Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.

Over half of the nearly 2 million healthcare-associated infections can be attributed to indwelling medical devices. In this review we highlight the difficulty in diagnosing implantable device related infection and how this leads to a likely underestimate of the prevalence. We then provide a length-scale conceptualization of device related infection pathogenesis. Within this conceptualization we focus specifically on biofilm formation and the role of host immune and coagulation systems. Using this framework, we describe how current and developing preventative strategies target specific processes along the entire length-scale. In light of the significant time horizon for the development and translation of new preventative technologies, we also emphasize the need for parallel development of in situ treatment strategies. Specific examples of both preventative and treatment strategies and how they align with the length-scale conceptualization are described.

The complement activation product, C5a, is a powerful phlogistic factor. Using antibodies to detect human or rat C5a, incubation at pH 7.4 of human blood neutrophils or rat alveolar macrophages (AMs) with C5 in the presence of phorbol 12-myristate 13-acetate (PMA) led to generation of C5a. Rat AMs activated with lipopolysaccharide also generated C5a from C5. With activated neutrophils, extensive cleavage of C5 occurred, whereas activated macrophages had much more selective proteolytic activity for C5. Peripheral blood human or rat mononuclear cells and rat alveolar epithelial cells when stimulated with phorbol ester all failed to demonstrate an ability to cleave C5, suggesting a specificity of C5 cleavage by phagocytic cells. With rat AMs, C5a generation was time-dependent and was blocked if AMs were pretreated with inhibitors of transcription or protein synthesis (actinomycin D or cycloheximide). Similar treatment of activated human polymorphonuclear leukocytes only partially reduced C5a generation after addition of C5. C5a generated by activated AMs was biologically (chemotactically) active. This generation was sensitive to serine protease inhibitors but not to other classes of inhibitors. These data indicate that phagocytic cells, especially lung macrophages, can generate C5a from C5. In the context of the lung, this may represent an important C5a-generating pathway that is independent of the plasma complement system. The complement system generating the complement activation products, C3a, C5a, and C5b-9 and the cellular defense system involving macrophages and neutrophils are known to form the first line of defense (innate immunity) against microorganisms and other tissue-damaging factors.

We developed a method to grow Staphylococcus epidermidis bacterial biofilms and characterize their rheological properties in situ in a continuously fed bioreactor incorporated into a parallel plate rheometer. The temperature and shear rates of growth modeled bloodstream conditions, a common site of S. epidermidis infection. We measured the linear elastic (G′) and viscous moduli (G″) of the material using small-amplitude oscillatory rheology and the yield stress using non-linear creep rheology. We found that the elastic and viscous moduli of the S. epidermidis biofilm were 11 ± 3 Pa and 1.9 ± 0.5 Pa at a frequency of 1 Hz (6.283 rad per s) and that the yield stress was approximately 20 Pa. We modeled the linear creep response of the biofilm using a Jeffreys model and found that S. epidermidis has a characteristic relaxation time of approximately 750 seconds and a linear creep viscosity of 3000 Pa s. The effects on the linear viscoelastic moduli of environmental stressors, such as NaCl concentration and extremes of temperature, were also studied. We found a non-monotonic relationship between moduli and NaCl concentrations, with the stiffest material properties found at human physiological concentrations (135 mM). Temperature dependent rheology showed hysteresis in the moduli when heated and cooled between 5 °C and 60 °C. Through these experiments, we demonstrated that biofilms are rheologically complex materials that can be characterized by a combination of low modulus (~10 Pa), long relaxation time (~103 seconds), and a finite yield stress (20 Pa). This suggests that biofilms should be viewed as soft viscoelastic solids whose properties are determined in part by local environmental conditions. The in situ growth method introduced here can be adapted to a wide range of biofilm systems and applied over a broad spectrum of rheological and environmental conditions because the technique minimizes the risk of irreversible, non-linear deformation of the microbial specimen before analysis.

We evaluated antibodies to different peptide regions of rat C5a in the sepsis model of cecal ligation and puncture (CLP) for their protective effects in rats. Rabbit polyclonal antibodies were developed to the following peptide regions of rat C5a: amino-terminal region (A), residues 1-16; middle region (M), residues 17-36; and the carboxyl-terminal region (C), residues 58-77. With rat neutrophils, the chemotactic activity of rat C5a was significantly inhibited by antibodies with the following rank order: anti-C > anti-M >> anti-A. In vivo, antibodies to the M and C (but not A) regions of C5a were protective in experimental sepsis, as determined by survival over a 10-day period, in a dose-dependent manner. The relative protective efficacies of anti-C5a preparations (in descending order of efficacy) were anti-C > anti-M >> anti-A. In CLP rats, a delay in infusion of antibodies, which were injected at 6 or 12 h after CLP, still resulted in significant improvement in survival rates. These in vivo and in vitro data suggest that there are optimal targets on C5a for blockade during sepsis and that delayed infusion of anti-C5a antibody until after onset of clinical evidence of sepsis still provides protective effects.

The current studies demonstrate protective and harmful effects of neutrophils (PMN) during experimental sepsis after cecal ligation and puncture (CLP). It is known that CLP induces signaling defects in blood PMN. When PMN were depleted 12 h after CLP, there were dramatic reductions in levels of bacteremia, evidence for reduced liver and renal dysfunction, sharp reductions in serum levels of cytokines (IL-1beta, IL-6, IL-10, TNF-alpha, and IL-2), and improved survival. In contrast, PMN depletion before CLP resulted in substantial increases in bacteremia and no evidence for attenuation of liver and renal failure dysfunction. These data suggest that at the onset of sepsis, PMN are important in regulating the levels of bacteremia, whereas after the onset of sepsis, as they lose innate immune functions, their presence is associated with higher levels of bacteremia and intensified organ dysfunction.

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We introduce a flexible microfluidic device to characterize the mechanical properties of soft viscoelastic solids such as bacterial biofilms. In the device, stress is imposed on a test specimen by application of a fixed pressure to a thin, flexible poly(dimethyl siloxane) (PDMS) membrane that is in contact with the specimen. The stress is applied by pressurizing a microfabricated air channel located above the test area. The strain resulting from the applied stress is quantified by measuring the membrane deflection with a confocal laser-scanning microscope. The deflection is governed by the viscoelastic properties of the PDMS membrane and of the test specimen. The relative contributions of the membrane and test material to the measured deformation are quantified by comparing a finite element analysis and an independent (control) measurement of the PDMS membrane mechanical properties. The flexible microfluidic rheometer was used to characterize both the steady-state elastic modulus and transient strain recoil of two soft materials: gellan gums and bacterial biofilms. The measured linear elastic moduli and viscoelastic relaxation times of gellan gum solutions were in good agreement with the results of conventional mechanical rheometry. The linear Young’s moduli of biofilms of Staphylococcus epidermidis and Klebsiella pneumoniae, which could not be measured using conventional methods, were found to be 3.2 kPa and 1.1 kPa, respectively, and the relaxation time of the S. epidermidis biofilm was 13.8 s. Additionally, strain hardening was observed in all the biofilms studied. Finally, design parameters and detection limits of the method show that the device is capable of characterizing soft viscoelastic solids with elastic moduli in the range of 102 – 105 Pa. The flexible microfluidic rheometer addresses a need for mechanical property characterization of soft viscoelastic solids common in fields such as biomaterials, food and consumer products. It requires only ~ 200 pL of test specimen.