Seven different DLA method modifications were tested, in terms of antibiotic addition placement and presence or absence of the base agar. The overall number of phage plaques per plate mainly depended on the antibiotic used. Differences in plaque quantity depended on the type of the DLA method modification. The largest total number of plaques was obtained by the addition of an antibiotic to a bottom agar with the presence of a top agar. This indicates that even though an antibiotic could manifest the PAS effect by a standard disk method, it would be worth examining if the effect is equally satisfactory when applying antibiotics directly into the agar, with regards to using the same bacteriophage and bacterial host.The research carried out so far for phage-antibiotic synergy (PAS) differs as regards the technique of modifying the double-layer agar (DLA) method to show the PAS effect on Petri plates, which may contribute to non-uniform research results. Therefore, there is a need to unify the method to effectively detect the PAS effect, at its most basic in vitro test. In this study, bacteriophage T4 With the increase in the frequency of antibiotic resistance and the decreasing numbers of new antibiotics being developed , alternaPhage therapy is based on the use of lytic phages to combat bacterial infections, including multidrug-resistant bacteria, and has many advantages, as phages persist as long as the targeted bacteria are present. Moreover, phages are very specific and efficient for their bacterial host. This reduces the destruction of natural flora and presents no detrimental effects on human cells . TherefoThis phenomenon is known as phage-antibiotic synergy (PAS), given this name by the authors who first described this effect . The PASP. aeruginosa produces an evolutionary trade-off, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes [The expected benefit of such a dual approach might be enhanced bacterial suppression, more efficient penetration into biofilms, and a decrease in the emergence of resistance . The las classes . Hypothe classes .Although the lytic activity of phages has been shown to be synergistically enhanced in the presence of antibiotics, PAS is still not fully understood. The research carried out to date differs as regards the method of modifying the double-layer agar (DLA) method to demonstrate the PAS effect ,16, whicThe purpose of the study was to standardize the double-layer agar (DLA) method on Petri plates for T4-like phages, in order to evaluate the most efficient variable for phage-antibiotic synergy (PAS) effect detection.5 plaques, in the bacterial inhibition zones caused by the antibiotic; the possibility of the occurrence of the PAS effect (+), by detecting the appearance of the larger plaques in the sub-lethal zones;  no PAS effect (/), when plaques had a standard size of T45 bacteriophage and plaque quantity had not changed; possible antagonist effect (-), when there were less morphologically standard plaques within the sub-lethal drug zones  and large, strong PAS-indicating plaques (1.01–1.5 mm) than in the presence of ampicillin but smaller amounts of small, standard T45 plaques (<0.5 mm) compared to AMP addition. Furthermore, the best results were also obtained during the addition of the antibiotic to the bottom agar with the presence of the top agar (“B with ctx + T”) and with the application of the antibiotic disk onto the standard soft-agar overlay with the bottom layer present (“B + T + D”). For ampicillin, “B + T with amp” and “B with amp + T” modifications were equally effective in terms of forming different diameter plaques. For big, PAS-indicating plaque (0.51–1 mm) visualizations, antibiotic disk modifications (“T + D” and “B + T + D”) showed equally best results. Only the “B + T + D” variable was characterized by a greater amount of medium-sized, standard phage plaques due to the presence of a bottom layer  and high (107 PFU/mL) T4 phage titers were added [Comeau et al.  also repphenicol . The resphenicol . On the phenicol  antibiot culture . The PASE. coli infections [Due to the results of other authors ,10 with fections . AntibioThe final experiment consisted of testing different DLA method variables in order to standardize and evaluate which was the most efficient for phage-antibiotic synergy (PAS) effect detection. Standard “double-layer agar method” or “double overlay agar plaque assay” is used for the determination of the concentration of functional bacteriophage particles (titre), usually expressed as plaque-forming units (PFU/mL). The method is based on mixing phage suspension (lysate) dilutions with host bacteria in “soft” agar, distributing this mixture on a standard agar plate to solidify. After incubation (with conditions adapted to the host bacterium), plaques are visualized as clear zones in the bacterial lawn . The DLAWithin the research, seven different DLA method modifications were tested in terms of antibiotic addition placement, as well as the presence or absence of the base agar with proper control plates. The addition of the antibiotic as antibiotic disks applied onto Petri plates was carried out as this was the first method used for the original PAS detection  and was The results of the study showed that the overall number of phage plaques per plate depended mainly on the antibiotic used. Despite triggering the PAS effect, the addition of ampicillin showed no significant differences in plaque enumeration in any of the method modifications that were used. However, in the case of cefotaxime, significant changes in the overall number of plaques were detected. Differences in the number of plaques depended on the type of the DLA method modification. The largest total number of plaques was obtained by the addition of an antibiotic to the bottom agar with the presence of the top agar, where bacterial and phage suspensions were mixed. This modification increased the number of plaques by 114% compared to the use of a top agar alone and by 37% compared to the standard DLA method containing bottom and top agar. In the case of the number of phage plaques divided into diameter ranges, experiments showed statistically significant differences between the type of method used and the antibiotic on plaque sizes. Cefotaxime incorporation resulted in a greater amount of large, strong PAS-indicating plaques than in the presence of ampicillin, with the best results also obtained with the addition of the antibiotic to the bottom agar with the presence of the top agar, beside the antibiotic disk modification with the bottom layer. For ampicillin, big PAS-indicating plaques were most visible with antibiotic disk modifications, regardless of the presence of a bottom layer.The results of this study indicate, that even though an antibiotic can manifest the PAS effect with T4-like phage by the standard disk method, further examination should be carried out to verify if the effect will be equally satisfactory when applying the antibiotic directly into the agar. One of the antibiotics used enhanced the total number of phage plaques (cefotaxime), where the other did not (ampicillin), despite belonging to the same antibiotic class (β-lactams). However, ampicillin is a member of the penicillins group, and cefotaxime belongs to the cephalosporins, and as reported by other authors, phage and antibiotic synergistic and antagonistic interactions depend highly on the mechanism of bacterial inhibition and stoichiometry of the pairing , and cefEscherichia coli K-12 C600 [5 . Phage and bacterium were part of a Collection of the Department of Microbiology and Biotechnology, at the West Pomeranian University of Technology, Szczecin. The bacterium was stored using the Microbank™ system at −20 °C prior to the experiments. In order to recover the strain, beads were streaked directly to Luria-Bertani (LB agar)  plates and incubated at 37 °C for 24 h. Next, a single colony was streaked onto a new LB agar plate with the same incubation conditions and then used to inoculate 50 mL of LB broth. Bacterial cells were incubated at 37 °C and 160 rpm in an orbital rotating shaker  to reach OD600nm = 0.5, corresponding to the exponential phase of growth and a titer of ~2 × 108 CFU. Optical density values were measured using an Infinite 200 PRO NanoQuant microplate reader . The E. coli bacteriophage T45 was added at this point, and samples were incubated for the further stage until the cultures cleared, or after 3 h. Following incubation, chloroform was added to the culture , and samples were vortexed for the next 5 min. The cultures were then centrifuged  at 3500× g for 15 min at 4 °C, and the supernatant was collected and stored at 4 °C. Phage activity was tested by a double overlay agar plaque assay [4 at pH 7.5). Isolated phages were purified through a triple transfer of single plaque and propagated until homologous plaques were obtained. Decimal dilutions of lysates in a TM buffer were prepared to define the titer of the bacteriophage and subjected to the plaque assay using the double agar layer method. Plaques were counted on a dilution plate in spots containing 30–300 plaques. Results were expressed in plaque-forming units per mL. Bacteriophage stock suspension was stored at 4 °C.-12 C600  was used600nm = 0.5) and 100 µL of phage lysate (of approx. 1 × 107 PFU/mL). After top agar solidification, the antibiotic disks were applied. Plates were incubated for 24 h at 37 °C and then analyzed for the presence of the PAS effect. The presence of the PAS effect was determined according to Comeau et al. [In order to detect which antibiotics may form the PAS effect, 43 different antibiotics were used . The tes600nm = 0.2 (which corresponds to 1 × 108 CFU/mL). The bacterial suspension was then diluted at 1:100 in the LB medium, which gave the final desired inoculum of 5 × 105 CFU/mL. The microtiter plate was incubated at 37 °C for 24 h. The MIC of the antibiotics for E. coli K-12 C600 bacterial strain was determined to be a concentration of antibiotic at which the optical density (OD600nm) was equal to that of a cell-free blank control.Based on the previous experiment, cefotaxime and ampicillin were chosen for further testing. Ampicillin and cefotaxime  stock solutions of 50 mg/mL were prepared by diluting them in deionized water and filter sterilizing . The MICs of antibiotics were determined by using a 96-well microplate dilution protocol with minor modifications . LB was 3 PFU/mL and 200 µL of E. coli was added when bacterial cells were in their exponential (log phase) of growth—at OD600nm = 0.5. The experiment was carried out in triplicate.For the experiment, different variables of the double-layer agar method  were usep ≤ 0.05.The results were analyzed statistically with Statistica 13.3 TIBCO Software Inc. . Shapiro–Wilk tests were used to evaluate the distribution of variables. One-way ANOVA was performed, and the significance of differences between the mean values of the number of phage plaques and the variables of the DLA method were calculated using a parametric Tukey test. Differences were considered significant at In this work we tried to unify the DLA method to effectively detect the PAS effect for T4-like bacteriophages, at its most basic in vitro test. Our studies have shown that the choice of the method modification has an impact on the final results. The selection of the DLA method variable seems to be mostly antibiotic type-dependent with regards to using the same bacteriophage and bacterial host. This indicates that even though an antibiotic could manifest the PAS effect by a standard disk method, it would be worth examining if the effect is equally satisfactory when applying antibiotics directly into the agar.


