muscle rigidity, myoglobinuria, hyperkalemia, elevated creatine kinase, severe myalgia, or tea-colored urine. Of 120 possibly MH-related findings (postoperative intensive care unit admissions, hyperthermia, arterial blood gas evaluation, hypercapnia, or tachycardia), 112 (93.3%) were deemed unlikely to be MH events; 8 (6.7%) had insufficient records to determine etiology. CONCLUSIONS: Results demonstrate a low frequency of classic intraanesthetic hypermetabolic phenotypes in an unselected population with actionable RYR1 variants. Further research on the actionability of screening for MH susceptibility in unselected populations, including economic impact, predictors of MH episodes, and expanded clinical phenotypes, is necessary.

Human transcriptional signature of protection after Plasmodium falciparum immunization and infectious challenge via mosquito bites.

The identification of immune correlates of protection against infectious pathogens will accelerate the design and optimization of recombinant and subunit vaccines. Systematic analyses such as immunoprofiling including serological, cellular, and molecular assessments supported by computational tools are key to not only identify correlates of protection but also biomarkers of disease susceptibility. The current study expands our previous cellular and serological profiling of vaccine-induced responses to a whole parasite malaria vaccine. The irradiated sporozoite model was chosen as it is considered the most effective vaccine against malaria. In contrast to whole blood transcriptomics analysis, we stimulated peripheral blood mononuclear cells (PBMC) with sporozoites and enriched for antigen-specific cells prior to conducting transcriptomics analysis. By focusing on transcriptional events triggered by antigen-specific stimulation, we were able to uncover quantitative and qualitative differences between protected and non-protected individuals to controlled human malaria infections and identified differentially expressed genes associated with sporozoite-specific responses. Further analyses including pathway and gene set enrichment analysis revealed that vaccination with irradiated sporozoites induced a transcriptomic profile associated with Th1-responses, Interferon-signaling, antigen-presentation, and inflammation. Analyzing longitudinal time points not only post-vaccination but also post-controlled human malaria infection further revealed that the transcriptomic profile of protected vs non-protected individuals was not static but continued to diverge over time. The results lay the foundation for comparing protective immune signatures induced by various vaccine platforms to uncover immune correlates of protection that are common across platforms.

Induction of antigen specific intrahepatic CD8+ T cell responses by a secreted heat shock protein based gp96-Ig-PfCA malaria vaccine.

Introduction: A highly efficacious and durable vaccine against malaria is an essential tool for global malaria eradication. One of the promising strategies to develop such a vaccine is to induce robust CD8+ T cell mediated immunity against malaria liver-stage parasites. Methods: Here we describe a novel malaria vaccine platform based on a secreted form of the heat shock protein, gp96-immunoglobulin, (gp96-Ig) to induce malaria antigen specific, memory CD8+ T cells. Gp96-Ig acts as an adjuvant to activate antigen presenting cells (APCs) and chaperone peptides/antigens to APCs for cross presentation to CD8+ T cells. Results: Our study shows that vaccination of mice and rhesus monkeys with HEK-293 cells transfected with gp96-Ig and two well-known Plasmodium falciparum CSP and AMA1 (PfCA) vaccine candidate antigens, induces liver-infiltrating, antigen specific, memory CD8+ T cell responses. The majority of the intrahepatic CSP and AMA1 specific CD8+ T cells expressed CD69 and CXCR3, the hallmark of tissue resident memory T cells (Trm). Also, we found intrahepatic, antigen-specific memory CD8+ T cells secreting IL-2, which is relevant for maintenance of effective memory responses in the liver. Discussion: Our novel gp96-Ig malaria vaccine strategy represents a unique approach to induce liver-homing, antigen-specific CD8+ T cells critical for Plasmodium liver-stage protection.

Exposure-related, global alterations in innate and adaptive immunity; a consideration for re-use of non-human primates in research.

