Republic of Singapore Air Force Helicopter Search-and-Rescue and Medical Evacuations: A 5-Year Review.

The Republic of Singapore Air Force (RSAF) provides Helicopter Search-and-Rescue (SAR) and Helicopter Medical Evacuation (Heli-Medevac) coverage for the Singapore Aeronautical Search and Rescue Region (ASSR) in the South China Sea, spanning 840,000 km2. This region contains busy international shipping lanes and air traffic routes. Each year, Singapore's Helicopter SAR and Heli-Medevac service is activated multiple times to rescue personnel lost at sea or to evacuate ill and injured ship sailors or passengers to tertiary hospitals in Singapore for stabilization and advanced care. This is a retrospective review on all civilian SAR and Heli-medevac activations by the RSAF over a 5-year period from 2016 to 2020. Case profiles, presenting conditions, in-flight treatment, and patient outcomes are reviewed and discussed. Key operational observations made from RSAF's SAR and Heli-Medevac, as well as lessons learned from these missions, are discussed in this article.

Controlling the cytokine storm in severe bacterial diarrhoea with an oral Toll-like receptor 4 antagonist.

Shigella dysenteriae causes the most severe of all infectious diarrhoeas and colitis. We infected rhesus macaques orally and also treated them orally with a small and non-absorbable polypropyletherimine dendrimer glucosamine that is a Toll-like receptor-4 (TLR4) antagonist. Antibiotics were not given for this life-threatening infection. Six days later, the clinical score for diarrhoea, mucus and blood was 54% lower, colon interleukin-8 and interleukin-6 were both 77% lower, and colon neutrophil infiltration was 75% less. Strikingly, vasculitis did not occur and tissue fibrin thrombi were reduced by 67%. There was no clinical toxicity or adverse effect of dendrimer glucosamine on systemic immunity. This is the first report in non-human primates of the therapeutic efficacy of a small and orally bioavailable TLR antagonist in severe infection. Our results show that an oral TLR4 antagonist can enable controlled resolution of the infection-related-inflammatory response and can also prevent neutrophil-mediated gut wall necrosis in severe infectious diarrhoeas.

Nebulised amphotericin B-polymethacrylic acid nanoparticle prophylaxis prevents invasive aspergillosis.

Aspergillus species are the major life threatening fungal pathogens in transplant patients. Germination of inhaled fungal spores initiates infection, causes severe pneumonia, and has a mortality of >50%. This is leading to the consideration of pre-exposure prophylaxis to prevent infection. We made a very low MWt amphotericin B-polymethacrylic acid nanoparticle. It was not toxic to lung epithelial cells or monocyte-derived-macrophages in-vitro, or in an in-vivo transplant immuno-suppression mouse model of life threatening invasive aspergillosis. Three days of nebuliser based prophylaxis delivered the nanoparticle effectively to lung and prevented both fungal growth and lung inflammation. Protection from disease was associated with >99% killing of the Aspergillus and a 90% reduction in lung TNF-α; the primary driver of tissue destructive immuno-pathology. This study provides in-vivo proof-of-principle that very small and cost-effective nanoparticles can be made simply, and delivered safely and effectively to lung by the aerosol route to prevent fungal infections. FROM THE CLINICAL EDITOR: Aspergillus is an opportunistic pathogen, which affects immunocompromised patients. One novel way to help fight against this infection is pre-exposure prophylaxis. The authors here made PMA based anionic hydrogels carrying amphotericin B, with mucoadhesive behavior. They showed that aerosol route of the drug was very effective in protecting against the disease in an in-vivo model and should provide a stepping-stone towards clinical trials in the future.

Partially glycosylated dendrimers block MD-2 and prevent TLR4-MD-2-LPS complex mediated cytokine responses.

