Objectives. This paper discusses the controversies surrounding the antibiotic prophylaxis preceding dental interventions within the following research question: how effective is dental antibiotic prophylaxis in preventing comorbidity and complications in those at risk? Methods. A synthesis of the available literature regarding antibiotic prophylaxis in dentistry was conducted under the lenses of Kazanjian's framework for health technology assessment with a focus on economic concerns, population impact, social context, population at risk, and the effectiveness of the evidence to support its use. Results. The papers reviewed show that we have been using antibiotic prophylaxis without a clear and full understanding of its benefits. Although the first guideline for antibiotic prophylaxis was introduced in 1990, it has been revised on several occasions, from 1991 to 2011. Evidence-based clinical guidelines are yet to be seen. Conclusions. Any perceived potential benefit from administering antibiotic prophylaxis before dental procedures must be weighed against the known risks of lethal toxicity, allergy, and development, selection, and transmission of microbial resistance. The implications of guideline changes and lack of evidence for the full use of antibiotic prophylaxis for the teaching of dentistry have to be further discussed. Because it is not possible to predict when a susceptible patient will develop an infection, prophylactic antibiotics are recommended when these patients undergo procedures that might produce bacteremia” , see Bacteria from the mouth can readily enter the bloodstream during daily oral hygiene activities like brushing and flossing . The potWhen 886 titles were limited to “evidence based,” 65 papers were found. When the same 886 titles were limited to “guidelines,” 218 papers were found. After combining these two sets, 41 publications were identified as potential evidence-based guidelines. Six were excluded because there was no full text available, and four because they were not related to dentistry. From the 31 publications left, two were erratum from Wilson's et al. work , 11, andThe development of guidelines for antibiotic prophylaxis has been driven by the perceived needs of those considered to be “at risk.” Until recently, those considered “at risk” would include individuals having one or more of the following: cardiac deformities, total joint replacement, or a weakened immune system . Not lonIt seems that the first recommendation for giving antibiotic prophylaxis to patients undergoing dental procedures was put forward by the American Heart Association in 1955. It was only in 1990 that such recommendations became an actual guideline adopted by the American Council on Clinical Affairs . Since tIn this regard, the American Council on Clinical Affairs (ACCA) made changes to the guidelines  based onThe Council recommended the conservative use of antibiotics to minimize the risk of developing resistance to current antibiotic regimens. They called for judicious use of antibiotics for the prevention of IE while keeping in mind that the awareness of the potential relationship between IE and dental treatment dates back to 1909,  a time win vitro susceptibility of infective endocarditis causing microorganisms, results of prophylactic studies in animal models, and retrospective and prospective studies of the prevention of infective endocarditis. They used MEDLINE database searches from 1950 to 2006 for English-language papers with the following key-words: endocarditis, infective endocarditis, prophylaxis, prevention, antibiotic, antimicrobial, pathogens, organisms, dental, gastrointestinal, genitourinary, streptococcus, enterococcus, staphylococcus, respiratory, dental surgery, pathogenesis, vaccine, immunization, and bacteremia. They also searched the reference lists of the identified papers. Although they based their arguments on most of the papers that would be included within the Clinical Affairs review three years later, they concluded that only an extremely small number of cases of infective endocarditis would be prevented by antibiotic prophylaxis for dental procedures even if such prophylactic therapy was 100% effective. Such conclusions were not explicitly stated at the Council's review. The Council on Clinical Affairs review is very similar to the work undertaken by Wilson and his 22 team members , 11 and As seen in the above discussion, the evidence for using antibiotic prophylaxis to prevent IE alone seems weak to say the least. Although the many revisions to the existing guidelines attest to the weakness of this association, the existence of so many different and yet overlapping guidelines is frustrating at best and confusing at worse. cardiac deformities and/or artificial devices in the circulatory system should receive antibiotic prophylaxis according to the current guidelines of the AHA. Consultation with the patient's physician may be required.Patients with immunocompromising conditions, such as those with HIV/AIDS, those who have had organ transplants, and those undergoing cancer treatment, should receive antibiotic prophylaxis using the current protocols of the AHA. Such patients would include those with a suppressed leukocyte count where the white blood cell count (WBC) is less than 3500 cells/mm3 (3.5 K/mm3) or the absolute neutrophil count (ANC) is less than 500 cells/mm3 (0.5 K/mm3).Patients with a variety of total joint replacement  should receive prophylactic antibiotics as well as those with recent (within two years) joint replacement or previous prosthetic joint infection. Consultation with the patient's orthopedic surgeon may be required.Patients with A similar situation happened in Canada. In February of 2005, the Canadian Dental Association (CDA) issued a position statement emphasising that patients at risk would include those under the following descriptions .Patients“in light of these new guidelines, CDA has now rescinded its 2005 position on Antibiotic Prophylaxis for Dental Patients at Risk. The withdrawn statement addressed the needs of patients at risk of infective endocarditis and those with total joint replacement. To ensure that Canadian dentists are not left without guidance on those topics while new material on antibiotic prophylaxis is being produced by its Committee on Clinical and Scientific Affairs, CDA has endorsed the new AHA guidelines and maintains its endorsement of the 2003 statement of the American Dental Association and the American Academy of Orthopaedic Surgeons concerning antibiotic prophylaxis for dental patients with total joint replacements.” The 2003 guideline was not found. The communication also had links to the AHA (New guidelines regarding antibiotics to prevent infective endocarditis) and to the American Dental Association—ADA and the American Academy of Orthopaedic Surgeons—AAOS . Unfortunately, the links were not functioning (“page not found”).Of interest from the three above points, the CDA only recommended consultation with the patients' health care provider in two, not for cases involving immunecompromising conditions. On July 3, 2007, the CDA  updated “was no longer” indicated for most dental patients with total joint replacements, nor for patients with orthopedic pins, plates, and screws  recommend that clinicians consider antibiotic prophylaxis for joint replacement patients prior to any invasive procedure that may cause bacteremia [” The same could be easily said about those at risk for any medical complication arising from bacteremia, although the CDA does not fully support such a recommendation. In one paper discussing the cost-effectiveness of IE associated with dental procedures, the authors evaluated the number of IE cases prevented and years of life saved [One of the major drivers of antibiotic prophylaxis has been to avoid burdening the health care system with treatment costs for conditions stemming from the presence of oral bacteremia. On that point, the American Academy of Orthopaedic Surgeons stated that cteremia .” The safe saved . They cofe saved . When pefe saved . Although there was no similar type of cost-effective analysis for immunocompromised individuals, Jacobson et al. (1991)  performeBeta-Lactams that are narrow spectrum bactericidals, which inhibit the building of the bacterial cell wall by interference with the synthesis of peptidoglycan  and Lincosamides (Clindamycin) and Macrolides (Claritromycin) that are narrow spectrum bacteriostatics, which inhibit protein synthesis at the ribosomal level. According to some studies, there has been an inadvertent use of antibiotics. Babbour and coworkers revised papers on antibiotic resistance [Clostridium difficile, and gastrointestinal disorders. Claritromycin might also cause gastrointestinal disorders, allergic reactions, and liver damage and have to be used with caution during pregnancy [Antibiotic prophylaxis usually involves a single dose of antibiotic often given to the patient close to the time of the appointment and differs from treatment that entails a course of antibiotics over a period of time. The oral, intramuscular, or intravenous recommended doses of antibiotics to be taken by those at risk before dental appointments are well known and do not seem to have changed . Dentistsistance  specificsistance . Amoxiciregnancy . Even moregnancy  includinregnancy . These fhttp://mdwhistleblower.blogspot.ca/2010/05/why-do-dentists-prescribe-prophylactic.html). Although the blog does discuss the role of the physicians on this issue, the emphasis is that “dentists irrationally prescribe antibiotics.” And this is what the lay public reads.Although any estimate of antibiotic consumption in Canada is difficult to make, Hutchinson and colleagues (2004)  believe In order to prevent bacteremia, an appropriate dose of a prophylactic antibiotic should be given prior to the procedure so that an effective tissue concentration of the drug is present to protect the patient from a bacteremia-induced periprosthetic sepsis . However“treatment and care should take into account patients' needs and preferences. Patients should have the opportunity to make informed decisions about their care and treatment.” It is worth mentioning that population at risk, in epidemiological terms, relates to those who would benefit the most from the intervention . This population, as discussed above, has changed throughout the years, from those with