Adsorption of Pathogens and Blockade of Sepsis Cascade.

Sepsis is caused by the host response to an infectious organism. It is common among hospitalized patients and is associated with significant morbidity and mortality. The current standard of care for sepsis is predominantly supportive, with early detection followed by prompt antibiotic administration. While this approach has undoubtedly improved patient outcomes, it has significant limitations. First, mortality from sepsis remains unacceptably high. Second, emerging pathogen resistance to antimicrobial therapies threatens a return to the pre-antimicrobial era of patient care. Lastly, the early stages of a pandemic (e.g., the recent coronavirus 19 pandemic) lack effective therapeutics. Given these limitations, novel treatment strategies are needed to advance the field and care for patients. One potential class of therapy is extracorporeal blood purification (EBP). While EBP is a broad classification, encompassing a wide range of techniques, this article will focus on three emerging EBP therapies that have been shown to bind and remove a wide variety of viral, bacterial, and fungal pathogens directly from circulation. These devices utilize different mechanisms of action for pathogen removal. The Seraph® 100 is composed of heparin coated beads. The Hemopurifier® combines the concept of plasma exchange with mannose-binding lectin (MBL). Lastly, the GARNET® utilizes a MBL fused to an IgG antibody. Via these mechanisms, these devices have been demonstrated to remove pathogens and pathogen-associated molecular patterns. The hope is that by directly removing pathogens, these EBP techniques may result in the biggest breakthrough in the management of sepsis since the advent of antibiotics almost 100 years ago.

Removal of Carbapenem-Resistant Enterobacteriaceae (CRE) from blood by heparin-functional hemoperfusion media.

Bloodstream infections due to Carbapenem-Resistant Enterobacteriaceae (CRE) are becoming more frequent and are associated with a high mortality. At present, combination antimicrobial therapy yields the best outcomes, but treatment options are limited. Many bacteria utilize heparan sulfate to bind to human cells. We studied the ability of a biomimetic device composed of polyethylene beads with endpoint-attached heparin to bind both sensitive and (CRE) E. coli and Klebsiella pneumoniae from spiked blood samples. Greater than 90% of susceptible, E. coli, CRE E. coli and CRE Klebsiella were removed by the beads. Future studies in human bacteremia with this technology are planned.

Affinity apheresis for treatment of bacteremia caused by Staphylococcus aureus and/or methicillin-resistant S. aureus (MRSA).

Staphylococcus aureus (SA) bacteremia is associated with high mortality, and often results in metastatic infections. The methicillin-resistant SA (MRSA) is an urgent health care issue, as nosocomial infections with these bacteria represent limited treatment alternatives. Samples of whole blood containing challenge inoculums of SA and MRSA strains were passed through columns packed with surface-heparinized polyethylene beads. The bound bacteria were eluted and quantitatively determined by culturing and by real-time PCR. Significant amounts of both SA and MRSA adhered to the heparinized beads (more than 65% of inoculated bacteria). After rinsing with buffer at high ionic strength, viable bacteria or bacterial DNA were eluted from the columns, indicating that the binding was specific. The conclusions that can be made from these experiments are that, as earlier reported in the literature, the high affinity of SA to heparin is retained in whole blood, and MRSA in whole blood binds to heparin with similar or higher affinity than SA. It should be possible to lower the amount of SA and/or MRSA from the blood of infected patients to levels that could be taken care of by the immune system. In previous studies, we have shown that passing blood from septic patients over beads coated with end-point-attached, biologically active heparin is a useful technique for regulating the levels of heparin-binding cytokine. These findings in combination with the present findings indicate the possibility of creating an apheresis technology for treatment of sepsis caused by SA and/or MRSA.

Assembly and structure of alpha-helical peptide films on hydrophobic fluorocarbon surfaces.