The use of POMs as chiral shift reagents represents an application of a class that is yet to be well established and opens avenues into aqueous host‐guest chemistry with self‐assembled recognition agents.Determining the relative configuration or enantiomeric excess of a substance may be achieved using NMR spectroscopy by employing chiral shift reagents (CSRs). Such reagents interact noncovalently with the chiral solute, resulting in each chiral form experiencing different magnetic anisotropy; this is then reflected in their NMR spectra. The Keplerate polyoxometalate (POM) is a molybdenum‐based, water‐soluble, discrete inorganic structure with a pore‐accessible inner cavity, decorated by differentiable ligands. Through ligand exchange from the self‐assembled nanostructure, a set of chiral Keplerate host molecules has been synthesised. By exploiting the interactions of analyte molecules at the surface pores, the relative configuration of chiral amino alcohol guests  in aqueous solvent was establish and their enantiomeric excess was determined by  Chiral resolution of racemic amino alcohol guests  has been achieved by using a chirally coordinated enantiopure polyoxometalate host, {Mo132(lactate)30}. NMR spectroscopic methods, including DOSY measurements, were applied to determine the enantiomeric excesses of guest mixtures in addition to the association constants of the host‐guest system binding process. Chiral shift reagents (CSRs) or chiral solvating agents (CSAs) allow the relative configuration or enantiomeric excess of a mixture of chiral molecules to be determined by NMR through the formation of noncovalent diastereomers. This is important as the specific stereochemistry of a molecule determines the chemical, physical and biological properties of the species. Structures including cyclodextrins,132} Keplerate‐type structure provides an interesting inorganic framework for use in the study of host‐guest interactions  acts as a container for regioselective reactions to occur, and has the ability to separate and stabilise entrapped species. (where L: (R)/(S)‐lactate) possessing chiral internal cavities facilitated by the affixation of chiral ligands, referred to generally as {Mo132(lactate)30}  to an aqueous solution of {Mo132(SO4)30}. Single crystals, suitable for X‐ray crystallographic analysis, were grown by slow evaporation of the solvent over five days. Although the ligand exchange process is generally straightforward, in this instance more careful control of pH was required (pH 2.8) as pH values outside a narrow range caused the formation of precipitate or poor crystal quality. Single‐crystal X‐ray structure analyses reveal that the typical spherical {Mo132} framework is retained for all products. This molybdate framework can be regarded as an icosahedron in which twelve {(MoVI)MoVI5O21(H2O)6} pentagonal units are placed at the vertices and are linked by thirty {MoV2O4}2+ dinuclear linkers , all structures crystallise in the R‐3 centrosymmetric space group, rather than a chiral space group.The enantiomerically pure {Mo1H NMR and 13C NMR spectra of {Mo132((R)‐lactate)30} displays two sets of well‐resolved signals corresponding to free (R)‐lactic acid and coordinated (R)‐lactate ligands. Although the ligands are found in two positions upon dissolution, the majority of ligands (approximately 26/30) are retained within the cavity. The broad signals belonging to encapsulated ligands are found upfield (2.9 ppm (CH(1)) and 0.1 ppm (CH3(2), respectively) with respect to the corresponding sharp signals of the free lactic acid species . The two signal sets, which are rapidly established, indicate that the ligand exchange process which is initiated upon crystal dissolution is slow on the timescale of the NMR measurements. The coordinated ligands are replaced by water ligands, as has been previously reported.1H DOSY NMR where the diffusion coefficient decreased from 520 pm2 s−1 for the solvated molecules to 111 pm2 s−1 for the coordinated (R)‐lactate ligands within {Mo132} . The presence of both encapsulated and free, solvated lactates demonstrates that the {Mo132} framework is still intact in the solution and a proportion of the lactates are retained inside the cavity, available as chiral centres for enantioselective recognition. Additionally, the simultaneous existence of both peak sets demonstrates that the ligands are in a slow exchange system between their solvated free state and their coordinated, encapsulated state. To confirm our assignment of the 1H NMR spectra, HSQC NMR was performed. Typical broad and sharp peaks related to the coordinated and solvated lactate ligands are observed, respectively, at: [1HFREE/1HENCAPSULATED:13C] (CH(1):C‐β) 3.9/2.8 ppm: 69 ppm, and (CH3(2):C‐γ) 1.2 ppm/0.0 ppm: 19 ppm .The 132} structure. The presence of (R)‐ or (S)‐lactate leads to a CD response of their respective structures upon crystal dissolution. The obtained spectra of {Mo132((R)‐lactate)30} and {Mo132((S)‐lactate)30} display opposite ellipticity of one another, with peaks centred at 214 nm originating from the lactate ligands, demonstrating the clusters’ opposing optical activities as a result of the coordinated enantiopure ligands . Compared with free lactate, there are slight differences in the CD signals, with absorption effects of the {Mo132} framework perturbing the obtained spectra, however the opposing ellipticity of the spectra indicates that the configuration of lactate is preserved upon coordination to {Mo132}. The CD signal corresponding to {Mo132} is, however, not detected at higher wavelengths due to the rather strong adsorption arising from intervalence charge‐transfer between MoV and MoVI centres in {Mo132}, which greatly suppresses the CD response transferred by the lactate ligands.Although the ligand structure could not be resolved in the solid‐state, we wanted to confirm that the stereostable ligands had indeed retained their stereochemistry upon their coordination within the {Mo132(lactate)30} structures were used to confirm the complete replacement of the sulfate ligands . The characteristic triplet pattern, due to the v3 stretching mode of the bidentate SO42− ligands (C2v) in the 1030–1190 cm−1 range, is only present in the {Mo132(SO4)30} spectrum and is not observed in the {Mo132(lactate)30} spectra, thus indicating that the replacement of these ligands has been achieved.Finally, the IR spectra of the {Mo132} structures in solution, we examined the extent of their chiral recognition of enantiopure guests. We selected amino alcohol guests due to their favorable solubility in aqueous solution, which our {Mo132} POM host is readily soluble in, and, as their amine group is positively charged at the pH studied here (ca pH 3), primary interaction at the {Mo2}‐formed surface pores was anticipated. In addition, a lack of coordination donors precludes the guest from replacing coordinated ligands of the {Mo132} host. The preferred affinity of positively charged species to the surface pores has been previously shown by Cadot et al.,After confirming the configuration and stability of the chirally decorated {MoS)‐propranolol is an active beta‐blocker, whereas the (R)‐ isomer is ineffective in this application but has utility as a potential contraceptive.Amino alcohols have uses in industrial and pharmacological applications. Enantiopurity of amino alcohols is important in this context as (2O solution of {Mo132((R)‐lactate)30} resulted in the presence of an additional single set of sharp peaks in the 1H NMR spectrum. These sharp peaks, which were downfield shifted in comparison to the spectrum of the amino alcohol in D2O only, indicate that the interaction of the guest with the {Mo132} structure occurs under a fast‐exchange regime, in contrast to the two‐set slow‐exchange mechanism exhibited by the ligand species, described previously. Twelve equivalents of guest per host  was chosen for NMR study after titrations indicated that the peak profile of the guests remained unchanged up to and including this number of equivalents, except for increasing guest peak intensities. Beyond this value the peaks broadened and the resulting solution began to form precipitate due to the initial formation of a surfactant encapsulated cluster .Introducing 12 equivalents of the phenylalaninol guest species to a 5 mM D2O only, are denoted Δδ  will be given for the guest R isomer cf. the S isomer. For example, a shift for an R guest that is further downfield than the S form will be given a negative value (−ΔΔδ), and where the shift separation is reversed, with the (R) isomer more upfield in comparison to the (S)‐guest isomer, the separation will be noted with a positive sign (+ΔΔδ).Any peak shifts of the guest isomers, from their positions in Dδ Figure c. To remR)‐ or (S)‐lactate‐containing {Mo132} host structures was first investigated. The phenylalaninol peak positions shift downfield between δ=0.05 (CH(D)) and 1 ppm (CH(A)), from those positions exhibited by the guests in D2O only ‐lactate)30} host was used: CH(D) ΔΔδ −0.02 ppm, CH(A) ΔΔδ −0.05 ppm, and CH2(B) ΔΔδ −0.03 ppm, with the (R)‐phenylalaninol isomer more downfield shifted ‐lactate)30} host is used, these peak positions show opposite behaviour: CH(D) ΔΔδ+0.02 ppm, CH(A) ΔΔδ+0.04 ppm, and CH2(B) ΔΔδ+0.01 ppm, respectively ), indicating that this position is most likely closest to the interaction site with the {Moδ 4.1 ppm, located at the chiral centre of the guest molecule, showed distinct separation with both host enantiomers when the sample temperature was lowered. The separation, as taken between the two highest peaks, was ΔΔδ −0.060 ppm for the {Mo132((R)‐lactate)30} host and ΔΔδ +0.058 ppm for the {Mo132((S)‐lactate)30} host. Although small, these values are comparable to those observed for amino alcohols in D2O with alternative CSR hosts, such as resorcinarenes (0.018–0.347 ppm).