BACKGROUND: Non-human primates (NHPs) play an important role in biomedical research, where they are often being re-used in multiple research studies over the course of their life-time. Researchers employ various study-specific screening criteria to reduce potential variables associated with subsequent re-use of NHPs. However, criteria set for NHP re-assignments largely neglect the impact of previous exposures on overall biology. Since the immune system is a key determinant of overall biological outcome, an altered biological state could be predicted by monitoring global changes in the immune profile. We postulate that every different exposure or a condition can generate a unique global immune profile in NHPs. METHODS: Changes in the global immune profile were evaluated in three different groups of rhesus macaques previously enrolled in dengue or malaria vaccine studies over six months after their last exposure. Naïve animals served as the baseline. Fresh blood samples were stained with various immune cell surface markers and analyzed by multi-color flow-cytometry to study immune cell dynamics in the peripheral blood. Serum cytokine profile in the pre-exposed animals were analyzed by mesoscale assay using a customized U-PLEX NHP biomarker panel of 12 cytokines/chemokines. RESULTS: Pre-exposed macaques showed altered dynamics in circulating cytokines and certain innate and adaptive immune cell subsets such as monocytes, HLA-DR+NKT cells, B cells and T cells. Some of these changes were transient, while some lasted for more than six months. Each group seemed to develop a global immune profile unique to their particular exposure. CONCLUSION: Our data strongly suggest that re-used NHPs should be evaluated for long-term, overall immunological changes and randomly assigned to new studies to avoid study bias.

Novel sporozoite-based ELISpot assay to assess frequency of parasite-specific B cells after vaccination with irradiated sporozoites.

BACKGROUND: Whole parasite vaccination is an efficacious strategy to induce sterile immunity and to prevent malaria transmission. Understanding the mechanism and response of immune cells to vaccines plays a critical role in deciphering correlates of protection against infection and disease. Immunoassays, such as ELISpot, are commonly used to assess the immunogenicity of vaccines towards T cells and B cells. To date, these assays only analyse responses to specific antigens since they are based on recombinant parasite-derived proteins or peptides. There is the need for an agnostic approach that allows the evaluation of all sporozoite-associated antigens. METHODS: ELISpot plates coated with a defined amount of lysed Plasmodium falciparum sporozoites were used to assess the frequency of sporozoite-specific B cells in peripheral blood mononuclear cells from donors immunized with either a recombinant malaria vaccine or irradiated sporozoites. RESULTS: This report describes the assay conditions for a specific and sensitive sporozoite-based B cell ELISpot assay. The assay development considers the quality of sporozoite preparation as well as the detection threshold of the frequency of antigen-specific B cells. The assay enables the detection of sporozoite-specific IgM and IgG-producing B cells. Moreover, the assay can detect sporozoite-reactive B cells from subjects that were either vaccinated with the radiation attenuated sporozoite vaccine or a recombinant pre-erythrocytic vaccine. CONCLUSION: The newly developed sporozoite-based B cell ELISpot enables the monitoring of changes in the frequency of sporozoite-specific B cells. Applying this assay to assess the potency of vaccination regimens or seasonal changes in B cell populations from subjects residing in malaria-endemic areas will provide an opportunity to gain insight into immune mechanisms involved in protection and/or disease.

The Selection of a Hepatocyte Cell Line Susceptible to Plasmodium falciparum Sporozoite Invasion That Is Associated With Expression of Glypican-3.

In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3-5% and early development of P. falciparum exoerythrocytic forms-a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax.

Inhibition of Clostridium perfringens epsilon toxin by ß-cyclodextrin derivatives.

Clostridium perfringens epsilon toxin (ETX) is considered as one of the most dangerous potential biological weapons. The goal of this work was to identify inhibitors of ETX using a novel approach for the inactivation of pore-forming toxins. The approach is based on the blocking of the target pore with molecules having the same symmetry as the pore itself. About 200 various ß-cyclodextrin derivatives were screened for inhibitors of ETX activity using a colorimetric cell viability assay. Several compounds with dose-dependent activities at low micromolar concentrations have been identified. The same compounds were also able to inhibit lethal toxin of Bacillus anthracis.

Blueberry necrotic ring blotch virus in Southern Highbush Blueberry: Insights into In Planta and In-Field Movement.

