The role of gamma globulin, complement component 1q, factor B, properdin, body temperature, C-reactive protein and serum amyloid alpha to the activity and the function of the human complement system and its pathways.

The complement system plays a crucial role in orchestrating the activation and regulation of inflammation within the human immune system. Three distinct activation pathways-classical, lectin, and alternative-converge to form the common lytic pathway, culminating in the formation of the membrane-attacking complex that disrupts the structure of pathogens. Dysregulated complement system activity can lead to tissue damage, autoimmune diseases, or immune deficiencies. In this study, the antimicrobial activity of human serum was investigated by using a bioluminescent microbe probe, Escherichia coli (pEGFPluxABCDEamp). This probe has previously been used to determine the antimicrobial activity of complement system and the polymorphonuclear neutrophils. In this study, blocking antibodies against key serum activators and components, including IgG, complement component 1q, factor B, and properdin, were utilized. The influence of body temperature and acute phase proteins, such as C reactive protein (CRP) and serum amyloid alpha (SAA), on the complement system was also examined. The study reveals the critical factors influencing complement system activity and pathway function. Alongside crucial factors like C1q and IgG, alternative pathway components factor B and properdin played pivotal roles. Results indicated that the alternative pathway accounted for approximately one third of the overall serum antimicrobial activity, and blocking this pathway disrupted the entire complement system. Contrary to expectations, elevated body temperature during inflammation did not enhance the antimicrobial activity of human serum. CRP demonstrated complement activation properties, but at higher physiological concentrations, it exhibited antagonistic tendencies, dampening the response. On the other hand, SAA enhanced the serum's activity. Overall, this study sheds a light on the critical factors affecting both complement system activity and pathway functionality, emphasizing the importance of a balanced immune response.

Indoor microbial exposure increases complement component C3a and C-reactive protein concentrations in serum.

Indoor exposure to microbial growth, caused by moisture damage, has been an established health risk for several decades. It is likely that a damp indoor environment contains biological pollutants that trigger both the innate and adaptive branches of the immune system. In this study, we investigated the association between moisture damage related microbial exposure and serum C3a, C5a and CRP concentrations in Finnish adults. Serum C3a and CRP concentrations were elevated in individuals exposed to moisture damage and microbial growth in an indoor air environment. The elevated concentrations may be due to environmental factors present in moisture-damaged buildings. Complement activation and the resulting proinflammatory cleavage products may be a driving factor in inflammatory responses following exposure to indoor moisture damage and related microbial growth.

Rapid detection of bacterial infection using a novel single-tube, four-colour flow cytometric method: Comparison with PCT and CRP.

BACKGROUND: A key factor behind the unnecessary use of antibiotics is the lack of rapid and accurate diagnostic tests. In this study, we developed a novel and fast flow cytometric single-tube method to detect bacterial infections within 30 minutes. METHODS: Quantitative flow cytometric four-colour analysis of host biomarkers CD35, CD64, CD329, and MHC class I expression on neutrophils and lymphocytes was performed on samples taken from 841 febrile patients with suspected infection. Obtained data was incorporated into the four-colour bacterial infection (FCBI)-index, using the developed bacterial infection algorithm. FINDINGS: In distinguishing between microbiologically confirmed bacterial (n = 193) and viral (n = 291) infections, the FCBI-index method was superior to serum C-reactive protein (CRP) and procalcitonin (PCT). In 269 confirmed viral respiratory tract infections, 43% (95% CI: 37-49%) of the patients had an increased FCBI-index, suggesting probable bacterial coinfection. INTERPRETATION: The proposed FCBI-index test might be a potent additional tool when assessing appropriateness of empiric antibiotic treatment. FUNDING: This study has been financially supported by Turku University Hospital (Turku, Finland) and The Finnish Medical Foundation.

Indoor-related microbe damage induces complement system activation in building users.

In this comparative study, serum complement system antimicrobial activity was measured from 159 serum samples, taken from individuals from microbe-damaged (70 samples) and from reference buildings (89 samples). Antimicrobial activity was assessed using a probe-based bacterial Escherichia coli-lux bioluminescence system and comparison was made at a group level between the experimental and reference group. The complement activity was higher in users of microbe-damaged buildings compared with the reference group and the significant (P < 0.001) increase in activity was found in the classical reaction pathway. This study strengthens our notion that exposure to indoor-related microbe damage increases the risk for systemic subclinical inflammation and creates a health risk for building users.

