Biofilms in Periprosthetic Orthopedic Infections Seen through the Eyes of Neutrophils: How Can We Help Neutrophils?

Despite advancements in our knowledge of neutrophil responses to planktonic bacteria during acute inflammation, much remains to be elucidated on how neutrophils deal with bacterial biofilms in implant infections. Further complexity transpires from the emerging findings on the role that biomaterials play in conditioning bacterial adhesion, the variety of biofilm matrices, and the insidious measures that biofilm bacteria devise against neutrophils. Thus, grasping the entirety of neutrophil-biofilm interactions occurring in periprosthetic tissues is a difficult goal. The bactericidal weapons of neutrophils consist of the following: ready-to-use antibacterial proteins and enzymes stored in granules; NADPH oxidase-derived reactive oxygen species (ROS); and net-like structures of DNA, histones, and granule proteins, which neutrophils extrude to extracellularly trap pathogens (the so-called NETs: an allusive acronym for "neutrophil extracellular traps"). Neutrophils are bactericidal (and therefore defensive) cells endowed with a rich offensive armamentarium through which, if frustrated in their attempts to engulf and phagocytose biofilms, they can trigger the destruction of periprosthetic bone. This study speculates on how neutrophils interact with biofilms in the dramatic scenario of implant infections, also considering the implications of this interaction in view of the design of new therapeutic strategies and functionalized biomaterials, to help neutrophils in their arduous task of managing biofilms.

Searching for Virulence Factors among Staphylococcus lugdunensis Isolates from Orthopedic Infections: Correlation of ß-hemolysin, hemolysin III, and slush Genes with Hemolytic Activity and Synergistic Hemolytic Activity.

Staphylococcus lugdunensis is an emerging high-virulent pathogen. Here, the presence and expression of virulence genes (icaA, fbl, vwbl, fbpA, slush A, B and C, and genes of the putative ß-hemolysin and hemolysin III) and the ability to induce synergistic hemolytic activity and hemolysis after 24, 48 and 72 h were investigated in a collection of twenty-two S. lugdunensis clinical isolates. The collection of isolates, mainly from implant orthopedic infections, had previously been grouped by ribotyping/dendrogram analysis and studied for biofilm matrices, biomasses and antibiotic resistances. Two isolates, constituting a unique small ribogroup sharing the same cluster, exhibited an amplicon size of the slush operon (S. lugdunensis synergistic hemolysin) which was shorter than the expected 977 bp. This outcome can predict the genetic lineage of the S. lugdunensis strains. One isolate (cra1342) presented two deletions: one of 90 bp in slush A and the other of 91 bp in slush B. Another isolate (N860314) showed a single 193 bp deletion, which encompassed part of the slush B terminal sequence and most of slush C. The isolate N860314 was devoid of hemolytic activity after 24 h, and the first consideration was that the deleted region deals with the coding of the active enzymatic site of the slush hemolysin. On the other hand, cra1342 and N860314 isolates with different slush deletions and with hemolytic activity after 24 and 48 h, respectively, could have replaced the hemolytic phenotype through other processes.

Antibacterial Activity on Orthopedic Clinical Isolates and Cytotoxicity of the Antimicrobial Peptide Dadapin-1.

In orthopedic surgery, biomaterial-associated infections represent a complication of serious concern. Most promising strategies to prevent these infections currently rely on the use of anti-infective biomaterials. Desirably, in anti-infective biomaterials, the antibacterial properties should be achieved by doping, grafting, or coating the material surfaces with molecules that are alternative to conventional antibiotics and exhibit a potent and highly specific activity against bacteria, without altering the biocompatibility. Antimicrobial peptides (AMPs) are among the most interesting candidate molecules for this biomaterial functionalization. Here, the potential expressed by the recently discovered peptide Dadapin-1 was explored by assaying its MIC, MBIC and MBC on clinical strains of relevant bacterial species isolated from orthopedic infections and by assessing its cytotoxicity on the human osteoblast-like MG63 cells. When appropriately tested in diluted Mueller Hinton Broth II (MHB II), Dadapin-1 exhibited significant antibacterial properties. MIC values were in the range of 3.1-6.2 µM for the gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus warneri, and 12.4-24.9 µM for the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Interestingly, the peptide was found non-cytotoxic, with an IC50 exceeding the highest concentration tested of 179 µM. Overall, Dadapin-1 expresses considerable potential for future application in the production of anti-infective biomaterials.

