To protect against aneuploidy, chromosomes must attach to microtubules from opposite poles (‘biorientation’) prior to their segregation during mitosis. Biorientation relies on the correction of erroneous attachments by the aurora B kinase, which destabilizes kinetochore-microtubule attachments that lack tension. Incorrect attachments are also avoided because sister kinetochores are intrinsically biased towards capture by microtubules from opposite poles. Here, we show that shugoshin acts as a pericentromeric adaptor that plays dual roles in biorientation in budding yeast. Shugoshin maintains the aurora B kinase at kinetochores that lack tension, thereby engaging the error correction machinery. Shugoshin also recruits the chromosome-organizing complex, condensin, to the pericentromere. Pericentromeric condensin biases sister kinetochores towards capture by microtubules from opposite poles. Our findings uncover the molecular basis of the bias to sister kinetochore capture and expose shugoshin as a pericentromeric hub controlling chromosome biorientation.DOI:http://dx.doi.org/10.7554/eLife.01374.001 When a cell divides to create two new daughter cells, it must produce a copy of each of its chromosomes. It is important that each daughter cell gets just one copy of each chromosome. If an error occurs and one cell gets two copies of a single chromosome, it can lead to cancer or birth defects. Fortunately, there are multiple checks to ensure that this does not happen.During cell division the chromosomes line up in a way that increases the likelihood that each daughter cell will have one copy of each chromosome. After this process—which is called biorientation—is completed, microtubules pull the chromosomes to opposite ends of the cell, which then divides.Proteins called shugoshin proteins are known to be involved in biorientation in many organisms. These proteins are found in a region called the pericentromere, which surrounds the area on the chromosomes that the microtubules attach to, but the details of their involvement in biorientation are not fully understood. Now Verzijlbergen et al. have exploited sophisticated genetic techniques in yeast to explore how shugoshin proteins work.These experiments showed that the shugoshin protein helps to recruit condensin—a protein that keeps the DNA organized within the chromosome—to the pericentromere to assist with biorientation. It also keeps aurora B kinase—one of the enzymes that helps to correct errors during cell division—in the pericentromere when a microtubule attaches to the wrong chromosome. These results help us understand how a ‘hub’ in the pericentromere ensures biorientation. The next challenge will be to understand how this hub is disassembled after biorientation to allow error-free cell division to proceed. As shugoshins have been found to be damaged in some cancers, understanding the workings of this hub could also shed new light on how they contribute to disease.DOI:http://dx.doi.org/10.7554/eLife.01374.002 The accurate segregation of chromosomes during mitosis relies on the capture of newly duplicated sister chromatids by microtubules emanating from opposite poles. This is possible because on each chromosome a multi-subunit kinetochore assembles on a region of DNA, known as a centromere, to mediate attachment to microtubules. The proper attachment of sister kinetochores to opposite poles, called biorientation, generates tension owing to sister chromatid cohesion . Sister Though fundamental for accurate chromosome segregation, the role of shugoshin in biorientation has remained unclear. Budding yeast has a single shugoshin, Sgo1, which protects pericentromeric cohesin during meiosis but does not regulate cohesion during mitosis . We havesgo1 null cells (sgo1Δ) together with three missense mutants: sgo1-100, sgo1-700 and sgo1-3A. The sgo1-3A mutant was engineered to disrupt the binding site for PP2A-Rts1 (sgo1-100 and sgo1-700 were isolated in a screen due to their inability to sense a lack of tension (sgo1Δ cells have previously been reported to affect biorientation after microtubule perturbation (sgo1-100 and sgo1-700 mutants is similar to that of wild-type cells (SPC42-tdTomato), the centromere of chromosome IV labeled with GFP (CEN4-GFP) and with CDC20 under control of the methionione-repressible promoter (pMET-CDC20), to enable metaphase arrest by addition of methionine. All strains were released from a G1 arrest into nocodazole- and methionine-containing medium to depolymerize microtubules and induce a metaphase arrest before nocodazole was washed out to allow metaphase spindle formation  containing aurora B kinase with centromeres . BuddingCEN4 . Early ICEN4 , the homCEN4 . The recCEN4 . Sgo1-inCEN4  can explrization . The Iplrization  and centrization  were alstub2-401 ) where E represents the specific primer efficiency value. Enrichment/ input value was obtained from the following formula: E^−ΔCT. qPCR was performed in triplicate, typically for each of three or more independent experimental repeats. Error bars represent standard error. For ChIP-Seq, purified chromatin was recovered using a PCR purification kit  followed by drying in a speedvac. Samples were sequenced on a HiSeq2000 instrument  by the EMBL Core Genomics Facility . The summary of data obtained is given in ChIP was performed as described using anti-HA 12CA5, anti-Myc 9E10 or anti-Pk(V5) antibody . Primershttps://github.com/AlastairKerr/Marston2013. Single reads were mapped using BWA (Version: 0.6.1-r104)   to the s.1-r104)  to remov.1-r104) . As thissgo1Δ cells, we were concerned that the small number of precipitating sequences would be biased for amplification. To avoid this problem, we removed PCR replicates from our analysis. However, since this would also remove bona fide identical sequences from our dataset, we simultaneously analyzed the data with these reads present and compared the two data sets where all 16 centromeres were considered together. To do this, 100 bp windows were examined that extended 25 kb in each direction of every centromere, and a local maximum of the number of mapped reads was taken. The comparison of this data with and without PCR replicates included can be seen in During the amplification step prior to sequencing, multiple identical reads will be generated. Due to the low yield of