[For fast‐exchange systems in NMR spectroscopy, lowering sample temperature generally leads to an increase in observed separations due to the reduced rate of exchange between the two possible guest environments. The above measurements were recorded at 300 K and therefore they were repeated at lower temperatures (278 K) to investigate this effect. The peak associated with proton CH(A)∼132(lactate)30} hosts as effective chiral shift reagents, the non‐equivalence of racemic guest peaks should be achieved. Addition of (R/S)‐phenylalaninol to either enantiopure {Mo132((R)‐lactate)30} or {Mo132((S)‐lactate)30} results in an expected similar overall shifting of peak positions, as observed for the addition of the enantiomerically pure guests. Comparison of the (R/S)‐phenylalaninol spectra with the enantiopure guest species and {Mo132((R)‐lactate)30} host highlights that the racemic guest mixture displays peaks which appear to be superimpositions of the two peak profiles exhibited for the enantiopure guests . The phenyl peak region (CH(D)∼δ 7.6 ppm) of the guest exhibits a peak profile which is different from those of the enantiopure guest species, appearing to contain two separate overlapping peaks, with minimal separation. The guest peaks relating to the two CH2 groups (B∼δ 3.8 ppm and C∼δ 3.2 ppm) show no significant differences to those observed for the enantiopure guests with {Mo132((R)‐lactate)30}. The peak related to the proton at the chiral centre (CH(A)∼δ 4.1 ppm) appears to contain two distinct peaks, which are related to the two isomer guest forms undergoing chiral recognition by the host. For {Mo132((S)‐lactate)30} with the same (R/S)‐phenylalaninol guest mixture, broadly similar peak positions and profiles were observed as described for the {Mo132((R)‐lactate)30} host . For each host species, there is no notable effect on the peak profiles of either the free or coordinated lactate ligands upon addition of the racemic guest species. The peak assignments and separations described were reflected in the HSQC spectrum at 278 K ‐lactate host and 63 pm2 s−1 for the (S)‐Lactate host. The free (R)‐ and (S)‐lactic acid possess diffusion coefficients of 282 and 275 pm2 s−1, respectively. Upon addition of (R/S)‐phenylalaninol to a solution of {Mo132((R)‐lactate)30}, the (R)‐guest molecule displays a diffusion coefficient of 91 pm2 s−1, whereas (S)‐phenylalaninol has a diffusion coefficient of 106 pm2 s−1. Similarly, addition of the guest to {Mo132(S)‐lactate)30} gives diffusion coefficients of 92 and 88 pm2 s−1 for the R and S guests, respectively. These results demonstrate significant guest interaction as well as a clear preference for homochiral recognition. That the (S)‐lactate host exhibits lower overall diffusion coefficients for both guests compared to the (R)‐lactate host, coupled with the greater downfield shift observed for the guest species with {Mo132((S)‐lactate)30} in the 1H NMR spectra, indicates that the strength of interaction of the phenylalaninol guests with {Mo132(S)‐lactate)30} is stronger than that of the same guests with the (R)‐lactate‐containing structure. However, the overall separating ability of each host structure is similar, although reversible in nature.In order to determine the feasibility of the {MoKa) using a single‐spectrum DOSY NMR method for the phenylalaninol guest species. This determination relied on an assumption of a 1 : 1 binding model between the guest and each of the host pore sites. Although analysis was performed to determine the binding ratio experimentally, a reliable figure could not be obtained due to a loss of spectral resolution above a 1 : 1 binding ratio. We expect the binding strength will be affected by allosteric effects of adjacent pore sites due to the concurrent sharing of individual ligands between two sites. For our calculation (see the Supporting Information for more details), the diffusion coefficient of the free guest species (Df) was taken as the value of the free lactic acid ligands, while the value corresponding to the {Mo132} host was taken as the value of the coordinated lactate ligands (Db). The observed diffusion coefficient of the guest (Do), the total guest concentration (GTOT), and the total host concentration (HTOT) are then used to determine the dissociation constant (Kd) and related association constant (Ka) according to Equation We were able to determine an approximate association constant (R and S guests with the {Mo132((R)‐lactate)30} host were 350(±5) and 190(±2) M−1, respectively. With the {Mo132((S)‐lactate)30} hosts, these values were effectively reversed, with 270(±2) and 390(±11) M−1 for the (R) and (S)‐phenylalaninol guests, respectively  1H NMR spectroscopy was applied to probe the resulting solutions  peak of the racemic amino alcohol guests arises, nonracemic  mixtures of the guest were added to the solutions. In a similar manner to those experiments with the racemic mixtures, the total number of guest equivalents added were maintained at twelve times the concentration of the {Mos Figure .132((R)‐lactate)30} host, when the ratio of R to S guest added was 3 : 1, the CH(A) peak related to the proton at the stereogenic centre (∼δ 4.1 ppm), displays the previously observed two peaks, with the more downfield shifted peak more intense than the other, attributing these to the R and S guest, respectively ‐lactate)30} host, the more downfield shifted peak is attributed to the (R)‐phenylalaninol guest, indicating that the extent of shift separation is (−ΔΔδ 0.06 ppm). This assessment can be confirmed by comparing this spectrum to that of the R : S guest in a 1 : 3 ratio, with the {Mo132((R)‐lactate)30} host. Here, the more intense peak is the more upfield shifted peak which is derived from (S)‐phenylalaninol.Firstly, for the {Moentre ∼δ .1 ppm, dR)‐ and (S)‐phenylalaninol scalemic mixture samples with the analogous (S)‐lactate‐containing {Mo132} host, was also performed ‐lactate)30} host is +ΔΔδ 0.06 ppm, with the S guest more downfield shifted. Again, by comparison of these results with the 1 : 3 (R)‐ to (S)‐phenylalaninol mixture, the more intense peak here is the more downfield shifted resonance, confirming that this peak relates to that of the (S)‐phenylalaninol guest. These measurements confirm that the separation of chiral amino alcohol guest species is entirely dependent upon the configuration of the ligands present on the {Mo132} host structure.Comparison of the above  and (S)‐2‐amino‐1‐phenylethanol guests, with the resulting spectra indicating that the most downfield shifted guest is the R isomer when the {Mo132((R)‐lactate)30} host is used, and the S guest is the most downfield shifted peak when the {Mo132((S)‐lactate)30} host is used .A similar measurement was performed for the (132((R)‐lactate)30} host, with the (R)‐ to (S)‐phenylalaninol guest added in a 3 : 1 ratio, the resulting integral ratios were 3:0.95, while for the 1 : 3 ratio the integral ratio was 0.96 : 3. These results therefore highlight the ability of the {Mo132(lactate)30} system to determine approximate enantiomeric excesses of these amino alcohol species.We were able to perform crude integration of the two peaks observed, roughly determining the enantiomeric excess of guest added to each sample. For the {Mo132(lactate)30} system, several control reactions were performed. The first of which involved the separate addition of enantiopure phenylalaninol guest isomers to a solution of D2O containing the enantiomerically pure (R) or (S)‐lactic acid ligands only . The phenylalaninol peak positions shifted downfield by CH(D) Δδ 0.0/0.0 ppm, CH2(B) Δδ +0.2/0.2 ppm, CH(A) Δδ +0.5 ppm (slightly obscured by the CH2(B) resonances), and CH2(C) Δδ +0.2/0.5 ppm, respectively, from those positions exhibited by the phenylalaninol guests in D2O only. A lack of enantiomer peak separation here indicated that the influence of the free lactic acid molecules is limited to non‐specific shifts exhibited by each guest isomer.After investigating the effect of racemic guest isomer addition to our {Moy Figure b, c. Thi132(SO4)30} in D2O only  Δδ +0.15/0.07 ppm, CH2(B) Δδ +0.4/0.5 ppm, CH(A) Δδ +0.8 ppm, and CH2(C) Δδ +0.45/0.75 ppm, respectively. All peaks of each isomer showed slight separation from one another of CH(D) ΔΔδ +0.02/+0.01 ppm, CH2(B) ΔΔδ +0.02/+0.02 ppm, CH(A) ΔΔδ +0.03 ppm, and CH2(C) ΔΔδ +0.02 ppm, with the (S)‐phenylalaninol isomer more downfield shifted for all peaks. This increased shift separation indicates that the {Mo132} host structure itself facilitates isomer separation, which may be caused by the formation of diastereoisomers either by interaction of guests with neighbouring pores or by guest facilitating stereoselective sulfate ligand hydrolysis to create an axially chiral pore. Unfortunately, an experimental basis to understand this phenomenon has not yet been achieved, however we are interested in understanding this behaviour as an ongoing endeavour. It should be noted that the overall shifting of the guest peaks is increased when either of the {Mo132(lactate)30} hosts are used, highlighting the increased interaction strength when the lactate ligands are present, which is subsequently manifested in the extent of peak separation.In another control experiment, the enantiopure phenylalaninol isomers were added separately to a solution containing {Moy Figure a. This w132} spherical‐type POM structures. In doing so, we have used extensive NMR methods to characterise the structures in the solution state. Following this, we used these newly functionalised species to enantioselectively recognise cationic amino alcohol guests in aqueous solvent, performing the role of a chiral shift reagent. Although solid‐state characterisation of the host‐guest interaction could not be achieved, we hypothesise that the electrostatic interaction between the cationic ammonium group of the guest and the electronegative {Mo132} host, coupled with hydrogen bond interactions between host‐bound chiral lactate ligands and guest isomers, facilitates the formation of diastereomeric complexes. The resulting separation process is comparable to previously reported methods that exclusively used organic species and were primarily limited to nonaqueous solvents, representing a new application of the POM species. The work completed here focused on the role of the {Mo2}‐formed pore as the site for interaction with chiral guests and therefore investigation of the internal cavity as a second site for chiral discrimination is a viable potential extension of this methodology, with a focus on the recognition of guests with more diverse functionalities.We have successfully isolated and characterised new chiral‐containing {MoThe idea for the work was conceived by L.C., the synthesis was done by Z.L.S., the main hypothesis development and work plan and NMR measurements by R.W.P. with help from N.W., N.L.B. and Y.M.A. Structural analysis was performed by Z.L.S. and D.L. D.L. and N.L.B. led the clusters team a with help from L.C. The manuscript was written by R.W.P. with input from all the authors.1898694 contains the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.All experimental procedures, data and equipment parameters can be found in the Supporting Information. Deposition Number The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Larger animal models are therefore a key step on the path to clinical translation of the technology. Herein, the current state of orthopedic gene therapy research in preclinical large animal models was investigated based on performed large animal studies. A summary and an outlook regarding current clinical studies in this sector are provided. It was found that the results found in the current research literature were generally positive but highly methodologically inconsistent, rendering a comparison difficult. Additionally, factors vital for translation have not been thoroughly addressed in these model systems, and the risk of bias was high in all reviewed publications. These limitations directly impact clinical translation of gene therapeutic approaches due to lack of comparability, inability to demonstrate non-inferiority or equivalence compared with current clinical standards, and lack of safety data. This review therefore aims to provide a current overview of ongoing preclinical and clinical work, potential bottlenecks in preclinical studies and for translation, and recommendations to overcome these to enable future deployment of this promising technology to the clinical setting.Bone defects and improper healing of fractures are an increasing public health burden, and there is an unmet clinical need in their successful repair. Gene therapy has been proposed as a possible approach to improve or augment bone healing with the potential to provide true functional regeneration. While large numbers of studies have been performed  While bone is highly competent at regeneration , a varieComplications in small fracture healing are commonly seen in older patients or those suffering with comorbidities, for example, disorders associated with systemic inflammation such as diabetes , 2012. TWhen