cardiac deformities, total joint replacement, weakened immune systems, diabetes type I, malnourishment and hemophilia to very specific cases in which professional judgment is deemed important. More often than not, however, patients prefer to take the prophylaxis as they perceive it as “safer” if they think complications from dental treatments are life-threatening (unlikely), without (or not wanting to have) full understanding of the risks of antibiotic use. The implications of such guideline changes and lack of evidence for the full use of antibiotic prophylaxis for the teaching of dentistry have to be further discussed. Kazanjian's framework offered an alternative view on the issue of antibiotic prophylaxis as a health technology in the light of its Economic Concerns, Population Impact, Social Context, Population at Risk, and the Effectiveness of the evidence to support its use. With focus on the latter, the guidelines and papers discussing this HT have emphasised that any perceived potential benefit from administering antibiotic prophylaxis before dental procedures must be weighed against the known risks of lethal toxicity, allergy, and development, selection, and transmission of microbial resistance. Although evidence-based clinical guideline is yet to be seen, one thing to keep in mind is that they are guidelines, and variation might occur as dentists must exercise their clinical judgment, communicate effectively with patients and other health care providers, and respect patients' autonomy in determining whether or not antibiotic prophylaxis is appropriate , 49. In


We previously reported that IgG4 antibodies restrict activation of immune effector cell functions and impair humoral responses in melanoma. Here, we investigate IgG4 as a predictor of risk for disease progression in a study of human sera  and peripheral blood B cells . IgG4  serum levels were elevated in melanoma. High relative IgG4 levels negatively correlated with progression-free survival (PFS) and overall survival. In early stage (I–II) disease, serum IgG4 was independently negatively prognostic for progression-free survival, as was elevation of IgG4+ circulating B cells (CD45+CD22+CD19+CD3−CD14−). In human tissues  IgG4+ cell infiltrates were found in 42.6% of melanomas, 21.4% of involved lymph nodes and 30% of metastases, suggesting inflammatory conditions that favor IgG4 at the peripheral and local levels. Consistent with emerging evidence for an immunosuppressive role for IgG4, these findings indicate association of elevated IgG4 with disease progression and less favorable clinical outcomes. Characterizing immunoglobulin and other humoral immune profiles in melanoma might identify valuable prognostic tools for patient stratification and in the future lead to more effective treatments less prone to tumor-induced blockade mechanisms.Emerging evidence suggests pathological and immunoregulatory functions for IgG4 antibodies and IgG4 LDHlactate dehydrogenaseCTCcirculating tumor cellSDstable diseasePDprogressive diseaseTregregulatory T cellCRPC-reactive proteinWBCwhite blood cell countROCreceiver operating characteristicAUCarea under the curvePFSprogression-free survival (PFS)OSoverall survivalHRhazard ratioCIconfidence intervalSNBsentinel lymph node biopsyTMAtissue microarrayPBMCperipheral blood mononuclear cellsMalignant melanoma remains a potentially lethal skin cancer despite emerging targeted therapies.The only serological biomarker for melanoma recommended in the American Joint Committee on Cancer guidelines is lactate dehydrogenase (LDH).Components of humoral immunity –including circulating antibodies– are emerging as biomarkers for autoimmune, inflammatory or allergic conditions and malignant disease. Monitoring antibodies in disease may provide 2 advantages: a) responses may occur at early points of pathogenesis indicating likely future disease onset or recurrence; and b) B cell differentiation, class-switching and antibody production are influenced by antigen recognition, or altered in response to inflammatory signals, with the resulting antibodies constituting a convenient readout. Protein array platforms like ‘immunosignature’ are now being developed to monitor circulating antibodies, and applications in Alzheimer's disease and in cancer are emerging.+ immune cell infiltrates and associations with regulatory elements such as regulatory T cells (Tregs).Consistent with the notion that components of the humoral response could be associated with malignancy, early data indicated that IgG4 antibody subclass serum levels are dysregulated in patients with melanoma.+ B cells and determine if there is an association with disease progression in melanoma.We previously reported that despite the presence of a tumor-reactive mature humoral compartment in patients, production of IgG4 subclass antibodies may be favored in melanoma and that IgG4 may contribute to defective antitumor immune responses.Table S1 and the study design is described in total) were detected in melanoma patients  as compared to healthy controls  as compared to patients who developed PD  than patients who developed PD  nor CRP (r = 0.14: P = 0.331: n = 50) correlated with IgG4 levels in this study cohort  were not significantly different between non-allergic individuals and those with a history of allergies or patients with known drug intolerances  and overall  . These findings suggest a prognostic value for IgG4 serum levels.Receiver operating characteristic (ROC) and area under the curve (AUC) analyses low, <0.034; n = 76 in IgG4high, ≥0.034). Using this cutoff point, IgG4 had a sensitivity of 57.14% (95%[CI] 45.35%–68.37%) and a specificity of 68.89% (95%[CI] 58.26%–78.23%), giving a likelihood ratio of 1.73.Since lactate dehydrogenase (LDH) is so far the only clinically-used serum biomarker used to predict disease progression,high group displayed statistically significantly lower OS (HR 95%[CI] 1.90 (1.17–3.29); log-rank P = 0.0116) and lower PFS (HR 95%[CI] 2.01 (1.34–3.35); log-rank P = 0.0016) as compared to the IgG4low group (P < 0.0001) and lower PFS (HR 95%[CI] 6.93 (43.34–427.5); log-rank P < 0.0001) as compared to those with normal LDH  and abnormal  sera concentrations. The thresholds for IgG4 and LDH were subsequently used to analyzed patient serum IgG4 and LDH  for PFS and OS using multivariate analysis  and 95% Confidence Intervals (CI) were calculated for the risk of progression-free survival. The adjusted HR for IgG4 was 1.23 (95%[CI] 0.76–1.98) versus 3.64 (95%[CI] 1.85–7.19) for LDH in Stage I–IV disease. In Stages III–IV, IgG4 had an adjusted HR of 0.99 (95%[CI] 0.55–1.77) whereas LDH had an adjusted HR of 5.47 (95%[CI] 2.47–10.92). However, in the sera of patients with Stage I–II melanoma, IgG4 had an adjusted HR of 2.46 (95%[CI] 1.01–6.02) for predicting progressive disease, whereas serum LDH had an adjusted HR of 12.29 (95%[CI] 1.18–128.00) (P = 0.0002) than LDH  or IgG4  individually .We next evaluated the prognostic potential of the combination of LDH and IgG4. When combined, these readouts had a higher prognostic value  in melanoma patients with melanoma compared to those from healthy volunteers were determined by flow cytometric analyses (+CD22+CD19+CD3−CD14−) in the blood of patients with melanoma compared with healthy volunteers  of melanoma patients was significantly elevated  compared with that of healthy volunteers'   and Stage III–IV  disease than those of healthy volunteers  (Since serum IgG4 levels were predictive of the risk of disease progression in earlier stages (Stages I–II) of melanoma, we further examined patient and healthy volunteer peripheral blood for the presence of corresponding circulating IgG4+ B cells from patients with melanoma diagnosed at different disease stages were further analyzed to examine whether they could predict the risk of disease progression . There were no statistically significant differences between SD and PD patient groups in Stages III–IV  or in Stages I–IV .The frequencies of circulating IgG4+ B cell compartment is elevated in the circulation of patients as compared to healthy volunteers, and further, that elevated IgG4+ B cell frequencies are predictive of the risk of disease progression in Stage I–II melanoma.Thus, similar to the prognostic value of elevated serum IgG4 levels in early stage (Stage I–II) melanoma, these data suggest that the IgG4+ B cells in melanoma were also reflected by tumor IgG4+ cell infiltration, we performed immunohistochemical analyses of tissue microarrays (n = 256) for the presence of tissue-resident IgG4+ cells. IgG4+ cell infiltration was detected in a proportion of melanoma tumors. Levels of infiltration were found in melanoma skin lesions (n = 108), melanoma lymph node metastases (n = 56) and distant organ metastases (n = 60) but only low levels of infiltration were found in healthy skin (n=32) specimens ; Stage III–IV: 40% ]. IgG4+ cell infiltrates were also found in skin tumor lesions of different thicknesses   melanoma, where an unmet need exists for predictors of disease progression.+ B cells may complement temporal immune markers such as serum proteins, cytokines and autoantibodies. From a clinical perspective, predicting which patients originally diagnosed with localized melanoma will develop metastatic disease remains a clinical challenge. Novel indicators of disease progression are required to improve disease management, especially at early disease stages. Presently, invasive interventions such as sentinel lymph node biopsy (SNB) are used to elucidate disease stage, to predict the risk of progressive disease and identify treatment options. In the future, comparing the predictive value of SNB with serum readouts, such as circulating IgG4, may permit less intrusive clinical tools for patient management.Associations of elevated serum IgG4 levels with increased risk of disease progression at early stages are supported by an elevated circulating IgG4+ B cell subset that is also predictive of the risk of disease progression in early stages (Stage I–II). We speculate that temporal and memory IgG4-biased immunity may be early indicators of active immunosuppression and indicate worse clinical outcomes, consistent with previous evidence that IgG4 antibodies contribute to impairment of effective immune responses. Therefore, immunomonitoring of circulating memory class-switched IgG4In our cohort, we confirm that the known serum biomarker LDH is an independent prognostic indicator in melanoma.