total DNA precipitating with Brn1-6HA in 600 = 0.2 and grown at room temperature for 6 hr. Cells were harvested by centrifugation and the cell pellet was resuspended in 2 μM of dithiobis(succunimidylpropionate)  crosslinker for 30 min at room temperature before cell pellets were drop-frozen in liquid nitrogen as described by tub2-401 allele, 4 L of media was inoculated to OD600 = 0.02, grown at 30°C for 8 hr with shaking and then the temperature was reduced to 18°C for 7 hr to induce the arrest before holding the cells at 4°C for up to 5 hr before harvesting, crosslinking, and freezing as described above. Pulldowns and mass spectrometry were performed as described in To grow cells for purification of Sgo1 from cycling cells, 4 L of YPDA culture were inoculated to ODFor co-immunoprecipitation of TAP-tagged Sgo1, 200 ml of nocodazole-treated culture was harvested and either firstly cross-linked using DSP as described in Western immunoblot was performed as described in Indirect immunofluorescence and FACS were performed as described in pMET-CDC20, CEN4-GFP and SPC42-tdTomato were arrested in G1 using alpha factor (4 μg/ml) in minimal medium lacking methionine (SD/-met) at room temperature for 3 hr. Alpha factor was washed out and cells were released from the G1 arrest into rich medium containing methionine and nocodazole (YPDA + Met + NOC) plus the appropriate drugs . Methionine represses pMET-CDC20 expression, resulting in a metaphase arrest and nocodazole depolymerizes microtubules. After 3 hr, a sample was extracted (t = 0) and nocodazole was washed out by filtering in the presence of methionine and cultures were released into YPDA + Met to allow spindles to reform while maintaining the metaphase arrest. Samples were taken at 20 min intervals. Samples were fixed in 3.7% formaldehyde for 10 min, before washing in PBS and resuspending in DAPI for microscopy. Samples with two separated SPC42-tdTomato foci were scored as containing one or two CEN4-GFP foci. Typically 200, and at least 100, cells were scored for each timepoint.Cells carrying For live cell imaging, cells were loaded onto the Onix Microfluidic Perfusion system  and visualized using a Deltavision Elite  coupled to a Cascade 2 EMCCD camera with temperature control to 30°C. Frames were grabbed at the indicated intervals and images were processed in ImagePro software .CEN4-GFP dot to nearest Spc42-tdTomato were obtained using this automated system.For image analysis, a custom-written macro was developed in ImagePro software (Media Cybernetics), details of which are available upon request. In brief, yeast are selected from a reference DIC image taken at each time point. The yeast are automatically thresholded and the centre pixel from each red and green spot is then calculated. The distances between the spots can then be calculated and the measurements output to a text file for analysis using microsoft excel. Measurements of inter-centromere distance and distance from CEN4 (CEN4-GFP) and SPB (SPC42-tdTomato) markers together with CDC20 under control of a methionine-repressible promoter (pMET-CDC20) were arrested in G1 using alpha factor and then loaded onto the microfluidic chamber. Cells were released in the chamber into medium containing nocodazole (to depolymerize microtubules) and methionine (to deplete CDC20 and induce a metaphase arrest) for 30 min. The addition of nocodazole to G1 cells prevents SPB separation because this requires microtubules. Therefore, under these conditions, microtubules do not separate which leads to a high rate of monoorientation once microtubules are allowed to reform. This leads to a strong reliance on the error correction machinery to establish biorientation , we began imaging at ∼74s intervals for a total of 21 frames.To test the efficiency of error correction, cells carrying entation . After 3CEN4-GFP foci. The total percentage of cells with two SPB foci that contained two GFP foci was calculated for all frames for each strain. The number of cell images analyzed was 1116 (wild type), 697 (sgo1Δ), 1251 (YCS5-aid) and 968 (ipl1-as). To mFor determination of the overall ability of strains to biorient in CEN4-GFP foci. Cells in which two SPB foci were detected for at least four consecutive frames were scored for the number of times that the number of CEN4-GFP foci changed from one to two or vice-versa for the frames in which two SPBs were consecutively visible. The ‘switching rate’ was calculated by dividing the number of times a cell alternated between one and two CEN4-GFP foci by the total time in which two SPB foci were consecutively visible. The average switching rate was determined for all cells in which two SPB foci were detected for at least four consecutive frames. In sgo1Δ cells (551 frames), 102 YCS5-aid cells (1046 frames) and 86 ipl1-as cells (748 frames).As a measure of the efficacy of the error correction machinery, we determined the ability of cells to switch between one and two visible CEN4 (CEN4-GFP) and SPB (SPC42-tdTomato) markers together with CDC20 under control of a methionine-repressible promoter (pMET-CDC20) were arrested in G1 using alpha factor and then released into medium containing methionine to deplete CDC20 and induce a metaphase arrest. Cells were loaded onto the microfluidics chamber and after approximately 1.5 hr, when around 90% of cells had 2 SPBs, cells were treated with YPDA containing nocodazole for 30 min. After 30 min, nocodazole was washed out after which cells were immediately imaged every ∼94 s for a total of 21 frames. We scored the percentage of cells in which separated GFP dots were observed at least once during the observation period.To test the bias on sister kinetochores to biorient, cells carrying  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  is important to promote biorientation through two mechanisms: through binding to the chromosome passenger complex, and through a newly identified association with the condensin complex. The authors show that Sgo1 is able to recruit condensin to centromeres or to ectopic sites. The authors identify a mutation in Sgo1 that perturbs binding to condensin and that this association is important for subsequent biorientation. The authors conclude that condensin is required to form a rigid back-to-back structure such that sister kinetochores are biased towards