considering orthopedic bone repair, gene therapy has several advantages over competing methods. The present standard of regenerative, intraoperative care for such defects is a bone autograft. Bone is removed from a healthy donor site on the patient  and then used to fill the original defect see  or spaceex vivo gene therapy, a combination of cell and gene therapy (CGT). In such approaches, cells are removed from the patient or donor, transduced or transfected in the lab, screened for successful modification, and then reintroduced to the patient  family of growth factors, due to their vital role in bone development, homeostasis, and repair . A populEach combination of delivery method, target cell population, and vector has a unique combination of positives and negatives, but common issues include targeting the correct cell population and establishing a suitable dose . New appResearch and development of regenerative approaches for bone repair does not differ in terms of its iterative preclinical approach from other biomedical research fields; in fact, the large majority of preclinical animal research has been performed in small rodent models. While small rodent models have many advantages, they often fail to recapitulate key aspects of human biology . ConsequWhile preclinical animal experiments are required for the large majority of new drugs and treatments developed in Europe and the United States , regulatThe PICO  question that the review attempts to answer is as follows: Has gene therapy been successfully applied to regenerate bone in large animal models? We also aimed to assess the various methodologies and approaches used across the field, making a thorough survey of methods that have been employed in large animal models. Finally, we assessed the risk of bias (RoB) of all of the publications using an established framework.The information gathered to answer these questions was then used to conduct a technology readiness assessment (TRA). A TRA aims to estimate the technology readiness level (TRL) of a technology, in this case gene therapy for orthopedic bone regeneration. TRAs were first developed by the National Aeronautics and Space Administration (NASA) to allow consistent discussion of technical maturity across various technologies but have now become a popular assessment technique in many diverse fields. Simply, TRAs rank a technology on a 9- or 10-point scale of TRLs, with higher values indicating technological maturity. In bioscience/medicine, these scores correspond to closeness to clinical translation. TRAs are still uncommon in the fields of biomedicine and biotechnology, and consequently, there is no widely accepted framework for their use. In this case, a slightly modified version of the United States Department of Defense TRA Deskbook guidelines for biomedical TRLs  has beenThe databases MEDLINE, EMBASE, and BIOSIS were searched in August 2019 for terms relating to animal models, gene therapy, and bone. Controlled or curated vocabulary was used for all databases, with an additional topic search added to the BIOSIS search due to a lack of sufficiently specific curated terms. The initial search returned 1,483 publications, which were then taken for filtering see .See the References were exported from the databases into the reference management software EndNote X7. EndNote’s duplicate detection function and a manual search were used to filter for duplicates. A relevance search was then conducted based on the listed inclusion and exclusion criteria.Inclusion criteria were as follows:•Use of gene therapy,•Focus on bone repair,•Use of large animal models, and•English language.Exclusion criteria were as follows:•Exclusive use of small animal models ;•in vitro; andExclusively •Review articles, letters to the editor, and other non-research article types.A first pass was made by searching the titles for terms associated with exclusion criteria . Identified titles were manually examined and excluded if a reasonable assumption that exclusion criteria were met could be made . The remaining publications were manually screened, with both the titles and abstracts consulted. Finally, an availability check was conducted. See https://github.com/intonumbers/pubmed-insights.In order to provide a summary overview of the most common and recurring topics shared by the selected systematic review publications, we have developed a Python script, which automatically creates a visual representation of the most common key phrases, involved authors, journals, and publication years. We provide and maintain the current version as Jupyter Notebook under the following GitHub Repository: First, we have queried the Entrez query and database system at the National Center for Biotechnology Information (NCBI) via their Entrez Programming Utilities (E-utilities) API for the corresponding 33 selected publications .To extract the most frequent key terms, we have included the values of the following keys per fetched publication dataset:•Title,•Abstract,•Results,•Keywords, and•Conclusions.1 built-in Morphy function. Next, we have formed all n-grams between word-size 2 and 5 for all key–value pairs per publication dataset except for the keywords key–value pair, where we have defined each entry as n-gram regardless of their size.Our data munging process’s first step was to remove all punctuation marks, special characters, and figures for each of the mentioned key–value pairs and replace all capital letters with their corresponding lowercase letters. We then removed common stop words (detailed stop-words list see GitHub repository) and lemmatized the remaining words using NLTK WordNet’sn-gram duplicates per publication dataset.Afterward, we have removed all n-gram occurs by the size of the corresponding n-gram and manually removed less meaningful n-grams , therapeutic gene/s, promoter of therapeutic gene/s, therapy dose , therapy delivery site, time between creation of defect and administration of therapy, length of experiment, methods used to investigate bone regeneration, and the methods used to investigate immune response to, persistence of, and localization of gene therapy.The following study characteristics were extracted from each publication: model species, number of animals, defect site/s, defect size and type, fixation method, inclusion and type of cells, inclusion and type of a scaffold/construct, vector, nature of therapy  RoB tool for animal studies . The tooAll publications were assessed independently by two reviewers (PW and TB) for both extracted study characteristics and the SYRCLE RoB tool. Results were then compared and discussed, with the consensus view presented here.Gene Therapy, the Journal of Orthopaedic Research, and Biomaterials.Automated machine reading data analysis of all 33 publication abstracts of the manuscripts selected for systematic review illustrates that the most common converging topic areas are centered around the use of bone marrow stromal cells (BMSCs) and BMP genes . FurtherThirty-three publications were identified as relevant during the search. These publications were found to have used five different species. Dogs were used by eight publications , goats bA wide variety of defect sites were investigated. Eight publications created defects in the maxillofacial region . The specific sites investigated were the mandible , inner oIn long-bone studies where fixation was potentially necessary, six used internal fixation methods; five used plates ; one useAll publications delivered their gene therapy directly into the defect. There was little variability in the length of time between the creation of the defect and the application of gene therapy. The large majority of publications applied their therapy immediately after defect creation, but five left several days between defect creation and therapy application. No chronic models were used. One publication applied their therapy 5 days post defect creation , while fL-lactide acid) (PLLA) (co-glycolic acid) (PLGA)-only scaffold  (PLLA) , and pol) (PLLA) , 2013 wescaffold . Two stuscaffold . Eight pscaffold . See Tabex vivo approaches  promoter or variants such as immediate-early (CMV-IE) . One pubin vivo or ex vivo application. Frequently, scaffold-based applications (largely ex vivo) did not provide information regarding multiplicity of infection and/or gene transfer efficiency. Sixteen publications failed to provide one or both of these pieces of information . All four scaffold-free viral in vivo approaches used roughly similar amounts of vector, between 1 × 1011 and 5 × 1011 particles (8 and ∼1.6 × 109 particles/kg (data available on request).Twenty publications used cells as part of their therapy. Autologous BMSCs were overwhelmingly the most popular option, being used by 11 publications . Variousormation . The pic was not . In a ha was not . Non-virlligrams , while tlligrams  or 1 mg lligrams . See Tabarticles . Dosage Four families of techniques were commonly used to investigate regenerated tissue. These were 2D radiograph, CT and μCT, histology and biomechanical testing see . Two-dimin vitro; however, only attempts to investigate persistence and the other factors in vivo were included at this stage. Eleven publications made some attempt to investigate the immune response to their therapy  of studies   . It is in = 11)  .p-values or histology or radiographic images for every time point and group. More serious failings included not clearly indicating numbers of animals used or group sizes, providing no radiographic images despite extensive use of the technique and providing no histology images despite extensive use of the technique. Only four publications were found to have provided all expected data and information. Several other sources of bias not directly covered by the assessment were identified across the publication set. Fourteen publications did not indicate that they had tested if their data were normally distributed prior to applying a parametric statistical test. Rarer issues included exceptionally small group sizes (n = 1 or 2) and use of animals for simultaneous experiments not included in the publication. The full results of the RoB assessment are available on request.The SYRCLE RoB assessment revealed that a large majority of the publications had not considered or mentioned most factors covered by the tool . Sequence generation  was only randomized by six publications, with none of these stating which randomization method was used. Six additional publications indicated they had randomized treatment sites within animals or used a blocking strategy, although again none stated which randomization method was used. The remaining 21 publications made no indication that randomization had been considered. Only one publication clearly indicated that animal baseline characteristics had been considered during group allocation. Two others stated that they had tried to balance groups based on only a subset of our defined baseline characteristics or additional factors. The remaining 30 publications did not address group balance. The assessment factors of group allocation concealment, random animal housing, investigator blinding at the treatment stage, and random assessment at the outcome stage were not considered by any publications. Investigator blinding at the analysis stage was performed by two publications for portions of their data. No publications blinded investigators for all of their data. The remaining 31 papers did not indicate that blinding at the data analysis