The paper presents an analysis of the possibilities of using glass waste from recycled lighting materials as aggregates for cement concrete. The research material was obtained from a company that utilizes electrical waste. Glass from pre-sorted elements was transported to the laboratory and crushed in a drum crusher. In this way, the aggregate obtained was subjected to the basic tests that are carried out for aggregates traditionally used in construction. The specific density of aggregate, bulk density, absorbability, crushing index, grain shape, texture type and aggregate flatness index were examined. In the next stage of research work, concrete mixtures were made in which crushed aggregate from crushed fluorescent lamps was used as a substitute for gravel aggregate. Mixtures containing 10%, 30%, 50% and 100% aggregate were made. A mixture containing only sand and gravel aggregate was made as a comparative mixture. Basic tests of both fresh concrete mix and hardened concrete were carried out for all concrete made. The consistency of the fresh concrete mix, the air content in the concrete mix, the density of hardened concrete, absorbability, water permeability under pressure and the basic compressive and tensile (flexular) strength tests were performed. The test results showed that the greater the addition of recycled glass aggregate, the less advantageous are the features of the concrete obtained with its participation. Microscopic analyses carried out in order to explain this phenomenon indicated an unfavorable influence of the grain shape of the aggregate thus obtained. Despite this fact, recycling of lighting waste in concrete composites is recommended as a pro-ecology measure; however, attention was paid to the benefits of using only 30% by mass of said waste in relation to the weight of the traditional aggregate used. Composite with such a quantity of waste retained the characteristics of cement concrete, which qualified its use for construction concrete. One of the most important issues of current science is the problem of rational waste management. Despite the fact that the recycled use of many exploited products is possible from a technical point of view, many methods and proposed solutions encounter economic limitations. In cases where raw materials for the production of new products are easily available and their acquisition is not expensive, recycling from the point of view of enterprise is unnecessary. If the recycled materials cannot be financially justified, they are not used. In many cases, manufacturing companies reserve the possibility of using worn-out products in new production provided that they are delivered for free. This is a sign of willingness to implement economically justified recycling. In this case, the financing of collection, segregation and transport becomes such an expensive undertaking that ultimately various types of waste, the recycling of which is technically possible, end up in illegal garbage dumps. An example of the situation described above is the recycling of selected glass waste. In the case of packaging glass, activities such as collection, selection, cleaning and transport of waste to glass-works are economically justified. Due to the large scale of packaging turnover and the possibility of obtaining payment for recyclable material, technical possibilities are appropriately used here. The situation of glass waste is different, the quantities of which are small and the adjustment processes of which are complicated. Products of this type include glass lighting materials such as light bulbs and fluorescent lamps. A fluorescent lamp is a discharge lamp in which light is emitted by a phosphor excited by ultraviolet radiation. It most often has the shape of a pipe, covered on the inside by the mentioned phosphor filled with mercury vapor and argon, in which the source of illumination is visible radiation emitted by the phosphor layer covering the inner surface of the tube. Due to the presence of mercury vapor, the utilization of lighting elements is carried out in a way that requires many treatments in which harmful substances are neutralized. The process of utilizing fluorescent lamps is carried out under negative pressure so that mercury vapors do not get into the atmosphere. First, the used fluorescent lamps are put in the feeder, which is tightly closed, and then its contents go to the chamber, in which the contents of the feeder are heated above 200 °C. As a result of heating, evaporation of mercury occurs, while glass and aluminum tips remain in the chamber on the bottom. After filling the chamber with glass and aluminum, it is emptied, and its contents are directed to the separator. Floating mercury vapor is directed to the next chamber, which has a temperature of −75 °C, which causes mercury to pass from its gaseous to liquid state. Separation of mercury leads to it being able to be reused. This process ensures that no mercury vapor remains in the glass material that could cause health problems. However, the glass is not yet devoid of steel elements. It goes into a separator in which parts other than steel are separated after fragmentation. Phosphor is also a problematic substance that is covered with glass elements. Its removal in the process of recovery of pure glass material is also a very costly procedure.As it can be seen in the above description the recovery of glass material is difficult and expensive, and the transport of small amounts to glass-works becomes completely economically unjustified. In such cases, it is necessary to look for other more local methods of recycling waste material. The solution proposed here is to include recycled glass in a different industry, such as the production of concrete. The possibility of including this waste in the local production of concretes would have mainly ecological benefits—the amount of matter in landfills would be reduced as well as the use of natural aggregates.The use of glass as an additive in cementitious composites is an issue analyzed by many research teams, e.g., ,2. BasedAnother proposed solution is the introduction of glass into concretes in the form of dust ,23,24,25A relatively new idea is the introduction of glass waste into cement mixtures as a substitute for coarse aggregates. Research carried out both on glass ,31,32,33Despite the fact that the recycling of glass waste in concrete composites is analyzed by many research teams, analyses carried out on recyclate from depleted fluorescent lamps in cement composites were not encountered. The only work devoted to this waste is , where tThe research material was obtained from a company dealing in the utilization of electric waste. Exploited bulbs and fluorescent lamps were deprived of harmful substances and equipment there, which was damaged during the process. The material ultimately collected for testing was mainly broken glass tubes. The pipes were transferred to a plastic container and transported to the laboratory. Crushed glass was made using a drum crusher. The crushing of the material was carried out until all aggregate grains were less than 8 mm. The research material is presented in The aggregate obtained in this way was subjected to an analysis of basic properties ,42,43,44To determine the specific density of aggregates, the standard method according to PN-EN1097-7: 2011  was usedThe properties of materials such as glass and gravel, presented in Gravel aggregate graining resulted in a tight arrangement of smaller sized grains between larger sized grains. This guaranteed the tightness of the crumb stack, which when bonded with leaven carried the load. In the case of glass aggregates, due to difficulties in their mutual arrangement between each other, the tightness of the crumb stack could not be maintained. Probably this fact was also the cause of the occurrence of air pores and lower strength parameters of composites with recyclate.The comparative method  was usedThe assessment of the type of grain texture of aggregates from waste glass materials was made on the basis of . The strAs a plasticizing admixture, CHRYSO Omega 132 was used. The admixture is manufactured using the latest hybrid polymer technology. This technology uses knowledge about the synthesis of molecules, and it enables a strong reduction of the amount of mixing water, long-term maintenance of the consistency of the concrete mix, and the homogeneity and cohesion of the concrete mix. The basic technical parameters of the admixture from its technological sheet are presented in 2 and Al2O3). The technical data sheet of the product ensured that it complies with the requirements of the EN 450-1 standard as class A in terms of roasting loss and N category in the fineness range and has pozzolanic properties. The basic characteristics of fly ash from its technical sheet are given in Fly ash was used as a concrete additive. It was a fine-grained dust consisting mainly of spherical, grained grains, obtained during the combustion of bituminous coal with the participation of co-combustion materials. The basic components of the ash were aluminosilicates (SiOAll components used in the study (including fly ash) are popularly used in Poland in the industrial production of concrete. CONTR concrete base mix presented in The theoretical and experimental method was used to determine the prescription of the concrete mix ,46. Due The design of the concrete mixture began with determining the qualitative components and determining their basic properties. Based on the technology sheets of the supplied substrates and own tests, the volume densities of the concrete constituents, natural aggregate moisture, aggregate sand points and a sand point of 54% were determined. On the basis of general formulas , the proAll prepared concrete mixtures were subjected to consistency tests according to EN 12350-2—Concrete mix tests, Part 2—Slump test . During All types of concrete were made on the basis of the same working recipe, replacing only the mixture of sand and gravel aggregates with glass aggregate. The dosage of water, chemical admixture, and additives were implemented in the same amounts. The observed precipitation results of the cone were different for different mixtures, the overall liquidity of the mixture increasing with the amount of glass additive. It is assumed that the glass, as a non-absorbent material, did not absorb water, which caused it to become plastic. This feature testified to the positive effect of the glass material on the concrete. Achieving the assumed consistency when dosing a smaller amount of mixing water positively affects the W/C index and improves all concrete properties. During the study, attention was also paid to the increasing variability index along with the addition of glass. This phenomenon proved that the addition of glass waste meant that the mixture was less homogeneous, and this feature is unfavorable from the point of view of concrete production.Another test that was carried out for all fresh concrete mixtures was the air content test  using