Colonization and Infection of Indwelling Medical Devices by Staphylococcus aureus with an Emphasis on Orthopedic Implants.

The use of indwelling medical devices has constantly increased in recent years and has revolutionized the quality of life of patients affected by different diseases. However, despite the improvement of hygiene conditions in hospitals, implant-associated infections remain a common and serious complication in prosthetic surgery, mainly in the orthopedic field, where infection often leads to implant failure. Staphylococcus aureus is the most common cause of biomaterial-centered infection. Upon binding to the medical devices, these bacteria proliferate and develop dense communities encased in a protective matrix called biofilm. Biofilm formation has been proposed as occurring in several stages-(1) attachment; (2) proliferation; (3) dispersal-and involves a variety of host and staphylococcal proteinaceous and non-proteinaceous factors. Moreover, biofilm formation is strictly regulated by several control systems. Biofilms enable staphylococci to avoid antimicrobial activity and host immune response and are a source of persistent bacteremia as well as of localized tissue destruction. While considerable information is available on staphylococcal biofilm formation on medical implants and important results have been achieved on the treatment of biofilms, preclinical and clinical applications need to be further investigated. Thus, the purpose of this review is to gather current studies about the mechanism of infection of indwelling medical devices by S. aureus with a special focus on the biochemical factors involved in biofilm formation and regulation. We also provide a summary of the current therapeutic strategies to combat biomaterial-associated infections and highlight the need to further explore biofilm physiology and conduct research for innovative anti-biofilm approaches.

Comparison of Automated Ribotyping, spa Typing, and MLST in 108 Clinical Isolates of Staphylococcus aureus from Orthopedic Infections.

108 isolates of Staphylococcus aureus, belonging to six large ribogroups according to the automated Ribo-Printer® system, were studied with two highly used molecular methods for epidemiological studies, namely multi-locus sequence typing (MLST) and spa typing, followed by BURP and eBURST v3 analysis for clustering spa types and sequence (ST) types. The aim was to evaluate whether automated ribotyping could be considered a useful screening tool for identifying S. aureus genetic lineages with respect to spa typing and MLST. Clarifying the relationship of riboprinting with these typing methods and establishing whether ribogroups fit single clonal complexes were two main objectives. Further information on the genetic profile of the isolates was obtained from agr typing and the search for the mecA, tst genes, and the IS256 insertion sequence. Automated ribotyping has been shown to predict spa clonal complexes and MLST clonal complexes. The high cost and lower discriminatory power of automated ribotyping compared to spa and MSLT typing could be an obstacle to fine genotyping analyzes, especially when high discriminatory power is required. On the other hand, numerous advantages such as automation, ease and speed of execution, stability, typeability and reproducibility make ribotyping a reliable method to be juxtaposed to gold standard methods.

Synthesis, Crystal Structure, and Antibacterial Properties of Silver-Functionalized Low-Dimensional Layered Zirconium Phosphonates.

New insoluble layered zirconium phosphate carboxyaminophosphonates (ZPs), with the general formula Zr2(PO4)H5[(O3PCH2)2N(CH2)nCOO]2·mH2O (n = 3, 4, and 5), have been prepared and characterized. The crystal structure for n = 3 and 4 samples was determined ab initio from X-ray powder diffraction data. The structure for n = 3 was monoclinic in space group C2/c with the following unit cell parameters: a = 34.346(1) Å, b = 8.4930(2) Å, c = 9.0401(2) Å, and ß = 97.15(1)°. The structure for n = 4 was triclinic in space group P1̅ with the following unit cell parameters: a = 17.9803(9) Å, b = 8.6066(4) Å, c = 9.0478(3) Å, α = 90.466(3)°, ß = 94.910(4)°, and γ = 99.552(4)°. The two structures had the same connectivity as Zr phosphate glycine diphosphonate (n = 1), as previously reported. By intercalation of short amines, these layered compounds were exfoliated in single lamella or packets of a few lamellae, which formed colloidal dispersions in water. After a thorough characterization, the dispersed lamellae were functionalized with Ag nanoparticles, which were grown in situ on the surface of exfoliated lamellae. Finally, their antimicrobial activity was tested on several Gram-positive and Gram-negative bacteria. All of these systems were found to be active against the four pathogens most frequently isolated from orthopedic prosthetic infections and often causative of nosocomial infections. Interestingly, they were found to express powerful inhibitory activity even against bacterial strains exhibiting a relevant profile of antibiotic resistance such as Staphylococcus aureus ATCC 700699.