The crystal structure of the TLR4-MD-2-LPS complex responsible for triggering powerful pro-inflammatory cytokine responses has recently become available. Central to cell surface complex formation is binding of lipopolysaccharide (LPS) to soluble MD-2. We have previously shown, in biologically based experiments, that a generation 3.5 PAMAM dendrimer with 64 peripheral carboxylic acid groups acts as an antagonist of pro-inflammatory cytokine production after surface modification with 8 glucosamine molecules. We have also shown using molecular modelling approaches that this partially glycosylated dendrimer has the flexibility, cluster density, surface electrostatic charge, and hydrophilicity to make it a therapeutically useful antagonist of complex formation. These studies enabled the computational study of the interactions of the unmodified dendrimer, glucosamine, and of the partially glycosylated dendrimer with TLR4 and MD-2 using molecular docking and molecular dynamics techniques. They demonstrate that dendrimer glucosamine forms co-operative electrostatic interactions with residues lining the entrance to MD-2's hydrophobic pocket. Crucially, dendrimer glucosamine interferes with the electrostatic binding of: (i) the 4'phosphate on the di-glucosamine of LPS to Ser118 on MD-2; (ii) LPS to Lys91 on MD-2; (iii) the subsequent binding of TLR4 to Tyr102 on MD-2. This is followed by additional co-operative interactions between several of the dendrimer glucosamine's carboxylic acid branches and MD-2. Collectively, these interactions block the entry of the lipid chains of LPS into MD-2's hydrophobic pocket, and also prevent TLR4-MD-2-LPS complex formation. Our studies have therefore defined the first nonlipid-based synthetic MD-2 antagonist using both animal model-based studies of pro-inflammatory cytokine responses and molecular modelling studies of a whole dendrimer with its target protein. Using this approach, it should now be possible to computationally design additional macromolecular dendrimer based antagonists for other Toll Like Receptors. They could be useful for treating a spectrum of infectious, inflammatory and malignant diseases.

Impaired interferon-gamma responses, increased interleukin-17 expression, and a tumor necrosis factor-alpha transcriptional program in invasive aspergillosis.

BACKGROUND: Invasive aspergillosis (IA) is the most common cause of death associated with fungal infection in the developed world. Historically, susceptibility to IA has been associated with prolonged neutropenia; however, IA has now become a major problem in patients on calcineurin inhibitors and allogenic hematopoietic stem cell transplant patients following engraftment. These observations suggest complex cellular mechanisms govern immunity to IA. METHODS: To characterize the key early events that govern outcome from infection with Aspergillus fumigatus, we performed a comparative immunochip microarray analysis of the pulmonary transcriptional response to IA between cyclophosphamide-treated mice and immunocompetent mice at 24 h after infection. RESULTS: We demonstrate that death due to infection is associated with a failure to generate an incremental interferon-gamma response, increased levels of interleukin-5 and interleukin-17a transcript, coordinated expression of a network of tumor necrosis factor-alpha-related genes, and increased levels of tumor necrosis factor-alpha. In contrast, clearance of infection is associated with increased expression of a number genes encoding proteins involved in innate pathogen clearance, as well as apoptosis and control of inflammation. CONCLUSION: This first organ-level immune response transcriptional analysis for IA has enabled us to gain new insights into the mechanisms that govern fungal immunity in the lung.

Polyvalent dendrimer glucosamine conjugates prevent scar tissue formation.

Dendrimers are hyperbranched macromolecules that can be chemically synthesized to have precise structural characteristics. We used anionic, polyamidoamine, generation 3.5 dendrimers to make novel water-soluble conjugates of D(+)-glucosamine and D(+)-glucosamine 6-sulfate with immuno-modulatory and antiangiogenic properties respectively. Dendrimer glucosamine inhibited Toll-like receptor 4-mediated lipopolysaccharide induced synthesis of pro-inflammatory chemokines (MIP-1 alpha, MIP-1 beta, IL-8) and cytokines (TNF-alpha, IL-1 beta, IL-6) from human dendritic cells and macrophages but allowed upregulation of the costimulatory molecules CD25, CD80, CD83 and CD86. Dendrimer glucosamine 6-sulfate blocked fibroblast growth factor-2 mediated endothelial cell proliferation and neoangiogenesis in human Matrigel and placental angiogenesis assays. When dendrimer glucosamine and dendrimer glucosamine 6-sulfate were used together in a validated and clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery, they increased the long-term success of the surgery from 30% to 80% (P = 0.029). We conclude that synthetically engineered macromolecules such as the dendrimers described here can be tailored to have defined immuno-modulatory and antiangiogenic properties, and they can be used synergistically to prevent scar tissue formation.

Short communication: pitfalls of culturing C8166 cells in serum-free media.