Indoor exposure to Streptomyces albus and Aspergillus versicolor elevates the levels of spore-specific IgG, IgG1 and IgG3 serum antibodies in building users - A new ELISA-based assay for exposure assessment.

Moisture-indicative microbes in buildings are associated with a variety of symptoms, ranging from mild irritation to severe clinical illnesses. These symptoms are caused principally by dried, dormant and dead microbe material like spores, mycelium and microbe metabolites, leading to the activation of the immune system and formation of the antigen-specific immunoglobulins. This activation presumably takes place through the respiratory track and is a normal immune reaction against pathogenic invaders. During continuous exposure, a prolonged state of inflammation will follow, and this forms a considerable health risk for a building's occupant. A new ELISA system utilizing spores from two species Streptomyces albus and Aspergillus versicolor as an antigen was developed to reveal the related immunological processes. In 159 persons, microbial exposure was observed to increase the levels of spore-specific IgG, IgG1 and IgG3 serum antibody levels of individuals residing in microbe-dense buildings compared with the control reference buildings. No differences were detected in the levels of S. albus- and A. versicolor-specific serum IgA or IgM levels.

A single-tube two-color flow cytometric method for distinguishing between febrile bacterial and viral infections.

The aim of this study was to develop a rapid single-tube two-color flow cytometric method to distinguish between febrile bacterial and viral infections. In this prospective comparative study, the quantitative flow cytometric analysis of CD35 and CD64 on isolated human leukocytes was obtained from 286 hospitalized febrile patients, of which 197 patients were found to have either a bacterial (n = 136) or viral (n = 61) infection. The data from infected patients was compared to 49 healthy controls and 23 patients in an inflammatory state. We developed a flow cytometric marker for bacterial infections, defined as the two-color bacterial infection index (TC-BI-index), by incorporating the quantitative analysis of CD35 and CD64 on isolated neutrophils, monocytes, and B-lymphocytes, which displayed 90% sensitivity and specificity in distinguishing between microbiologically confirmed bacterial (n = 77) and viral infections (n = 61) within 45 min. We propose that TC-BI-index test will be useful in assisting physicians to ascertain whether antibiotic treatment is required, thus limiting unnecessary antimicrobial usage.

Bacterial infection (BI)-INDEX: an improved and simplified rapid flow cytometric bacterial infection marker.

The aim of this study was to develop a rapid and simple flow cytometric bacterial infection marker. In this prospective comparative study, quantitative flow cytometric analysis of CD10, CD35, CD66b, CD282, and MHC Class I molecules on human neutrophils, monocytes, and B-lymphocytes from 141 hospitalized febrile patients with suspected infection and from 50 healthy controls was performed. We developed a flow cytometric marker of local and systemic bacterial infections, designated "bacterial infection (BI)-INDEX", incorporating the quantitative analysis of CD10, CD35, MHCI, CD66b, and CD282 on neutrophils, monocytes, and B-lymphocytes, which displayed 90% sensitivity and 96% specificity in distinguishing between microbiologically confirmed bacterial (n = 31) and viral infections (n = 27) within a 1-h time-frame. We propose that our novel rapid BI-INDEX test will be useful in assisting physicians to ascertain whether antibiotic treatment is required, thus limiting unnecessary antimicrobial usage.

Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1ß. Some bacterial species can alter their physiological properties as a result of sensing IL-1ß. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1ß sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1ß through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1ß than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1ß, but this binding was not specific, as a control protein for IL-1ß also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1ß-binding surface-exposed lipoprotein that may be part of the bacterial IL-1ß-sensing system.

Resistance to bacterial infectious diseases in rainbow trout (Oncorhynchus mykiss).

Individually tagged rainbow trout representing 15 full-sibling families were sequentially challenged twice with Aeromonas salmonicida causing furunculosis: first as cohabitation and then as injected intraperitoneally. The bleeding procedure prior to challenges caused the outbreak of cold water disease by Flavobacterium psychrophilum. Before and after the outbreak and challenges, 11 immunological parameters were measured from blood samples. The immunological responses predicted the fate of the fish since nearly all the initial responses were lower in individuals which later died from cold water disease than in survivors. Fish died from furunculosis had impaired respiratory burst (RB) response to A. salmonicida. Fish that had initially the highest responses survived in the outbreak and challenges. The outbreak and challenges resulted in these individuals higher and faster responses compared with initial values. Unlike in mammals, the number of monocytes, but not that of granulocytes, in rainbow trout blood correlated well with the whole blood RB activity. The fish families differed markedly from each other in capacity to resist the induced diseases.