thAnalysis of the results of an air content test in a concrete mix proved that with the same components, the air content in the mixture increased significantly. This was an unexpected phenomenon because the quantities of all components were the same. Also, aeration agents were not added to the mixture preparation. Two hypotheses were put forward in order to explain this phenomenon. Firstly, assuming that the glass is a neutral material that does not react chemically with the components of the concrete, it was assumed to react with the components of the concrete that the phosphor layer could be coated with fluorescent tubes. As a result of the chemical reaction, a gas could appear that caused differences in the results of the air content test. The second hypothesis was that air gaps were formed between the irregular shaped particles, which could not leave the mixture during the test. The explanation and confirmation of hypotheses was planned to be carried out during microscopic examination.The next study was a study of the volume density of concrete samples. The volume density was tested on 10 × 10 × 10 cm cubic samples. Six samples of each concrete were prepared. The test was carried out in accordance with EN 12390-7 . The res3, CG10–2290 kg/m3, CG30-2180 kg/m3, CG50-1960 kg/m3, and CG100-1660 kg/m3. The results of this study show that this property was different for all concrete types. The density of concrete was lower, the more glass aggregate was used. The lower density of the crushed aggregate itself could have an impact on this feature. Probably, however, the differences in density were largely caused by the increasing air content in the mixture.The mean bulk densities measured for the concrete tested were, respectively, CONTR-2300 kg/mWater absorption was tested on identical samples as volumetric density. Six samples of each concrete were also tested. The samples were immersed in water and they remained there until their mass stabilized. The absorbability was calculated according to  as the rThe water absorption varied, and for the mixtures CONR, CG10, CG30 CG50 and CG100 was 3.52%, 3.73%, 4.73%, 5.89% and 8.93%, respectively. This proved, therefore, that with the increasing amount of glass aggregate, the absorption of concrete increased, which probably also caused the occurrence of air voids in the concrete volume. Analyzing the results of this study, attention was also paid to the volatility index increasing along with the content of glass waste. This was another proof that the samples with the increasing amount of waste are more heterogeneous.Another test was the tensile (flexular) strength test of hardened concrete. As the largest dimension of the aggregate used to make the concrete, the 8 mm dimension for the strength tests was prepared in the form of bars measuring 4 × 4 × 16 cm. After forming, the samples were subjected to moisturizing care in a closed container partially filled with water. After the third day after the molding, the samples were removed from the molds and then they were still in the described environment for the following days. The tests were carried out in accordance with EN 12390-3 . For thiAnalysis of the test results has shown that samples with a higher content of glass waste had lower tensile strength. Comparing the tested values for CONTR concrete strength, the decrease in strength was found for concretes: CG-10-17%, CG-30-10.5%, CG-50-45.2% and CG-100-53.8%. In the obtained results, similarly to in the case of previous studies, attention was drawn to the very high coefficient of variation obtained especially for samples with the addition of 100% glass aggregate. Interesting was also the increase in tensile strength for a composite containing 30% recyclate. With reference to , it can Another test was the test of crushing strength of hardened concrete. The halves of the samples obtained during the tensile (flexular) strength test were applied. The test was carried out on the same testing machine as the previous one. The results are presented in The most favorable compressive strength test results were observed for CONTR concrete, i.e., without glass content in its composition. The worst for the series were observed when the glass content of fluorescent lamps in the aggregate was 100%. Analyzing the results again, it was found that the addition of glass negatively affects the characteristics of concrete, and it is a linear relationship, i.e., that the higher the glass content from exemplary glass in the aggregate, the lower the compressive strength. It should be emphasized that the difference in extreme values was as much as 10-fold. The negative impact of glass from fluorescent lamps on the final compressive strength of concrete is visible. Referring to the concrete strengths of subsequent series, i.e., CG-10, CG-30, CG-50 and CG-100, CONTR concrete strength was estimated to mean that the percentage decrease in strength was 13.5%, 32.5%, 69.3%, and 90.8%, respectively. During the tests, some anomalies were also noted. Samples with the glass content were more damaged by peeling than cracking. A sample of CG-100 concrete during the compressive strength test is shown in The last stage of the conducted research work was microscopic analysis, carried out using an optical microscope. These analyses were conducted in order to clarify the phenomenon of lowering the quality of concretes due to the addition of lighting glass. The glass material itself, tested by methods such as traditional cracks, showed advantageous features as aggregate. Microscopic analyses were planned to confirm one of the hypotheses regarding the increasing amount of air in concrete resulting from the addition of glassware. The first stage of microscopic analysis was the observation of phosphor-coated glass surfaces. Fragments of concrete samples after destruction became detached during the test. Microscopic analysis showed that the luminous phosphor remained on the surface of the glass elements. This was proof that there was no chemical reaction with the cement components. Subsequent microscopic analyses were carried out on the cracked edges of the samples that were formed after testing the tensile strength (flexular) of concrete. Exemplary samples are shown in In This fact explains both the unfavorable parameters of the tested concretes (compressive strength of the glass itself and its low water absorption) and the linear relationship between deterioration of properties and the content of glass aggregate. As this phenomenon cannot be controlled and depends mainly on the way in which the grains are arranged in a mixture of concretes with a high content of glass aggregates, they have a large heterogeneity. This was confirmed by high variability indices of the examined properties.Despite the obtained results confirming the general adverse effect of ceramic glass aggregates on the performance parameters of cement composites, the proposed solution for implementation in industrial activities is recommended. Special attention is paid here to the fact that substituting 30% of aggregate for recyclable aggregate makes it possible to obtain a concrete composite with high technical parameters. Such a composite has a compressive strength of 31.7 MPa and a tensile (flexular) strength of 7.43 MPa, as well as other positive properties that qualify it for the constructional composite. This action is recommended especially for pro ecological reasons. Thanks to such solutions, local glass waste can be used for traditional concrete production, and this has a doubly positive environmental effect: unwanted material is utilized, and at the same time, the consumption of natural aggregates is reduced.Among the specific applications, it should be particularly emphasized that:1. It is possible to obtain glass material in the form of crushed glass grains from which the concrete aggregate can be made from worn-out lighting materials.2. Aggregate formed from the crushing of glass waste examined by methods for traditional aggregates has favorable parameters—no worse than those tested for traditional aggregates such as sand and gravel.3. The concretes made with the use of glass aggregate have less advantageous characteristics with greater amounts of glass aggregate being used in their production. The reason for the deterioration of the features is the flat shape of the grains of the recycled aggregate derived from lighting waste. Flat grains cause air bubbles during vibrations to accumulate under them, creating voids, and the high content of air voids formed under the grains of the flat aggregate are the cause of the deterioration of concrete features and their high heterogeneity.4. A composite containing 30% glass aggregate has technical features that qualify it as a construction material. The use of glass recyclates for concrete composites in the amount of 30% of aggregate mass gives a satisfactory effect and is in line with environmental trends. Higher percentages are generally less recommended, as the occurrence of air voids will unavoidably grow. To some extent, this can be overcome by increasing the fluidity of the mix, extending the vibration time for the automatic arrangement of close grains, or using less liquid mix and introducing vibro pressing.5. This research raised some safety concerns. The aggregate used in the tests has numerous sharp edges and may cause injury when in contact with human skin. This fact emphasizes how difficult this waste is in respect to its utilization and disposal. Technological pollution by phosphor does not allow direct use of waste in the production of new glass. Using it in crushed form, e.g., as aggregate for hardening embankments, similarly to processes that use concrete or ceramic destruct, is also not possible. Tests of this kind caused injury to car tires. On the other hand, waste disposal by landfill is unfavorable from an environmental point of view. It is a devastation of the material and energy that were used in the production process of products. All the above aspects prompted the authors of the article to attempt to use this material for the production of composites in which it would be a substitute for natural aggregates. One of the authors of the article has extensive engineering experience in the production of concrete composites and their use in industrial conditions. These experiences show that both machine operators at concrete mixing plants in the production of concrete and employees laying the composite in formwork  do not have direct contact with the cement composite. In these processes, employees’ skin is not allowed to come into contact with the concrete mix—also due to the adverse effects of the cement paste itself on human skin. In specific cases, e.g., in the event of a breakdown or experimental work, special precautions should be taken, and the possibility of injury should be taken into account. Also, as described in the introduction, the initial treatment of glass must ensure that no mercury vapor remains in the glass material that could cause health problems.


