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BACKGROUND: The complement and coagulation systems share an evolutionary origin with many components showing structural homology. Certain components, including complement factor H (FH) and coagulation factor XII (FXII), have separately been shown to have auxiliary activities across the two systems.OBJECTIVES: The interaction between FXII and FH was investigated.METHODS: Using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) complex formation between different FXII forms and FH was investigated. The presence of α-FXIIa:FH complexes upon contact activation in plasma was evaluated by ELISA and immunoprecipitation.RESULTS: We identified and characterized a direct interaction between the components and demonstrated that among different forms of FXII, only the activated α-FXIIa formed complexes with FH, with an apparent binding strength K d of 34 ± 9 nmol/L. The complex formation involved the kringle domain of the heavy chain of FXII. C1-inhibitor induced inhibition of α-FXIIa did not alter the binding of α-FXIIa toward FH. We further demonstrated the presence of α-FXIIa:FH complexes in normal human plasma upon contact activation, indicating formation of α-FXIIa:FH complexes as a consequence of α-FXIIa generation. Complex formation between α-FXIIa and FH was also assessed in hereditary angioedema (HAE) patients with C1-inhibitor deficiency as well as rheumatoid arthritis (RA) patients with high levels of anti-cyclic citrullinated peptide (anti-CCP) upon contact activation. We observed elevated levels of α-FXIIa:FH complexes in HAE patients, and equal levels of complexes in RA patients and healthy individuals upon contact activation. CONCLUSION: A direct interaction between α-FXIIa and FH is demonstrated. Our findings represent a new crosstalk between these systems, potentially important in the onset and pathology of inflammatory vascular diseases.

The developmental speed of new antimicrobials does not meet the emergence of multidrugresistant bacteria sufficiently. A potential shortcut is assessing the antimicrobial activity of already approved drugs. Intrudingly, the antibacterial action of glatiramer acetate (GA) has recently been discovered. GA is a well-known and safe immunomodulatory drug particular effective against Gram-negative bacteria, which disrupts biological membranes by resembling the activity of antimicrobial peptides. Thus, GA can potentially be included in treatment strategies used to combat infections caused by multidrug-resistant Gram-negatives. One potential application is chronic respiratory infections caused by Pseudomonas aeruginosa, however the safety of GA inhalation has never been assessed. Here, the safety of inhaling nebulized GA is evaluated in a preclinical pig model. The potential side effects, i.e., bronchoconstriction, respiratory tract symptoms and systemic- and local inflammation were assessed by ventilator monitoring, clinical observation, biochemistry, flowcytometry, and histopathology. No signs of bronchoconstriction assessed by increased airway peak pressure, P peak, or decreased oxygen pressure were observed. Also, there were no signs of local inflammation in the final histopathology examination of the pulmonary tissue. As we did not observe any potential pulmonary side effects of inhaled GA, our preliminary results suggest that GA inhalation is safe and potentially can be a part of the treatment strategy targeting chronic lung infections caused by multidrug-resistant Gram-negative bacteria.

The current treatment regimen for nontuberculous mycobacteria (NTM) involves long courses of antibiotic cocktails that demonstrate limited efficacy and cause frequent and serious side effects. Mycobacterium abscessus, in particular, is difficult to treat, motivating studies to identify new therapeutic targets. Experiments using zebrafish and human cell culture lines have demonstrated that M. abscessus is more virulent when aggregated into cord-like biofilms, at least in part because of the decreased ability of phagocytes to efficiently engulf and kill corded M. abscessus cells.

Classic drug development strategies have failed to meet the urgent clinical needs in treating infections with Gram-negative bacteria. Repurposing drugs can lead to timely availability of new antibiotics, accelerated by existing safety profiles. Glatiramer acetate (GA) is a widely used and safe formulation for treatment of multiple sclerosis. It contains a large diversity of essentially isomeric polypeptides with the cationic and amphiphilic character of many antimicrobial peptides (AMP). Here, we report that GA is antibacterial, targeting Gram-negative organisms with higher activity towards Pseudomonas aeruginosa than the naturally-occurring AMP LL-37 in human plasma. As judged from flow cytometric assays, bacterial killing by GA occurred within minutes. Laboratory strains of Escherichia coli and P. aeruginosa were killed by a process of condensing intracellular contents. Efficient killing by GA was also demonstrated in Acinetobacter baumannii clinical isolates and approximately 50% of clinical isolates of P. aeruginosa from chronic airway infection in CF patients. By contrast, the Gram-positive Staphylococcus aureus cells appeared to be protected from GA by an increased formation of nm-scale particulates. Our data identify GA as an attractive drug repurposing candidate to treat infections with Gram-negative bacteria.