Use of complement regulators, CD35, CD46, CD55, and CD59, on leukocytes as markers for diagnosis of viral and bacterial infections.

Several complement regulatory proteins exist on self-cells to prevent damage by the serum complement system. In the present study, we aimed to perform quantitative analysis of membrane-bound complement regulators, CR1 (CD35), MCP (CD46), DAF (CD55), and MIRL (CD59), on peripheral blood neutrophils, monocytes, and lymphocytes from healthy controls (n=36) and febrile patients diagnosed with either bacterial (n=21) or viral (n=26) infections. Our results show that: (a) increased CD35 and CD55 levels on neutrophils and monocytes present potent markers of bacterial infection, (b) increased expression of CD46 on monocytes is an indicator of viral infection, and (c) increased CD59 expression on neutrophils and monocytes is a general infection marker. Additionally, CD19-positive B-lymphocytes represent practically the only lymphocyte population capable of expressing CD35. We further developed two novel clinical flow cytometric markers (indices), specifically, clinical mononucleosis (CM)-INDEX (incorporating CD35, CD55, and CD59 expression on lymphocytes) and clinical bacterial infection (CBI)-INDEX (incorporating CD35 and CD55 expression on neutrophils and lymphocytes), for the effective detection of viral mononucleosis and bacterial infection, respectively. In summary, bacterial and viral infections induce different expression patterns of membrane-bound complement regulators in human leukocytes, which may be effectively exploited in clinical differential diagnosis.

A rapid flow cytometric method for distinguishing between febrile bacterial and viral infections.

Antibiotic resistance due to the inappropriate use of antimicrobials is one of the most critical public health problems worldwide. A major factor underlying the unnecessary use of antibiotics is the lack of rapid and accurate diagnostic tests. Therefore, we aimed to develop a novel rapid flow cytometric method for distinguishing between febrile bacterial and viral infections. In this prospective comparative study, quantitative flow cytometric analysis of FcγRII/CD32, CR1/CD35, MHC Class I receptor (MHCI), and C5aR/CD88 on human phagocytes was performed in 286 hospitalized febrile patients with suspected infection. After using microbiological and serological detection methods, or clinical diagnosis, 205 patients were identified with either bacterial (n=136) or viral (n=69) infection. Receptor data from patients were compared to those of 50 healthy controls. We developed a flow cytometric marker of local and systemic bacterial infections designated "bacterial infection score (BIS)" incorporating the quantitative analysis of FcγRII/CD32, CR1/CD35, C5aR/CD88 and MHCI on neutrophils and/or monocytes, which displays 91% sensitivity and 92% specificity in distinguishing between microbiologically confirmed bacterial (n=77) and serologically confirmed viral infections (n=61) within 1h. The BIS method was effectively applied to distinguish between bacterial and viral (pandemic H1N1 influenza) pneumonia cases with 96% sensitivity and 92% specificity. We propose that the rapid BIS test can assist physicians in deciding whether antibiotic treatment is necessary, thus reducing unnecessary antimicrobial use.

Bacterial infections, DNA virus infections, and RNA virus infections manifest differently in neutrophil receptor expression.

Treating viral illnesses or noninfective causes of inflammation with antibiotics is ineffective and contributes to the development of antibiotic resistance, toxicity, and allergic reactions, leading to increasing medical costs. A major factor behind unnecessary use of antibiotics is, of course, incorrect diagnosis. For this reason, timely and accurate information on whether the infection is bacterial in origin would be highly beneficial. In this paper we will present our recent studies on the expression of opsonin receptors on phagocytes. The analysis of the expression levels of FcγRI, CR1, and CR3, along with CRP and ESR data, provides a novel application to the diagnosis of infectious and inflammatory diseases. The best clinical benefit will be obtained when the individual variables are combined to generate the CIS point method for a bacterial infection marker, DNAVS point for differentiating between DNA and RNA virus infections, and CRP/CD11b ratio for a marker of Gram-positive sepsis.

Trimeric form of intracellular ATP synthase subunit ß of Aggregatibacter actinomycetemcomitans binds human interleukin-1ß.

Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1ß, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1ß receptor has been identified, current knowledge of the bacterial IL-1ß sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1ß than inactive ones. The effect of IL-1ß on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1ß to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1ß, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1ß and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1ß slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1ß. Our results suggest that IL-1ß might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit ß interacted with IL-1ß, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1ß during inflammation.