microtubule capture from opposite spindle poles.Overall this is an interesting study that reveals new insights into how budding yeast chromosomes biorent, and uncover a new role for condensin in centromere and kinetochore behaviour.There are, however, a number of substantive concerns that preclude publication in its present form. In particular, the conclusion that condensin recruitment alters kinetochore geometry to promote orientation towards opposite poles requires further supporting evidence. Moreover, the authors themselves do not appear to be certain whether there is a direct link between Sgo1 and condensin recruitment. They argue that Sgo1 is sufficient for condensin targeting to the pericentromere by over-expressing Sgo1 and measuring Brn1 accumulation. However, they previously state that Sgo1's effect on condensin is indirect, mediated through Scc2/4 (cohesin). There are also concerns regarding the validity of the approach the authors use to measure bi-orientation and the presentation of the ChIP analyses. The latter prevents a critical evaluation of the data quality and missing information will need to be added.Concerns to be addressed:1) The evidence for a direct interaction between Sgo1 and condensin is limited. Only a small number  of condensin peptides can be identified in the mass-spec analysis of Sgo1 purifications. In fact, the highest scoring condensin subunit  ranks number 137 in the list of proteins identified . Moreover, only a minute fraction of condensin . The authors should at least test whether ectopic recruitment can be measured at both sides of the TetO repeats. Yet a much better test whether condensin recruitment by Sgo1 is significant would be to employ the TetO-TetR approach to tether to centromeres an Sgo1 mutant that can otherwise not bind  and assay whether this restores condensin levels and chromosome bi-orientation.An explanation for the altered binding profiles of condensin measured by ChIP-seq might be changes in cohesin accumulation at centromeres in the Sgo1 mutants. The authors need to check by ChIP-qPCR  whether cohesin levels at centromeres are affected in the Sgo1 mutant.2) The authors monitor the splitting of a sister centromere pair as a measure for sister chromatid bi-orientation . The redOne possibility to distinguish whether chromatid pairs have attached to spindle microtubules from only one spindle pole or whether chromatids have bi-oriented but their centromeres cannot separate would be to score centromere-spindle pole body (SPB) distances in the live cell microscopy assay . If centThe data in 3) Most of the graphs that represent the results from ChIP-qPCR experiments  lack infTaking into account the possible variability between individual ChIP-qPCR experiments, careful statistical analysis will be required to evaluate the significance of the observed differences. For example, the values measured at CEN4 for the same wild-type strain (AM3513) in two independent Ipl1 HA-ChIP experiments vary 4-fold compare , calling 1) The evidence for a direct interaction between Sgo1 and condensin is limited. Only a small number  of condensin peptides can be identified in the mass-spec analysis of Sgo1 purifications. In fact, the highest scoring condensin subunit  ranks number 137 in the list of proteins identified .The Excel file submitted with the original manuscript was unsorted and did not take the contaminant proteins that also co-purify with the “no tag” control into account. When contaminants are taken into account, the condensin subunits rank number 8 and 26 in the list of proteins identified, in cycling and arrested cells, respectively. We have revised the file with the list of proteins sorted to exclude contaminant proteins.Moreover, only a minute fraction of condensin (less than 0.01%? See) can be detected by Western blotting in Sgo1 immunoprecipitations. Since both Sgo1 and condensin are enriched at centromeres, it is very likely that a small amount of condensin co-precipitates with Sgo1 because both proteins are bound to the same stretch of chromatin. In addition, the authors add amine-reactive crosslinkers to the cell extracts in both experiments (a fact they should mention at least in the figure legends and not only in the methods section), which will result in the formation of protein-chromatin crosslinks. The authors will need to repeat the pull-down experiments after DNase digestion and in the absence of crosslinker in order to rule out these concerns.We thank the reviewers for raising this important point. We performed the experiment suggested by the reviewers and found that the amount of the Ycs4 condensin subunit co-purifying with Sgo1 was independent of treatment with cross-linker or DNase. This shows that the interaction between Sgo1 and condensin is not dependent on DNA. This data is now presented in A second argument for a role of Sgo1 in condensin loading is that centromeric condensin levels increase upon Sgo1 overexpression. The increase in condensin binding could, however, result from the delay in anaphase onset caused by Sgo1 overexpression. Would condensin levels remain unchanged if anaphase onset were delayed by other means?SGO1-overexpressing cells was a consequence of the metaphase arrest caused by high levels of Sgo1, we overexpressed SGO1 in cells arrested in mitosis by treatment with nocodazole. Measurement of Brn1 association with two centromeric sites and a pericentromeric site by ChIP-qPCR showed that SGO1 overexpression also increased Brn1 levels in cells arrested in mitosis, arguing against the idea that the centromeric enrichment of Brn1 is solely due to the cell cycle block caused by SGO1 overexpression. This data is presented as To exclude the possibility that Brn1’s chromosomal enrichment in Ectopic recruitment of condensin by Sgo1 fused to TetR seems rather inefficient, with a two-fold enrichment (and considerable variation?). The authors should at least test whether ectopic recruitment can be measured at both sides of the TetO repeats. Yet a much better test whether condensin recruitment by Sgo1 is significant would be to employ the TetO-TetR approach to tether to centromeres an Sgo1 mutant that can otherwise not bind  and assay whether this restores condensin levels and chromosome bi-orientation.tetO repeats. Following sonication during the ChIP protocol, the