Blueberry necrotic ring blotch virus (BNRBV) causes an emerging disease of southern highbush blueberry (SHB) in the southeastern United States. Disease incidence and severity vary considerably from year to year within the same planting. Experiments were conducted to determine how the virus spreads in the field. Leaf tissue from symptomatic field plants tested positive for BNRBV in 2011, whereas the same plants were asymptomatic in 2012 and tested negative for the virus. Symptomatic and asymptomatic leaves from individual shoots were tested for the presence of the virus, and symptomatic leaves tested positive (100%), whereas 65.4% of the asymptomatic leaves from the same shoots tested negative. Leaves were selected in which half the leaf blade was symptomatic and the other half was not; symptomatic leaf halves tested positive (100%), whereas 76.0% of the asymptomatic halves from the same leaf tested negative for the virus. When virus-free, potted trap plants were interspersed in the field among established plants that had shown disease symptoms the previous year, disease onset in trap plants was observed 2 to 3 weeks after disease onset in field plants. In a separate experiment, asymptomatic softwood cuttings were collected from mother plants symptomatic for BNRBV, rooted, and monitored for symptom development for a period of 12 to 27 months. No BNRBV symptoms were observed in the progeny, whereas disease incidence was high for cuttings taken at the same time from plants infected with Blueberry red ringspot virus used as a control. Collectively, these studies suggest that BNRBV does not infect SHB plants systemically and is not transmitted through vegetative propagation, and that the virus likely does not persist in plants after natural defoliation in the fall.

Symmetry requirements for effective blocking of pore-forming toxins: comparative study with alpha-, beta-, and gamma-cyclodextrin derivatives.

We compared the abilities of structurally related cationic cyclodextrins to inhibit Bacillus anthracis lethal toxin and Staphylococcus aureus α-hemolysin. We found that both ß- and γ-cyclodextrin derivatives effectively inhibited anthrax toxin action by blocking the transmembrane oligomeric pores formed by the protective antigen (PA) subunit of the toxin, whereas α-cyclodextrins were ineffective. In contrast, α-hemolysin was selectively blocked only by ß-cyclodextrin derivatives, demonstrating that both symmetry and size of the inhibitor and the pore are important.

Symmetry complementarity-guided design of anthrax toxin inhibitors based on ß-cyclodextrin: Synthesis and relative activities of face-selective functionalized polycationic clusters.

Three new series of potential anthrax toxin inhibitors based on the ß-cyclodextrin (ßCD) scaffold were developed by exploiting face-selective Cu(I)-catalyzed azide-alkyne 1,3-cycloadditions, amine-isothiocyanate coupling, and allyl group hydroboration-oxidation/hydroxy → amine replacement reactions. The molecular design follows the "symmetry-complementarity" concept between homogeneously functionalized polycationic ßCD derivatives and protective antigen (PA), a component of anthrax toxin known to form C7-symmetric pores on the cell membrane used by lethal and edema factors to gain access to the cytosol. The synthesis and antitoxin activity of a collection of ßCD derivatives differing in the number, arrangement, and face location of the cationic elements are reported herein. These results set the basis for a structure-activity relationship development program of new candidates to combat the anthrax threat.

In vivo efficacy of beta-cyclodextrin derivatives against anthrax lethal toxin.

We evaluated the in vivo efficacy of three beta-cyclodextrin derivatives that block the anthrax protective antigen pore. These compounds were at least 15-fold more potent than previously described beta-cyclodextrins in protecting against anthrax lethal toxin in a rat model. One of the drugs was shown to protect mice from bacterial infection.

Inhibition of S. aureus alpha-hemolysin and B. anthracis lethal toxin by beta-cyclodextrin derivatives.

Many pathogens utilize the formation of transmembrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered to be important virulence factors, which makes them attractive targets for the discovery of new therapeutic agents. Our research is based on the idea that compounds designed to block the pores can inhibit the action of virulence factors, and that the chances to find high affinity blocking agents increase if they have the same symmetry as the target pore. Recently, we demonstrated that derivatives of beta-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. To test the broader applicability of this approach, we sought beta-cyclodextrin derivatives capable of inhibiting the activity of Staphylococcus aureus alpha-hemolysin (alpha-HL), which is regarded as a major virulence factor playing an important role in staphylococcal infection. We identified several amino acid derivatives of beta-cyclodextrin that inhibited the activity of alpha-HL and LeTx in cell-based assays at low micromolar concentrations. One of the compounds was tested for the ability to block ion conductance through the pores formed by alpha-HL and PA in artificial lipid membranes. We anticipate that this approach can serve as the basis for a structure-directed drug discovery program to find new and effective therapeutics against various pathogens that utilize pore-forming proteins as virulence factors.

Search for cyclodextrin-based inhibitors of anthrax toxins: synthesis, structural features, and relative activities.

Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of beta-cyclodextrin inhibited anthrax lethal toxin action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. In the present study, we evaluate a series of new beta-cyclodextrin derivatives with the goal of identifying potent inhibitors of anthrax toxins. Newly synthesized hepta-6-thioaminoalkyl and hepta-6-thioguanidinoalkyl derivatives of beta-cyclodextrin with alkyl spacers of various lengths were tested for the ability to inhibit cytotoxicity of lethal toxin in cells as well as to block ion conductance through PA channels reconstituted in planar bilayer lipid membranes. Most of the tested derivatives were protective against anthrax lethal toxin action at low or submicromolar concentrations. They also blocked ion conductance through PA channels at concentrations as low as 0.1 nM. The activities of the derivatives in both cell protection and channel blocking were found to depend on the length and chemical nature of the substituent groups. One of the compounds was also shown to block the edema toxin activity. It is hoped that these results will help to identify a new class of drugs for anthrax treatment, i.e., drugs that block the pathway for toxin translocation into the cytosol, the PA channel.

Beta-cyclodextrin derivatives that inhibit anthrax lethal toxin.

Recently, we demonstrated that simultaneous blocking of bacterial growth by antibiotics and inhibition of anthrax toxin action with antibodies against protective antigen were beneficial for the treatment of anthrax. The present study examined the hypothesis that blocking the pore formed by protective antigen can inhibit the action of anthrax toxin. The potential inhibitors were chosen by a structure-based design using beta-cyclodextrin as the starting molecule. Several beta-cyclodextrin derivatives were evaluated for their ability to protect RAW 264.7 cells from the action of anthrax lethal toxin. Per-substituted aminoalkyl derivatives displayed inhibitory activity and were protective against anthrax lethal toxin action at low micromolar concentrations. These results provide the basis for a structure-based drug discovery program, with the goal of identifying new drug candidates for anthrax treatment.

Prevention of lethal respiratory vaccinia infections in mice with interferon-alpha and interferon-gamma.

The antiviral efficacy of interferons (IFNs) was evaluated using a vaccinia intranasal infection model in mice in this study. We provide evidence that intranasal administration of IFN-alpha and IFN-gamma (days -1 to +3) resulted in 100 and 90% survival against a lethal respiratory vaccinia infection (8 LD50) in mice, respectively; whereas no animals in the placebo group survived through the study period (21 days). The IFN treatment consisted of a single daily dose of 5x10(3) U per mouse for 5 consecutive days. The efficacy of IFN-gamma was evident even when the IFN-gamma treatments started 1-2 days after infection and when a lower dose (2x10(3) U per mouse) was used. The treatment of IFN-alpha and IFN-gamma reduced the virus titers in the lungs of infected mice by 1000-10,000-fold, when the administration started 1 day after infection. Our data suggest that IFN-alpha and IFN-gamma are effective in protecting vaccinia-infected mice from viral replication in lungs and mortality, and may be beneficial in other human orthopoxvirus infections.

A culturally competent intervention of education and care for black women with type 2 diabetes.

This article reports on the development and pilot feasibility testing of a culturally competent intervention of education and care for black women with type 2 diabetes mellitus (T2DM). Using a one group, pretest posttest quasi-experimental design, the intervention was tested with a convenience sample of 25 community black women with T2DM. The conceptual basis, process, and content of the intervention as well as the feasibility and acceptability of study materials and methods are described. Significant improvements from baseline to 3 months were observed in measures of glycemic control, weight, body mass index, and diabetes-related emotional distress. The findings suggest that a culturally sensitive intervention of nurse practitioner diabetes care and education is beneficial for black women with T2DM, resulting in program attendance, kept appointments, improved glycemic control and weight, and decreased diabetes-related emotional distress.

Treatment of anthrax infection with combination of ciprofloxacin and antibodies to protective antigen of Bacillus anthracis.

Currently there is no effective treatment for inhalational anthrax beyond administration of antibiotics shortly after exposure. There is need for new, safe and effective treatments to supplement traditional antibiotic therapy. Our study was based on the premise that simultaneous inhibition of lethal toxin action with antibodies and blocking of bacterial growth by antibiotics will be beneficial for the treatment of anthrax. In this study, we tested the effects of a combination treatment using purified rabbit or sheep anti-protective antigen (PA) antibodies and the antibiotic ciprofloxacin in a rodent anthrax model. In mice infected with a dose of Bacillus anthracis Sterne strain corresponding to 10 LD(50), antibiotic treatment with ciprofloxacin alone only cured 50% of infected animals. Administration of anti-PA IgG in combination with ciprofloxacin produced 90-100% survival. These data indicate that a combination of antibiotic/immunoglobulin therapy is more effective than antibiotic treatment alone in a rodent anthrax model.