The novel applications of the quantitative analysis of neutrophil cell surface FcgammaRI (CD64) to the diagnosis of infectious and inflammatory diseases.

PURPOSE OF REVIEW: Several studies show that the number of FcgammaRI (CD64) on the surface of neutrophils increases in infections. However, in spite of increased research interest in recent years, there is no clear general view on the usability of neutrophil FcgammaRI in clinical infection diagnostics. This review tries to bring the clarity to this matter. RECENT FINDINGS: It is shown here that although the high number of FcgammaRI on neutrophils is a sensitive marker of bacterial infection, it is highly expressed also in DNA virus infections. As a consequence, neutrophil FcgammaRI cannot be used in distinguishing between bacterial and viral infections. It is also clear that FcgammaRI on neutrophils cannot be used in distinguishing between Gram-positive and Gram-negative bacterial infections or between microbiologically confirmed and clinically diagnosed bacterial infections. In addition, neutrophil FcgammaRI cannot be used to reliably detect RNA virus infections, inflammatory diseases, or cancer. SUMMARY: The best clinical benefit from the quantitative analysis of FcgammaRI on neutrophils will be obtained when it is used simultaneously with a reliable bacterial infection marker. DNA virus score point is an efficient novel method in differentiating between DNA and RNA virus infections.

Simultaneous quantitative analysis of Fc gamma RI (CD64) and CR1 (CD35) on neutrophils in distinguishing between bacterial infections, viral infections, and inflammatory diseases.

A flow cytometric quantitative analysis of receptors on neutrophils can be exploited in distinguishing between inflammatory and infectious diseases. In this prospective comparative study, simultaneous quantitative analysis of CD64 and CD35 on peripheral blood neutrophils was performed in febrile patients in order to differentiate between bacterial infections (n=89), viral infections (n=46), and inflammatory diseases (n=21). The patient data was compared to 60 healthy controls. We could divide patients into three groups depending on how they express CD35 and CD64 on neutrophils: (1) patients with a high probability of viral infection (low CD35/low CD64 and low CD35/high CD64), (2) patients with a high probability of inflammatory disease (high CD35/low CD64), and (3) patients with a high probability of bacterial infection (high CD35/high CD64). In summary, simultaneous quantitative analysis of CD64 and CD35 on neutrophils could potentially assist physicians to distinguish between inflammatory and infectious diseases.

CRP/CD11b ratio: a novel parameter for detecting gram-positive sepsis.

To commence proper antibiotic treatment in sepsis, timely knowledge of whether the cause of systemic infection is gram-negative (gram(-)) or gram-positive (gram(+)) bacteria in origin would be beneficial for clinicians. In this clinical prospective study, our objective was to develop a method for distinguishing between gram(+) and gram(-) bacterial infection. In gram(-) bacterial infection (n = 21), the average amount of CD11b on neutrophils was significantly higher than in gram(+) bacterial infection (n = 22). On the contrary, serum C-reactive protein (CRP) level was significantly higher in gram(+) than in gram(-) bacterial infection. By dividing the serum CRP value by the amount of CD11b on neutrophils, we derived a novel marker of gram(+) sepsis, CRP/CD11b ratio, which displayed 76% sensitivity and 80% specificity for the detection of gram(+) sepsis (n = 17) among febrile patients with microbiologically confirmed or clinically diagnosed bacterial infection. The detection of gram(+) sepsis is possible after the combination of neutrophil CD11b data and serum CRP level. In conclusion, our findings indicate that the proposed CRP/CD11b ratio test could potentially assist physicians in determining an appropriate antibiotic treatment in patients with severe bacterial infection.

Comparison of degranulation of easily mobilizable intracellular granules by human phagocytes in healthy subjects and patients with infectious diseases.