Long-term culture of C8166 cells in serum-free media can result in changes in their level of expression of immunologically important cell surface makers and a loss of infectivity by HIV-1. We have now demonstrated that these phenotypic changes are due to an outgrowth of a very small number of contaminating cells of mouse origin. Our observations emphasize the importance of carefully recharacterizing any cells that have been adapted to grow in a serum-free culture media.

Identification of two mutually exclusive groups after long-term monitoring of HIV DNA 2-LTR circle copy number in patients on HAART.

BACKGROUND: Anti-retroviral drug therapy reduces but does not abolish HIV transmission and replication throughout the body. HIV DNA 2-long terminal repeat (2-LTR) circles have been shown in point-based studies to persist in some patients whose plasma HIV RNA was undetectable. However, the degree of fluctuation of circle copy number over time has not been determined. METHODS: A reliable, reproducible and robust quantitative LightCycler (LC qPCR)-based assay for HIV DNA 2-LTR circles in peripheral blood mononuclear (PBMN) cells was established. A prospective, longitudinal study of these circles was undertaken in HIV-1-positive patients on anti-retroviral therapy whose plasma HIV RNA was undetectable at < 50 copies/ml. Patients starting therapy for the first time were also monitored. RESULTS: A cohort of 60 patients whose plasma HIV RNA was undetectable for 32 +/- 2 months were monitored for circles for 15 +/- 2 months. The circle copy number ranged from < 10 to 620 copies/106 PBMN cells. The circle-negative (< 10 copies/1 x 106 PBMN) cells group of 36 patients and the circle-positive (> 10 copies/106 PBMN cells) group of 24 patients were mutually exclusive (P < 0.0001). The mean circle half-life in seven of the 10 patients starting anti-retroviral therapy for the first time was 5.7 days. CONCLUSION: The circle assay is useful for identifying those patients in whom transmission of infectious virus continues despite prolonged periods of time during which plasma HIV RNA is undetectable. New drug combinations and new therapeutic approaches should be aimed at those patients whose plasma HIV RNA is undetectable but who remain positive for 2-LTR circles.

Optimisation of the degree of sulfation of a polymer based construct to block the entry of HIV-1 into cells.

Blocking the entry of HIV-1 into CD4+ cells is an important new therapeutic target for the development of novel vaginal microbicides. In this study, sulfated derivatives of the linear polysaccharide dextrin were synthesised whose percentage sulphation increased incrementally from 7.4 to 48.3%. Their anti-HIV-1 activity in C8166 cells was first seen when percentage sulfation reached 33.2%, but it was only seen in peripheral blood mononuclear cells when it reached 36.3%. It did not increase further when sulfation reached 40.2%. Primary viruses with a V3 loop charge of greater than +5 were blocked by 80 microg/ml of dextrin 2 sulfate but primary viruses with a V3 loop charge of less than +3 required 1,600 microg/ml to block viral entry effectively. Our results identify the relative contribution of the percentage sulfation of a polymer based construct for optimising its anti-HIV-1 activity whilst minimising its toxicity. A better understanding of these structure-function relationships will inform the design and development of novel vaginal microbicides to effectively block the sexual transmission of all primary viral isolates of HIV-1.

Preventing acute gut wall damage in infectious diarrhoeas with glycosylated dendrimers.

Intestinal pathogens use the host's excessive inflammatory cytokine response, designed to eliminate dangerous bacteria, to disrupt epithelial gut wall integrity and promote their tissue invasion. We sought to develop a non-antibiotic-based approach to prevent this injury. Molecular docking studies suggested that glycosylated dendrimers block the TLR4-MD-2-LPS complex, and a 13.6 kDa polyamidoamine (PAMAM) dendrimer glucosamine (DG) reduced the induction of human monocyte interleukin (IL)-6 by Gram-negative bacteria. In a rabbit model of shigellosis, PAMAM-DG prevented epithelial gut wall damage and intestinal villous destruction, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. Computational modelling studies identified a 3.3 kDa polypropyletherimine (PETIM)-DG as the smallest likely bioactive molecule. In human monocytes, high purity PETIM-DG potently inhibited Shigella Lipid A-induced IL-6 expression. In rabbits, PETIM-DG prevented Shigella-induced epithelial gut wall damage, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. There was no change in ß-defensin, IL-10, interferon-ß, transforming growth factor-ß, CD3 or FoxP3 expression. Small and orally delivered DG could be useful for preventing gut wall tissue damage in a wide spectrum of infectious diarrhoeal diseases.