To explore the clinical patterns of patients with IgG4-related disease (IgG4-RD) based on laboratory tests and the number of organs involved.Twenty-two baseline variables were obtained from 154 patients with IgG4-RD. Based on principal component analysis (PCA), patients with IgG4-RD were classified into different subgroups using cluster analysis. Additionally, IgG4-RD composite score (IgG4-RD CS) as a comprehensive score was calculated for each patient by principal component evaluation. Multiple linear regression was used to establish the “IgG4-RD CS” prediction model for the comprehensive assessment of IgG4-RD. To evaluate the value of the IgG4-RD CS in the assessment of disease severity, patients in different IgG4-RD CS groups and in different IgG4-RD responder index (RI) groups were compared.PCA indicated that the 22 baseline variables of IgG4-RD patients mainly consisted of inflammation, high serum IgG4, multi-organ involvement, and allergy-related phenotypes. Cluster analysis classified patients into three groups: cluster 1, inflammation and immunoglobulin-dominant group; cluster 2, internal organs-dominant group; and cluster 3, inflammation and immunoglobulin-low with superficial organs-dominant group. Moreover, there were significant differences in serum and clinical characteristics among subgroups based on the CS and RI scores. IgG4-RD CS had a similar ability to assess disease severity as RI. The “IgG4-RD CS” prediction model was established using four independent variables including lymphocyte count, eosinophil count, IgG levels, and the total number of involved organs.Our study indicated that newly diagnosed IgG4-RD patients could be divided into three subgroups. We also showed that the IgG4-RD CS had the potential to be complementary to the RI score, which can help assess disease severity. IgG4-related disease (IgG4-RD) is a multi-organ immune-mediated condition characterized by tumefactive lesions, a dense lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells, storiform fibrosis, and, often but not always, elevated serum IgG4 concentrations –3. The cAn increasing number of studies on the outcomes and prediction of disease relapse in IgG4-RD showed that high baseline serum IgG4, IgE, and eosinophilia could predict IgG4-RD relapse independently ; eosinopTo our knowledge, no classification of IgG4-RD based on diversified routine blood tests has been proposed. In this study, we aimed to explore the baseline clinical patterns of IgG4-RD based on 19 different types of blood tests and the number of organs involved. We also tried to establish a prediction model for a comprehensive score , which was expected to assess disease severity and partly predict outcomes in IgG4-RD patients.ClinicalTrials.gov (NCT01670695). All the patients fulfilled the 2011 comprehensive diagnostic criteria for definite, probable, or possible IgG4-RD [This study was approved by the Ethics Committee of Peking Union Medical College Hospital (PUMCH). All the patients provided written and informed consent. The prospective cohort of patients with IgG4-RD in the PUMCH  was regiAge at onset refers to the age at which the patient first noticed the symptoms attributed to IgG4-RD or to the timepoint at which the disease was first recognized (whichever was earlier) . DiseaseAll obtainable baseline data was recorded before initial treatment. Laboratory tests consisted of complete blood count, urinalysis, liver, and renal function tests, erythrocyte sedimentation rate (ESR), hyper-sensitivity C-reactive protein (hsCRP), complement 3 (C3), C4, serum immunoglobulin (Ig) levels, and total IgE and IgG subclasses. To facilitate application, 19 variables from the laboratory tests involved in this study were the most common routine tests in IgG4-RD clinical practice, including white blood count, lymphocyte count, lymphocyte percentage, eosinophil count, eosinophil percentage, platelet count (Plt), hemoglobin (Hb), ESR, hsCRP, C3, C4, IgG, IgA, IgM, IgE, IgG1, IgG2, IgG3, and IgG4.Disease response after initial treatment was assessed after 3 months. According to the IgG4-RD RI, disease response was defined as an improvement of the IgG4-RD RI ≥ 2 compared to the baseline , and comThe initial 6-month period was defined as the remission induction stage, and the therapeutic goals of this stage were defined as fulfilling each of the following: (1) ≥ 50% decline in the IgG4-RD RI, (2) GC tapered to maintenance dose (prednisone ≤ 10 mg/day), and (3) no relapse during GC tapering (within 6 months) .Relapse was defined by a review of the new progress or recurrence of symptoms and signs and the new development or return of abnormal findings on physical examination, laboratory tests that reflected IgG4-RD activity within specific organs, or imaging studies , 15, 16.P value < 0.05 was considered significant for all statistical testing.All statistical tests were performed using IBM SPSS (version 24). The continuous normally distributed data is presented as mean ± standard deviation, non-normally distributed data is presented as median , and categorical data is presented as a percentage (%). Comparisons of continuous data such as levels of IgG4 among 3 groups were tested using the one-way ANOVA or the Kruskal-Wallis test. Multiple comparisons were performed using the Student-Newman-Keuls test. Categorical variables were assessed using the chi-square or Fisher’s exact tests. We also used the chi-square tests to compare the proportion of patients with disease relapse among the different subgroups. We calculated the correlation coefficients among the blood test results such as correlations between IgE and eosinophil count using the Pearson correlation coefficient  or the Spearman correlation coefficient (nonparametric data). A two-sided λ > 1). According to Component Matrix and eigenvalue, PC scores  were also calculated in each patient as new variables  for further cluster analysis. Cluster analysis was performed using a hierarchical and agglomerative clustering algorithm with the Ward method [For easy exploration and visualization of 22 baseline variables, which contained 19 variables from laboratory tests and the number of total, internal, and superficial organs involved, and for estimation of the correlation between variables, we used PCA to statistically aggregate these variables, reducing the number of observed variables into a smaller number of principal components (PCs) and reducing the dimensionality of baseline phenotyping data. According to eigenvalues and cumulative contribution rate, we selected the appropriate number of PCs  with high eigenvalues  as weight coefficient for each patient (IgG4-RD SC = C1F1 + C2F2 + ... + CiFi), which enabled us to group patients according to their IgG4-RD CS grades by separating the IgG4-RD CS range into trisection averages. Finally, a mathematical model for the “IgG4-RD CS” was established using multivariate linear regression.Calculation of IgG4-RD CS involved two steps. First, PCA was applied to 22 variables to derive PCs representing large fractions of variance. Second, IgG4-RD CS was computed by summing individual PC scores  with the contribution rate. The average age at onset was 53.35 years . The median duration of disease was 11.5 months (range 10 days to 20 years). There were 68 (44.16%) patients with a history of allergies . There were 87 (56.49%), 3 (1.95%), and 64 (41.56%) cases, which were diagnosed as definite, probable, and possible IgG4-RD, respectively.Of the 154 patients, the median number of the total organs involved was 3 (range 1–8). About 30% (46/154) had only superficial organ involvement, and 31% (48/154) had only internal organ involvement. The median IgG4-RD RI was 11.5 (range 3–28). The detailed demographic and clinical characteristics are shown in Additional file r = 0.604, P < 0.001), IgE , and IgG3  and negative correlations with IgA , IgM , C3 , and C4 .With regard to the correlations between serological variables, we mainly focused on IgG4, IgG, IgE, ESR, and CRP. Serum IgG4 concentration showed positive correlations with serum IgG concentration , ESR , CRP , as well as Plt  and negatively with C3 , C4 , and Hb .Serum IgG concentration correlated positively with IgE , as well as eosinophil proportion .As for serum IgE levels, positive correlations were found between IgE and eosinophil count , IgG , IgE , IgG3 , and eosinophil count  as well as eosinophil proportion  and negative correlations with IgA , C3 , and C4 .We also investigated the correlations between RI and serological variables. RI scores showed positive correlations with serum IgG4  were extracted from 22 baseline variables by PCA, and the cumulative score was up to 73.34% . IgA and CRP were statistically close and showed positive correlation . The results of the PCA indicated that