majority of fragments are much shorter than this (in the range of ∼200 bp), so a primer set at this distance is expected to only inefficiently detect proteins recruited to the tetO repeats themselves. As suggested by the reviewers, we have now analyzed a site on the other side of the tetO repeats which is much closer (∼50bp) to the tethering site. As can be seen in In the original manuscript, we used a primer set ∼800 bp to the left of the SGO1. As we showed in sgo1-700 mutant loses the bias to sister kinetochore biorientation , it was necessary to perform this experiment in diploid cells and therefore we cannot presently test the ability of these tethered constructs to retain sister kinetochore bias (we cannot easily arrest and release the cells from G1 as they are not sensitive to mating pheromone). Moreover, other unpublished findings in the lab have suggested that the dynamicity of Sgo1 on centromeres is key to its regulation and important for accurate segregation so it is likely that Sgo1 tethering affects its function. Indeed, even tethered wild type Sgo1-tetR-GFP is not fully able to rescue the mis-segregation of chromosome IV in our experiment  whether cohesin levels at centromeres are affected in the Sgo1 mutant.sgo1? cells.We performed both these experiments and the data is shown in Overall, we feel our study now argues strongly for a direct role of Sgo1 in condensin recruitment to the pericentromere.2) The authors monitor the splitting of a sister centromere pair as a measure for sister chromatid bi-orientation . The reduction of sister centromere splitting observed in the Sgo1 and condensin mutants could, however, equally well result from an increase in centromeric sister chromatid cohesion. An increase in centromeric cohesion would prevent splitting of bi-oriented sister chromatids.One possibility to distinguish whether chromatid pairs have attached to spindle microtubules from only one spindle pole or whether chromatids have bi-oriented but their centromeres cannot separate would be to score centromere-spindle pole body (SPB) distances in the live cell microscopy assay . If centromeres were bi-oriented but merely failed to split, they should be positioned at equal distances to both SPBs. If sister centromeres failed to bi-orient, they should frequently be found close to one SPB. Alternatively, the authors could release cells from the Cdc20 arrest and allow them to enter anaphase, then score whether sister centromeres segregate to one pole or to opposite poles .sgo1? and YCS5-aid cells is not significantly different to that of wild type cells . The situation is further complicated by the fact that due to the partial functionality of the error correction machinery in condensin mutants  Most of the graphs that represent the results from ChIP-qPCR experiments  lack information that is essential to evaluate the data. What do the column plots represent? Do they show mean values, and if yes, from how many PCR runs and how many independent immunoprecipitation experiments? What do the error bars represent? The legend foris missing completely.We apologize for this oversight. We have now included a more detailed description of the procedures used in the Materials and methods and detailed figure legends throughout, explaining how each experiment was carried out, the procedures used to process the data and the meaning of the figures presented. As detailed in the figure legends, the vast majority of ChIP-qPCR experiments show mean values of three independent repeats. qPCR was always performed in triplicate . The number of independent immunoprecipitation experiments is given in the figure legends . Error bars represent standard error. The exception to this are time course experiments where the day-to-day variability of cell cycle progression means data from different days cannot be combined usefully. Here, experiments are repeated, typically 3 times in total, and a representative experiment is shown. Overall, the pattern does not vary, though the absolute values at each timepoint can.Taking into account the possible variability between individual ChIP-qPCR experiments, careful statistical analysis will be required to evaluate the significance of the observed differences. For example, the values measured at CEN4 for the same wild-type strain (AM3513) in two independent Ipl1 HA-ChIP experiments vary 4-fold (compare), calling into question whether the 2-fold reduction in ChIP signals in the Sgo1 mutants  is significant. Similarly, the ChIP signal at CEN4 measured for the Sgo1-100 mutant in G1 phase is more than 2-fold higher than the signal for wildtype Sgo1 . Is the 4-fold increase in Sgo1 ChIP signals during mitosis  then still significant?Sgo1-100 mutant proteins is likely due to slight changes in the timing of alpha factor release in the handling of these cultures (see above about the interpretation of time course experiments). The main point of these experiments was to show that residual association to centromeres occurs even though it is not maintained in nocodazole-arrested cells. We do not think it is appropriate to take any further interpretation based on the absolute values from these experiments as due to the inherent variability of time course experiments; to do so would require exquisitely controlled conditions and much more closely spaced sampling.Again, we apologize: these discrepancies are due to our failure to provide adequate figure legends describing the procedures used to obtain the data and can all be accounted for by different growth conditions. In particular, the ChIP-qPCR signals obtained in experiments where nocodazole was directly added to cycling cells are typically much higher than experiments where cells were first arrested in G1 before being released into nocodazole. The fraction of cells that were approaching, or in, mitosis at the time of nocodazole will spend a much greater time in mitosis, where our proteins of interest are presumably continually recruited, thereby accounting for these differences. Of course, repeats are carried out using exactly the same conditions, with all strains within one repeat grown and processed on the same day, allowing direct comparison for those conditions. Specifically, for the experiment shown in