+ cell infiltrate in a proportion of melanomas across tumor locations, tumor thicknesses and stages of disease. Together with elevated IgG4 serum levels and increased circulating IgG4+ B cells, this may indicate that immunosuppressive mechanisms are present and active at both the peripheral and local levels throughout disease stages. Although it was not feasible here to associate these infiltrates with patient history or clinical outcomes, in future studies IgG4+ infiltrates may be examined along with immune infiltrates associated with tumor-induced suppressive mechanisms, such as Tregs.. Levels of IgG4+ cell infiltration may also be evaluated along with clinicopathological features such as ulceration and mitotic rate, or combined with other tissue markers such as S-100B.Clinical practice has benefited by the companion diagnostic test for BRAF V600 mutations, used to select patients who may benefit from targeted pathway inhibitor drugs like vemurafenib.in vitro and in restricting tumor growth in vivo as compared to IgG1. Importantly, both antigen-specific and nonspecific IgG4 antibodies can impair IgG1-mediated tumoricidal functions and this IgG4 blockade is mediated through interfering with IgG1 engaging activity at Fcγ receptors.+ immune cell infiltrates in affected tissues; and 3) associations of tissue-resident IgG4+ cells with regulatory cells such as FoxP3+ Tregs. In concordance with previous reports, our data here provide rationale for further evaluation of dysregulated IgG4 antibody immunity as a prognostic indicator in melanoma, perhaps along with other immunoregulatory components. Clinical applications in regards to other malignant and inflammatory diseases or in monitoring host responses to vaccination and immunotherapies may also warrant future investigation.Dysregulated antibody profiles are associated with a variety of inflammatory pathologies, including malignant melanoma and other types of cancer. The production of IgG4 subclass antibodies is promoted in IL-10-driven alternative T helper type 2 (Th2)-biased immune conditions and in response to prolonged antigen exposure. This results in class-switching and production of IgG4, a subclass known to trigger ineffective Fc-mediated cell activation due to distinctive conformation features.Monitoring of dysregulated components of host immune surveillance, such as IgG4 or cell subsets that indicate immunosuppression,The immunological mechanisms that govern the nature of humoral responses in cancer remain only partly elucidated, including those underlying biased IgG4 subclass expression in patient circulation and melanoma tumors. Although IgE and IgG4 are promoted by Th2 conditions, preferential expression of IL-10 in melanoma tumors could polarize a classical Th2-type immunity toward IgG4.de novo IgE or class switching from IgG4 to IgE.In the future, it can be envisaged that antibodies less prone to IgG4 immune impairment, treatments that counteract IL-10-driven inflammation, or interventions that promote classical rather than alternative Th2 immunity could help refocus antitumoral immune signals. Strategies may include induction of protective Th2 responses that could perhaps drive In summary, we confirm that IgG4, a purported indicator of immunosuppressive forces in melanoma is elevated in the circulation of melanoma patients, is associated with worse clinical outcomes, and is a negative prognostic indicator at early stages of melanoma. Broader applications for IgG4 monitoring may be complimented by other clinical tools, such as LDH, or upon incorporation into immune profiling algorithms. Larger prospective studies will ascertain the clinical significance of IgG4, perhaps along with other immunoglobulin or immune cell signatures, in facilitating more accurate prediction of progressive disease. This may pave the way for improved patient stratification and optimal personalized therapies.Human specimens were collected with written informed consent . Patients were staged and classified according to the American Joint Committee on Cancer Melanoma Staging and Classification criteria.Luminex bead array assay kits for assessments of IgG4 subclass antibodies in human sera were used according to the manufacturers' instructions. Data analyses to assess IgG subclass antibodies were conducted using Milliplex® MAP  and data were acquired and analyzed using FlexMap3D (Luminex Corporation). LDH serum levels were measured at the central laboratories of the associated hospital.+ cell infiltrates were detected using the VECTOR Red Alkaline Phosphatase Substrate Kit with levamisole . All sections were mounted in DPX mounting solution and analyzed on a Zeiss Axiophot microscope using a 10x magnification lens (Carl Zeiss) and NIS-Elements imaging software (Nikon). Cell density of sections stained for IgG4+ B cell infiltration was evaluated by 2 independent researchers to identify the occupancy of infiltration per high power field (10x) using the following criteria for occupancy: “negative” = 0% infiltration; “low” < 25% infiltration; and “high” > 25% infiltration.Tissue microarrays  of paraffin embedded sections were cut at 6–8 μm thickness on a microtome (Leica) and dried overnight at 60°C. Prior to staining, sections were deparaffinized in xylene for 20 min and rehydrated by serial incubations in alcohol. Heat-induced antigen retrieval was performed in a 95°C water bath using a citric acid solution (pH = 6.0). Sections were subsequently blocked with human FcR blocking reagent  according to the manufacturer's instructions and stained using a mouse anti-human IgG4 antibody , followed by a rabbit anti-mouse IgG antibody conjugated to biotin . IgG4+ cells were analyzed based on gating with an isotype control. All sample analyses were performed using FlowJo software (Treestar).Peripheral blood mononuclear cells (PBMC) were isolated from venous blood by density centrifugation over Ficoll-Paque Plus . Cells were placed in RPMI 1640 medium  with 11.25% human serum albumin , frozen in 10% DMSO  and stored in liquid nitrogen. Prior to analyses, cells were thawed and stained with fluorophore-conjugated antibodies against the following surface markers : CD45, CD22, CD19, CD3, CD14, IgG4. Prior to use, the anti-IgG4 antibody was pre-labeled with R-phycoerythrin  following the manufacturer's instructions. Dead cells were excluded by staining with LIVE/DEAD® Fixable Dead Cell Stains . Samples were subsequently acquired on a 5-laser SORP Fortessa (BD Bioscience) flow cytometer and data analyzed using the same instrument application settings across different experiments. IgG4Human serum and cell populations were assessed for normal Gaussian distribution with D'Agostino and Pearson omnibus normality test, followed by Kruskal–Wallis one-way analysis of variance with post-hoc Dunn's test. For comparisons between 2 groups and where populations were not normally distributed  statistical significance between groups was calculated using the Mann-Whitney-U-test. For correlation and patient survival analyses, Spearman and Mantel-Cox analyses were applied. Receiver Operating Characteristic (ROC) analyses and area under the curve (AUC) analyses were performed to assess the prognostic capacity of IgG4 in patient sera. The prognostic capacity of LDH and IgG4 combined serum tests was evaluated by applying a ROC analysis and calculating the area under the curve (AUC3). Threshold values were calculated via the Youden's IndexComparative IgG4 serum level analyses among melanoma patients with subsequent stable (SD) or progressive disease (PD) were conducted. Patients were segregated into: a) those with stable disease (neither progressive disease nor subsequent relapse during study period); and b) those with progressive disease . Patients could only be up-staged from their initial staging during the study period.P values are derived from 2-sided comparisons with values of less than 0.05 considered to indicate statistical significance.Error bars in all figures represent standard error of mean (SEM). All statistical tests were performed using GraphPad Prism Version 6, with the exception of the calculation of hazard ratio (HR), which was performed using the SAS software version 9.2 (SAS Institute Inc.). All reported


Background: The human IgG4 antibody subclass does not activate complement and forms half-antibodies.Results: Ultracentrifugation and x-ray/neutron scattering together with atomistic modeling revealed asymmetric concentration-dependent IgG4 solution structures.Conclusion: The complement and receptor Fc binding sites are hindered by the Fab regions, explaining loss of activity.Significance: These solution structures clarify IgG4 function and its therapeutic applications. 222) and a hinge mutant IgG4(Pro222) in different buffers and temperatures where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients sw20,0 of 6.6–6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the x-ray radius of gyration gR was unchanged with concentration in 50–250 mm NaCl buffers, whereas the neutron gR values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves (P(r)) revealed two peaks, M1 and M2, that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures in addition to IgG4 dimer formation at high concentration in heavy water. Constrained x-ray and neutron scattering modeling revealed asymmetric solution structures for IgG4(Ser222) with extended hinge structures. The IgG4(Pro222) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement and clarify aspects of its stability and function for therapeutic applications.Human IgG4 antibody shows therapeutically useful properties compared with the IgG1, IgG2, and IgG3 subclasses. Thus IgG4 does not activate complement and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we studied the solution structure of wild-type IgG4(Ser IgG4 plasma levels rise after prolonged antigen exposure, leading to anti-inflammatory effects  . Cys220 and Cys223 in the core hinge form disulfide bridges between the heavy chains. Mutation of Ser222 to Pro222 by genetic engineering gives an IgG1-like core hinge and eliminates half-mer formation (222) hinge is more flexible than the mutant IgG4(Pro222) hinge, perhaps explaining the formation of stable intrachain disulfide bonds in the mutant   in whichormation . Conforme mutant . The eque mutant . The kin409 to be crucial for stabilizing