stage had been considered. Thirteen publications clearly addressed the subject of incomplete experimental outcomes, either stating none had occurred or listing their number and nature. Only one publication clearly stated that there had been incomplete outcomes during the study but did not provide the number or nature of these events. Nineteen publications did not mention incomplete outcomes. While it is possible none occurred in these studies, it cannot be safely assumed. Selective outcome reporting  was common, with 29 publications identified to have failed to provide some information. The large majority of these cases were minor, with common omissions including failing to provide non-significant The publications reviewed here cover a wide range of approaches to gene therapy for bone regeneration in large animal models. This is both a blessing and a curse. It is clearly beneficial to attempt different approaches to establish which methods may be the most effective. However, there is currently such little consistency between studies that it is very difficult to compare effectiveness. Some simple trends across the literature can be clearly seen, for example, aspects of vector design, the use of orthotopic test sites, and bone analysis methods. However, almost all other elements of experimental design are highly variable, including crucially model species, defect sites, and specifics of analysis techniques.ex vivo approaches. Consequently, a direct comparison of results was not possible between the large majority of publications in the set, and even among those that were relatively similar, it was thought to be unproductive due to the numerous differences in experimental design. This made it impossible to thoroughly answer the sub-questions posed at the start of the review.While it is difficult to make generalizations, it seems that species choice was primarily influenced by practicality and researcher familiarity rather than advantages of the model system. Defects in horses were typically small and never larger than 15 mm , while dDespite difficulties in the comparison, there is a general trend of positive results among the set. Near universally, some form of improvement versus control groups was seen, with only two publications showing unambiguously negative results . It is oThe generally positive results found by the first portion of the review are called into question by the RoB assessment, which found that investigators are universally doing a poor job of avoiding bias. Of the nine core factors covered by the assessment, no publication satisfactorily addressed more than two, and there was no indication that many of the factors were considered by any of the publications at all. The only two factors considered by a substantial fraction of the set were incomplete outcome reporting and group randomization; and even so, no publications reported randomization methods satisfactorily. In terms of additional factors, the most serious was thought to be potential statistical errors. Though many publications used parametric tests, many failed to check if such approaches were appropriate for their data. Overall, it would seem that researchers are either unaware of or unconcerned by the risks of bias, which can potentially lead to erroneous positive results and cumulatively do great harm to the progress of the field if widespread. For example, in the field of stroke, it has been reported that apparent substantial improvements in outcome can be largely attributed to bias, leading to a great deal of wasted time and resources pursuing treatments that were actually much less effective than first thought . Bias isIn terms of technology readiness and closeness to translation, it seems that there is still some way to go. While results were generally positive, factors vital for gene therapy translation were routinely ignored. For the purposes of this review, the three factors of immune response, persistence of transgene expression, and biodistribution were taken as a yardstick for closeness to translation. Very few publications attempted to investigate these factors in any capacity, and those that did typically did not perform a thorough investigation see . ConsequOutlook and Current Status Regarding Clinical Studies and Authorized Clinical Gene Therapeutics for Orthopedics section, it is essential to clearly classify the type of drug developed and applicable translational pathway as well as to perform early engagement with regulatory authorities to inform translation-enabling preclinical studies, ensuring that study design and data procurement would either support or directly feed into preclinical data packages required for investigational new drug (IND)-enabling studies prior to clinical trials.To improve the comparability of research in the field and help bring gene therapy for bone regeneration closer to translation, we suggest that several principles of experimental design, large animal models, and investigative approaches should be defined and broadly adhered to by the research community. This would help to reduce bias and allow comparisons to be more easily drawn between different studies without restricting investigators to particular therapeutic approaches. In the following section, we propose potential standards in these areas. Our recommendations here are summarized in In Vivo Experiments” (ARRIVE) guidelines laid out by the United Kingdom National Centre for the replacement, refinement, and reduction of animals in research (NC3Rs)  . Additio (NC3Rs) . While ATo investigate non-inferiority in comparison with current treatment approaches, we recommend that investigators always include a group treated with the current clinical gold standard, autologous bone graft. Investigators could also include a group treated with a recombinant version of their transgene as an additional positive control.We propose two different model systems, for load-bearing and non-load-bearing defects.Load bearing: Sheep, femoral mid-diaphysis full-thickness osteotomy/segmental defect.Non-load bearing: Mini-pig, calvarial full-thickness oval defect.For load-bearing defects, we have selected the sheep femoral defect. While it could be argued that pigs are more representative of human bone in some regard , sheep aAs we have previously stated in this review, there is no clearly superior animal model for bone regeneration research, and as such, our recommendations here are fairly arbitrary. We are aware of the irony of suggesting our own arbitrary guidelines after criticizing authors for not providing adequate justification for model choice; however, we hope that the very act of standardization could prove highly beneficial to the field. If these suggestions prove to be impractical, we hope they will at least spur discussion of more appropriate standardized options.We recommend authors place a greater emphasis on quantitative techniques such as biomechanical testing and μCT imaging and reduce their reliance on qualitative and semi-quantitative approaches such as histology and 2D radiographs. Quantitative techniques are vital for formally demonstrating the non-inferiority of novel approaches to the existing clinical gold standard and would allow a more valuable comparison of data between studies. Qualitative and semi-quantitative methods still have value as supporting information, and standardization of approaches would further improve their usefulness. Histology can provide extremely useful information that is not provided by other methods if good standards of practice, blinding, and evaluation are maintained. Novel, automated methods in digital histology and machine learning approaches certainly have the potential to revolutionize this approach and provide reliable results in the future . UndoubtIn summary, we would like to suggest possible standards for improving the quality, comparability, and reproducibility of outcome measures and procedures for future preclinical work in the field of orthopedic gene therapy  for the treatment of chronic lower limb ischemia. After three clinical trials , the drug was approved in the Russian Federation in 2011 . This peculiarity of the Russian regulatory environment for combination products allowed a phased approach wherein an authorized gene therapy drug can be subsequently combined with a biomaterial and repurposed as a GAM. Thereby, for the new indication of orthopedic gene therapy, an accelerated path to translation can be pursued wherein the medical device pathway can be followed even if the resulting product is a cATMP. This means, however, that for new approaches that are developed, all components would need to follow separate translational pathways . Therefore, this can only be regarded as an advantage for accelerated translation if there is already a separate gene therapeutic that can be repurposed and integrated to a biomaterial for the subsequent indication.Gene therapy for bone regeneration is now clearly on its way to clinical practice; however, the clinical data are currently limited, and no human trials have been performed outside the Russian Federation. Continued successful clinical translation of regenerative orthopedic gene therapy products is limited by several factors closely associated with the particular complexities of different regulatory environments. An excellent review of EMA and FDA frameworks for advanced therapy medicinal products (ATMPs) has been provided by  in 2011  as a genThis example illustrates that there are still significant worldwide differences in regulatory approaches to ATMP translation and that orthopedic gene therapy in particular encompasses a complex array of different approaches that can be classed as either GTMPs/CGTs or combination approaches with different terminologies, definitions, and regulatory requirements. Even relatively closely aligned regulatory frameworks such as EMA and FDA exhibit significant differences that result in the requirement of independent translational regulatory pathways to market authorization if one desires to bring an orthopedic gene therapy to the clinic for patient benefit. Ultimately, it would be highly desirable to facilitate harmonization and convergence  of diffeThe original contributions presented in the study are included in the article/PW wrote the major part of the manuscript and performed the systematic review. IB wrote the clinical trials part and edited and reviewed the manuscript. TB was the second reviewer performing the systematic review. PJ wrote the program and performed the machine reading analysis. EJ contributed to the manuscript text and edited and reviewed. RD performed the systematic analysis of histological methods, contributed to the manuscript text, and reviewed and edited the manuscript. PG participated in conceptualization, contributed to the clinical aspects of the manuscript and reviewed and edited the manuscript. GF proposed the manuscript, conceptualized its contents, supervised PW and TB, wrote the translational part of the manuscript, and reviewed and edited and handled submission. All authors contributed to the article and approved the submitted version.pro bono, non-salaried consultant for Histograft, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.IB is the CEO of the company Histograft, LLC. PJ is the CEO of the company Into Numbers Data Science GmbH. GF works as