Tracing the origins of extracellular DNA in bacterial biofilms: story of death and predation to community benefit.

Extracellular DNA (eDNA) is a macromolecule copiously found in various natural microenvironments, but its origin and significance still remain partly mysterious phenomena. Here, the multifaceted origins of eDNA in bacterial biofilms are explored. The release of eDNA can follow a suicidal programmed bacterial apoptosis or a fratricide-induced death, under the control of quorum sensing systems or triggered by specific stressors. eDNA can be released into the extracellular space or as a free macromolecule or enclosed within membrane vesicles or even through an explosion of bubbles. eDNA can also be derived from host tissue cells through bacterial cytolytic/proapoptotic toxins or stolen from neutrophil extracellular traps (NETs). eDNA can alternatively be produced by lysis-independent mechanisms. Sub-inhibitory doses of antibiotics, by killing a fraction of bacteria, result in stimulating the release of eDNA. Even phages appear to play a role in favoring eDNA release. Unveiling the origins of eDNA is critical to correctly address biofilm-associated infections.

Extracellular DNA (eDNA). A Major Ubiquitous Element of the Bacterial Biofilm Architecture.

After the first ancient studies on microbial slime (the name by which the biofilm matrix was initially indicated), multitudes of studies on the morphology, composition and physiology of biofilms have arisen. The emergence of the role that biofilms play in the pathogenesis of recalcitrant and persistent clinical infections, such as periprosthetic orthopedic infections, has reinforced scientific interest. Extracellular DNA (eDNA) is a recently uncovered component that is proving to be almost omnipresent in the extracellular polymeric substance (EPS) of biofilm. This macromolecule is eliciting unprecedented consideration for the critical impact on the pathogenesis of chronic clinical infections. After a systematic review of the literature, an updated description of eDNA in biofilms is presented, with a special focus on the latest findings regarding its fundamental structural role and the contribution it makes to the complex architecture of bacterial biofilms through interactions with a variety of other molecular components of the biofilm matrix.

Exploring the anticancer effects of standardized extracts of poplar-type propolis: In vitro cytotoxicity toward cancer and normal cell lines.

Propolis was shown to exert antimicrobial, antioxidant, anti-inflammatory, and anticancer activities. Its composition is influenced by seasonal, climatic and phytogeographic conditions. Further variability derives from the extraction methods. Multi Dynamic Extraction Method (MED) has been recently proposed to improve extracts reproducibility. Here, the cytotoxic/anticancer activity of three MED extracts of poplar-type propolis was assayed on human promyelocytic leukaemia HL60, human monocytic leukaemia THP-1, human osteosarcoma MG63, murine fibroblast L929 and human mesenchymal cells (hMSCs). As far as we are aware of, MG63 cells have never been challenged with propolis before, while few studies have so far addressed the effects of propolis on non-tumor cell lines. Consistent results were observed for all propolis preparations. The extracts turned out mildly cytotoxic toward cancer cells, in particular osteosarcoma cells (IC50: 81.9-86.7 µg/ml). Nonetheless, cytotoxicity was observed also in non-tumor L929 cells, with an even lower IC50. hMSCs demonstrated the lowest sensitivity to propolis (IC50: 258.3-287.2 µg/ml). In THP-1 cells, extracts were found to stimulate apoptosis caspase 3/7 activity. The IC50 values observed with osteosarcoma and leukaemia cells do not support a relevant cytotoxicity (as the figures abundantly exceeded 30 µg/ml), despites some selective activity exhibited with HL60 cells. The results confirm the validity of the extraction method, emphasizing the need to assess the selectivity of the interaction with cancer cells when screening for anticancer-drug candidates.

Various biofilm matrices of the emerging pathogen Staphylococcus lugdunensis: exopolysaccharides, proteins, eDNA and their correlation with biofilm mass.