the CH3-CH3 interaction   –16. Fab-exchange . Interesexchange . The abi222) had been studied by ultracentrifugation and x-ray scattering to show that IgG4 undergoes a concentration-dependent Fab-arm rearrangement (222) and IgG4(Pro222). We show that IgG4(Ser222) and IgG4(Pro222) are principally monomeric, but IgG4(Ser222) shows self-associative behavior in heavy water. Both forms display concentration-dependent structural changes corresponding to Fab-arm rearrangements at low concentrations. Both the IgG4(Ser222) and IgG4(Pro222) solution structures display extended asymmetric arrangements of the Fab regions relative to the Fc region. Our three independently determined IgG4 solution structures showed that the Fab regions were close enough to the Fc region to hinder the C1q and FcR binding sites at the top of the Fc region in IgG4. The implications of this result for the reduced immune reactivity of IgG4 are discussed.An understanding of IgG4 would be facilitated by its atomistic solution structure. Atomistic antibody structures are determined using our joint strategy of x-ray and neutron scattering coupled with analytical ultracentrifugation and constrained modeling based on crystal structures –21. Join–angement . This co222, was generously supplied by Dr. Jim Davies (Lonza Biologics) . Other studies named Ser222 as Ser228 (229 (241 (222 was generously supplied by Dr. Bryan Smith (UCB) (222) and IgG4(Pro222) were further purified by gel filtration to remove nonspecific aggregates using a Superose 6 10/300 column , concentrated using an Amicon Ultra spin concentrator (50-kDa-molecular mass cutoff), and dialyzed at 4 °C against the measurement buffer (see below). The sequence identity between both IgG4 molecules was 100% for the CH1, CH2, and CH3 domains (except for the S222P substitution). Different identities were found for the VH (67.5%), VL (59.3%), and CL (96.2%) domains, giving an overall identity between the two IgG4 sequences of 86.7% . The N-linked oligosaccharides at Asn291 on the CH2 domains  were assumed to be a complex-type biantennary oligosaccharide structure with a Man3-GlcNAc2 core and two NeuNAc-Gal-GlcNAc antennae (222) molecular mass was calculated to be 147.0 kDa, its unhydrated volume was 188.7 nm3, its hydrated volume was 248.7 nm3 (based on a hydration of 0.3 g of water/g of glycoprotein and an electrostricted volume of 0.0245 nm3 per bound water molecule), its partial specific volume v was 0.728 ml/g, and its absorption coefficient was 14.5 . For IgG4(Pro222), the molecular mass was 149.5 kDa, v was 0.728 ml/g, and the absorption coefficient was 14.1 . Buffers containing 50 mm NaCl or 250 mm NaCl in place of 137 mm NaCl were termed PBS-50 and PBS-250, respectively. The buffer densities were measured using an Anton Paar DMA 5000 density meter for comparison with the theoretical values from SEDNTERP  samples in PBS-50, PBS-137, and PBS-250 at 20 °C; in PBS-137 at 6, 20, and 30 °C; and in PBS-137 with 100% 2H2O. Data were collected at rotor speeds of 40,000 and 50,000 rpm in two-sector cells with column heights of 12 mm. Sedimentation velocity data were acquired for IgG4(Pro222) in PBS-137 at 20 °C only. Data were collected at rotor speeds of 30,000, 40,000, 50,000, and 60,000 rpm. Sedimentation analysis was performed using direct boundary Lamm fits of up to 700 scans using SEDFIT (version 12.1) (c(s) that assume all species to have the same frictional ratio f/f0. The final SEDFIT analyses used a fixed resolution of 200 and optimized the c(s) fit by floating f/f0 and the baseline until the overall root mean square deviation and visual appearance of the fits were satisfactory. The percentage of oligomers in the total loading concentration was derived using the c(s) integration function.Analytical ultracentrifugation data for both IgG4 forms were obtained on two Beckman XL-I instruments equipped with AnTi50 rotors. Sedimentation velocity data were acquired for IgG4(Seron 12.1) , 30. SED222) in PBS-50, PBS-137, and PBS-250 was studied at 20 °C at eight concentrations between 0.48 and 6.91 mg/ml (3.3–47 μm) in which 100-μl samples were measured in a polycarboxylate capillary (2-mm diameter) that avoids protein deposits during beam exposures. The sample was moved continuously during beam exposure to eliminate radiation damage. Sets of 10 time frames, each of duration 0.1 or 0.2 s/frame, were acquired in quadruplicate as a control of reproducibility. Online checks during data acquisition confirmed the absence of radiation damage after which the 10 frames were averaged. In the same beam session, IgG4(Pro222) in PBS-137 at 20 °C was studied at concentrations between 0.5 and 2.0 mg/ml (3.3-13.4 μm).X-ray scattering data were obtained during a single beam session in 16-bunch mode on Instrument ID02 at the European Synchrotron Radiation Facility, Grenoble, France, operating with a ring energy of 6.0 GeV . Data we222) samples were measured in 2-mm-path length circular banjo cells in a thermostated rack at 20 °C as well as at 6 and 37 °C. Data acquisitions lasted ∼1–2 h for four samples at concentrations between 2.0 (14 μm) and 8.0 mg/ml (55 μm) in PBS-137 prepared in 100% heavy water.Neutron scattering data were obtained on Instrument SANS2D at the ISIS pulsed neutron source at the Rutherford Appleton Laboratory, Didcot, UK . A pulsegR is a measure of structural elongation if the internal inhomogeneity of scattering densities within the protein has no effect. Guinier analyses at low Q  gives the gR and the forward scattering at zero angle I(0) Q]Q→0 is obtained from RXS-1 and RXS-2, respectively. The gR and RXS analyses were performed using an interactive PERL script program SCTPL7I(Q) in reciprocal space into real space to give the distance distribution function P(r) was carried out using the program GNOM (P(r) corresponds to the distribution of distances r between all volume elements. This yields the maximum dimension of the macromolecule L and its most commonly occurring distance vector M in real space. To calculate this, the x-ray I(Q) curve utilized up to 341 data points in the Q range between 0.03 and 1.60 nm−1. The neutron I(Q) curve utilized up to 112 data points in the Q range between 0.01 and 2.0 nm−1.In a given solute-solvent contrast, the radius of gyration gle I(0) . This et region , identifram GNOM .P(r) c222) models were created by joining molecular structures for the IgG4 Fab and Fc regions. A homology model for human IgG4 was constructed from crystal structures for Fab B72.3 and human IgG1 (Protein Data Bank codes 1BBJ and 1HZH)  , 37 as dI(Q) was calculated using the Debye equation adapted to spheres  or 1142 spheres (neutrons) were discarded. Of the 20,000 hydrated models, 82% showed no steric overlap. The x-ray gR, RXS-1, and RXS-2 values were calculated from the modeled curves in the same Q ranges used for the experimental Guinier fits. Models that passed gR and RXS filters of ±5% of the experimental value were then ranked using a goodness-of-fit R-factor  in the Q range extending to 1.6 nm−1. For the neutron modeling, 84% of the 20,000 unhydrated sphere models showed no steric overlap. The neutron models were assessed as for the x-ray scattering models above with corrections for wavelength spread and beam divergence, but no correction was required for a flat background caused from incoherent scattering because of the extrapolation to zero concentration.The scattering curve was calculated from each IgG4 model using sphere models. A cube side of 0.557 nm and a cutoff of four non-hydrogen atoms was used to convert the atomic coordinates into 1142 spheres that corresponded to the unhydrated structure seen by neutron scattering. Because hydration shells are visible by x-rays, a hydration shell corresponding to 0.3 g of water/g of protein was created using HYDRO , giving  spheres . Details spheres , 40. Stesw20,0) were calculated directly from the hydrated Debye sphere models using the program HYDRO  using a Python script. The three angles between the Fab and Fc regions were also calculated; these were defined in a Python script as the angle of intersection from the dot product between two vectors. Each vector was defined as the long axis through each Fab or Fc region, each defined as the line passing through the centers of gravity between each cluster of four cysteine α-carbon atoms at the two ends of the Fab and Fc regions. One cluster at each end of each Fab or Fc region corresponded to the conserved disulfide bridge in each immunoglobulin fold domain. Artwork was prepared using PyMOL . The Fc regions in the 10 best fit IgG4 models were superimposed using the align function within PyMOL. To dock the crystal structures for the Fc region and the C1q head , the websupplemental material. They have been deposited in the Protein Data Bank under accession codes 4PTO (IgG4(Ser222) in PBS-137), 4PTQ (IgG4(Ser222) in PBS-137 in 2H2O), and 4PTR (IgG4(Pro222) in PBS-137).The three sets of 10 best fit models are currently available as 222) and its mutant IgG4(Pro222) were subjected to gel filtration to ensure monodispersity immediately prior to the ultracentrifugation and scattering experiments. Both molecules eluted as a symmetric main peak at 16 ml  and showed a single band between 200 and 116 kDa in non-reducing SDS-PAGE that corresponds to the expected masses of 147.0 and 149.5 kDa for intact IgG4(Ser222) and IgG4(Pro222), respectively. Under reducing conditions, the heavy chains for both IgG4 molecules were observed at an apparent molecular mass of 55 kDa, and their light chains were between 31 and 21.5 kDa, both as expected .Wild-type IgG4(Ser222) between 0.5 and 8 mg/ml and IgG4(Pro222) between 0.5 and 2.5 mg/ml. The SEDFIT analyses in different NaCl buffers in H2O and at different temperatures in 2H2O buffers involved fits of as many as 700 scans, and the good agreement between the experimental boundary scans and fitted lines is clear (A–C). A major monomer peak was observed at sw20,0 values of 6.8 S for IgG4(Ser222) and 6.6 S for IgG4(Pro222). These sw20,0 values are consistent with the range of values (6.2–6.9 S) reported previously for human IgG4 (–222) and IgG4(Pro222) were predominantly monomeric in solution and were accompanied by a minor dimer peak.Sedimentation velocity experiments examined the size and shape of IgG4 analyses, the molecular masses of the monomer peak for IgG4(Ser222) were reported as 144 (PBS-50), 145 (PBS-137), and 