The aim of this study was to compare degranulation of easily mobilizable secretory vesicles (SVs) or secretory vesicle-like granules (SVLGs) in neutrophils, monocytes, and eosinophils of healthy controls (n = 60) and febrile patients with microbiologically confirmed or clinically diagnosed bacterial (n = 89) and viral (n = 46) infections. For this purpose, flow cytometric immunophenotyping of isolated phagocytes was performed using monoclonal antibodies against the phagocytosis receptors CR1 (CD35) and CR3 (CD11b) that are predominantly stored in the SVs of resting neutrophils. Similar to neutrophils, monocytes contain easily mobilizable SVLGs that constitute the main intracellular reservoir of CD35 and CD11b. In both neutrophils and monocytes, activation mechanisms leading to degranulation of SV and SVLG appeared dependent on both intra- and extracellular calcium levels. The kinetics of degranulation of SVLGs in control monocytes was significantly faster than that of SVs of control neutrophils. We conclude that phagocytes in patients with bacterial infections can be arranged in order of decreasing magnitude of SV or SVLG degranulation as follows (from left to right): neutrophils > monocytes " eosinophils. However, in viral infections, the corresponding degranulation order is monocytes > neutrophils approximately eosinophils.

A novel method for distinguishing between dsDNA and ssRNA virus infections.

BACKGROUND: To commence proper antiviral treatment, timely knowledge of whether the infection is caused by DNA or RNA virus would be beneficial for the clinician. OBJECTIVES: Our objective was to develop a method for distinguishing between DNA and RNA virus infections. STUDY DESIGN: In this prospective study, total and differential count of leukocytes, serum C-reactive protein level, erythrocyte sedimentation rate, and quantitative flow cytometric analysis of FcgammaRI (CD64) on neutrophils and monocytes were obtained from 289 hospitalized febrile patients. After microbiological confirmation, 89 patients (31%) were found to have either bacterial (n=46) or viral (n=43) infection. The patient data was compared to 60 healthy controls. RESULTS: For the first time ever, it was noticed that in dsDNA virus infections (n=21) the average amount of CD64 on neutrophils was over five-fold compared to ssRNA virus infections (n=22). CONCLUSIONS: DNA virus score (DNAVS) point, which incorporates quantitative analysis of CD64 on neutrophils and total and differential count of leukocytes, varied between 0 and 8, and displayed 95% sensitivity and 100% specificity in distinguishing between dsDNA and ssRNA virus infections [average (S.D.); DNAVS points: 5.4 (2.5) vs. 0.3 (0.4); p<0.001].

Simultaneous quantitative analysis of FcgammaRI (CD64) expression on neutrophils and monocytes: a new, improved way to detect infections.

We performed simultaneous quantitative flow cytometric analysis of neutrophil and monocyte FcgammaRI (CD64) in 289 hospitalized febrile patients. Microbiological evaluation or clinical diagnosis confirmed bacterial (n=89) or viral (n=46) infection in 135 patients. Patient data were compared with data from 60 healthy controls. The average number of FcgammaRI on the surfaces of both neutrophils and monocytes was significantly increased in patients with febrile viral and bacterial infections, compared to healthy controls. Furthermore, we describe a novel marker of febrile infection, designated 'CD64 score point', which incorporates the quantitative analysis of FcgammaRI expressed on both neutrophils and monocytes, with 94% sensitivity and 98% specificity in distinguishing between febrile infections and healthy controls. By contrast, analysis of FcgammaRI expression on neutrophils and monocytes displayed poor sensitivity (73% and 52%) and specificity (65% and 52%) in distinguishing between bacterial and viral infections, and the levels did not differ significantly between systemic (sepsis), local, and clinically diagnosed bacterial infections. In summary, our results clearly show that the increased number of FcgammaRI on neutrophils and monocytes is a useful marker of febrile infection, but cannot be applied for differential diagnosis between bacterial and viral infections or between systemic and local bacterial infections.

Distinction between bacterial and viral infections.

PURPOSE OF REVIEW: To commence proper treatment as rapidly as possible and to reduce unnecessary antibiotic treatments, timely knowledge of whether the infection is bacterial or viral in origin would be beneficial for the clinician. As a reliable prediction of the causative agent of bacterial infection is not possible based on clinical features, there is an ongoing need for sensitive and specific markers of bacterial infection. RECENT FINDINGS: The most common differential diagnosis methods are reviewed here. It is also demonstrated that the measurement of the expression of complement receptors, particularly CR1 (CD35), on neutrophils can be a useful preliminary test to differentiate between bacterial and viral infections. In addition, a novel marker of local and systemic bacterial infections designated 'clinical infection score (CIS) point', which incorporates quantitative analysis of complement receptors on neutrophils and standard clinical laboratory data and displays 98% sensitivity and 97% specificity in distinguishing between bacterial and viral infections, is presented. SUMMARY: We conclude that the diagnostic yield of measured individual variables in distinguishing between bacterial and viral infections increases upon combination.