Acupuncture is commonly performed at acupoints. No comparisons of quantitative physiological alterations in the brachial artery (BA) induced by the stimulation of different acupoints in the lower limbs have been performed in humans. Therefore, we investigated changes in blood flow volume (BFV) in the BA as an indicator of the physiological effects induced by stimulation at 3 points. Seventy-five healthy participants aged 33 ± 9 years (mean ± SD) were enrolled and randomly assigned to 3 groups; they received stimulation at 3 different points located on the lower limbs: ST36, LR3, and a non-acupoint. Stimulation was performed bilaterally with manual rotation of the needles. Using ultrasonography, BFV was measured continuously from rest to 180 seconds after stimulation. LR3 stimulation significantly increased BFV compared to that before needle insertion. Meanwhile, stimulation at ST36 and the non-acupoint significantly decreased BFV compared to that before needle insertion. Stimulation at LR3 elicited a significant increase in BFV compared to that at ST36 and the non-acupoint. The results suggest that the stimulation of different points on the lower limbs causes distinct physiological effects on BFV in the BA. Acupuncqi . The treqi . The acuqi , 4. The qi , 4. One Blood flow volume (BFV) is an important index for representing the condition of organs and tissues. Therefore, we employed BFV as a quantitative indicator of the effects of traditional interventions on the human body. There are some reports in the literature about the hemodynamic responses to acupuncture stimulation at several acupoints –9. Acupu Seventy-five healthy adult volunteers (45 men and 30 women) aged 33 ± 9 years  were enrolled in this study. No participants had cardiovascular disease. None of the participants took any medicine 1 month before the experiment. All participants were examined after fasting and abstaining from alcohol and caffeine for at least 3 hours. The study protocol was approved by the Ethics Committee of Tohoku University, Graduate School of Medicine. All participants provided written informed consent prior to the beginning of the experiment.α10®, Hitachi-Aloka Medical Ltd., Tokyo, Japan) on the right arm; this system contains a high-resolution linear array transducer (13 MHz) and a computer-assisted analysis software . The software automatically detected the vessel edge and measured the vessel diameter and BFV continuously [−1·m−2), and (4) blood flow volume (mL·min−1·m−2). The participants rested in the supine position in a quiet, air-conditioned room (temperature 25-26°C). Three monitoring electrocardiographic electrodes were attached to the anterior part of the chest of each participant. Four electrodes for impedance cardiography (ICG)  were placed at the base of the neck and the level of the xiphoid process in the midaxillary line. ICG utilizes 4 dual sensors on the neck and chest to apply low-amplitude high-frequency alternating electrical current to the participant's thorax. Pulsatile changes in BFV and velocity are measured as changes in impedance. These changes are subsequently synchronized with the electrocardiogram to automatically calculate hemodynamic parameters such as stroke volume and the cardiac index (CI) . ICG is inuously . The cominuously . The riginuously , 22. BFVinuously –23. To einuously , 25. Ultinuously . Thus, tinuously . Therefon = 75) were randomly assigned to 1 of 3 groups receiving the following treatments: (1) needle stimulation at ST36     . The parAfter the ultrasonograph was positioned, the participants rested in the supine position for 10 minutes. We then measured the BA hemodynamics, blood pressure, heart rate, and the CI at rest , during needle stimulation, and 30, 60, and 180 seconds after needle stimulation. Needle stimulation was performed by a licensed acupuncturist. Disposable fine stainless-steel needles  were inserted bilaterally on the target point and maintained at a depth of 10 mm during the test to ensure they reached the muscle. After the needles were inserted, the stimulation was performed for 18 seconds by rotating the needles <90° manually. The needles were retained for the duration of the test following stimulation and were then removed.P < 0.05. Individual variations were analyzed as percent changes. Statistical analysis was performed with PASW software . Comparisons between the ST36, LR3, and non-acupoint groups were performed by two-way analysis of variance (ANOVA) with a post hoc Tukey test. Repeated-measures one-way ANOVA with a post hoc Dunnett's test were used for statistical comparison between pre- and post-needle insertion values. Mean values between groups were compared using the Kruskal-Wallis H test. The level of statistical significance was set at  One participant assigned to the LR3 group was excluded from the analysis because of arrhythmia. There were no significant differences among the clinical profiles of the 3 groups .No significant differences were observed among the 3 groups or with respect to the percent change in systolic or diastolic blood pressure in each test before and after needle insertion. No significant differences were observed among the 3 groups with respect to the percent change in heart rate in each test. Regarding the percent change in heart rate, stimulation at LR3 elicited a significant decrease in heart rate measured during needle stimulation compared to that before needle insertion. The heart rate tended to decrease during needle stimulation. No significant differences were observed among the 3 groups with respect to the percent change in the CI in each test. Regarding the percent change in the CI, the stimulation at the non-acupoint elicited a significant increase in the CI in the BA 180 seconds after needle stimulation compared to that before needle insertion.No significant differences were observed among the 3 groups with respect to the percent change in SVRI in each test . RegardiTo our knowledge, this is the first study comparing the differences in BFV in the BA induced by needle stimulation at 3 acupoints as assessed by ultrasonography. The results show that BFV in the BA increased significantly after needle stimulation at LR3, whereas it decreased significantly after needle stimulation at ST36 and the non-acupoint. Furthermore, after needle stimulation at the non-acupoint, the CI increased significantly while SVRI decreased