Staphylococcus lugdunensis is an emerging high-virulent pathogen causative of hospital-acquired infections. Biofilm formation is a complex pathogenic process that leads to well-established bacterial communities. There is a paucity of data on the composition of the biofilm matrix among S. lugdunensis strains. Here, twenty-two S. lugdunensis clinical isolates, mainly from orthopaedic infections but also from other clinical sources, were sub-grouped by ribotyping and dendrogram analysis. Biofilms were analysed by fluorimetric methods based on FITC-Wheat Germ Agglutinin, SYPRO Ruby and TOTO-1 dyes to detect exopolysaccharides, proteins and extracellular DNA (eDNA), respectively. Biofilm morphology was investigated under confocal laser scanning microscopy (CLSM). Isolates displayed intriguing diversities in biofilm mass and matrix composition. The content of exopolysaccharides was found to be to be strongly associated with the biofilm mass (R2 = 0.882), while the content of proteins turned out to be weakly (R2 = 0.465) and that of eDNA very weakly associated (R2 = 0.202) to the biofilm mass.

Hijacking of immune defences by biofilms: a multifront strategy.

Biofilm formation by pathogens and opportunistic bacteria is the basis of persistent or recurrent infections. Up to 80% of bacterial infections in humans are associated with biofilms. Despite the efficiency of the evolved and complex human defence system against planktonic bacteria, biofilms are capable of subverting host defences. The immune system is not completely effective in opposing bacteria and preventing infection. Increasing attention is being focussed on the mechanisms enabling bacterial biofilms to skew the coordinate action of humoral and cell mediated responses. Knowledge of the interactions between biofilm bacteria and the immune system is critical to effectively address biofilm infections, which have multiplied over the years with the spread of biomaterials in medicine. In this article, the latest information on the interactions between bacterial biofilms and immune cells is examined and the areas where of information is still lacking are explored.

Antibacterial Properties of a Novel Zirconium Phosphate-Glycinediphosphonate Loaded with Either Zinc or Silver.

A novel compound consisting of a zirconium phosphate-glycinediphosphonate (ZPGly) has recently been introduced. This 2D-structured material forming nanosheets was exfoliated under appropriate conditions, producing colloidal aqueous dispersions (ZPGly-e) which were then loaded with zinc (Zn/ZPGly) or silver ions. Silver ions were subsequently reduced to produce metallic silver nanoparticles on exfoliated ZPGly nanosheets (Ag@ZPGly). In the search for new anti-infective materials, the present study investigated the properties of colloidal dispersions of ZPGly-e, Zn/ZPGly, and Ag@ZPGly. Ag@ZPGly was found to be a bactericidal material and was assayed to define its minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) on the five most prevalent pathogens of orthopaedic implant infections, namely: Staphylococcus aureus ATCC25923, Staphylococcus epidermidis RP62A, Enterococcus faecalis ATCC29212, Escherichia coli ATCC51739, and Pseudomonas aeruginosa ATCC27853. MIC and MBC were in the range of 125-250 µg/mL and 125-1000 µg/mL, respectively, with E. coli being the most sensitive species. Even colloidal suspensions of exfoliated ZPGly nanosheets and Zn/ZPGly exhibited some intrinsic antibacterial properties, but only at greater concentrations. Unexpectedly, Zn/ZPGly was less active than ZPGly-e.

Implant infections: adhesion, biofilm formation and immune evasion.

Medical device-associated infections account for a large proportion of hospital-acquired infections. A variety of opportunistic pathogens can cause implant infections, depending on the type of the implant and on the anatomical site of implantation. The success of these versatile pathogens depends on rapid adhesion to virtually all biomaterial surfaces and survival in the hostile host environment. Biofilm formation on implant surfaces shelters the bacteria and encourages persistence of infection. Furthermore, implant-infecting bacteria can elude innate and adaptive host defences as well as biocides and antibiotic chemotherapies. In this Review, we explore the fundamental pathogenic mechanisms underlying implant infections, highlighting orthopaedic implants and Staphylococcus aureus as a prime example, and discuss innovative targets for preventive and therapeutic strategies.