the baseline phenotypes of IgG4-RD basically consisted of the inflammation-related axis, disease severity-related axis, and allergy-related axis abnormalities.Seven PCs  . Cluster 1 had the highest percentage of patients with low-level complement (45%), while most patients in cluster 2 and cluster 3 had normal-level complement .To further clarify the situation of complement in clusters, we divided the levels of C3 and C4 into 3 grades respectively: low, normal, and high-levels  as the comprehensive reflection of 22 baseline variables. All 154 patients had a median CS of − 0.1149 (range − 1.223 to 1.996).n = 51, n = 90, n = 13) accordingly. Then, we compared 22 baseline variables among CS1–3. As expected, we found statistical differences in RI, the number of internal organs involved, the number of total organs involved, lymphocyte count, eosinophil count, ESR, Hb, IgG, IgG1, IgG3, IgG4, C4, and other variables among CS1–3  and divided 154 patients into 3 groups . We explored the treatment response, remission, and disease relapse. There were 5 patients with no change to treatment, 51 patients with partial responses, and 64 patients with complete responses. Twelve patients (13.3%) failed to achieve remission among those with GC therapy. There were 31 patients (20.1%) who had experienced relapse or were in recurrent states.n = 71, n = 74, n = 9, respectively) again according to the RI score by dividing the range into trisection by average  and explored the differences in the treatment regimens among different subgroups using chi-square tests , and both CS1 and RI1 had the highest proportion of patients without GC treatment . Interestingly, we also found IgG4-RD CS had superior ability to guide treatment in female patients. In male patients, IgG4-RD CS and IgG4-RD RI shared the similar trends corresponding to clusters; cluster 1 and cluster 2 showed higher disease severity scores (both RI and IgG4-RD CS), which was consistent with higher probability of receiving active treatments (GC or GC+IM) , and subgroup CS1 had the highest proportion of patients with CR as well as subgroup RI1  + 0.247*lymphocyte count (109/L) + 0.135*total organs involved number − 1.852”. The 95% confidence interval (95% CI) of these four independent variables and constants were shown as follows: IgG , eosinophil count , lymphocyte count , total number of organs involved , and constant . The model had excellent adjusted R square  and Durbin-Watson value (DW = 1.842). The plot of residuals also indicated that the model was a statistical success  + 0.385*eosinophil count .Additional file 2. Eigenvalue and variance contribution.Additional file 3. Correlation coefficient of each phenotype after extracting 3 PCs.Additional file 4. Other differences among Clusters. a, Distribution of the grades of complement in clusters. Normal values of C3 and C4 were 0.73-1.46 and 0.1-0.4(g/L), respectively. b, Distribution of the grades of IgG4-RD CS in clusters. . c, IgG4-RD CS was compared between clusters. *, P value <0.05; ***, P value <0.001.Additional file 5. Three clusters of patients identified by cluster analysis in male and female IgG4-RD patients. cluster1, inflammation and immunoglobulin-dominant group; cluster2, internal organs-dominant group; cluster3, superficial organs-dominant group.Additional file 6. Comparisons of baseline characteristics among three clusters of male IgG4-RD patients. *, P value <0.05; **, P value <0.01; ***, P value <0.001.Additional file 7. Comparisons of baseline characteristics among three clusters of female IgG4-RD patients. Inter.ratio, proportion of internal organs to total organs involved. *, P value <0.05; **, P value <0.01; ***, P value <0.001.Additional file 8. Disease severity scores (mean ± SD) and percentages of different treatments in male and female IgG-RD patients. a, IgG4-RD CS and IgG4-RD RI in male patients; b, IgG4-RD CS and IgG4-RD RI (mean±SD) in female patients; c, Percentages of different treatments in male patients; d, Percentages of different treatments in female patients.Additional file 9. Comparisons of baseline characteristics among CS1-3 grouped by IgG4-RD composite score. *, P value <0.05; **, P value <0.01; ***, P value <0.001.Additional file 10. Baseline characteristics of patients with IgG4-RD grouped by IgG4-RD CS.Additional file 11. Comparisons of remission induction and disease relapse among subgroups. a-b, the remission induction and disease relapse among different subgroups were shown in pie charts.Additional file 12. Comparisons of relapse rate among subgroups. a-c, No difference in cumulative relapse rate by Kaplan-Meier curves. d-i, Real-time ratio of non-relapse patients in follow-up patients. The horizontal axis showed follow-up time (month), the left vertical axis showed the number of patients, the right vertical axis showed the real-time ratio of non-relapse patients in follow-up patients. Black dot, the real-time number of patients in following up; Red dot, the real-time number of relapse patients in following up. The blue curve showed the dynamic changes of the real-time ratio of non-relapse patients in follow-up patients.Additional file 13. Residuals plot of the IgG4-RD CS prediction model by multiple linear regression. a, Residuals were shown with histogram; b, Residuals appeared completely random showed homoscedasticity.


Cathodic protection (CP) is being increasingly used on reinforced concrete structures to protect steel reinforcing bars from corrosion in aggressive conditions. Due to the complexity of environmental conditions, the design specifications in national and international standards are still open to discussion to achieve both sufficient and efficient protection for reinforced concrete structures in engineering practices. This paper reports an experimental research to investigate the influence of chloride content on concrete resistivity, rebar corrosion rate and the performance of CP operation using different current densities. It aims to understand the correlation between the chloride content and concrete resistivity together with the CP current requirement, and to investigate the precision of the CP design criteria in standards. The corrosion of steel reinforcements has been recognised as the major cause for the premature deterioration of reinforced concrete structures worldwide . ExtensiAdequate protection provided by CP for the steel reinforcement in concrete depends on many factors. In addition to the steel composition and the nature of concrete components, the physical conditions, such as concrete porosity, degree of carbonation, water and chloride contents, and environmental temperature, play the important roles affecting the effectiveness of CP operation. CP arrangement and the applied current densities are all related to the above conditions , 10. AddIn general, there are two acceptable criteria in CP performance appraisal. One relates to the instant-off potential  of the reinforcement. The other one relates to the potential decay (depolarization) of the reinforcement , 17. The2 on the rebars is sufficient for protection, while for the structures that have already suffered from reinforcement corrosion, a current density in the range of 5–20 mA/m2 is recommended [2 were also suggested when reinforcements are exposed to severe environmental conditions [Applying an adequate current density to ensure sufficient current across the critical areas of the protected reinforcement  but at aommended . Higher nditions .Based on the discussion above, it is noted that some uncertainty still exist on the topic of defining the current specification for CP design for reinforced concrete structures for varied and complex application conditions. As an effort to obtain more detailed specific information for the CP design for chloride contaminated reinforced concrete structures, this paper reports an experimental study on the effect of concrete chloride contamination degree on the corrosion evaluation parameters that are employed for reinforcement cathodic protection assessment. Specifically, this work investigates the correlation between the chloride content and concrete resistivity, and the relationship of these two parameters with the rebar corrosion rate. These studies enable identification of more precise characteristic relationships between concrete chloride content, the applied current density and the instant-off potential. Thus, the experimental results provide a direct guidance for the specification of the CP current density requirements for atmospherically exposed concrete structure at different levels of chloride contamination.2. Locally produced limestone Portland cement (CEM II/A-LL in British standard BS EN 197-1: 2011) was used at 390 kg/m3. Natural sands of the maximum size of 4.75 mm and a specific gravity of 2.47 