From sequence to 3D structure of hyperbranched molecules: application to surface modified PAMAM dendrimers.

The molecular modeling of hyperbranched molecules is currently constrained by difficulties in model building, due partly to lack of parameterization of their building blocks. We have addressed this problem with specific relevance to a class of hyperbranched macromolecules known as dendrimers by describing a new concept and developing a method that translates monomeric linear sequences into a full atomistic model of a hyperbranched molecule. Such molecular-modeling-based advances will enable modeling studies of important biological interactions between naturally occurring macromolecules and synthetic macromolecules. Our results also suggest that it should be possible to apply this sequence-based methodology to generate hyperbranched structures of other dendrimeric structures and of linear polymers.

Computational design principles for bioactive dendrimer based constructs as antagonists of the TLR4-MD-2-LPS complex.

The cell surface interaction between bacterial lipopolysaccharide (LPS), Toll-like receptor 4 (TLR4) and MD-2 is central to bacterial sepsis syndromes and wound healing. We have shown that a generation (G) 3.5 polyamidoamine (PAMAM) dendrimer that was partially glycosylated with glucosamine inhibits TLR4-MD-2-LPS induced inflammation in a rabbit model of tissue scaring. However, it was a mixture of closely related chemical species because of the polydispersity of the starting PAMAM dendrimer. Generation 2 triazine dendrimers with single chemical entity material status are available at low cost and at the kilogram scale. PAMAM dendrimer can be synthetically grafted onto this triazine core dendrimer to make new triazine-PAMAM hybrid dendrimers. This led us to examine whether molecular modelling methods could be used to identify the key structural design principles for a bioactive lead molecule that could be synthesized and biologically evaluated. We describe our computer aided molecular studies of several dendrimer based constructs and the key design principles identified. Our approach should be more broadly applicable to the biologically focused, rational and accelerated design of molecules for other TLR receptors. They could be useful for treating infectious, inflammatory and malignant diseases.

Structural studies of biologically active glycosylated polyamidoamine (PAMAM) dendrimers.

The partial modification of carboxylic acid terminated polyamidoamine (PAMAM) dendrimers with glucosamine has been reported to give dendrimer glucosamine conjugates novel immuno-modulatory and anti-angiogenic properties. Experimental analysis of these glycosylated dendrimers showed that, on average, eight glucosamine molecules were covalently bound to each dendrimer. In order to better understand the surface loading and distribution of these glucosamine molecules, molecular reactivity was determined by evaluation of electronic properties using frontier molecular orbital theory (FMOT) and molecular dynamics simulations. It was shown that the surface loading and distribution of zero length amide bond-conjugated glucosamine molecules was determined by both electronic effects and by the different dynamic conformations adopted by the modified dendrimer during the incremental addition of glucosamine. Importantly, the structural features and the dynamic behavior of the partially glycosylated generation 3.5 PAMAM dendrimer showed that its flexibility and polarity changed with the incremental addition of glucosamine. These peripheral glucosamine molecules remained available on the dendrimer's surface for interaction with the biological target.

Accelerated healing of cutaneous leishmaniasis in non-healing BALB/c mice using water soluble amphotericin B-polymethacrylic acid.

Cutaneous leishmaniasis (CL) is a neglected tropical disease that causes prominent skin scaring. No water soluble, non-toxic, short course and low cost treatment exists. We developed a new water soluble amphotericin B-polymethacrylic acid (AmB-PMA) using established and scalable chemistries. AmB-PMA was stable for 9 months during storage. In vitro, it was effective against Leishmania spp. promastigotes and amastigote infected macrophages. It was also less toxic and more effective than deoxycholate-AmB, and similar to liposomal AmB. Its in vivo activity was determined in both early and established CL lesion models of Leishmania major infection in genetically susceptible non-healing BALB/c mice. Intradermal AmB-PMA at a total dose of 18 mg of AmB/kg body weight led to rapid parasite killing and lesion healing. No toxicity was seen. No parasite relapse occurred after 80 days follow-up. Histological studies confirmed rapid parasite clearance from macrophages followed by accelerated fibroblast mediated tissue repair, regeneration and cure of the infection. Quantitative mRNA studies of the CL lesions showed that accelerated healing was associated with increased Tumour Necrosis Factor-α and Interferon-γ, and reduced Interleukin-10. These results suggest that a cost-effective AmB-PMA could be used to pharmacologically treat and immuno-therapeutically accelerate the healing of CL lesions.