IgG4-related ophthalmic disease (IgG4-ROD) is one of the phenotypes of IgG4-related disease (IgG4-RD), and its lesions are mainly located in the ocular. Currently, there are few studies on IgG4-ROD and no study has compared the phenotypic differences between IgG4-ROD and non IgG4-ROD (nIgG4-ROD). Thus, it is difficult to establish the optimal treatment strategy for IgG4-ROD. The aim of this study was to identify the disparities between the two groups and to clarify the risk factors for IgG4-ROD relapse.434 IgG4-RD patients met comprehensive diagnostic criteria and diagnosed at Peking University People’s Hospital between January 2009 and January 2020 were recruited in this study. Patients were divided into IgG4-ROD and nIgG4-ROD group according to the ophthalmic involvement. Demographic, clinical, and laboratory data of two groups were collected and compared. Cox regression analysis was used to identify the independent risk factors for IgG4-ROD relapse.255 IgG4-ROD patients were identified in this study. IgG4-ROD group had almost equal sex ratio, younger age of disease onset and diagnosis comparing with nIgG4-ROD patients. As compared to nIgG4-ROD group, higher percentage of IgG4-ROD patients met the 2019 American College of Rheumatology/European League Against Rheumatism classification criteria (AECC) for IgG4-RD; moreover, IgG4-ROD patients had higher AECC scores and IgG4-RD responder index (RI). Allergic diseases and multiorgan involvement were more common in IgG4-ROD group. IgG4-ROD was frequently associated with salivary gland, paranasal sinus, lung, and lymph node involvement, while retroperitoneal fibrosis and biliary system lesions were more common in nIgG4-ROD. IgG4-ROD patients had higher serum IgG4 levels, IgG4/IgG ratio, IgE levels, and lower CRP levels. The initial glucocorticoid plus immunosuppressant was a protective factor for IgG4-ROD relapse. IgG4-ROD patients treated with initial glucocorticoid plus immunosuppressant had longer relapse-free survival time than patients treated with initial glucocorticoid monotherapy.IgG4-ROD patients had distinctive clinical features compared with nIgG4-ROD patients. The initial glucocorticoid plus immunosuppressant was a protective factor for IgG4-ROD relapse, which could prolong the relapse-free survival time of IgG4-ROD patients. These findings may have important implications for understanding and management of IgG4-ROD. IgG4-related disease (IgG4-RD) is a new clinicopathological entity, originally proposed by Kamisawa et al. in 2003 . AlthougIn the past, different terms have been used to name IgG4-RD with ocular lesions –14. HoweThe use of systemic glucocorticoids as first-line therapy for IgG4-ROD usually leads to an effective initial response . HoweverIn this study, we focused on comparing the clinical features between IgG4-ROD and nIgG4-ROD patients and exploring the risk factors of IgG4-ROD patients. The aim was to identify the disparities between the two groups and to clarify the risk factors for IgG4-ROD relapse, so as to further improve the understanding of IgG4-RD with different phenotypes and help to better formulate diagnosis and management strategies.This retrospective study was conducted in the Peking University People’s Hospital, a total of 434 patients with IgG4-RD who have attended at our hospital between January 2009 and January 2020 were recruited. Patients with infections, tumors, or other autoimmune diseases were excluded from this study. All enrolled patients met the IgG4-RD comprehensive diagnostic criteria : (1) cliAll patients’ clinical data including age, sex, history of allergic disease, initial lesion site, number of organs involved, and ophthalmic symptoms were collected from medical records. IgG4-RD responder index (RI)  and 2019t test or Wilcoxon rank sum test for quantitative variables, and chi-square test or Fisher’s exact test for categorical variables. Univariate and multivariate Cox regression analyses (Enter method) were performed to identity the possible risk factors for IgG4-ROD relapse. Variables with P < 0.2 in the univariate analysis were included in the multivariate analysis. The Kaplan-Meier survival curve and log-rank test were used to compare the difference of the initial treatment options on the relapse of IgG4-ROD. All the data were processed by the IBM SPSS Statistics version 23.0 software. Significant differences were defined as P < 0.05.The quantitative variables were described as mean ± standard deviation (SD) or median . Categorical variables were presented as proportions. Differences between groups were analyzed using two independent samples P < 0.001). The mean age of disease onset in IgG4-ROD group and nIgG4-ROD group was 51.1 ± 13.2 years and 55.5 ± 13.5 years, respectively, suggesting the former had an earlier disease onset (P = 0.001). The mean age of disease diagnosis in IgG4-ROD group was 54.4 ± 12.6 years, which was younger than that in nIgG4-ROD group, but there was no statistical difference between the two groups , 28 (6.5%), and 194 (44.7%) patients were diagnosed as definite, probable, and possible IgG4-RD, according to the IgG4-RD comprehensive diagnostic criteria. Among these patients, 255 cases were identified with IgG4-ROD and another 179 cases were diagnosed with nIgG4-ROD. The demographic characteristics of the patients are displayed in Table P < 0.001); moreover, IgG4-ROD patients had higher AECC scores (P < 0.001) and IgG4-RD RI (P = 0.045). Notably, the prevalence of allergic diseases  in IgG4-ROD group was higher than that in nIgG4-ROD group (P < 0.001)  had multiple organ involvement. By comparison, it was found that the median number of involved organs and the percentage of ≥ 4 organs involved in IgG4-ROD group were significantly higher than those in nIgG4-ROD group  and IgG4/IgG ratio (P < 0.001) than nIgG4-ROD patients. IgG4-ROD group also had higher serum IgE levels and the higher proportion of patients with elevated IgE than nIgG4-ROD group . While nIgG4-ROD patients had higher CRP levels and the higher proportion of patients with elevated CRP . In addition, higher ESR levels were also found in nIgG4-ROD group, but there was no statistical difference between two groups. In our study, no significant differences were found in the eosinophilia, complement, globulin, IgG, ANA, and RF results between the two groups.The laboratory parameters of the two groups are summarized in Table P < 0.001).Univariate Cox regression analysis revealed that sex, age of disease onset, and age of disease diagnosis; levels of serum C4, ESR, CRP, and IgG4; and initial treatment options were associated with IgG4-ROD relapse Table . MultivaIgG4-RD is an immune-mediated systemic fibroinflammatory disease featuring tumefactive lesions, which can affect almost every organ system in the body. IgG4-ROD refers to IgG4-RD with ophthalmic manifestations and is one of the phenotypes of IgG4-RD. There are currently few studies focusing on IgG4-ROD, and no research has explored the differences in the clinical features between IgG4-ROD and nIgG4-ROD. Thus, it is difficult to establish the best treatment strategy for IgG4-ROD. Therefore, we conducted a large retrospective cohort study to compare demographic characteristics, clinical presentations, and laboratory parameters between IgG4-ROD patients and nIgG4-ROD patients. In addition, the risk factors for IgG4-ROD relapse were also identified.Currently, there is no exact data on the epidemiology of IgG4-ROD due to the lack of well recognition and wide understanding of IgG4-ROD. Several previous studies conducted in Japanese and US populations have reported that the frequency of IgG4-ROD ranges 4 to 34% of IgG4-RD patients –24. WhilOur research showed that IgG4-ROD patients had almost equal sex ratio, younger age of disease onset and diagnosis, and higher IgG4-RD RI. In addition, the initial lesion sites in majority of IgG4-ROD patients were mainly located in the ocular (41.6%) and salivary gland (34.9%), which were directly exposed to the external environment and lack barrier protection. The damage of tissue and immune tolerance to autoantigen in genetically susceptible individuals after exposure to certain environmental factors  is considered to be a plausible mode of pathogenesis of IgG4-RD . The reaIgG4-RD RI as a tool to evaluate the disease activity of IgG4-RD was used in this study. The higher IgG4-RD RI in IgG4-ROD group suggested that IgG4-ROD may have higher disease activity and may require more aggressive treatment. PET/CT is also considered to facilitate the management of IgG4-ROD, due to its good performance in detecting organ involvement, differentiating active and inactive lesions, and monitoring treatment response and relapse during IgG4-RD diagnosis and follow-up , 26; howWith regard to the affected ocular anatomic structure in IgG4-ROD, we found that the lacrimal gland and extraocular muscle were most frequently involved, while other anatomic structures were rarely involved, which is highly in line with previous researches , 17. SimIn terms of laboratory parameters, IgG4-ROD patients had lower levels of ESR and CRP, which may reflect the absence of a higher activity of acute inflammation in IgG4-ROD. As we all know, elevated serum IgG4 concentration is one of the hallmark features of IgG4-RD. Previous studies have confirmed that serum IgG4 tends to be higher in IgG4-RD patients with the lacrimal or salivary gland , 28 and It is currently believed that abnormal immune response against infection, allergen, or tissue injury may be the initiating factors in the pathogenesis of IgG4-RD, and then triggers can activate T follicular helper (Tfh) and T follicular regulatory (Tfr) cell immune response, and the subsequent production of IL-4/IL-10 cytokines can promote the production and class switch of IgG4 and IgE in plasmablasts, eosinophil recruitment, and fibroblast activation , 30, 31 The phenotypic differences of IgG4-RD have been more clearly understood after IgG4-RD was classified into four different groups: pancreato-biliary (group 1), retroperitoneum/aortitis (group 2), head and neck limited (group 3), and Mikulicz/systemic (group 4) . When grThere are currently no clinical treatment guidelines to instruct the diagnosis and treatment of IgG4-ROD. The use of systemic glucocorticoids as first-line treatment for majority of IgG4-ROD patients usually results in the improvement of symptoms and decrease of serum IgG4 levels; however, the relapse is not uncommon during glucocorticoid tapering or withdrawal , 18. In Our study has some limitations. First, our study suffers from the limitation of its retrospective design, non-randomized controlled investigation, and treatment protocol. Second, this study was conducted in a single rheumatology center, thus possibly leading to a selection bias that cases with multiple organ involvements were reported.In conclusion, our study demonstrated and confirmed on the largest cohort of IgG4-ROD patients that IgG4-ROD showed younger age of disease onset and diagnosis, and almost equal sex ratio. IgG4-ROD group had higher AECC scores and IgG4-RD RI. Allergic diseases and multiorgan involvement were more frequent in IgG4-ROD group. Moreover, IgG4-ROD patients had higher serum IgG4 levels, IgG4/IgG ratio, and serum IgE levels. The salivary gland, paranasal sinus, lung, and lymph node involvement were more frequent in IgG4-ROD patients. In addition, initial glucocorticoid plus immunosuppressant was a protective factor, which could prolong the relapse-free survival time of IgG4-ROD patients. These findings may have important implications for the understanding and management of IgG4-ROD.