significantly. These results indicate that the physiological effects on the BFV in the BA triggered by needle stimulation vary depending on the acupoint stimulated. Needle stimulation at the lower limbs is known to elicit systemic visceral responses via supraspinal reflexes –32. HoweStimulation at ST36 and the non-acupoint elicited significant decreases in BFV in the BA during needle stimulation compared to that before needle insertion. The physiological mechanism involved in the decrease in BFV in the BA induced by the stimulations applied to the skin is a peripheral vascular resistance caused by an instantaneous increase in sympathetic tone . During Stimulation at LR3 elicited a significant increase in BFV in the BA 60 and 180 seconds after needle stimulation compared to that before needle insertion. In addition, for LR3 stimulation, a systemic reaction was elicited such that the SVRI decreased significantly 180 seconds after needle stimulation without any changes in blood pressure, heart rate, or the CI compared to those before needle insertion. This reaction suggests that the increase of BFV in the BA after stimulation at LR3 depends on the decrease of SVRI. A previous report suggests that the changes in BFV in the BA caused by needle stimulation at LR3 depend on the decrease of the resistive index of the peripheral artery after needle stimulation . This reThe physiological reactions mentioned earlier may support the idea that stimulations at ST36, LR3, and the non-acupoint have different impacts on the autonomic nervous system, which regulates BFV in the BA. A recent report also suggested that opposite cardiovascular effects were shown in the response to stimulation of LR3 and ST36 in rats . We can  The present study was a human observational study. Measuring sympathetic and parasympathetic activities in human subjects is invasive and difficult. Therefore, we observed the effects of stimulation at 2 acupoints and 1 non-acupoint on BFV and speculated on the mechanism using previous experimental studies as a basis. In a previous study with 25 participants per group, we reported the changes in BFV in the BA measured by ultrasonography when acupuncture was performed at a single acupoint , 13. Acc The present study revealed that BFV in the BA increased after needle stimulation at LR3 without changes in the CI. Needle stimulation at ST36 and the non-acupoint affected the hemodynamics of the BA. These physiological effects may be dependent on acupoint location.


Spontaneous death appeared to be due to irreversible renal failure, because > 85% of glomeruli in kidneys from mice that died spontaneously were obliterated by glomerulosclerosis.The gamma3 genotype (+/+ vs. +/- vs. -/-) did not appear to significantly affect serum titers of IgG auto-antibodies specific for double-stranded DNA (dsDNA) or α-actinin. However, while substantial serum titers of IgG3 auto-antibodies specific for double-stranded DNA (dsDNA) or α-actinin were seen in gamma3 +/+ mice, somewhat lower serum titers of these IgG3 auto-antibodies were found in gamma3 +/- mice, and gamma3 -/- mice exhibited baseline concentrations of these auto-antibodies. Analysis of immunoglobulins eluted from snap-frozen kidneys obtained from mice of all three gamma3 genotypes at ~18 weeks of age revealed much higher quantities of IgG in the kidneys from gamma3 +/+ than gamma3 -/- mice, and most IgG eluted from +/+ mice was IgG3. The serum creatinine levels in gamma3 +/+ mice substantially exceeded those of age-matched gamma3 -/- mice after ~21 weeks of age. Histopathological examination of kidneys from mice sacrificed at pre-determined ages also revealed more extensive glomerulosclerosis in gamma3 +/+ or +/- mice than in -/- mice beginning at 21 weeks of age. Survival analysis for IgG3-deficient and IgG3-producing MRL/lpr mice against glomerulonephritis-associated morbidity and mortality by slowing or arresting the progression to glomerulosclerosis.The available evidence suggests that IgG3 deficiency partially protects MRL/This article was reviewed by Pushpa Pandiyan, Irun Cohen, and Etienne Joly. Tnfrsf6lpr (MRL/lpr) inbred strain are genetically predisposed to a spontaneous autoimmune syndrome that resembles human systemic lupus erythematosus (SLE or lupus) in several key features. As in lupus patients, a major cause of morbidity and mortality in both male and female MRL/lpr mice is progressive glomerulonephritis [Mice of the MRL/MpJ-ephritis -3. The pephritis .lpr mice [lpr or other inbred strains prone to autoimmune disease [lpr background was associated with increased titers of serum auto-antibodies of the IgG3 (as well as the IgG1 and IgG2a subclasses) specific for dsDNA and more severe glomerulonephritis and earlier mortality [lpr mice [Analysis of antibodies specific for auto-antigens or those deposited in the affected kidneys has revealed that IgG antibodies are the dominant isotype . In termlpr mice . Several disease -9. Conve disease . Similarortality . In all lpr mice -14, the In the course of characterizing the J606 myeloma protein, the first identified murine IgG3 antibody, the investigators noted that the J606 Fc regions tended to self-associate at concentrations in the mg/ml range . SubsequPrevious studies with IgG3-deficient mice of the BALB/c background demonstrated that such mice were more susceptible to mortality following pneumococcal infection than wild-type BALB/c mice . These min vivo. In addition to the circumstantial evidence, recounted above, implicating antibodies of the IgG3 subclass in the pathogenesis of glomerulonephritis in murine models of lupus, antibody aggregation has long been regarded as a possible cause of inflammation and tissue damage [Irrespective of the potentially beneficial consequences of antibody self-association, there are reasons to wonder if the unusual physical properties of IgG3 antibodies have any potentially negative consequences e damage . It is oe damage .lpr mice, we sought to assess the consequences of genetic deficiency in the capacity to synthesize IgG3 antibodies. In this report, we compare titers of serum auto-antibodies to dsDNA and α-actinin, renal deposition of auto-antibodies, renal function, kidney histopathology, and survival among mice of the three γ3 heavy chain genotypes: +/+, +/-, and -/-.Therefore, to address more definitively