141 kDa (PBS-250) in light water and 171 (PBS-137 at 6 °C), 149 (PBS-137 at 20 °C), and 145 kDa (PBS-137 at 30 °C) in heavy water. These agree well with the composition-calculated mass of 147 kDa with the exception of PBS-137 in heavy water at 6 °C. However, for PBS-137 in heavy water at 6 °C, a reaction boundary was observed from the concentration dependence of its monomer peak (B). The molecular mass of the IgG4(Pro222) monomer peak was measured as 143 kDa (PBS-137) in agreement with the composition-calculated mass of 150 kDa.From the mer peak B. The moA–C). The monomer s20,w value decreased slightly with rotor speed. For IgG4(Ser222), the monomer sw20,0 value was 6.76 S for 40,000 rpm, decreasing to 6.68 S for 50,000 rpm for PBS-137 at 20 °C. For IgG4(Pro222), the monomer sw20,0 value was 6.64 S for 30,000 rpm, decreasing to 6.59 S for 40,000 rpm, 6.56 S for 50,000 rpm, and 6.53 S for 60,000 rpm. All the IgG4 data reported in this study are for 40,000 rpm. Extrapolation of the s20,w values to zero concentration gave monomer sw20,0 values of 6.71, 6.76, and 6.64 S for the PBS-50, PBS-137, and PBS-250 buffers, respectively, in light water (A). In PBS-137 in heavy water, the monomer sedimented at apparent rates of 2.75, 4.07, and 5.07 S at 6, 20, and 30 °C, respectively (B). When corrected for the buffer density and viscosity of heavy water, sw20,0 values of 6.49, 7.22, and 7.35 S, respectively, were obtained. Given that the partial specific volume v for proteins is affected by the hydration shell , the sw20,0 value was 6.59 S for PBS-137 at 20 °C, which is not significantly different from that for IgG4(Ser222).The apparent sedimentation rates of the IgG4 monomer depended on the buffer composition and temperature , A–C. Thht water A. In PBSectively B. When con shell , 40 and c(s) analyses for IgG4(Ser222) revealed a minor dimer peak at sw20,0 values between 9 and 10 S in the size distribution analyses c(s) (222) dimer. IgG4(Pro222) also showed a small dimer peak of size 2–3% with a sw20,0 value of 9.25 ± 3.0 S and a molecular mass of 276 ± 15 kDa. This agreed well with the predicted mass of 300 kDa for its dimer. The monomer and dimer c(s) peaks were integrated. The amount of dimer did not alter with concentration or buffer composition (B) and totaled between 1 and 3% dimer. The monomer/dimer ratio corresponded to weak dissociation constants (DK) in a range of 600–3000 μm. In comparison, dimer formation for rabbit IgG was noticeably higher with the corresponding DK values ranging from 70 μm for PBS-137 at 20 °C in heavy water to 350 μm in PBS-137 at 30 °C , 9.78 ± 0.32 (PBS-137), and 9.71 ± 0.36 S (PBS-250) at 20 °C. They were slightly larger but similar at 10.27 ± 0.70, 10.17 ± 0.81, and 10.02 ± 0.23 S at 6, 20, and 30 °C, respectively, in PBS-137 in heavy water.The ses c(s)  and 2. Tposition B and totat 30 °C . The s20The solution structure of IgG4 was jointly analyzed by both x-ray and neutron scattering for reproducibility. X-rays in light water buffers monitor the hydration shell as well as the protein structure, whereas neutrons in heavy water buffers monitor the unhydrated structure (see below).222) was carried out between 0.48 and 6.91 mg/ml using time frame analyses to ensure the absence of radiation damage effects. Guinier analyses resulted in high quality linear plots in three distinct regions of the I(Q) curves, as expected for antibodies, from which the gR, RXS-1, and RXS-2 values were obtained within satisfactory Q·gR and Q·RXS limits  in PBS-50, PBS-137, and PBS-250 showed no concentration dependence with mean values of 4.90, 4.92, and 4.96 nm, respectively (A). The I(0)/c values for IgG4, which were similar at 0.0192, 0.0181, and 0.0179, respectively, also showed no concentration dependence (A). Each of the RXS-1 and RXS-2 values were unchanged among PBS-50, PBS-137, and PBS-250 with mean RXS-1 values of 2.53, 2.56, and 2.51 nm, respectively, and mean RXS-2 values of 1.40, 1.37, and 1.33 nm, respectively. These values agree with the gR, RXS-1, and RXS-2 values of 4.99, 2.56, and 1.40 nm, respectively, for this same human IgG4(Ser222) molecule in PBS-137 (222), IgG4(Pro222) was studied between 0.5 and 2.0 mg/ml in PBS-137 . The x-ray gR values showed no concentration dependence with a mean value of 4.90 nm (C). Similarly, the I(0)/c values for IgG4(Pro222) were 0.0191, showing no concentration dependence and in agreement with those for IgG4(Ser222) (C). The mean RXS-1 and RXS-2 values were unchanged at 2.60 and 1.37 nm, respectively (C).X-rays were most effective for looking at IgG4 at 20 °C in three different NaCl concentrations. X-ray data collection of IgG4(Serectively A. The I(pendence A. Each o 4.90 nm C. Simila(Ser222) C. The meectively C.222) in 100% 2H2O buffer was analyzed between 2.0 and 8.0 mg/ml. The Guinier analyses revealed high quality linear fits for the same three parameters, gR, RXS-1, and RXS-2, as described above for x-rays . The neutron gR values increased with increasing concentration from 5.16 to 5.82 nm at 6 °C, from 4.87 to 5.36 nm at 20 °C, and from 4.62 to 4.85 nm at 37 °C (B). These gR changes were comparatively large, indicating that heavy water promoted IgG4 dimer formation. Because the neutron Guinier fits go to smaller Q values than the x-ray Guinier fits, the neutron fits were more sensitive to dimer formation than the x-ray fits. Extrapolations to zero concentration resulted in gR values of 4.99 (6 °C), 4.77 (20 °C), and 4.67 nm (37 °C). The corresponding I(0)/c values also increased (B). The neutron RXS-1 values decreased slightly with concentration from 2.50 to 2.28 nm at 6 °C, from 2.43 to 2.30 nm at 20 °C, and from 2.50 to 2.33 nm at 37 °C (B). The RXS-2 values showed no concentration dependence with mean values of 1.23, 1.23, and 1.17 nm at 6, 20, and 37 °C, respectively (B). The neutron gR, RXS-1, and RXS-2 values were slightly smaller than the x-ray values, such as that of 4.92 nm for the gR value in PBS-137, and this reduction is attributed to the near invisibility of the surface hydration shell in heavy water  provides structural information on IgG4 in real space. The x-ray P(r) analyses gave gR and I(0)/c values for IgG4(Ser222) that were similar to those from the x-ray Guinier analyses, showing that the two analyses were self-consistent . The maximum length L of IgG4 was determined from the value of r when the P(r) curve intersects zero to be 16 nm for PBS-50, PBS-137, and PBS-250 (A). The maxima in the P(r) curves correspond to the most frequently occurring interatomic distances within the structure. For IgG4(Ser222), two peaks, M1 and M2, were identified in many P(r) curves except for that of the 0.5 mg/ml P(r) curve where only M1 was identified. The M1 peak corresponds mostly to distances within each Fab and Fc region, whereas the M2 peak corresponds mostly to distances between pairs of Fab and Fc regions. No buffer dependence in the positions of peaks M1 and M2 was observed. However, the positions of the M2 peaks depended on the IgG4 concentration below 2 mg/ml (A). The more extensive data sets of this study confirmed this dependence seen previously with more limited data (222) in which a 1–2-nm change in the average separation between the Fab-Fc pairs was deduced (A). The IgG4(Pro222) mutant also showed two M1 and M2 peaks with a similar pronounced shift in the M2 peak (but an unchanged M1 peak) with change in concentration, and the L value was unchanged at 15 nm (C and 5C).The distance distribution function  PBS-250 A. The ma 2 mg/ml A. The moted data . The con deduced . At conc deduced A. The Igat 15 nm C and 5C.P(r) analyses reflected IgG4 dimer formation in heavy water. The gR and I(0)/c values for IgG4(Ser222) at 6, 20, and 37 °C increased with increasing concentration (B). This was attributed to dimer formation. This result is consistent with the sedimentation velocity experiments where a reaction boundary between monomer and dimer was observed in heavy water at 6 and 20 °C (B) but not in light water. The neutron L values were between 17 and 21 nm at 6 °C, 16 and 19 nm at 20 °C, and 15 and 16 nm at 37 °C (B). Thus the greater proportion of dimer at lower temperatures resulted in increased dimensions. Because low amounts of dimer are present, the maximum length of the dimer is probably greater than 21 nm. The two peaks M1 and M2 were again identified at ∼4 and 7 nm, respectively, in the neutron P(r) curves (B). The positions of M1 and M2 were unchanged between 2 and 8 mg/ml in agreement with the x-ray P(r) data.The neutron ntration B. This wnd 20 °C B but notat 37 °C B. Thus t) curves B. The po213ESKYGPPCPSCPAPEFLGGP232 . The IgG4 hinge contains six Pro residues and two Cys220 and Cys223 disulfide bridges. The asymmetric modeling considered only the upper hinge, 212VESKYGPPC220, with Val212 and Cys220 acting as tethers. As this hinge is located asymmetrically relative to the Fc region, these 10,000 models do not have 2-fold symmetry. However, the two interchain disulfide bonds are intact. The symmetric modeling considered the upper, middle, and lower hinge, and this resulted in a 21-residue peptide, 212VESKYGPPCPSCPAPEFLGGP232. Because the two Pro232 residues were located in the middle of the Fc region, this approach generated both symmetric and asymmetric models. For these 10,000 further models, the Cys220 and Cys223 interchain disulfide bonds were not kept intact.The human IgG4 homology models were created from crystal structures for B72.3 Fab  and humaFLGGP232  in which222) and IgG4(Pro222) solution structures, 20,000 conformationally randomized IgG4 structures were created from the Fab and Fc structures starting from four conformational libraries of randomized hinge peptides of lengths 2.2–3.1 and 3.1–3.15 nm (asymmetric) and 5.0–7.0 and 7.0–7.35 nm (symmetric) . Each modeled scattering curve was compared with the experimental x-ray and neutron scattering curves. The nine x-ray curves included IgG4(Ser222) in PBS-137 at concentrations of 0.48, 0.96, 1.93, 3.86, and 5.79 mg/ml and IgG4(Pro222) in PBS-137 at concentrations of 0.5, 1.0, 1.5, and 2.0 mg/ml. These curve fits investigated whether molecular models could rationalize the conformational change seen in IgG4 at below 2 mg/ml for the wild-type and mutant forms of IgG4 (A and C). Because IgG4 dimerizes in heavy water, neutron curve fits were based on linear extrapolation of the four experimental curves for IgG4(Ser222) to zero concentration (B). This curve fit examined a single conformation for the IgG4 monomer only. The 10 fit searches were assessed in R-factor gversus R graphs  A ±5% filter for the modeled gR values  identified the models that agreed best with the experimental x-ray or neutron gR values. (iii) The models that passed the n and gR filters were arranged in order of their lowest R-factors. The resulting 10 best fit models for IgG4 were found as a single cluster at the R-factor minimum in each of the 10 searches (green).Filters were used to identify the 10 best fit models for each search. (i) A ±5% filter for steric overlap eliminated models in which the Fab and Fc regions sterically overlapped with each other because of inappropriate hinge conformations. To match the composition-calculated volume of IgG4, sphere models needed a minimum number searches , green.220 and Cys223 were explicitly conserved in the asymmetric models. For the symmetric models, the pairs of Cys220 and Cys223 residues may no longer be proximate. Their α-carbon separations were 1.02–3.29 nm for Cys220 and 1.20–2.43 nm for Cys223 in IgG4(Ser222) by x-rays, 0.75–2.18 nm for Cys220 and 0.98–1.99 nm for Cys223 in IgG4(Pro222) by x-rays, and 2.02 nm for Cys220 and 1.98 nm for Cys223 for IgG4(Ser222) by neutrons. These α-carbon separations were comparable with an expected α-carbon separation of 0.4–0.75 nm between two bridged Cys residues (The two interchain disulfide bonds at Cysresidues .I(Q) and P(r) curve fits in all 10 cases with the experimental curves  showed slightly higher proportions of asymmetric structures compared with the asymmetric and symmetric arrangements seen for IgG4(Pro222), there were no significant differences between the wild-type and mutant structures. Unlike our previous study (222) x-ray and neutron models and in the IgG4(Pro222) x-ray models . The x-ray R-factor values for the best fit IgG4 models (pink) were acceptable at 2.5–4.0% for IgG4(Ser222) and 3.0–3.6% for IgG4(Pro222)  . The neuing fits .sw20,0 values of each of the nine sets of scattering best fit hydrated IgG4 models were calculated for comparison with the experimental values of 6.76 S for IgG4(Ser222) and 6.59 S for IgG4(Pro222) (A). For the best fit hydrated sphere models, the sw20,0 values were 6.76–6.99 and 6.72–7.02 S for IgG4(Ser222) and IgG4(Pro222), respectively, using HYDRO  and IgG4(Pro222), respectively, using HYDROPRO  A. For thng HYDRO . For theHYDROPRO . Given tHYDROPRO , both seThe large body of x-ray scattering data permitted a detailed appraisal of the solution structure of human IgG4, and this was supported by complementary neutron scattering and ultracentrifugation experiments. The scattering data enabled atomistic conformational analyses of IgG4 that resulted in three independent determinations of an asymmetric IgG4 solution structure. The combination of the resulting three IgG4 solution structures with (i) a recent docking model for the interaction between human Fc and the crystal structure of the C1q globular head , 50 and IgG4 has the lowest IgG serum concentration of the four IgG subclasses at an average level of 0.4 mg/ml (in a range of 0.2–1 mg/ml). The data collection of our IgG4 study utilized a concentration range of 0.4–8.0 mg/ml, which spanned the serum range and above. IgG1 is the most common subclass at 8 mg/ml (in a range of 5–12 mg/ml) followed by 4 mg/ml for IgG2 (in a range of 2–6 mg/ml) and 0.8 mg/ml for IgG3 (in a range of 0.5–0.1 mg/ml) . IgG4 al222), our x-ray data collection involved measurement of 192 curves (or 1920 curves if time frames are included) within 2 h (–For IgG4(Serthin 2 h . This hithin 2 h . All sixthin 2 h . In contthin 2 h –48.222) data collection indicated no observable change in the gR, M1/M2, or sw20,0 values in concentrations between 2 and 8 mg/ml in six buffers and temperatures (222) and IgG4(Pro222) showed reproducible concentration-dependent changes in the M2 peak that monitors the mean separation between the Fab-Fab and Fab-Fc pairs (222)  the IgG4 hinge structures are extended in solution and (ii) the arrangement of the Fab and Fc regions is unaffected by different buffers. IgG4 was modeled starting from the intact IgG1 b12 molecule in which the lower hinge was asymmetrically arranged . To avoiFigs. 6ilibrium ; therefovelocity . These aty (1HZH ), murineZH (1IGT ), and muGT (1IGY ). A fourGY (1MCO ). Our reGY  best fit models was compared with the IgG4 Fc crystal structures. The root mean square difference in α-carbon positions when superimposed was low at 0.0937 and 0.0841 nm . When the Fc region in the scattering best fit model for IgG4(Ser222) was replaced with the Fc crystal structures, the resulting R-factors of 2.7 (Protein Data Bank code 4C54) and 2.9% (Protein Data Bank code 4C55) were almost unchanged compared with that of 2.8% for the best fit model  has a slightly shorter overall length at 15 nm compared with 16 nm for IgG4(Ser222), its sw20,0 value is slightly smaller at 6.6 S compared with 6.8 S for IgG4(Ser222). Both forms showed decreased sw20,0 values when the concentration went below 2 mg/ml. Experimentally both forms were indistinguishable within error.Wild-type and mutant IgG4 differ through the S222P substitution in its hinge. This change to Pro abolishes half-mer formation and presumably results from the reduced flexibility of the hinge , 12. OurS values . Althoug222) are predominantly asymmetric, especially at the highest concentrations (A). At lower concentrations, both IgG4(Ser222) and IgG4(Pro222) displayed a combination of asymmetric and symmetric best fit structural models. Although the IgG4(Pro222) form is thought to be less flexible (212 and Pro232 with a maximum possible length of 7.0 nm. The five IgG4(Ser222) hinge lengths decreased slightly with increasing concentration (A). In fact, each of the three final best fit structures gave similar hinge lengths of 4.99–5.73 ± 1.08 nm for IgG4(Ser222) and 4.81–5.94 ± 1.08 nm for IgG4(Pro222) in PBS-137 and 5.58 ± 1.10 nm for IgG4(Ser222) in PBS-137 in heavy water.The best fit structures for IgG4 and IgG4(Pro222) models are positioned close to the IgG4 binding site for C1q, potentially hindering C1q binding. Docking studies were performed using a shape complementarity method by the PatchDock server . In contrast to the rabbit IgG-C1q interaction, our docked IgG4-C1q structures (A and B) show that the C1q head is sterically hindered by the Fab regions from interacting with the top of the Fc region in IgG4. In addition, biochemical studies using domain-switched chimeric antibodies showed that the differential complement activation of the subclasses resided in the COOH-terminal CH2 domain  , FcγRIIB/FcγRIIC, FcγRIIIA , and FcγRIIIB , NA2 , and SH ). The affinity constants (AK) for binding of the four IgG subclasses with all nine FcγR variants have been established , IgG4 binding is slightly weaker (3.4 × 107m−1), and IgG2 displayed no measureable binding. For the remaining FcγRII and FcγRIII subclasses, IgG4 showed low affinity binding with AK values near 2 × 107m−1 for FcγRIIA (His/Arg131), FcγRIIB/FcγRIIC, and FcγRIIIA  and no measureable binding to FcγRIIIB . In contrast, IgG1 and IgG3 bound to all the FcγRII and FcγRIII subclasses and allelic variants often with higher AK values ranging between 1.2 × 105 and 9.8 × 106m−1. Conversely, IgG2 showed mostly lower affinities than IgG1, IgG3, and IgG4 for all FcγRs, including no measurable binding for FcγRIIIB  and IgG4(Pro222) solution structures on these crystal structures. This showed that FcγR binding appears to be permitted. Given that the Fab regions in our IgG4 models are close to the top of the Fc region where the FcγRs bind, these Fab regions may offer steric hindrance to binding (C and D). However, as IgG4 is able to bind most FcγRs , a small movement of the Fab region in our IgG4 models may permit binding to take place, and the strength of binding between Fc and FcγR may be primarily determined by the residues present in the hinge and CH2 domains. In this context, IgG4 possesses Phe234 in comparison with IgG1 and IgG3 in which this is Leu234 and with IgG2, which has a shorter lower hinge region with Val234 and Ala236 substitutions and the deletion of Leu235. IgG4 also has a shorter overall hinge length, which may lead to weaker FcγR binding  would be greater than the relaxation rate, leading to a deformed IgG4 structure. The upper limit for the relaxation rate is therefore defined by the collision rate constant kcoll for 2 mg/ml. The collisional frequency Z can be calculated using the diffusion coefficient from SEDFIT and the radius of IgG4 from N is Avogadro's number (6.02214 × 10−23), D is the diffusion coefficient (4.07 × 10−11 m2 s−1), and r is the radius for two IgG4 monomers A and B. This gives a collision frequency of 6300 m−1 s−1. The kcoll value can be calculated from p is 1  and Eact is 0, then the equation is simplified to kcoll = Z. At 2 mg/ml (14 μm), the upper limit for the relaxation rate is obtained by multiplying kcoll by the concentration to give 88 s−1. Because the calculation assumes that all collisions lead to a shape change, which is unlikely, the true rate of relaxation will be lower than 88 s−1.To understand better how a diffusion-collision process might lead to conformational change in IgG4, this process can be considered as an equilibrium where collisions between IgG4 molecules at higher concentrations lead to a deformed shape that can relax back to the unperturbed shape in dilute solution. Above 2 mg/ml, the rate of collisions (in vivo (222) and IgG4(Pro222) by non-reducing SDS-PAGE as a faint band at ∼75 kDa . However, these bands have been shown to be artifacts formed under the harsh conditions of SDS-PAGE and depend on heating times and the SDS buffers  in heavy water, especially at reduced temperature (B). This indicated fast on/fast off association rates between monomer and dimer. Neutron scattering confirmed the temperature-dependent self-association in heavy water (B). Greater dimer formation in heavy water is attributed to weaker hydrogen bonding in this solvent, meaning that protein-protein interactions are promoted. In other studies, IgG4 has been shown to associate with the Fc fragments of other IgG classes, probably occurring between their CH3 domains (H3 domains may be an intermediate before half-mer swapping. IgG4 can also associate with IgG1 following a conformational change of IgG1 that exposes the IgG1 CH3 domains (Another aspect relevant to antibody bioprocessing relates to IgG4 dimer formation and self-association. Extensive dimer formation may lead to nonspecific antibody aggregation. Indeed, rabbit IgG was observed to form as much as 25% dimer in heavy water buffers, possibly arising through Fab-Fab pairings between the two monomers . The lowperature B. This ivy water B. Greate