Streptococcus agalactiae Non-Pilus, Cell Wall-Anchored Proteins: Involvement in Colonization and Pathogenesis and Potential as Vaccine Candidates.

Group B Streptococcus (GBS) remains an important etiological agent of several infectious diseases including neonatal septicemia, pneumonia, meningitis, and orthopedic device infections. This pathogenicity is due to a variety of virulence factors expressed by Streptococcus agalactiae. Single virulence factors are not sufficient to provoke a streptococcal infection, which is instead promoted by the coordinated activity of several pathogenicity factors. Such determinants, mostly cell wall-associated and secreted proteins, include adhesins that mediate binding of the pathogen to host extracellular matrix/plasma ligands and cell surfaces, proteins that cooperate in the invasion of and survival within host cells and factors that neutralize phagocytosis and/or modulate the immune response. The genome-based approaches and bioinformatics tools and the extensive use of biophysical and biochemical methods and animal model studies have provided a great wealth of information on the molecular structure and function of these virulence factors. In fact, a number of new GBS surface-exposed or secreted proteins have been identified (GBS immunogenic bacterial adhesion protein, leucine-rich repeat of GBS, serine-rich repeat proteins), the three-dimensional structures of known streptococcal proteins (αC protein, C5a peptidase) have been solved and an understanding of the pathogenetic role of "old" and new determinants has been better defined in recent years. Herein, we provide an update of our current understanding of the major surface cell wall-anchored proteins from GBS, with emphasis on their biochemical and structural properties and the pathogenetic roles they may have in the onset and progression of host infection. We also focus on the antigenic profile of these compounds and discuss them as targets for therapeutic intervention.

New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells.

Complete eradication of bacterial infections is often a challenging task, especially in presence of prosthetic devices. Invasion of non-phagocytic host cells appears to be a critical mechanism of microbial persistence in host tissues. Hidden within host cells, bacteria elude host defences and antibiotic treatments that are intracellularly inactive. The intracellular invasiveness of bacteria is generally measured by conventional gentamicin protection assays. The efficiency of invasion, however, markedly differs across bacterial species and adjustments to the titre of the microbial inocula used in the assays are often needed to enumerate intracellular bacteria. Such changes affect the standardisation of the method and hamper a direct comparison of bacteria on a same scale. This study aims at investigating the precise relation between inoculum, in terms of multiplicity of infection (MOI), and internalised bacteria. The investigation included nine Staphylococcus aureus, seven Staphylococcus epidermidis, five Staphylococcus lugdunensis and two Enterococcus faecalis clinical strains, which are co-cultured with MG63 human osteoblasts. Unprecedented insights are offered on the relations existing between MOI, number of internalised bacteria and per cent of internalised bacteria. New parameters are identified that are of potential use for qualifying the efficiency of internalization and compare the behaviour of bacterial strains.

Serratiopeptidase reduces the invasion of osteoblasts by Staphylococcus aureus.

Finding new strategies to counteract periprosthetic infection and implant failure is a main target in orthopedics. Staphylococcus aureus, the leading etiologic agent of orthopedic implant infections, is able to enter and kill osteoblasts, to stimulate pro-inflammatory chemokine secretion, to recruit osteoclasts, and to cause inflammatory osteolysis. Moreover, by entering eukaryotic cells, staphylococci hide from the host immune defenses and shelter from the extracellular antibiotics. Thus, infection persists, inflammation thrives, and a highly destructive osteomyelitis occurs around the implant. The ability of serratiopeptidase (SPEP), a metalloprotease by Serratia marcescens, to control S. aureus invasion of osteoblastic MG-63 cells and pro-inflammatory chemokine MCP-1 secretion was evaluated. Human osteoblast cells were infected with staphylococcal strains in the presence and in the absence of SPEP. Cell proliferation and cell viability were also evaluated. The release of pro-inflammatory chemokine MCP-1 was evaluated after the exposure of the osteoblast cells to staphylococcal strains. The significance of the differences in the results of each test and the relative control values was determined with Student's t-test. SPEP impairs their invasiveness into osteoblasts, without affecting the viability and proliferation of bone cells, and tones down their production of MCP-1. We recognize SPEP as a potential tool against S. aureus bone infection and destruction.