Summary The pathogenesis of sepsis involves a dual inflammatory response, with a hyperinflammatory phase followed by, or in combination with, a hypoinflammatory phase. The adhesion molecules lymphocyte function-associated antigen (LFA-1) (CD11a/CD18) and macrophage-1 (Mac-1) (CD11b/CD18) support leucocyte adhesion to intercellular adhesion molecules and phagocytosis through complement opsonization, both processes relevant to the immune response during sepsis. Here, we investigate the role of soluble (s)CD18 in sepsis with emphasis on sCD18 as a mechanistic biomarker of immune reactions and outcome of sepsis. sCD18 levels were measured in 15 septic and 15 critically ill non-septic patients. Fifteen healthy volunteers served as controls. CD18 shedding from human mononuclear cells was increased in vitro by several proinflammatory mediators relevant in sepsis. sCD18 inhibited cell adhesion to the complement fragment iC3b, which is a ligand for CD11b/CD18, also known as Mac-1 or complement receptor 3. Serum sCD18 levels in sepsis non-survivors displayed two distinct peaks permitting a partitioning into two groups, namely sCD18 ‘high’ and sCD18 ‘low’, with median levels of sCD18 at 2158 mU/ml [interquartile range (IQR) 2093–2811 mU/ml] and 488 mU/ml (IQR 360–617 mU/ml), respectively, at the day of intensive care unit admission. Serum sCD18 levels partitioned sepsis non-survivors into one group of ‘high’ sCD18 and low CRP and another group with ‘low’ sCD18 and high C-reactive protein. Together with the mechanistic data generated in vitro, we suggest the partitioning in sCD18 to reflect a compensatory anti-inflammatory response syndrome and hyperinflammation, respectively, manifested as part of sepsis.

The formulation glatiramer acetate (GA) is widely used in therapy of multiple sclerosis. GA consists of random copolymers of four amino acids, in ratios that produce a predominantly positive charge and an amphipathic character. With the extraordinary complexity of the drug, several pharmacological modes-of-action were suggested, but so far none, which rationalizes the cationicity and amphipathicity as part of the mode-of-action. Here, we report that GA rapidly kills primary human T lymphocytes and, less actively, monocytes. LL-37 is a cleavage product of human cathelicidin with important roles in innate immunity. It shares the positive charge and amphipathic character of GA, and, as shown here, also the ability to kill human leukocyte. The cytotoxicity of both compounds depends on sialic acid in the cell membrane. The killing was associated with the generation of CD45 + debris, derived from cell membrane deformation. Nanoparticle tracking analysis confirmed the formation of such debris, even at low GA concentrations. Electric cell-substrate impedance sensing measurements also recorded stable alterations in T lymphocytes following such treatment. LL-37 forms oligomers through weak hydrophobic contacts, which is critical for the lytic properties. In our study, SAXS showed that GA also forms this type of contacts. Taken together, our study offers new insight on the immunomodulatory mode-of-action of positively charged co-polymers. The comparison of LL-37 and GA highlights a consistent requirement of certain oligomeric and chemical properties to support cytotoxic effects of cationic polymers targeting human leukocytes. The formulation glatiramer acetate (GA) is widely used in therapy of multiple sclerosis. GA consists of random copolymers of four amino acids, in ratios that produce a predominantly positive charge and an amphipathic character. With the extraordinary complexity of the drug, several pharmacological modes-of-action were suggested, but so far none, which rationalizes the cationicity and amphipathicity as part of the mode-of-action. Here, we report that GA rapidly kills primary human T lymphocytes and, less actively, monocytes. LL-37 is a cleavage product of human cathelicidin with important roles in innate immunity. It shares the positive charge and amphipathic character of GA, and, as shown here, also the ability to kill human leukocyte. The cytotoxicity of both compounds depends on sialic acid in the cell membrane. The killing was associated with the generation of CD45 + debris, derived from cell membrane deformation. Nanoparticle tracking analysis confirmed the formation of such debris, even at low GA concentrations. Electric cell-substrate impedance sensing measurements also recorded stable alterations in T lymphocytes following such treatment. LL-37 forms oligomers through weak hydrophobic contacts, which is critical for the lytic properties. In our study, SAXS showed that GA also forms this type of contacts. Taken together, our study offers new insight on the immunomodulatory mode-of-action of positively charged co-polymers. The comparison of LL-37 and GA highlights a consistent requirement of certain oligomeric and chemical properties to support cytotoxic effects of cationic polymers targeting human leukocytes.