Substantial progress has been made in China in using traditional Chinese medicine (TCM) to treat acquired immune deficiency syndrome (AIDS). Our objective was to review the latest developments in TCM treatment of AIDS in China between 2004 and 2014. We reviewed the content of original articles investigating the efficacy and safety of TCM for treating AIDS published in Chinese and English language journals. Relevant references from 2004 to 2014 were found using PubMed and the China National Knowledge Infrastructure Database. We found that TCM has been widely used for treating AIDS and its complications in China. The number of TCM studies has increased, which indicates efficacy and safety. Measures of efficacy in the reviewed articles included the alleviation of human immunodeficiency virus (HIV)-related signs and symptoms, improvements in quality of life, improvements in long-term survival, counteraction of the adverse side effects of antiviral drugs, promotion of immune reconstitution, and improvement of laboratory results. In sum, the literature indicates that TCM is safe. TCM plays an important role in the treatment of AIDS. Some studies have attempted to measure the efficacy and safety of TCM for treating AIDS, but more evidence is needed. Therefore, more research on this topic is required in the future.The online version of this article (doi:10.1186/s40249-015-0093-6) contains supplementary material, which is available to authorized users. Please see Additional file Acquired immune deficiency syndrome (AIDS) is a type of communicable disease caused by human immunodeficiency virus (HIV) infection . AIDS waFortunately, methods such as community-based interventions have improved knowledge, attitudes, and practice outcomes for AIDS , 4. MortAt present, cART is the most effective treatment strategy for AIDS, and has the advantage of immunohematologic and virologic outcomes. However, the lifelong medication involved in cART is often limited by drug toxicity, poor adherence to therapy, and drug resistance . It is tCAM has been used worldwide to treat AIDS and its complications, and includes traditional Chinese medicine (TCM), acupuncture and moxibustion, and qigong. Some clinical research has demonstrated that TCM is effective in treating AIDS . Since 2TCM and Western medicine (WM) use different strategies to treat AIDS. WM theory is mainly based on physiological structures or anatomy, and conceives the therapy goal as the elimination or removal of the pathogen. TCM emphasizes the holistic nature of the body and the subjective experiences of patients and practitioners. TCM disorders arise from external pathological factors or an imbalance of yin and yang, and the therapy goals are the harmonization of internal function and external natural environmental changes. Thus, the main goals of WM, especially cART, are to eliminate, diminish, or suppress the viral load levels in the body and to improve the immune system. CD4 + T cell counts and HIV loads are gold standard evaluation criteria for AIDS therapy as recommended by the World Health Organization (WHO). In contrast, TCM places less emphasis on objective laboratory examination results, such as CD4 + T cell counts and levels of HIV loads, and focuses more on AIDS patients’ clinical complaints, such as abnormal symptoms and signs, lower QOL, complications, opportunistic infections, and cART side effects , 13.In this paper, we summarize the findings of studies on the efficacy and safety of TCM for treating HIV/AIDS in the 10 years from 2004 to 2014.The signs and symptoms of AIDS result from conditions that do not normally develop in individuals with healthy immune systems. Patients infected with HIV often complain of many signs and symptoms  , especiaOne of the advantages of TCM is that it can alleviate HIV-related signs and symptoms. At present, there are two main TCM approaches for this. One targets a single symptom and uses a corresponding fixed herbal preparation or strategy to relieve or ease it. The other aims to prevent the occurrence of self-reported symptoms by using fixed-combination herbal preparations combined with syndrome differentiation.Galla chinensis, Myristica eragrans, Cortex dictamni, and garlic. In a randomized, double-blinded, double-dummy, controlled clinical trial ending in 2011 to test Xie Li Kang capsule on HIV-related diarrhea [HIV/AIDS patients often report diarrhea; this has many potential causes, including intestinal parasites. Xie Li Kang capsule is a fixed-combination Chinese herbal hospital preparation with the TCM properties of warming the mid-section, invigorating the spleen, producing an insecticidal action, and relieving diarrhea with astringents. Its main ingredients include diarrhea , the priAstragalus membranaceus, ginseng, Ligusticum chuanxiong, Radix Paeoniae alba, and Scutellaria baicalensis. It has been used to treat HIV/AIDS in Henan Province, China, and more than 4,500 people with the disease take this preparation every day [Yi Ai Kang capsule is a Chinese hospital herbal preparation that strengthens the spleen, dries damp, invigorates vital energy, and nourishes blood. Its main ingredients include very day . One cohvery day . The resAstragali mongolici, Fructus lycii, Panax notoginseng, Poria cocos, and Radix et Rhizoma glycyrrhizae. One randomized, double-blinded, placebo-controlled clinical trial tested Aining granule plus cART on 100 HIV/AIDS cases [Aining granule tonifies qi, nourishes yin, and invigorates the spleen; it consists of Radix DS cases . AlthougClassic clinical TCM treatment based on syndrome differentiation has been used to treat HIV-related signs and symptoms. A multicenter, stratified, randomized, parallel-group trial of 164 subjects evaluated the effects of TCM on clinical symptoms of AIDS with pulmonary inflammation . A randoIn summary, Chinese medicine professionals (CMPs) have used TCM to alleviate HIV-related signs and symptoms, and many observations and trials have been reported. However, the quality of this research is not high; therefore, the effectiveness of TCM for HIV-related signs and symptoms is still uncertain.QOL has been defined as “an individual’s perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns” . QOL is Treatment with Chinese herbal medicine based on TCM syndrome differentiation might improve QOL for people with AIDS. A prospective TCM study into QOL in asymptomatic HIV-infected persons was carried out from June 2009 to December 2010 in China . This waPanax quinquefolius, Dioscorea opposita, Astragalus membranaceus, Poria cocos, Atractylodes macrocephala, Radix Rehmanniae preparata, Angelica sinensis, Colla corii asini, Radix Paeoniae alba, and Ophiopogon japonicus; this capsule tonifies qi and activates blood circulation, strengthens the spleen, tonifies the lung, dries damp, expels wind, and removes dampness. In a randomized, single-blind, parallel, placebo-controlled study on 102 HIV/AIDS patients not taking cART [Fixed-combination Chinese herbal hospital preparations may also improve QOL in people with AIDS. Aikang capsule contains 21 Chinese herbs, including ing cART , the NotTo summarize, QOL has been used as an indicator to assess TCM treatment for HIV/AIDS in China and other countries. However, research to date has been limited and more evidence is needed to confirm TCM’s effectiveness.AIDS is still a fatal disease with limited therapeutic options. Among infectious diseases, HIV/AIDS has become the leading cause of death in many countries. Survival is an important prognostic index and can be used to express the serious lethality of the disease and to evaluate long-term curative effects . RecentlIn a prospective cohort study, 165 of 385 AIDS patients used a 16-herb Chinese medicine anonymity formula for 14 days to 9 months . The 8-yIn a 6-year retrospective cohort study , 1,666 pThe existing research into the effects of TCM treatment on long-term survival of AIDS patients might be biased toward the null without accounting for selective bias. In particular, there are no results from randomized, controlled trials; thus, the effect of TCM on the survival of HIV/AIDS patient is still uncertain.Although cART can reduce the morbidity and mortality of AIDS, it inevitably has adverse effects. The main adverse effects of antiviral drugs are myelosuppression, liver dysfunction, digestive discomfort , anaphylactic reaction, dyslipidemia, and sleep disorders. Patients in developing countries have a limited choice of antivirus strategies. Antiretroviral treatment is lifelong, and therefore demands good treatment adherence. However, some patients cannot endure the treatment’s adverse effects or cannot continue with cART because of other serious diseases. It is therefore very important to find methods to reduce drug toxicity or increase tolerance of antiviral drug toxicity. Research and clinical observations indicate that TCM can alleviate or improve some adverse effects.Inula japonica, Pericarpium citri reticulatae, Pinellia ternata, Atractylodes macrocephala, Semen raphani, Radix codonopsis, and Coptis chinensis. This preparation regulates stomach qi and reduces phlegm, clears heat and dries damp, and eliminates vomiting and warms the stomach. A randomized, placebo-controlled trial investigated the effect of Wendan granule on alleviating gastrointestinal symptoms caused by antiretroviral therapy in 100 patients [The main ingredients of Wendan granule include patients . The incRehmannia glutinosa, Angelica sinensis, Herba Epimedii, Astragalus membranaceus, and Spatholobus suberectus. It tonifies qi, promotes blood circulation, strengthens the spleen, and reinforces the kidney. A randomized, double-blinded trial on 116 AIDS patients found that 6 weeks of systematic administration of Jing yuan kang capsule had a good therapeutic effect on peripheral leucopenia in the treatment group compared with the control group [Jing yuan kang capsule is an herbal formulation composed of six Chinese herbs; the main ingredients are ginseng, ol group .Angelica