The structure, orientation, and formation of amphiphilic alpha-helix model peptide films on fluorocarbon surfaces has been monitored with sum frequency generation (SFG) vibrational spectroscopy, near-edge x-ray absorption fine structure (NEXAFS) spectroscopy, and x-ray photoelectron spectroscopy (XPS). The alpha-helix peptide is a 14-mer of hydrophilic lysine and hydrophobic leucine residues with a hydrophobic periodicity of 3.5. This periodicity yields a rigid amphiphilic peptide with leucine and lysine side chains located on opposite sides. XPS composition analysis confirms the formation of a peptide film that covers about 75% of the surface. NEXAFS data are consistent with chemically intact adsorption of the peptides. A weak linear dichroism of the amide pi( *) is likely due to the broad distribution of amide bond orientations inherent to the alpha-helical secondary structure. SFG spectra exhibit strong peaks near 2865 and 2935 cm(-1) related to aligned leucine side chains interacting with the hydrophobic surface. Water modes near 3200 and 3400 cm(-1) indicate ordering of water molecules in the adsorbed-peptide fluorocarbon surface interfacial region. Amide I peaks observed near 1655 cm(-1) confirm that the secondary structure is preserved in the adsorbed peptide. A kinetic study of the film formation process using XPS and SFG showed rapid adsorption of the peptides followed by a longer assembly process. Peptide SFG spectra taken at the air-buffer interface showed features related to well-ordered peptide films. Moving samples through the buffer surface led to the transfer of ordered peptide films onto the substrates.

Cytokines in blood from septic patients interact with surface-immobilized heparin.

When passing blood from septic patients through a column packed with surface heparinized beads, we were able to significantly reduce concentrations of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha from initially very high levels. Passage of blood over nonheparinized beads did not affect the TNF levels. Meanwhile, concentrations of the regulated on activation, normal T-cells expressed, and secreted leukocyte activating cytokine (RANTES) remained unchanged following passage through the heparinized column, but rose significantly after passage through a column packed with the nonheparinized control beads. We conclude that surface heparinization may be a useful technique for selectively regulating the levels of heparin-binding cytokines from whole blood. This may have potential implications for the treatment of hyper-inflammatory conditions such as severe sepsis. Our data also suggests that surface activation and its associated inflammatory response may be avoided by using heparinization of the extracorporeal circuit.

Multi-technique Characterization of Adsorbed Peptide and Protein Orientation: LK310 and Protein G B1.

The ability to orient biologically active proteins on surfaces is a major challenge in the design, construction, and successful deployment of many medical technologies. As methods to orient biomolecules are developed, it is also essential to develop techniques that can an accurately determine the orientation and structure of these materials. In this study, two model protein and peptide systems are presented to highlight the strengths of three surface analysis techniques for characterizing protein films: time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum-frequency generation (SFG) vibrational spectroscopy, and near-edge x-ray absorption fine structure (NEXAFS) spectroscopy. First, the orientation of Protein G B1, a rigid 6 kDa domain covalently attached to a maleimide-functionalized self-assembled monolayer, was examined using ToF-SIMS. Although the thickness of the Protein G layer was similar to the ToF-SIMS sampling depth, orientation of Protein G was successfully determined by analyzing the C2H5S+ intensity, a secondary ion derived from a methionine residue located at one end of the protein. Next, the secondary structure of a 13-mer leucine-lysine peptide (LK310) adsorbed onto hydrophilic quartz and hydrophobic fluorocarbon surfaces was examined. SFG spectra indicated that the peptide's lysine side chains were ordered on the quartz surface, while the peptide's leucine side chains were ordered on the fluorocarbon surface. NEXAFS results provided complementary information about the structure of the LK310 film and the orientations of amide bonds within the LK310 peptide.

Hydration of sulphobetaine and tetra(ethylene glycol)-terminated self-assembled monolayers studied by sum frequency generation vibrational spectroscopy.