An amphotericin B-based drug for treating experimental Leishmania major infection.

There is an urgent need for a non-toxic and low-cost treatment for cutaneous leishmaniasis. We synthesised and tested in vivo an amphotericin B-poly(methacrylic acid) drug (AmB-PMA) that had previously shown in-vitro activity against Leishmania major and L. donovani parasites. Efficacy was determined using L. major footpad infection in 30 non-healing BALB/c mice. Three subcutaneous injections of AmB-PMA at days 7, 14 and 21 post-infection resulted in a reduction of ∼80% in lesion size by day 35 post-infection in 18 treated mice compared with six untreated controls, and complete healing of lesions by day 50 with no lesion relapse seen at day 80 post-infection in six treated mice. Healing was associated with decreased IL-10 (P=0.002) and increased IFN-γ (P=0.005) in the footpad.

Site-specific PEGylation of protein disulfide bonds using a three-carbon bridge.

The covalent conjugation of a functionalized poly(ethylene glycol) (PEG) to multiple nucleophilic amine residues results in a heterogeneous mixture of PEG positional isomers. Their physicochemical, biological, and pharmaceutical properties vary with the site of conjugation of PEG. Yields are low because of inefficient conjugation chemistry and production costs high because of complex purification procedures. Our solution to these fundamental problems in PEGylating proteins has been to exploit the latent conjugation selectivity of the two sulfur atoms that are derived from the ubiquitous disulfide bonds of proteins. This approach to PEGylation involves two steps: (1) disulfide reduction to release the two cysteine thiols and (2) re-forming the disulfide by bis-alkylation via a three-carbon bridge to which PEG was covalently attached. During this process, irreversible denaturation of the protein did not occur. Mechanistically, the conjugation is conducted by a sequential, interactive bis-alkylation using alpha,beta-unsaturated beta'-monosulfone functionalized PEG reagents. The combination of (a) maintaining the protein's tertiary structure after disulfide reduction, (b) the mechanism for bis-thiol selectivity of the PEG reagent, and (c) the steric shielding of PEG ensure that only one PEG molecule is conjugated at each disulfide bond. PEG was site-specifically conjugated via a three-carbon bridge to 2 equiv of the tripeptide glutathione, the cyclic peptide hormone somatostatin, the tetrameric protein l-asparaginase, and to the disulfides in interferon alpha-2b (IFN). SDS-PAGE, mass spectral, and NMR analyses were used to confirm conjugation, thiol selectivity, and connectivity. The biological activity of the l-asparaginase did not change after the attachment of four PEG molecules. In the case of IFN, a small reduction in biological activity was seen with the single-bridged IFN (without PEG attached). A significantly larger reduction in biological activity was seen with the three-carbon disulfide single-bridged PEG-IFNs and with the double-bridged IFN (without PEG attached). The reduction of the PEG-IFN's in vitro biological activity was a consequence of the steric shielding caused by PEG, and it was comparable to that seen with all other forms of PEG-IFNs reported. However, when a three-carbon bridge was used to attach PEG, our PEG-IFN's biological activity was found to be independent of the length of the PEG. This property has not previously been described for PEG-IFNs. Our studies therefore suggest that peptides, proteins, enzymes, and antibody fragments can be site-specifically PEGylated across a native disulfide bond using three-carbon bridges without destroying their tertiary structure or abolishing their biological activity. The stoichiometric efficiency of this approach also enables recycling of any unreacted protein. It therefore offers the potential to make PEGylated biopharmaceuticals as cost-effective medicines for global use.

Site-specific PEGylation of native disulfide bonds in therapeutic proteins.

Native disulfide bonds in therapeutic proteins are crucial for tertiary structure and biological activity and are therefore considered unsuitable for chemical modification. We show that native disulfides in human interferon alpha-2b and in a fragment of an antibody to CD4(+) can be modified by site-specific bisalkylation of the two cysteine sulfur atoms to form a three-carbon PEGylated bridge. The yield of PEGylated protein is high, and tertiary structure and biological activity are retained.