sinensis, Radix Paeoniae alba, Atractylodes macrocephala, Poria cocos, Alisma orientale, Radix Curcumae, and Hedyotis diffusa. In one cohort study, a decoction based on Danggui Shaoyao San was used to treat cART-induced liver dysfunction in 48 patients [Danggui Shaoyao San is a famous Chinese herbal formula that nourishes blood, regulates the liver, nourishes the spleen, disinhibits dampness, and nourishes qi. Its main ingredients are patients . The resAlthough there is some evidence that TCM can mitigate the adverse effects of cART, research in this field is still preliminary.HIV infection usually results in immunodeficiency. Immune reconstitution (IR) is observed when the immune response recovers to normal or near normal levels after cART or other treatments. This reduces the incidence of various AIDS-related opportunistic infections and tumors and reduces mortality and morbidity in AIDS patients. IR evaluation indexes include the number and functional recovery of CD4 + T cells, the stability of the immune activation state, and the restoration of lymph node structure. In addition to cART, adoptive immune cell therapy, bone marrow transplantation, and cytokine infusion can benefit IR . HoweverThe holistic framework of TCM, its use of syndrome differentiation, and rich lore of herbal medicine treatment, mean that it is uniquely placed to promote IR. According to TCM theory, the essence of clinical manifestation is a deficiency of vital qi at the middle-late stage of AIDS. Therefore, strengthening qi is a fundamental TCM treatment therapy. Some research has demonstrated the important role of TCM in IR.Panacis quinquefolii, Astragalus membranaceus, and Viola yedoensis. It tonifies qi, nourishes yin, and invigorates primordial qi. In a randomized, double-blinded, placebo-controlled clinical trial, Immune No.2 was used to promote IR in 233 patients with HIV/AIDS after cART [Immune No.2 is a Chinese hospital herbal preparation that includes Radix ter cART . The stuThe two main goals of AIDS treatment are to eliminate viral loads and to raise CD4 + T cell counts. These two indexes have also been adopted in TCM treatment of AIDS, and a few studies have demonstrated that TCM is very promising in reducing HIV viral loads, increasing CD4 + T cell counts, or affecting other immunological indicators. Compared with the index of HIV viral loads, the CD4 + T cell count is currently the more popular measure of efficacy in TCM clinical trials for AIDS treatment.Fructus lycii, Radix Trichosanthis, Viola yedoensis, and Salvia miltiorrhiza. It supplements qi, nourishes yin, clears heat, and detoxifies. In a randomized, double-blinded, parallel, placebo-controlled clinical trial, Zhongyan-4 was tested on 72 HIV/AIDS patients in the early or middle stage of the disease [Zhongyan-4 is a Chinese hospital herbal preparation that includes ginseng,  disease . The finAstragalus membranaceus, ginseng, roasted Rhizoma Atractylodis macrocephalae, Poria cocos, Pericarpium Citri reticulatae, Fructus Amomi, Semen coicis, Radix Morindae officinalis, Herba Cistanche deserticola, Herba Epimedii, Forsythia suspensa, Angelica sinensis, and Radix et Rhizoma glycyrrhizae. It tonifies qi, nourishes yin, invigorates the spleen, clears heat, and detoxifies. A 6-month cohort study of 34 outpatients with HIV/AIDS tested the granule’s effects on regulation of immune activation molecules CD38 and human leukocyte antigen-D related to CD4 + T and CD8 + T cells [Fuzheng Paidu granule consists of  T cells . The resThe combined use of herbal medicines and chemical medicines is common in clinical practice, since this kind of combination can not only improve treatment efficacy but also help to reduce drug dosages and adverse reactions. Herbal medicines and their effective ingredients probably induce or inhibit the action of drug metabolic enzymes and cause drug interactions. Therefore, the interaction between antiviral medicines and herbal medicines is an issue of concern.Research has demonstrated that some herbal medicines may interact with antiviral drugs , which cAlthough the discovery of cART has changed the prognosis of HIV-infected patients, and those treated with cART have a life expectancy similar to that of uninfected subjects, TCM has been used worldwide to treat AIDS and its complications.This review revealed some new aspects of TCM clinical research on AIDS: use of the distinctive features of TCM to meet clinical demands, a focus on clinical signs and symptoms, and the use of different research designs that are methodologically robust. Research has also examined the pathogenesis, mechanism, efficacy, and safety of TCM. We believe that additional research will help to reveal the importance of TCM for treatment of AIDS.


While patients with rapid virologicalresponse (RVR) do not require triple therapy, patients with a decline<1logTo develop a tool to predict first 4 weeks’ viral response in patientswith hepatitis C genotype 1&4 treated with P+R.In this prospective and multicenter study, HCV mono-infected (n=538) andHCV/HIV co-infected (n=186) patients were included. To develop and validatea prognostic tool to detect RVR and D1L, we segregated the patients as anestimation cohort (to construct the model) and a validation cohort .D1L was reached in 509 (80.2%) and RVR in 148 (22.5%) patients. Multivariateanalyses demonstrated that HIV co-infection, Forns’ index, LVL,IL28B-CC and Genotype-1 were independently related to RVR as well as D1L.Diagnostic accuracy (AUROC) for D1L was: 0.81 (95%CI: 0.76 — 0.86) inthe estimation cohort and 0.71 (95%CI: 0.62 — 0.79) in the validationcohort; RVR prediction: AUROC 0.83 (95%CI: 0.78 — 0.88) in theestimation cohort and 0.82 (95%CI: 0.76 — 0.88) in the validationcohort. Cost-analysis of standard 48-week treatment indicated a saving of30.3% if the prognostic tool is implemented.The combination of genetic (IL28B polymorphism) and viral genotype togetherwith viral load, HIV co-infection and fibrosis stage defined a tool able topredict RVR and D1L at week 4. Using this tool would be a cost-savingstrategy compared to universal triple therapy for hepatitis C. RVR was higher in HCV genotype 4 than in HCV genotype 1 . Thus, HCVgenotype 4 reached RVR in 45.2% (33/73) vs. 23.6% (98/416) inHCV genotype 1 (p < 0.001) in mono-infected patients (vs. 28.8% (38/132); p < 0.05)].Conversely, cirrhotic patients had less RVR than non-cirrhotic patients . FIB-4, APRI and Forns’ index were significantly decreased (p< 0.001) in patients with RVR.RVR was achieved in 22.5% (148/659) of the overall cohort. Age was lower inpatients with RVR  and the cut-off 0.248 showed asensitivity of 76%, specificity of 75%, positive predictive value of 47% andnegative predictive value of 91%. This model was confirmed in the validationcohort, except for the HCV genotype., with an AUROC of 0.82  ; p < 0.001], HIV co-infection , IL28B-CC , Genotype 1 , Forns’ Index  . The AUR< 0.001)  and a cuThe final formula derived was: RVR. The AUROC for RRVR was 0.79  in the estimation cohort and was 0.77  in the validation cohort.Additionally, we obtained the AUROC in mono-infected HCV genotype 1 patients forRThe final formula derived was: vs. 83.7% (391/467); p < 0.001]. IL28B-CC had 93.8%(211/225) D1L, while IL28B-CT/TT had 72.4% (284/392) D1L (p < 0.001). D1Lwas higher in HCV genotype 1 than in HCV genotype 4 . FIB-4, APRI andForns’ index were significantly lower (p < 0.001) in patients withD1L. Significant fibrosis was related to less D1L .D1L was reached in 80.2% (509/635) of the overall cohort. Co-infected patientsshowed a lower prevalence of D1L than mono-infected patients , HIV co-infection , IL28B-CC , Genotype 1 ,Forns’ Index . The AURThe final formula was: SVR for RVR was 76.5% (101/132) and was 67% (146/218) for predicted RVR (p< 0.05). On the other hand, SVR for D1L was 51.7% (238/460) and was 54.3%(215/396) for predicted D1L (p > 0.05).Total cost for hepatitis C therapy per patient was estimated as €35,233for the 48 weeks. The implementation of the proposed prognostic tool wasassociated with €10,665 saving/patient, in the base case scenario.The total savings per patient in alternative scenarios ranged from €12,298to €9,627 . AssuminThe combination of IL28B genotype with viral load, HCV-genotype, Forns’ Index(a non-invasive marker of fibrosis) and the presence of HIV co-infection enabled usto construct a tool able to predict virological response at week 4 in patientstreated with peginterferon alfa-2a+ribavirin.In the course of boceprevir research & development for the treatment ofpatients with chronic hepatitis C genotype 1, a lead-in phase over 4 weeks wasincluded in the design of the majority of clinical trials. Theoretically, thisapproach was to improve sustained virological response rate  and lowehttp://www.optimtool.com/formula.html) for decision making regardinghepatitis C treatment and could confidently predict achieving D1L and RVR inpatients with hepatitis C genotypes 1&4. This is possible irrespective ofco-infection by HIV, or measurement of viral load, or the non-invasive estimation offibrosis using the Forns’ Index. Although our study was designed to developthis tool in treatment-naïve patients, a similar approach could be exploredin patients with previous treatment failure. Indeed, the Realize study demonstratedthat, in the lead-in phase arm, SVR rate was lower in previously-treatednon-responders without D1L. This supports the concept that, in these patients withfunctional mono-therapy, triple therapy should not be initiated since it is doomedto be ineffective or superfluous. The implementation of our predictive tool of viralresponse at week 4 could be useful in the assessment of those potential candidatesfor antiviral treatment  we did not considered eitherresponse-guided therapy or futility rules of triple therapy in our model. This wasbecause of lack of individual data regarding the relevance of D1L in short treatmentregimens in the pivotal studies of boceprevir- and telaprevir-based treatments,26. ThisIn conclusion, the combination of genetic (IL28B polymorphism) and viral genotypetogether with viral load, HIV co-infection and fibrosis stage defined a tool topredict RVR and D1L at week 4. The implementation of this tool in clinical practicecould be a cost-saving strategy, compared to the universal triple therapy forhepatitis C. As such, it could contribute to a more efficient allocation of limitedresources.