Antibiotic stewardship continues to be health concern that physicians often acknowledge, but whose real-life practices do not reflect that awareness. There is a wide range of opinions on the efficacy of the type of modality that is most effective to teach stewardship. Our project addresses resident needs specifically, with coverage in four topics—proper antibiotic dosing, IV to PO transitioning, duplicate coverage, and antibiotic time outs.Categorical Internal Medicine residents in PGY 1-3 were sent an optional 48-question Likert survey querying needs in the above four topics.General Demographics. Resident response was 35%, with equal representation from all PGY years. Over half reported no ID or stewardship elective exposure and 74% agreed they could benefit from further education on stewardship . Proper Dosing Educational Needs. Of residents, 68% reported feeling confident about where to find information on dosing antibiotics for a given condition/organism , but only 37% were comfortable with establishing an initial dose. When a range was suggested, 55% of respondents admitted to at least “sometimes, often, or always” choosing the highest suggested dose by default. IV to PO transition. Residents preferred (76%) and used (89%) IV antibiotics by default in an inpatient setting. Nearly 45% of respondents reported “sometimes or rarely” feeling comfortable in making an IV to PO transition, and 40% “often or always” avoid PO transition until discharge . Duplicate Coverage. Over 70% of residents reported they “sometimes, rarely, or never” felt confident in stopping double coverage themselves when started by the primary team . Antibiotic Time Out. Only 17% of respondents had heard of an antibiotic timeout, and only 8% have ever used one ; 80% of residents had no structured way to review usage and 53% reported “sometimes or often” forgetting about assessing for de-escalation daily.Figure 1. Resident DemographicsOur anonymous, optional survey attracted a 35% response rate from the categorical residents at our suburban program spread over two tertiary hospitals with >1200 beds total. Most had not received prior training in infectious disease or stewardship, yet most recognized antibiotic overuse and resistance as a major, ongoing problem.Figure 2. Resident responses on proper dosing and IV to PO questions.(A) Residents appear most uncomfortable with initial antibiotic dosing and seeking additional sources for best dosage when commonly used sources suggest a range of possible doses. (B) Majority of residents preferred and used IV antibiotics, and commonly transitioned to PO only at patient discharge. Some residents reported discomfort with establishing equivalent IV to PO transition dosages.Figure 3. Resident responses to questions regarding duplication of therapy and antibiotic time outs.(A) Though many could and had recognized duplication of therapy on the wards, several participants reported at least some discomfort in independently stopping double coverage. (B) Most residents had not heard of or utilized an antibiotic time-out or any other structured method to re-assess their antibiotic use on daily rounds. As such, 41% of respondents admitted they would likely just continue initial, broad-spectrum therapy.Our analysis aimed to establish resident educational needs in four major topics in stewardship. Gaps in knowledge include timing transition from IV to PO, initial antibiotic dosing, stopping double-coverage, and lack of awareness of timeouts. This needs assessment will be used to build an antibiotic stewardship curriculum for IM residents.All Authors: No reported disclosures


The study was devoted to the numerical modelling of concrete-to-concrete interfaces. Such an interface can be found in many modern composite structures, so proper characterisation of its behaviour is of great importance. A strategy for calibration of a model based on cohesive finite elements and the elastic-damage traction–separation constitutive law available by default in the Abaqus code was proposed. Moreover, the default interface material model was enhanced with the user-field-variables subroutine to include a real strength envelope for such interfaces. Afterwards, the modelling approach was validated with numerical simulation of the most popular tests for determining the strength characteristics of concrete-to-concrete interfaces: three-point bending beam with a notch, splitting bi-material cubic specimens, and slant-shear tests. The results of own pilot studies were used as well as those reported by other researchers. The performed simulations proved the accuracy of the proposed modelling strategy . Furthermore, the presented examples allowed us to better understand the basic test methods for concrete interfaces and the observed mechanisms of failure during them. Numerical modelling of concrete, cf. ,2,3, andThe present study puts forward concrete-to concrete interfaces. Interfaces of different types of concretes, whose strength properties vary significantly, can often be found in modern structures, cf. Concrete-to-concrete interfaces usually have lower strength properties than the concrete layers that form it. Therefore, the proper and robust numerical model for the interface is crucial to simulate the behaviour of many composite structures. It has to precisely predict the strength of the interface  as well as its post-cracking behaviour. Such models are implemented in popular FE codes like ATENA , ANSYS , or AbaqTo determine the mechanical parameters of the interface model, the results of different strength tests can be used. They were widely described in many references ,17,18,19αint ,29 whichThe bond strength between layers of concrete cast at different times is affected by many factors described in numerous references, cf. ,25,30,31There are a few recent studies concerning the calibration of a numerical model for the concrete-to-concrete interface. Frenzel and Cubarch  performeUSDFLD user subroutine coded in FORTRAN, which facilitates the correct prediction of the interface strength subjected to compressive normal forces. The issue of fracture energy in two modes of fracture (tensile and shear) was discussed and implemented in the analyses. The results of experimental tests found in the literature , The major novel elements presented within the article are as follows: a complete strategy for calibration of interface traction–separation laws for concrete-to-concrete interfaces available by default in the Abaqus code and the detailed non-linear simulation of the most popular strength tests of concrete-to-concrete interfaces. They took into account: advanced constitutive models for concretes and their interfaces as well as detailed geometry of the loading devices. It is worth mentioning that the progress of delamination of bi-material specimens is hard to follow with modern optical measurement systems due to their brittle failure modes, so only numerical studies could give a wider insight into this process at this point ,27.Chen et al.  performeformulas ,36. The formulas . They obeference ).A pilot study, which facilitates the determination strength of the interface, was performed at the Faculty of Civil Engineering WUT. The research program consisted of slant-shear and splitting tests. The slant-shear specimens were of a rectangular shape with dimensions 100 × 100 × 500 mm, and the shear plane was placed at 30 degrees with the vertical direction. The splitting tests were performed using cubic specimens of 150 mm edge. The assumed class for the stronger concrete was C30/37 (concrete A) and for the stronger one was C55/67 (concrete B). The mix composition was summarized in During each casting, six additional 150 mm cubic specimens were prepared in order to determine the cubic compressive (three specimens) and splitting tensile strength (three specimens). The cubic compressive and tensile strengths were determined in accordance with their standards, respectively, ,39. FirsThe following well-known formula for determining the splitting tensile strength was used: follows :(3)σult=Santos and Julio performed the wide laboratory test programme, which aimed at assessing the shear strength between concrete layers ,25. TheyThe interface was modelled using cohesive elements, whose symbol is COH3D8 according to programme documentation —see Figuelastic regime (before damage initiation), the traction–separation law neglecting normal-shear states coupling has the following form:n, s, and t mean normal, first, and second tangential direction, respectively, e.g., In a 3D domain in the ce plane ,41.a—the biggest dimension of the connected parts and E and G are Young’s and Kirchhoff’s modules of the weaker concrete, respectively.Equation  can be rferences ,40. The eference . Howevereference . It recodamage initiation criterion. Among the few available ones, the quadratic nominal stress criterion was selected. It can be represented as:n and s , respectively. The inelastic part of the traction–separation law implemented in the Abaqus code was formulated in the framework of the damage theory. The onset to the damage state is indicated by the c—the cohesion. The standard Abaqus damage traction–separation interface model was enhanced by a user subroutine USDFLD to make the failure envelope pressure-dependent:Therefore, damage factors are not activated in a pure compression state, and allowable shear stresses do not depend on normal stress—see USDFLD subroutine allowed us to modify the parameters of models implemented in the Abaqus code by default at the beginning of each increment of the Newton scheme. Consequently, it is important to set a small value of increment load size when one uses such an approach. On the other hand, the problem is highly non-linear, so small increments are necessary for the convergence of the incremental-iterative procedure. The proposed algorithm reads the normal traction and calculates the admissible shear stress according to Carol’s formula. All the discussed criteria are shown in The post-cracking behaviour. After reaching the damage-initiation criterion, tractions are calculated taking into account the damage factor D:The last issue connected with a traction–separation interface model is its The above equation shows that in the case of interface compression, there is no reduction in normal stiffness. The evolution of the damage parameter under a combination of normal and shear deformation is described using an effective displacement:D is described by the relation:In the present modelling strategy, exponential damage evolution was used, in which damage variable l to 7.0 ). The moThe post-peak behaviour of the concrete-to-concrete interface differed significantly for tension and shear states. In the present study, the effective displacement at failure was related to the fracture energy for the mode I type of fracture  a,37 aGf,iformulas ,46:(17)GThe whole calibration strategy for a concrete-to-concrete interface was summarised in concrete region was modelled with C3D8 continuum elements with selective integration [The egration . The conegration ,51. The egration ,53,54,55In general, the model is formulated in the framework of the theory of plasticity with an additive strain tensor decomposition:Lubliner  and isotLubliner ), whereaLubliner ). DistriIn all presented case studies, the shape