the importance of IgG3 antibodies in the spontaneous renal disease of MRL/lpr Tnfrsf6(MRL/lpr) inbred strain were created through 12 generations of backcrossing mice carrying the defective γ3 heavy chain constant region gene [lpr strain. Mice of all three γ3 heavy chain constant region genotypes  were generated by crosses between heterozygotes or crosses between γ3 +/- and either -/- or +/+ mice. Figure IgG3-deficient mice possessing the genetic background of the MRL/MpJ-ion gene  to mice lpr origin for six of six mice (data not shown). One SNP, located on the distal end of chromosome 12 near the γ3 heavy chain constant region locus, was typed as of donor origin in each mouse. A chromosome 12 locus, sbb2, identified by Bolland et al. [sbb2 locus is flanked on both sides by SNPs of MRL/lpr origin. Similarly, the Fas locus on chromosome 19 is surrounded on both sides by SNPs of MRL/lpr origin, strongly suggesting that the γ3 -/- mice have the lpr Fas allele.Two hundred SNPs were typed by single base extension reactions and sequencing by Jax Services  for each of six mice typed by us as γ3-deficient. Of the 200 SNPs typed, 199, on chromosomes 1-19, were determined to be of MRL/d et al.  as a sitlpr strain are known to contain significant numbers of cells exhibiting a T cell phenotype  that is unusual for cells in the periphery in other mouse strains. As revealed in Figure lpr γ3 -/- mice also have these cells in numbers roughly comparable to the +/+ and +/- mice, consistent with an authentic MRL/lpr genetic background.Characterization of major T-lymphocyte subsets in the spleen was performed by flow cytometry. This analysis provided an additional test of the success of the backcrossing, as mice of the MRL/Titers of serum auto-antibodies with specificity for dsDNA and α-actinin were determined by ELISA using samples from four month-old mice of all three γ3 genotypes Figure . The serEluates from the kidneys of γ3 +/+ mice contained more IgG  than those from -/- mice . As expected, the IgG3 concentrations in eluates from γ3 +/+ mice far exceeded those in -/- mice (data not shown). Most (> 80%) of the IgG antibodies susceptible to elution from the +/+ kidneys at 21 weeks were IgG3 subclass, and the increased IgG content in renal eluates from +/+ mice at 21 versus 18 weeks of age was almost exclusively of the IgG3 subclass. In contrast, < 12% of the IgG in eluates from -/- mice was IgG3. The eluates from +/- mice were between these extremes, but IgG3 represented only ~10% of the total IgG eluted from +/- mice at either age. Overall, there is considerably more IgG deposited in +/+ than in -/- kidneys, and IgG3 antibodies are a significant fraction of the IgG eluted from the +/+ mice at 21 weeks.All mice, regardless of the γ3 genotype, exhibited pathologic and heavy proteinuria throughout the period of timed sacrifices (data not shown). Urinary protein excretion increased from 2.7, 0.8 or 1.7 mg/d in the γ3 +/+, +/- and -/- mice, respectively, at 18 weeks of age to 15.3, 7.4 or 7.6 mg/d in the respective groups at 26 weeks. As a more standardized measure of glomerular permselectivity, the ratio of protein to creatinine in urine exhibited a similar pattern, exceeding 15 in +/+ mice at all times points observed, while increasing from 4.58 to 21.1 in +/- and from 5.5 to 11.9 in -/- mice , also suggests that the absence of IgG3 antibodies influences survival by altering the progression of glomerular damage. Specifically, mice of all three genotypes at 18 weeks of age had a minority of glomeruli with no or only minor mesangial lesions Figure , and a lIn both human and murine glomerulonephritides associated with lupus or lupus-like disease, glomerular crescents, tubulointerstitial inflammation and interstitial fibrosis are frequent concomitants. A minority of the glomeruli in the mice examined in this work exhibited small crescents, principally cellular at 18 or 21 weeks of age, but mixed fibrocellular or fibrous changes at 21 or 26 weeks of age. The overall frequency and size of the crescents did not differ significantly among the genotypes at any age. Whereas the character of crescents in γ3 +/+ and +/- mice shifted from cellular to increasingly fibrous collaterally with increased age and increased incidence of glomerulosclerosis, fibrous crescents were unusual in γ3 -/- mice, even at 26 weeks old (data not shown). Kidneys from most mice exhibited patchy lymphohistiocytic infiltrates within the tubulointerstitium, and the frequency of such involvement did not differ among genotypes. However, the extent of the interstitial infiltrates and the appearance of tracts of interstitial fibrosis were closely correlated to the extent of glomerulosclerosis, as is typically the case in spontaneous lupus in humans and in other animal models of chronic glomerulonephritis. Therefore, the extent of interstitial inflammation and the extent of interstitial fibrosis were both significantly greater in γ3 +/+ and +/- mice than in the -/- littermates (data not shown).lpr mice, including littermates of all genotypes, revealed that γ3 +/+ (n = 28) and +/- (n = 42) mice died at significantly younger ages than γ3 -/- (n = 16) mice  to IgG3-deficient (γ3 -/-) mice of the MRL/lpr background. The current analysis was undertaken with the recognition that even after twelve generations of backcrossing to minimize the chances of cryptic but relevant genetic differences between IgG3-deficient (γ3 -/-) and IgG3-producing (γ3 +/- and +/+) mice of the MRL/lpr background, there remains some finite, if low, probability of such allelic disparities [lpr mice of the three genotypes deriving from the same litters of crosses between γ3 heterozygotes. Genetic analysis of single nucleotide polymorphisms at 200 loci on chromosomes 1-19 (data not shown) strongly suggested that donor origin DNA was restricted to the distal end of chromosome 12 near the γ3 heavy chain constant region locus. Furthermore, the biologic behavior of the γ3 +/+ mice resembled closely that of mice of the parental MRL/lpr strain in several characteristic features.While the evidence implicating IgG3 antibodies in the pathogenesis of the spontaneous glomerulonephritis in MRL/parities . TherefoWhile the presence of two defective γ3 heavy chain constant region alleles did not greatly reduce the titers of auto-antibodies of