were used for the fine aggregates at 1125 kg/m3. The coarse aggregates were limestone of maximum size of 10 mm and a specific gravity of 2.49, and were used at 580 kg/m3 in proportion. Pure NaCl  was added as contaminant into the mix water, to prepare specimens with different chloride contamination contents. The concrete mixes had a water to cement ratio of 0.4.Concrete specimens used in this study were prepared following the method recommended by the British Building Research Establishment (BRE)  to give 3, illustrated in Fig. π × 10 × 73 = 6880 mm2. A layer of a woven (CF) sheet was embedded in each specimen to be used as the anode. The nominal surface area of the embedded carbon fibre anode is 144 × 93 mm2. The carbon fibre anode extended about 30 mm out of the specimens for electrical connection. All the cast reinforced concrete specimens had the entire part of the steel bars exposed to the atmosphere coated again using epoxy resin after the concrete set.Ten reinforced concrete specimens (2 specimens for each chloride content) with the size of Length × Height × Depth = 90 × 93 × 150 mm3 and with two parallel embedded woven carbon fibre (CF) sheets as shown in Fig. Another ten rectangular concrete specimens of the size of Length × Height × Depth = 100 × 100 × 70 mm3 and cured in the same conditions were analysed using potentiometric titration method described in ASTM C1152/C1152M-12 [All the prepared concrete specimens were placed in water with the same chloride concentration as that of the mix water used and cured for 28 days. Such method aims to ensure an even chloride distribution. Thereafter all concrete specimens were taken out and exposed to an atmosphere of a relative humidity of 50 ± 5% and a temperature of 20 ± 3 °C for 5 weeks, i.e. until they attained a stable weight before conducting all the experiments. To obtain the accurate total chloride contents in the specimens, another ten concrete specimens of all the same mixtures (two specimens for each designed chloride content) with the size of 100 × 100 × 100 mm1152M-12 .icorr. A small potential shift, ΔE, was applied on rebars of an open circuit potential, Ecorr. The potential shift varied from − 20 to + 20 mV [The corrosion rates of rebars in the reinforced concrete specimens were assessed before the implementation of CP using the linear polarization method described by Stern and Geary . The pot + 20 mV , 29 at aRp, was then determined according to the slope of the plot of the applied voltage versus the measured current at the point of zero current. The same method was applied for all the specimens for the sake of comparison [B = (βaβc/2.3(βa + βc)) is a constant in mV, βa and βc are the anodic and cathodic Tafel constants, Rp is the polarization resistance in kΩ (ΔE/ΔI), Icorr is the corrosion current in mA.Polarization resistance, mparison . The corB for the chloride contaminated specimens [icorr, in (mA/m2) is determined in terms of the Eq.  were connected in series in each test at the same time as shown in Fig. Galvanostatic polarization technique was adapted to apply ten different CP current densities on the rebars in each specimen. They are 5, 10, 15, 20, 25, 35, 45, 55, 65, and 75 mA/mEach test had a certain CP current density applied for 24 h and afterwards switched off for more than one day (24 h) to ensure a sufficient depolarization of the rebars. In the time, the potential of rebars was continuously recorded from the start and until 4 h after the interruption of the CP current in the time of depolarization. Based on the recorded data, the instant-off potential, and 4-h potential decay can be obtained.The measured chloride contents in the cured specimens of each mixes with different added NaCl and the corresponding electrical resistivity of the concrete are listed in the Table 2, moderate when in the range of 5–10 mA/m2, and high when greater than 10 mA/m2. In terms of the classification, reinforcements will have a low corrosion rate if the total chloride content is less than 0.45% by the mass of cement and this chloride content may be taken as a threshold for the risk posed by reinforcement corrosion. If chloride content is over 1.4%, reinforcements present a high corrosion rate. The threshold value is in agreement with that recommended in literature [Figure terature , in whicterature  concludeterature  also conterature , 39.Fig.2, the measurement were conducted for up to 120 h (5 days), while for the other two cases the measure were interrupted after 25 h. It can been seen that, for all the three cases, the instant-off potential displays a significant change in the first 3 h of CP operation under all the applied current densities. After 3 h, all the curves become flat, showing a very slight variation with time, suggesting that the system had reached a stable state. According to the results in the Fig. The potential of the reinforcements instantly after the interruption of CP is called the instant-off potential which, in the present study, was automatically measured in 1 s after the CP was switched off , 21. Figto 120 h  days, wh2) for all the different chloride contaminated specimens the − 720 mV criterion is still far away to be achieved.Instant-off potential is one of the important criteria used to evaluate CP performance , for exaI = 0). Comparing the Figs. 2 is sufficient to provide the required protection for the reinforcement in the concrete of 0.814% chloride in terms of the 100 mV potential decay criterion. However, current density of 75 mA/m2 is not enough to protect the reinforcement even in chloride free concrete in terms of the − 720 mV instant-off potential criterion.The potential difference between the instant-off potential and the potential measured at the 4 h after switching off the CP current is called the 4-h potential decay which is another important parameter used to evaluate the effectiveness of CP operation , 41. GenFigure Figure Figure Similarly, Fig. The initial formation of passive layer was not taken into account, as chloride was added into the mix water to accelerate corrosion. Without considering the initial passivation, the measurement obtained in this work can only provide the guidance for the reinforcements that have already been experiencing active corrosion, such as, for example, those in chloride contaminated concrete with a low pH pore solution.All measurements are based on the hypothesis that corrosion on the rebars is homogeneously distributed, i.e. even if the corrosion might be localized on the microscopic scale, on a macroscopic scale there are no regions where corrosion is substantially more severe compared to others. In order to work under this assumption, in preparing specimens, efforts were made to enhance even chloride distribution in concrete by (1) adding Cl into mixing water as the most conventional measure –46 but aFinally, for the results obtained in this study, there are two specific concerns that should be clarified:A total chloride content of 0.31% by weight of cement or 17 kΩ cm concrete electrical resisitivity may be set as a threshold for CP implementation to protect the reinforcements in Portland concrete from corrosion.An instant-off potential of − 500 mV with respect to Ag/AgCl/0.5KCl electrode can provide adequate protection, in relation to the 100 mV depolarization criterion, for the reinforced concrete of up to 3.4% chloride contamination by weight of cement, or concrete resistivity is no less than 6.7 kΩ cm.A clear correlation between CP current requirement and chloride content and concrete resistivity were obtained, and characterisation modelling has been suggested.This paper has produced a number of experimental data for CP performance on the reinforcement of Portland cement concrete exposed to chloride contamination under atmospheric condition. From the reported work, the following conclusions can be obtained:


Am-POSS, prepared by hydrolytic condensation, contains two types of ammonium side-chain groups, where the numbering of x and y represents the type of ammonium ions in the POSS structure, corresponding to primary (1), secondary (2), tertiary (3), and quaternary (4) ammonium ions. Mixtures of the two starting materials selected from organotrialkoxysilanes containing primary, secondary, and tertiary amines and a quaternary ammonium salt  were dissolved in dimethyl sulfoxide (DMSO). The hydrolytic condensation was performed in the presence of bis(trifluoromethansulfonyl)imide (HNTf2) and water. All Am-POSS structures consisted of a cage-type octamer (T8-POSS), as confirmed by 29Si NMR spectrometry and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analyses indicated that Am-POSS, Am-POSS, and Am-POSS had amorphous structures. These POSSs have two or three differences in the number of methyl groups between the two types of ammonium side-chains. Conversely, Am-POSS, Am-POSS, and Am-POSS had crystalline structures. The difference in the number of methyl groups between the two types of ammonium side-chains in these POSSs is only one. Therefore, the crystalline-amorphous structure of Am-POSS is controlled by the side-chain group combinations. Furthermore, Am-POSS, Am-POSS, and Am-POSS are protic ionic liquids with relatively low flow temperatures.Polyhedral oligomeric silsesquioxanes (POSSs),  Polyhedral oligomeric silsesquioxanes (POSSs), which are generally prepared by the hydrolytic condensation of trifunctional silane compounds, such as organotrialkoxysilanes and organotrichlorosilanes, have attracted much attention in recent years as compounds having both inorganic and organic characteristics. In addition to the remarkable thermal and chemical stability derived from siloxane-bond frameworks, good dispersibility and solubility are provided due to regulated cage structures and suitable side-chain groups ,2,3,4. PWell-known POSSs are typically crystalline, cage-type octamers, because of their isotropic cubic structure. The amorphization of POSSs is one of the recent interesting topics in basic research and material applications. For example, amorphous POSS dimers  and polyTm) ammonium chloride (TMTESPAC), respectively, in the presence of bis(trifluoromethansulfonyl)imide (HNTf2) and water (the feed molar ratio of amine:HNTf2:H2O was 1.0:1.5:5.0) (Am-POSS(x) are summarized in Am-POSS(1), was also insoluble in water.1.5:5.0) . Solubil1H NMR spectra of Am-POSS(x) in DMSO-d6 showed the signals attributable to the side-chains of the corresponding POSSs (Am-POSS(4) contained no peaks originating from Cl (ca. 2.6 and 2.8 keV). The Si:S atom ratios were estimated to be 1.00:1.95 for Am-POSS(1), 1.00:2.02 for Am-POSS(2), 1.00:1.96 for Am-POSS(3), and 1.00:2.00 for Am-POSS(4), respectively, indicating that the molar ratios of ammonium cations to NTf2 anions in all products were ca. 1:1  in DMSO-d6 at 40 °C showed only the sharp signals due to the T3 structure (CSi(OSi)n(OH)n3–, n = 3) in the range between δ −66.6 and −67.3 ppm. These peaks are attributable to cage-type octamer (T8-POSS)  was performed on these POSSs. The MALDI-TOF MS of Am-POSS(1), Am-POSS(2), Am-POSS(3), and Am-POSS(4) showed several peaks corresponding to the masses of T8-POSS  and cage-type dodecamer (T12-POSS), were present in the products.The T8-POSS) . To supp T8-POSS . These rAm-POSS(x) showed sharp diffraction peaks, indicating their crystalline structures (Am-POSS(1), Am-POSS(2), Am-POSS(3), and Am-POSS(4), the endothermic peaks due to Tms were observed at 186 and 209 °C (Am-POSS(4), potentially caused by the different crystalline forms of these POSSs. A more detailed study on the crystalline forms of these POSSs containing one type of ammonium side-chain group is currently in progress.The X-ray diffraction (XRD) patterns of all ructures . In addid 209 °C a, −1, 13d 209 °C b, 94 andd 209 °C c, and 17d 209 °C d, respecAm-POSSs were prepared by the hydrolytic condensation of a mixture of two starting materials selected from APTMS, MAPTMS, DMAPTMS, and TMTESPAC. DMSO was used as a solvent together with HNTf2 and water (the feed molar ratio of amine: HNTf2:H2O was 1.0:1.5:5.0)  are summarized in Am-POSS were soluble in highly polar organic solvents but insoluble in water and low polarity organic solvents.1.5:5.0) . Solubil1H NMR spectra of Am-POSS in DMSO-d6 showed the signals attributable to the side-chains of both the two selected organotrialkoxysilane components  was estimated to be ca. 1:1, considering the integrated ratio of the signals in the 1H NMR spectrum, i.e., (a + a′):e′ (4.00:2.96) for Am-POSS, (a + a″):c″ (4.00:2.01) for Am-POSS, c:c‴ (2.00:2.07) for Am-POSS, (a′ + a″):c″ (4.00:1.99) for Am-POSS, c′:c‴ (2.00:2.00) for Am-POSS, and (a″ + a‴):c‴ (4.00:2.02) for Am-POSS. The EDX patterns of Am-POSS, Am-POSS, and Am-POSS contained no peaks originating from Cl (ca. 2.6 and 2.8 keV). The Si:S atom ratios were estimated to be 1.00:1.98 for Am-POSS, 1.00:2.03 for Am-POSS, 1.00:2.00 for Am-POSS, 1.00:2.06 for Am-POSS, 1.00:2.00 for Am-POSS, and 1.00:1.98 for Am-POSS, respectively, indicating that the molar ratios of ammonium cations to NTf2 anions in all products were ca. 1:1  in DMSO-d6 at 40 °C showed only the sharp signals in the range between δ −66.6 and −67.3 ppm, corresponding to the T3 structure (8-POSS). To support these 29Si NMR results, MALDI-TOF MS was performed on these POSSs. The MALDI-TOF MS of all Am-POSS showed several peaks ascribed to the masses of T8-POSS , Am-POSS, and Am-POSS, baseline shifts due to Tgs were observed at −6 °C , Am-POSS, and Am-POSS had Tms at 154 °C   and Am-POSS , Am-POSS, and Am-POSS are crystalline compounds. On the other hand, Am-POSS, Am-POSS, and Am-POSS did not show any diffraction peaks, indicating that these compounds have amorphous structures. These XRD results did support the DSC results.In the DSC curves of at −6 °C , 15 °C  a. RepeatOSS d,f. AlthAm-POSS, Am-POSS, and Am-POSS have two or three differences in the number of methyl groups between two types of ammonium side-chains. Because of the low molecular symmetry of the POSSs containing two different randomly distributed side-chain groups, crystallization was suppressed, leading to amorphous structures. Conversely, the molecular symmetry of Am-POSS, Am-POSS, and Am-POSS were maintained because of the difference in the numbers of methyl groups between the two types of ammonium side-chains was only one. Therefore, it seems difficult to suppress the crystallization of these POSSs.Based on the DSC and XRD results, we concluded that crystalline and amorphous POSSs were selectively prepared, depending on the combination of side-chain groups. ILs are generally defined as salts that melt below 100 °C . The fundamental thermodynamic properties of ILs have been extensively studied ,23,24 foTm) was 23 °C. Another IL containing a POSS structure was reported by Feng, Zhang, and co-workers. It contained cationic imidazolium side-chains and dodecyl sulfate anions as counterions and had a Tm of 18 °C , and tetramethylsilane (TMS) were purchased from Tokyo Chemical Industry Co., Ltd. . HNTf2 was purchased from Kanto Chemical Co., Inc. . 2,5-Dihydroxybenzoic acid (DHB) was purchased from Bruker Daltonik Gmbh . Other reagents and solvents were purchased from FUJIFILM Wako Pure Chemical Co., Ltd. . All reagents and solvents were used without further purification.APTMS, MAPTMS, DMAPTMS, TMTESPAC, trischromium(III) ). 1H NMR : δ 7.75–7.42 , δ 2.81–2.65 , δ 1.62–1.40 , δ 0.65 . 29Si NMR : δ −66.6 .A mixture of a 0.5 mol·LAm-POSS(2) was prepared similar to Am-POSS(1), except that the starting material was MAPTMS . A white solid product (0.679 g) was obtained 3NH2CH3 (CF3SO2)2N, FW = 405.4]). 1H NMR : δ 8.35–8.09 , δ 2.89–2.75 , δ 2.57 , δ 1.65–1.46 , δ 0.65 . 29Si NMR : δ −66.8 .Am-POSS(3) was prepared similar to Am-POSS(1), except that the starting material was DMAPTMS . A white solid product (0.807 g) was obtained 3NH(CH3)2 (CF3SO2)2N, FW = 419.4]). 1H NMR : δ 9.37–9.05 2), δ 3.03–2.90 2), δ 2.83–2.65 2), δ 1.71–1.46 2), δ 0.64 2). 29Si NMR : δ −67.0 .Am-POSS(4) was prepared similar to Am-POSS(1), except that the starting material was TMTESPAC . A white solid product (0.855 g) was obtained 3N(CH3)3 (CF3SO2)2N, FW = 433.5]). 1H NMR : δ 3.26–3.15 3), δ 3.03 3), δ 1.76–1.54 3), δ 0.64 3). 29Si NMR : δ −67.3 .Am-POSS was prepared similar to Am-POSS(1), except that the starting material was a mixture of APTMS  and MAPTMS . A white solid product (0.761 g) was obtained 3NH3 (CF3SO2)2N, FW = 391.4] × 0.50 +  × 0.50 = 398.4}). 1H NMR : δ 8.34–8.09 , δ 7.74–7.44 , δ 2.89–2.78 , δ 2.78–2.68 , δ 2.57 , δ 1.68–1.40 , δ 0.76–0.47 . 29Si NMR : δ −66.6 and −66.8 .Am-POSS was prepared similar to Am-POSS(1), except that the starting material was a mixture of APTMS  and DMAPTMS . A brown and viscous solid product (0.804 g) was obtained 3NH3 (CF3SO2)2N, FW = 391.4] × 0.50 +  × 0.50 = 405.4}). 1H NMR : δ 9.38–9.05 2 of the DMAPTMS component), δ 7.79–7.40 , δ 3.03–2.90 2 of the DMAPTMS component), δ 2.83–2.65 2 of the DMAPTMS component and 2H, br, –SiCH2CH2CH2NH3 of the APTMS component), δ 1.70–1.41 2 of the DMAPTMS component and –SiCH2CH2CH2NH3 of the APTMS component), δ 0.77–0.42 2 of the DMAPTMS component and –SiCH2CH2CH2NH3 of the APTMS component). 29Si NMR : δ −66.6 and −67.0 .Am-POSS was prepared similar to Am-POSS(1), except that the starting material was a mixture of APTMS  and TMTESPAC . A dark brown and viscous solid product (0.809 g) was obtained 3NH3 (CF3SO2)2N, FW = 391.4] × 0.49 +  × 0.51 = 412.9}). 1H NMR : δ 7.75–7.43 , δ 3.25–3.13 3 of the TMTESPAC component), δ 3.08–2.92 3 of the TMTESPAC component), δ 2.81–2.68 , δ 1.78–1.60 3 of the TMTESPAC component), δ 1.60–1.42 , δ 0.78–0.44 3 of the TMTESPAC component and –SiCH2CH2CH2NH3 of the APTMS component). 