Sum-frequency generation (SFG) vibrational spectroscopy is used to study the surface and the underlying substrate of both homogeneous and mixed self-assembled monolayers (SAMs) of 11-mercaptoundecyl-1-sulphobetainethiol (HS(CH(2))(11)N(+)(CH(3))(2)(CH(2))(3)SO(3)(-)) and 1-mercapto-11-undecyl tetra(ethylene glycol) (HS(CH(2))(11)O(CH(2)CH(2)O)(4)OH) with an 11-mercapto-1-undecanol (HS(CH(2))(11)OH) diluent. SFG results on the C-H region of the dry and hydrated SAMs gave an in situ look into the molecular orientation and suggested an approach to maximize signal-to-noise ratio on these difficult to analyze hydrophilic SAMs. Vibrational fingerprint studies in the 3000-3600 cm(-1) spectral range for the SAMs exposed serially to air, water, and deuterated water revealed that a layer of tightly bound structured water was associated with the surface of a nonfouling monolayer but was not present on a hydrophobic N-undecylmercaptan (HS(CH(2))(10)CH(3)) control. The percentage of water retained upon submersion in D(2)O correlated well with the relative amount of protein that was previously shown to absorb onto the monolayers. These results provide evidence supporting the current theory regarding the role of a tightly bound vicinal water layer in the protein resistance of a nonfouling group.

A new optical parametric amplifier based on lithium thioindate used for sum frequency generation vibrational spectroscopic studies of the amide I mode of an interfacial model peptide.

We describe a new optical parametric amplifier (OPA) that employs lithium thioindate, LiInS2 (LIS), to create tunable infrared light between 1500 cm(-1) and 2000 cm(-1). The OPA based on LIS described within provides intense infrared light with a good beam profile relative to similar OPAs built on silver gallium sulfide, AgGaS2 (AGS), or silver gallium selenide, AgGaSe2 (AGSe). We have used the new LIS OPA to perform surface-specific sum frequency generation (SFG) vibrational spectroscopy of the amide I vibrational mode of a model peptide at the hydrophobic deuterated polystyrene (d8-PS)-phosphate buffered saline interface. This model polypeptide (which is known to be an alpha-helix in the bulk solution under the high ionic strength conditions employed here) contains hydrophobic leucyl (L) residues and hydrophilic lysyl (K) residues, with sequence Ac-LKKLLKLLKKLLKL-NH2. The amide I mode at the d8-PS-buffer interface was found to be centered around 1655 cm(-1). This can be interpreted as the peptide having maintained its alpha-helical structure when adsorbed on the hydrophobic surface, although other interpretations are discussed.

In situ adsorption studies of a 14-amino acid leucine-lysine peptide onto hydrophobic polystyrene and hydrophilic silica surfaces using quartz crystal microbalance, atomic force microscopy, and sum frequency generation vibrational spectroscopy.

The adsorption of a 14-amino acid amphiphilic peptide, LK14, which is composed of leucine (L, nonpolar) and lysine (K, charged), on hydrophobic polystyrene (PS) and hydrophilic silica (SiO2) was investigated in situ by quartz crystal microbalance (QCM), atomic force microscopy (AFM), and sum frequency generation (SFG) vibrational spectroscopy. The LK14 peptide, adsorbed from a pH 7.4 phosphate-buffered saline (PBS) solution, displayed very different coverage, surface roughness and friction, topography, and surface-induced orientation when adsorbed onto PS versus SiO2 surfaces. Real-time QCM adsorption data revealed that the peptide adsorbed onto hydrophobic PS through a fast (t < 2 min) process, while a much slower (t > 30 min) multistep adsorption and rearrangement occurred on the hydrophilic SiO2. AFM measurements showed different surface morphologies and friction coefficients for LK14 adsorbed on the two surfaces. Surface-specific SFG spectra indicate very different ordering of the adsorbed peptide on hydrophobic PS as compared to hydrophilic SiO2. At the LK14 solution/PS interface, CH resonances corresponding to the hydrophobic leucine side chains are evident. Conversely, only NH modes are observed at the peptide solution/SiO2 interface, indicating a different average molecular orientation on this hydrophilic surface. The surface-dependent difference in the molecular-scale peptide interaction at the solution/hydrophobic solid versus solution/hydrophilic solid interfaces (measured by SFG) is manifested as significantly different macromolecular-level adsorption properties on the two surfaces (determined via AFM and QCM experiments).