Molecular Characterization of a Prevalent Ribocluster of Methicillin-Sensitive Staphylococcus aureus from Orthopedic Implant Infections. Correspondence with MLST CC30.

Staphylococcus aureus is the leading etiologic agent of orthopedic implant infections. Here a ribocluster of 27 S. aureus strains underwent further molecular characterization and subtyping by multilocus sequence typing (MLST) and spa-typing. This cluster had been detected by automated ribotyping (with the EcoRI restriction enzyme) of 200 S. aureus isolates from periprosthetic infections of patients who underwent revision at the Rizzoli Orthopaedic Institute. The ribocluster, consisting of agr type III strains, with a 74% co-occurrence of bone sialoprotein-binding (bbp) and collagen-binding (cna) genes, lacked mecA and IS256, and exhibited a high prevalence of the toxic shock syndrome toxin gene (tst, 85%). Strains' relatedness was analyzed by BURP and eBURST. Two predominant spa types, t012 (32%) and t021 (36%), and one predominant sequence type, ST30 (18/27, 67%) were identified: a S. aureus lineage spread worldwide belonging to MLST CC30. Two new sequence types (ST2954, ST2960) and one new spa type (t13129) were detected for the first time. Interestingly, the 27-strain cluster detected by ribotyping corresponded exactly to MLST CC30, the sole CC identified by eBURST.

Orthopedic implant infections: Incompetence of Staphylococcus epidermidis, Staphylococcus lugdunensis, and Enterococcus faecalis to invade osteoblasts.

Septic failure is still the major complication of prosthetic implants. Entering host cells, bacteria hide from host immune defenses, shelter from extracellular antibiotics, and cause chronic infection. Staphylococcus aureus, the leading etiologic agent of orthopedic implant infections, is able to enter bone cells and induce osteoblast apoptosis, osteoclast recruitment, and highly destructive osteomyelitis. Staphylococcus epidermidis, Staphylococcus lugdunensis, and Enterococcus faecalis are opportunistic pathogens causative of implant-related infections. This study investigated the ability to internalize into osteoblastic MG63 cells of 22 S. epidermidis, 9 S. lugdunensis, and 21 E. faecalis clinical isolates from orthopedic implant infections. Isolates were categorized in clusters by ribotyping. Internalization assay was carried out by means of a microtiter plate-based method. S. epidermidis, S. lugdunensis, and E. faecalis strains turned out incompetent to enter osteoblasts, exhibiting negligible internalization into MG63 cells, nearly three orders of magnitude lower than that of S. aureus. Osteoblast invasion does not appear as a pathogenetic mechanism utilized by S. epidermidis, S. lugdunensis, or E. faecalis for infecting orthopedic implants. Moreover, it can be inferred that intracellularly active antimicrobials should not be necessary against implant infections caused by the three bacterial species. Finally, implications with the uptake of biomaterial microparticles by nonphagocytic cells are enlightened. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 788-801, 2016.

In vitro effect of temperature on the conformational structure and collagen binding of SdrF, a Staphylococcus epidermidis adhesin.

Staphylococcus epidermidis is the leading etiologic agent of device-related infections. S. epidermidis is able to bind, by means of the adhesins of its cell wall, the host matrix proteins filming the artificial surfaces. Thence, bacteria cling to biomaterials and infection develops. The effect of temperature on integrity, structure, and biological activity of the collagen-binding adhesin (SdrF) of S. epidermidis has been here investigated. By cloning in E. coli XL1-Blue, a recombinant of the SdrF binding domain B (rSdrFB), carrying an N-terminal polyhistidine, was obtained. Purification was by HiTrap(TM) Chelating HP columns. Assessment of purity, molecular weight, and integrity was by SDS-PAGE. The rSdrFB-collagen binding was investigated by ELISA. A full three-dimensional reconstruction of rSdrFB was achieved by small-angle X-ray scattering (SAXS). At 25 °C, rSdrFB bound to type I collagen in a dose-dependent, saturable manner, with a Kd of 2.48 × 10(-7) M. When temperature increased from 25 to 37 °C, a strong conformational change occurred, together with the abolition of the rSdrFB-collagen binding. The rSdrFB integrity was not affected by temperature variation. SdrFB-collagen binding is switched on/off depending on the temperature. Implications with the infection pathogenesis are enlightened.