The existence of the ocular microbiota has been reported but functional analyses to evaluate its significance in regulating ocular immunity are currently lacking. We compared the relative contribution of eye and gut commensals in regulating the ocular susceptibility to Pseudomonas aeruginosa–induced keratitis. We find that in health, the presence of microbiota strengthened the ocular innate immune barrier by significantly increasing the concentrations of immune effectors in the tear film, including secretory IgA and complement proteins. Consistent with this view, Swiss Webster (SW) mice that are typically resistant to P. aeruginosa–induced keratitis become susceptible due to the lack of microbiota. This was exemplified by increased corneal bacterial burden and elevated pathology of the germ free (GF) mice when compared to the conventionally maintained SW mice. The protective immunity was found to be dependent on both eye and gut microbiota with the eye microbiota having a moderate, but significant impact on the resistance to infection. These events were IL-1ß–dependent as corneal IL-1ß levels were decreased in the infected GF and antibiotic-treated mice when compared to the SPF controls, and neutralization of IL-1ß increased the ocular bacterial burden in the SPF mice. Monocolonizing GF mice with Coagulase Negative Staphylococcus sp. isolated from the conjunctival swabs was sufficient to restore resistance to infection. Cumulatively, these data underline a previously unappreciated role for microbiota in regulating susceptibility to ocular keratitis. We predict that these results will have significant implications for contact lens wearers, where alterations in the ocular commensal communities may render the ocular surface vulnerable to infections., Author Summary Contact lens wear is associated with frequent Pseudomonas aeruginosa–induced keratitis, however the reasons for this association remain unclear. Recent genomics–based approaches revealed that contact lens wearers harbor altered ocular commensal communities when compared to non-lens wearers raising important questions, namely, does wearing of contact lenses increase the frequency of keratitis in patients due to contamination of the contact lenses with species derived from the skin or does ocular microbiota exert immune functions that are required for the maintenance of ocular health? We demonstrate a clear role for ocular microbiota in regulating protection against Pseudomonas aeruginosa–induced infections. At the ocular surface, commensal bacteria provide signals that regulate the magnitude of neutrophil recruitment during infection. These events may be driven by a frequent gram-positive commensal–Coagulase Negative Staphylococcus (CNS) sp. In addition to the impact of ocular microbiota, there is an important contribution of gut microbiota that stimulate neutrophil development in the bone marrow, thereby regulating the pool of mature neutrophils and their activation state. Cumulatively, these data show for the first time a role for microbiota in regulating the susceptibility to P. aeruginosa–keratitis.

The present invention provides a positively charged co-polymer for use as an antimicrobial agent, wherein said positively charged co-polymer is composed of amino acids and/or derivatives thereof and wherein at least 75 molar percent of said amino acids are selected from the group consisting of alanine, lysine, glutamate, arginine and tyrosine and/or derivatives thereof. The present invention also provides methods for treating, preventing or ameliorating a microbial infection comprising administration of positively charged random co-polymers as well as a pharmaceutical composition comprising said co-polymer. The invention further provides a kit of parts comprising the positively charged random co-polymer. The present invention provides a positively charged co-polymer for use as an antimicrobial agent, wherein said positively charged co-polymer is composed of amino acids and/or derivatives thereof and wherein at least 75 molar percent of said amino acids are selected from the group consisting of alanine, lysine, glutamate, arginine and tyrosine and/or derivatives thereof. The present invention also provides methods for treating, preventing or ameliorating a microbial infection comprising administration of positively charged random co-polymers as well as a pharmaceutical composition comprising said co-polymer. The invention further provides a kit of parts comprising the positively charged random co-polymer.

Influenza is a major challenge to healthcare systems world-wide. While prophylactic vaccination is largely efficient, long-lasting immunity has not been achieved in immunized populations, at least in part due to the challenges arising from the antigen variation between strains of influenza A virus as a consequence of genetic drift and shift. From progress in our understanding of the immune system, the mode-of-action of vaccines can be divided into the stimulation of the adaptive system through inclusion of appropriate vaccine antigens and of the innate immune system by the addition of adjuvant to the vaccine formulation. A shared property of many vaccine adjuvants is found in their nature of water-insoluble precipitates, for instance the particulate material made from aluminum salts. Previously, it was thought that embedding of vaccine antigens in these materials provided a "depot" of antigens enabling a long exposure of the immune system to the antigen. However, more recent work points to a role of particulate adjuvants in stimulating cellular parts of the innate immune system. Here, we briefly outline the infectious medicine and immune biology of influenza virus infection and procedures to provide sufficient and stably available amounts of vaccine antigen. This is followed by presentation of the many roles of adjuvants, which involve humoral factors of innate immunity, notably complement. In a perspective of the ultrastructural properties of these humoral factors, it becomes possible to rationalize why these insoluble precipitates or emulsions are such a provocation of the immune system. We propose that the biophysics of particulate material may hold opportunities that could aid the development of more efficient influenza vaccines.

Antimicrobial resistance (AMR) defines the ability of a microbeto resist a medicine, which once could be effectively treatedusing the drug. This situation is emerging as a major problemin health care globally. A closer look at the recent literature,in particular with a clinical microbiology perspective in mind,quickly points to AMR as multifaceted in origins, and hence, nota single problem. This has consequence for how to approachthe treatment of infections and especially how and when tofocus on new drug therapies.