29Si NMR : δ −66.6 and −67.3 .Am-POSS was prepared similar to Am-POSS(1), except that the starting material was a mixture of MAPTMS  and DMAPTMS . A brown and viscous product (0.779 g) was obtained 3NH2CH3 (CF3SO2)2N, FW = 405.4] × 0.50 +  × 0.50 = 412.4}). 1H NMR : δ 9.34–9.09 2 of the DMAPTMS component), δ 8.34–8.10 , δ 3.03–2.90 2 of the DMAPTMS component), δ 2.88–2.79 , δ 2.79–2.65 2 of the DMAPTMS component), δ 2.57 , δ 1.71–1.39 2 of the DMAPTMS component and –SiCH2CH2CH2NH2CH3 of the MAPTMS component), δ 0.75–0.43 2 of the DMAPTMS component and –SiCH2CH2CH2NH2CH3 of the MAPTMS component). 29Si NMR : δ −67.0 and −67.1 .Am-POSS was prepared similar to Am-POSS(1), except that the starting material was a mixture of MAPTMS  and TMTESPAC . A dark brown and viscous product (0.828 g) was obtained 3N H2CH3 (CF3SO2)2N, FW = 405.4] × 0.50 +  × 0.50 = 419.5}). 1H NMR : δ 8.36–8.07 , δ 3.25–3.14 3 of the TMTESPAC component), δ 3.10–2.95 3 of the TMTESPAC component), δ 2.89–2.77 , δ 2.60–2.53 , δ 1.76–1.60 3 of the TMTESPAC component), δ 1.60–1.46 , δ 0.78–0.45 3 of the TMTESPAC component and –SiCH2CH2CH2NH2CH3 of the MAPTMS component). 29Si NMR : δ −66.8 and −67.3 .Am-POSS was prepared similar to Am-POSS(1), except that the starting material was a mixture of DMAPTMS  and TMTESPAC . A dark brown and viscous product (0.799 g) was obtained 3N H(CH3)2 (CF3SO2)2N, FW = 419.4] × 0.50 +  × 0.50 = 426.5}). 1H NMR : δ 9.35–9.12 2 of the DMAPTMS component), δ 3.25–3.15 3 of the TMTESPAC component), δ 3.09–3.00 3 of the TMTESPAC component), δ 3.00–2.91 2 of the DMAPTMS component), δ 2.81–2.68 2 of the DMAPTMS component), δ 1.77–1.47 3 of the TMTESPAC component and –SiCH2CH2CH2NH(CH3)2 of the DMAPTMS component), δ 0.77–0.44 3 of the TMTESPAC component and –SiCH2CH2CH2NH(CH3)2 of the DMAPTMS component). 29Si NMR : δ −67.2 and −67.3 .1H and 29Si NMR spectra were recorded using an ECX-400 spectrometer . The absence of Cl and the elemental ratios of Si:S in the products were confirmed by EDX analyses using an XL30 scanning electron microscope . The MALDI-TOF MS analyses of the products were performed using a Voyager Biospectrometry Workstation Ver. 5.1  with 2,5-dihydroxybenzoic acid (DHB) as the matrix under positive ion mode. The DSC analyses were performed on a DSC-60 Plus . The samples placed in an aluminum capsule were cooled to −140 °C at a rate of 20 °C min−1 under a nitrogen flow (300 mL min−1) and then heated from −140 °C to 240 °C at the same rate. The Tg and Tm values were determined as the onset of the baseline shift and as the tops of the endothermic peaks, respectively, in the curves of the third scan (from −140 °C to 240 °C at a rate of 20 °C min−1) to eliminate the heat history in the samples. The XRD patterns were recorded at a scanning speed of 2θ = 9.0° min−1 using an X’Pert Pro diffractometer  with Ni-filtered Cu Kα radiation (0.15418 nm). The TGA was performed on a TGA-50  at a heating rate of 10 °C min−1 up to 1000 °C under nitrogen flow (100 mL min−1). The pyrolysis temperatures were determined from the 5% (Td5) and 10% (Td10) weight losses.The Am-POSS were successfully prepared by the hydrolytic condensation in DMSO of mixtures of two starting materials, which were selected from the organotrialkoxysilanes containing primary, secondary, and tertiary amines and a quaternary ammonium salt, in the presence of HNTf2 and water. The products consisted of T8-POSS only. The DSC curves of Am-POSS, Am-POSS, and Am-POSS did not show the endothermic peaks due to Tms, indicating their amorphous structures. Conversely, those of Am-POSS, Am-POSS, and Am-POSS showed the endothermic peaks due to Tms, indicating their crystalline structures. XRD data also supported these results. These results indicate that the crystalline or amorphous structure of Am-POSS can be controlled by the side-chain group combinations. Am-POSS, Am-POSS, and Am-POSS were PILs with relatively low flow temperatures, while Am-POSS, Am-POSS, and Am-POSS were PILs with relatively high flow temperatures.


While endemic normals (EN) remain free of infection despite constant exposure to the infective larvae, a small group of patients, generally microfilaria free (Mf-) develops severe pathology (CP) such as lymphedema or hydrocele. Another group of infected individuals remains asymptomatic while expressing large amounts of microfilariae (Mf+). This Mf+ group is characterized by an immune-suppressed profile with high levels of anti-inflammatory cytokines and elevated IgG4. This particular immunoglobulin is unable to activate the complement. The complement system plays a critical role in both innate and adaptive immunity. However, its importance and regulation during LF is not fully understood. Using affinity chromatography and solid-phase-enzyme-immunoassays, we investigated the ability of antibody isotypes from LF clinical groups to bind C1q, the first element of the complement’s classical pathway. The results indicate that while C1q is similarly expressed in all LF clinical groups, IgG1–2 in the plasma from Mf+ individuals presented significantly lower affinity to C1q compared to EN, Mf−, and CP. In addition, selective depletion of IgG4 significantly enhanced the affinity of IgG1–2 to C1q in Mf+ individuals. Strikingly, no effect was seen on the ability of IgG3 to bind C1q in the same conditions. More interestingly, papain-generated IgG4-Fc-portions interacted with Fc portions of IgG1–2 as revealed by far-western blot analysis. These data suggest that while being unable to bind C1q, IgG4 inhibits the The online version of this article (10.1007/s00436-019-06451-2) contains supplementary material, which is available to authorized users. Lymphatic filariasis (LF) is a major public health concern in tropical and subtropical countries. Despite 13 years of mass drug administration (MDA) programs, 68 million LF cases remain worldwide, of which 37 million suffer from severe pathology (lymphedema and hydrocele)   (n = 22), clinically asymptomatic microfilaraemic  and amicrofilaraemic  subjects, positive for circulating filarial antigen and a group of chronic pathological individuals termed “CP” (n = 18), negative for filarial antigen.Samples include EN, residing in the endemic region but free of infection and negative for circulating filarial antigen (CFA) and microfilaraemia (Mf)  and then transferred to liquid nitrogen until used.Plasma samples from endemic normals and LF-infected individuals were collected using a ficoll gradient centrifugation method . Microtiter plates provided in the kit were pre-coated with an anti-human C1q antibody. After thorough washing of the wells, 100 μl of prediluted plasma samples (1:100) and prepared standards were added to their respective wells and plates were allowed to incubate for 2 h at room temperature on a microplate shaker  set at 400 rpm. Wells were then washed 6 times and each well was filled with 100 μl of prepared biotin-conjugated detection antibody and incubated for 1 h on a microplate shaker. Thereafter the plates were washed and 100 μl of streptavidin-HRP was added to the wells for 1 h with shaking in the dark. After a final washing step, wells were incubated with 100 μl of TMB substrate solution for 30 min and substrate reaction stopped with acidic stop solution. The absorbance of each microwell was read at 450 nm using the SpectraMAX ELISA reader  and the concentration of C1q in samples determined.IgG4 antibodies were depleted from plasma samples of EN, Mf+, Mf−, and CP individuals using the CaptureSelect Human IgG4 affinity matrix , containing an antibody fragment recognizing human IgG4, according to the manufacturer’s instructions. Briefly, CaptureSelect affinity matrix was carefully packed and equilibrated in 10 ml affinity chromatography column with PBS (pH 7.3). One hundred microliters of plasma samples was diluted with 1400 μl binding buffer (1× PBS) and loaded onto the column with a linear flow rate of 150 cm/h. Since the antibody fragment in the affinity matrix binds to IgG4 antibodies, non-IgG4 plasma components were washed out with PBS and collected as IgG4-negative plasma. Bound IgG4 was eluted in 1 ml fractions using Elution Buffer (0.1 M Glycine/HCl pH 3.0) and immediately neutralized with 1:10 volume of saturated Tris-HCl (pH 9.0). The total protein concentration in IgG4-positive and IgG4-negative plasma was then assessed at 280 nm using a NanoDrop 1000 spectrophotometer  and concentrations of immunoglobulin isotypes IgG1–4, IgA, IgE, and IgM were assessed by Luminex.To determine the immunoglobulin isotypes composition in IgG4-positive and IgG4-negative plasma of EN and LF patients, ProcartaPlex Human Antibody Isotyping Panels  were used according to