of the loading device was modelled precisely. In order to reduce the computational effort, they were discretized with R3D4 rigid elements. Between the surfaces of the loading device and the specimens, the contact with Coulomb friction was assumed with the friction coefficient equal to 0.6. Due to convergence issues, the simulations were displacement-driven, i.e., as the boundary conditions, displacements of loading devices were assumed.The non-linear problem was solved using an incremental-iterative Newton–Raphson algorithm. Two default convergence criteria were used, which are precisely described in reference . The toldisplacement-driven tension (pure mode I), i.e., displacement-driven pure shear (pure mode II), i.e., at the first step, the pressure that induces compressive normal traction; at second-step displacement—driven shear, i.e.,  The first example concerns a numerical model consisting of one C3D8 continuum and one COH3D8 interface element—see The results of all performed analyses are shown in The next example is a simulation of a three-point bending test of a beam with a notch described in The results of the performed analyses are shown in The following case study concerns the simulation of our own laboratory tests: splitting and slant-shear of a concrete prism. A detailed analysis of the one-material splitting test, sometimes called “a Brazilian test,” can also be found in reference . The modThe ultimate forces obtained from laboratory tests and numerical analyses are shown in A deformed mesh and a map of equivalent plastic strain in tension (PEEQT according to the programme documentation ) for therocedure ,59. ThisA deformed mesh and map of equivalent plastic strain in compression (PEEQ according to the programme documentation ) for theThe last CS concerns the simulation of Santos and Julio ,25 testsA comparison of results obtained with the numerical analysis and from experimental tests is shown in D for an interface is presented as maps for three load levels: 0.6 In Traction distributions along the selected cross-section (in the middle of the specimen) are shown in USDFLD user procedure, which enabled us to introduce the strength envelope, which is dependent on a normal traction value. The model took into account the different fracture energy values for modes of fractures I and II. The proposed approach was verified and validated with four case studies concerning: one element test, simulation of three-point bending of the bi-material notched beam [The strategy for adjusting an interface model available by default in the Abaqus code to the behaviour of concrete-to-concrete interface was proposed in hed beam , simulathed beam ,25. The Besides, the fully non-linear simulation of the most popular laboratory tests aimed at determining the interface strength gave a better insight in the specimen’s behaviour under loading. The simulation for the three-point bending test of the notched beam showed the progressive delamination in the interface, cf. UMAT or UEL user procedure [The limitation of the presented approach is the inability of the model to cover the residual strength envelope caused by the shear–friction phenomenon and the irreversible (plastic) slip since the default traction–separation material model was formulated in the framework of damage-elasticity without the plastic part. These issues can be overcome by the implementation of the traction–separation law within the rocedure .USDFLD user subroutine coded in FORTRAN, which enabled us to relate a shear strength with tractions normal to the interface. Different values of fracture energy in fracture modes I and II were also included. The results presented in the article can be used in the analyses of complex composite structures made of concrete layers cast at different times as well as in the assessment of various methods of repairing deteriorated structures. The presented modelling approach can also be useful in the simulation of masonry structures—the interface model can be easily adjusted to the behaviour of mortar [The article presented the complete strategy for calibration of the concrete-to-concrete interface model in the Abaqus code. The default traction–separation implemented in this system does not take into account the strength envelope for such interfaces. This issue was overcome with the f mortar .The method of loading specimens, which is fully consistent with the standards for splitting tests , can incIn the case of small specimens, the mesh density of 5 mm was sufficient to obtain satisfactory accuracy of the FEA results.The numerical model was able to cover the chipping of sharp edges for slant-shear specimens see .The value of fracture energy has a noticeable influence on the ultimate force predicted by the model see , so thisThe traction stress distribution is not homogenous along the interface during the whole loading history in the case of slant-shear specimens made of concrete, which are of different classes see  should bFurthermore, very detailed simulation of the most popular strength tests’ methods for concrete-to-concrete interfaces was presented in four case studies. The following observations can be made on their basis:Further studies will be concentrated on the determination of the fracture energy for different concrete-to-concrete interfaces (different concrete classes and surface preparation) and its dependency on the bond efficiency coefficient. Additionally, an attempt will be made to implement more of the sophisticated interface models—examples of such models can be found in references ,42,57.


IgG4-related disease (IgG4-RD) is a recently recognized systemic fibro-inflammatory disease of unknown cause involving many organs including pancreas, salivary glands, and lymph nodes. Chronic tuberculosis (TB) infection has been reported in IgG4-RD, but the prevalence of TB infection has not been evaluated in IgG4-RD.Characterization of a patient with IgG4-RD by physical examination, laboratory tests, magnetic resonance imaging (MRI) and histological examination. TB infection was evaluated by medical history, radiological examinations, sputum examinations, tubercular skin test (TST) and interferon gamma (IFN-γ) release assay test (IGRA). Medical records of IgG4-RD patients were reviewed in our institute from February 2015 to September 2020 to explore the prevalence of TB infection in IgG4-RD.We described a 40-year-old Chinese man presented with headache and diplopia. Physical examination revealed bitemporal hemianopsia and limited abduction of both eyes. MRI revealed uniformly enhancing mass overlying clivus with dural tail sign. Laboratory data revealed elevation of IgG4 (1.9g/L), and TB-IGRA demonstrated significantly elevated IFN-γ (414.21 pg/ml). The clivus lesion was subtotally removed and IgG4 was strongly positive on immunohistochemical staining. The diagnosis of IgG4-RD was established, and the patient received treatment of corticosteroids, methotrexate, and cyclophosphamide with isoniazid prophylaxis. Consequently, the mass shrank remarkably within 3 months. A similar concurrence of TB disease or latent TB infection (LTBI) and IgG4-RD was present in 17/47 (36.2%) patients in our institute.High frequency of TB/LTBI presented in patients with IgG4-RD. Patients with IgG4-RD and LTBI should be closely monitored for resurgence of TB. Whether TB represents a risk for IgG4-RD should be further investigated in prospective cohort. IgG4-related disease (IgG4-RD) is a novel syndrome that forms an inflammatory pseudotumor with increasing IgG4 positive plasma cells and lymphocytes infiltrating tissues, such as lacrimal gland, salivary gland, pancreas, bile duct, kidney, and retroperitoneum . HistoloHelicobacter pylori infection (Mycobaterium tuberculosis (M. tb) infection in IgG4-RD have been reported consecutively  or may have no clinical evidence of disease   . LTBI isn, LTBI) . The dianfection . IGRA renfection .Here we reported a case of IgG4-related clivus lesion in a patient with LTBI. The co-occurrence of IgG4-RD and TB infection in the same patient prompted us to explore whether this phenomenon is frequent in a retrospective IgG4-RD cohort.A 40-year-old man presented with intermittent headache and diplopia. 6 years before admission, he had recurrent headache at top and frontal area. He had been diagnosed with tubercular meningitis in the local hospital and received HRZE regimen for 2 months followed by HRE regimen for 27 months. His headache was not alleviated and then he went to our hospital 3 years ago. Contrast-enhanced magnetic resonance imaging (MRI) demonstrated an enhancing mass overlying clivus with dural tail sign. The lesion was initially considered to be a meningioma based on its imaging appearance. The patient then received a gamma knife surgery. The headache mitigated a little but persisted to date. 7 months before returning to our institution, he developed diplopia, hoarseness, and dysphagia progressively. On examination, the patient had bitemporal hemianopsia and limited abduction of both eyes (2/month for 3 months) was initiated and was well tolerated by the patient. Isoniazid was also started to prevent TB recurrence. In a follow-up of 3 months, he had attenuated bitemporal hemianopsia and better abduction of both eyes, and MRI showed the clivus mass shrank remarkably , IgG4 subclass 1.9 g/L . Serum levels of IgA, IgM, C3, and C4 were normal. Antinuclear antibody revealed 1:100 titer, while anti-neutrophil cytoplasmic antibodies, anti-SS-A, anti-SS-B, anti-dsDNA, anti-Smith, anti-Scl-70 as well as other anti-ENA were all negative. HCV antibody, HBV surface antigen and HBV-DNA were negative. TST was positive (induration ≥10mm) and TB-IGRA demonstrated significantly elevated IFN-γ . Repeated sputum tests were negative. A computed tomography scan of her chest showed neither lung lesions suggestive of TB nor hilar or mediastinal lymph node swelling. Investigation of other systemic manifestation of IgG4-RD was negative. We diagnosed the patient with IgG4-RD complicated with LTBI. Treatment of corticosteroids combined with methotrexate (10mg/week) and cyclophosphamide   , 18 was Patients with rheumatic diseases are vulnerable to infections because of the intrinsic immune dysregulation associated with the disease processes, the immunosuppressive therapies used, and other associated comorbidities. T-SPOT.TB-positive rates in patients with rheumatic diseases were higher than healthy controls and an 11.2% positive conversion rate was present in those receiving biologics . Per ourM. tb infection had been reported in IgG4-RD  for the publication of any potentially identifiable images or data included in this article.PQ, ZLin, and YM: case report data collection and analysis, writing of the original draft. ZLiu, XW, and BC: retrospective search for IgG4-RD patients and data analysis, manuscript review/editing. CL: methodology for the database search, data collection, and manuscript review/editing. YZ, YL, and CT: conceptualization, methodology, data visualization, writing of the original draft, and supervision. PQ and CL have contributed equally to this article. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (No. 81501412) and the 1·3·5 project for disciplines of excellence, West China Hospital, Sichuan University .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.