manufacturer’s instructions. Briefly, antibody-coated magnetic bead mixtures were incubated with 25 μl of assay buffer, kit standards, or diluted plasma samples (1:20000) in ProcartaPlex 96-well plates at room temperature for 1 h. Twenty-five microliters of detection antibody mixture was then added, and the plates were incubated on an orbital shaker  at 500 rpm for 30 min. After that, each well was incubated with 50 μl of diluted Streptavidin-Phycoerythrin for 30 min. All incubations were performed at room temperature in the dark (plate covered with black microplate lid) and the plates washed using a hand-held magnetic plate washer. Afterward, samples were suspended in 120 μl reading buffer. Data were acquired using a MAGPIX Luminex system (Luminex Cooperation) and analyzed with ProcartaPlex Analyst software 1.0.To measure the levels of total IgG bound to complement C1q in plasma from EN, Mf+, Mf−, and CP, an enzyme immunoassay (EIA) was used as previously described . Optical density was measured at 450 nm using the SpectraMAX ELISA reader and the results were expressed as arbitrary units (AU) relative to a unique plasma sample with the highest OD used as standard on all plates and arbitrarily set at 5 AU.To evaluate the binding capacity of immunoglobulin isotypes in IgG4-positive and IgG4-negative plasma from EN, Mf+, Mf−, and CP individuals to C1q, high binding ELISA plates  were coated with 50 μl recombinant human complement C1q  at the concentration of 1 μg/ml and incubated at 4 °C overnight. The plates were then washed 5 times with PBS/0.05% Tween 20 and blocked with PBS/1% BSA for 1 h at room temperature. The wash step was repeated and subsequently, plates were incubated overnight at 4 °C with 50 μl/well of IgG4-positive and IgG4-negative plasma samples at various dilutions . Wells were further washed and diluted biotin-conjugated anti-IgG1, IgE (1:1000); IgG2, IgG4 (1:15000), and IgG3, IgM (1:4000)  were added, followed by incubation at room temperature for 2 h. Finally, the plates were again washed and then incubated with 50 μl/well of Streptavidin-HRP for 45 min in the dark. After a final washing step, 50 μl/well TMB substrate solution were added to the plates and the reaction was stopped 15 min later with 25 μl/well 2 N HTo purify IgG1–2 fractions, total IgG was first isolated from IgG4-negative plasma using prepacked HiTrap™ Protein G columns  according to the manufacturer’s instructions. Briefly, 100 μl of plasma samples were diluted with 1400 μl PBS and passed through a pre-equilibrated protein G-Sepharose column . After Protein G binds to human IgG1–3 subclasses, non-IgG plasma components were washed out from the column. Bound IgG1–3 were eluted in 1 ml fractions using IgG Elution Buffer  and neutralized with saturated Tris-HCl (pH 9.0). Thereafter, IgG1–2 antibodies were isolated from IgG1–3 fractions of Mf+ and Mf− using prepacked Protein A columns  following the manufacturer’s recommendations. Briefly, diluted IgG1–3 samples were passed through a pre-equilibrated protein A column. Since Protein A highly binds to all human IgG subclasses, except IgG3, non-IgG1–2 components were washed out from the column and collected. Bound IgG1–2 antibodies were eluted in 1 ml fractions using Elution Buffer  and neutralized with saturated Tris-HCl (pH 9.0). The protein concentration was then assessed at 280 nm using a NanoDrop 1000 spectrophotometer .Fc fragments of IgG4 and IgG1–2 antibodies from Mf+ and Mf− individuals were separately generated by papain digestion using Pierce Fab preparation kit  following the manufacturer’s instructions. Digested IgG4 and IgG1–2 antibodies were further separated by centrifugation from immobilized papain. Thereafter, purification of the Fc fragments was accomplished using a Protein A affinity chromatography. Briefly, the digest was passed through Protein A spin column provided in the kit. Protein A column binds the Fc fragments and undigested IgG, allowing the Fab fragments to pass through the column. The Fc fragments from IgG4 and IgG1–2 were then eluted using IgG Elution Buffer  and neutralized with Tris-HCl (pH 9.0). The purity of eluted fractions was analyzed by western blot, and the interaction of IgG4-Fc fragments with the Fc portions of IgG1–2 from Mf+ and Mf− was investigated using ELISA and western blot.For the western blot analysis of the purity of the fractions, 1 μg of total IgG, digested IgG1–2 and IgG4 fractions, and the corresponding Fc fragments from Mf+ and Mf− plasma were loaded onto different lanes of a polyacrylamide gel (10–12%) and resolved by SDS-PAGE . The resolved proteins were transferred onto nitrocellulose membranes  using a Bio-Rad Trans-Blot Turbo Transfer System . The membranes were then blocked with 3% BSA in Tris Buffered Saline (TBS)  for 1 h at room temperature prior incubation with the primary antibodies (mouse anti-human IgG4 Fc and IgG Fab (1:1000) ) for 1.5 h at room temperature. The nitrocellulose membranes were then washed with TBS/0.05% Tween 20 before incubation for 1 h with alkaline phosphatase-conjugated goat anti-mouse IgG (1:500) . Immune complexes were finally detected with NBT (nitro blue tetrazolium) and BCIP .To examine the IgG4 Fc-IgG1–2 Fc interactions, ELISA and specific western blot were used.2SO4 solution . Optical density was measured at 450 nm using the SpectraMAX ELISA reader, and the results were expressed as arbitrary units (AU) relative to a unique plasma sample with the highest OD used as standard on all plates and arbitrarily set at 5 AU.For ELISA, high-binding ELISA plates  were coated with 100 μl of purified IgG1–2 Fc fragments at the concentration of 1 μg/ml in PBS and incubated at 4 °C overnight. Control wells were incubated with PBS. The plates were washed 5 times with PBS/0.05% Tween 20 and blocked with PBS/1% BSA for 1 h at room temperature. After repeated washes, 100 μl/well of either IgG4-positive and IgG4-negative fractions of Mf+ and Mf− were added to the respective plates overnight at 4 °C. Wells were further washed and diluted biotin-conjugated mouse anti-human IgG4 (1:500) and IgG (1:20000)  were added to the corresponding plates. After 2 h incubation at room temperature, plates were washed and revealed with 50 μl/well of Streptavidin-HRP for 45 min in the dark. After a final washing step, 50-μl/well TMB substrate solution was added to the plates and the reaction was stopped 15 min later with 25-μl/well 2N HFor western blotting, 1 μg of purified total IgG1–2, IgG1–2 Fc, and IgG1–2 Fab fragments from Mf+ and Mf− plasma were loaded onto 4–12% SDS-PAGE gels. After separation  and transfer using a Bio-Rad Trans-Blot Turbo Transfer System , the nitrocellulose membranes  were blocked with 3% BSA in Tris Buffered Saline (TBS))  for 1 h at room temperature before incubation with 1 μg/ml of IgG4 Fc and IgG4 Fab fragments from Mf+ and Mf− overnight at 4 °C. The membranes were thereafter washed and incubated with the primary antibodies (mouse anti-human IgG4 Fc and IgG4 Fab (1:1000) ) for 1.5 h followed by the secondary antibody, alkaline phosphatase-conjugated goat anti-mouse IgG (1:500) , for 1 h. Fc-Fc interactions between IgG4 and IgG1–2 fragments were detected using NBT (nitro blue tetrazolium) and BCIP .U test was used. Significance was accepted when p < 0.05.To determine statistical differences between the different groups, the software PRISM 5.03  was used. Comparative analyzes among groups were conducted using a Kruskal-Wallis test with a Dunn’s nonparametric post hoc test (> 2 groups). In case of two groups, a Mann-Whitney To find out whether differences exist in the affinity of the antibody isotypes from the plasma of EN, Mf+, Mf−, and CP individuals to C1q, we investigated the capacity of each IgG subclass antibodies, IgE, and IgM in the plasma of individuals of the clinical groups to interact with C1q. While no difference was observed in C1q and IgG-C1q levels in EN, Mf+, Mf−, and CP . We then investigated the interaction between IgG4-positive and IgG4-negative fractions of IgG4 and Fc portions of IgG1–2 from Mf+ and Mf−. We observed that binding did not occur with IgG4-negative fraction but rather with the positive fraction of Mf+ Fig.  and Mf− Since IgG4-positive fraction interacted with the Fc part of IgG1 and IgG2, we further discriminated between the involvement of the Fc and Fab portions of IgG4 in this interaction by western blot. On Fig. The complement system plays an essential role in the immune response against parasitic worms through interactions of C1q with antigen-bound immunoglobulins, primarily IgGs (IgG1–3), and IgM  infections and recently confirmed by Lubbers et al. suggesting that C1q expression is increased in the peripheral blood of patients with active tuberculosis compared to healthy controls and individuals with latent TB infection