all IgG subclasses, this genotype was associated with highly significant reductions in IgG3 auto-antibodies specific for dsDNA and α-actinin. Perhaps the most crucial point is that γ3 +/+ mice, and to a lesser extent +/- mice, exhibit substantial serum titers of these IgG3 auto-antibodies that could contribute to progressive kidney damage, whereas in γ3 -/- mice such progression is diminished. If the auto-antibodies of the IgG3 subclass effectively contribute to scarring of kidney tissue, then this difference in the concentrations of these antibodies could account for the observed differences among the mice of the three γ3 heavy chain constant region genotypes. The results from the kidney elution studies are also generally supportive of the importance of IgG3 antibodies in mediating the destruction of functional renal tissue in γ3 +/+ mice.lpr mice [The mechanism(s) by which IgG3 antibodies might contribute to the pathogenesis of glomerulonephritis were not directly addressed in the present study. Additional investigation will be necessary to determine the relative importance of direct versus indirect pathways in the mediation of tissue damage by IgG3 antibodies. Observations indicating that both IgG2a and IgG3 antibodies are contributory to kidney disease raise the possibility that IgG3 rheumatoid factors (i.e. antibodies able to bind to IgG Fc regions), which can exhibit specificity for IgG2a Fc regions and are present at high frequency in MRL/lpr mice ,17, exaclpr mice ,36. Anotlpr mice -39. In tlpr mouse strain. Nevertheless, it is also clear that even in the absence of IgG3 production, kidney damage ensues, as reflected in endocapillary proliferation.Among mice sacrificed at defined ages, the glomerular pathology in γ3 +/+ and +/- mice evolved from endocapillary proliferation to frank sclerosis far more frequently than in γ3 -/- mice. Histopathologic examination of kidneys recovered from mice dying spontaneously is also consistent with the notion that the absence of IgG3 antibodies attenuates the irreversible renal damage associated with the MRL/lpr +/+ mice, we believe that the γ3 -/- mice have an authentic MRL/lpr background, and that it is very unlikely that an unidentified allelic difference between +/+ (or +/-) and -/- mice accounts for the phenotypic differences described herein.The comparison of the γ3 +/+, +/- and -/- mice with respect to long-term survival revealed dramatic, and highly statistically significant differences between either +/+ or +/- and -/- mice (p = 0.0006) in the average age of mortality. These results are reasonably, and most simply, interpreted as suggesting that antibodies of the IgG3 subclass contribute significantly to the more rapid mortality of the γ3 +/+ and +/- mice by causing the immune complex-mediated renal disease to progress to end stage renal failure. Given the SNP typing, the nearly identical mortality curves for male and female mice of all γ3 genotypes, and the presence in the γ3 -/- mice of the unusual CD3+, CD4-, CD8- lymphoid population that characterizes MRL/Finally, it is of interest to note that the γ3 heavy chain constant region locus has yet to be associated with the murine lupus phenotype in genetic screening -42. Thislpr strain but are not the sole contributors to this renal tissue damage. In the absence of such antibodies, kidney disease still occurs but, on average, fails to progress to the same extent as in mice able to produce IgG3 antibodies. These results also demonstrate that gene products encoded at loci at which there are no naturally-occurring disease-associated allelic variants can nevertheless contribute significantly to disease pathogenesis.Antibodies of the IgG3 subclass contribute to the spontaneously occurring glomerular disruption and dysfunction that arises in mice of the inbred MRL/lpr) background. Female and male MRL/lpr mice used for the backcrossing were obtained from The Jackson Laboratory . Mice of all three γ3 heavy chain constant region genotypes were then bred at Taconic Farms  for the present study. Genotyping was done in our lab  for genomic single nucleotide polymorphism (SNP) typing, with 200 SNPs, by single base extension reactions and sequencing.Tail samples from six MRL/lpr γ3 +/+, +/- and -/- mice as described [Immunoblots for serum IgG3 were performed using samples from MRL/escribed . The IgGlpr γ3 +/+, +/-, and -/- mice and C3H control mice were processed into single-cell suspensions for analysis by flow cytometry. We used the following antibodies to mouse T cells: a cocktail of anti-CD3epsilon-PE-Cy7, anti-CD4-PE, anti-CD8alpha-APC, . Mouse B cells were identified by anti-CD19-PE . A multi-laser flow cytometer, BD LSR (BD Biosciences), was used to analyze about 1 × 10e6 cells per spleen sample. Isotype-matched control antibodies were used in all analyses.Spleens from 3 month-old MRL/p-nitrophenyl phosphate (Sigma), and the OD was monitored at 405 nm using a MRX Revelation ELISA Reader .Concentrations of serum antibodies specific for dsDNA or α-actinin were measured by ELISA as described -46. BrieOne-half of a snap-frozen kidney from each mouse was thawed and minced with a clean razor blade. Kidneys from mice of a given cohort were pooled, and Ig was eluted . The eluRandomly selected mice of all three genotypes were sacrificed by exsanguination under ether anesthesia at approximately 18, 21 or 26 weeks of age (within a window of 3 days); serum and kidney tissue were harvested. On the day prior to sacrifice, individual mice were placed into metabolic cages for collection of urine samples over ~16 hours. Proteinuria was quantified by a proprietary protein assay kit  based on a dye-binding procedure  in a micUpon sacrifice  or discovery of spontaneous death, each kidney was removed and divided in the transverse plane into three portions. The central portion of each kidney was fixed by immersion in 10% neutral buffered formalin, dehydrated through a graded series of ethanol solutions to anhydrous, washed twice in xylene, and infiltrated with molten paraffin  in an automated tissue processor . Tissue blocks were embedded into molten paraffin, sectioned at 2 μm in a Leica microtome, and stained with periodic acid-Schiff reagent with a hematoxylin counterstain. Sections were coded to prevent observer bias, and evaluated by a renal pathologist.During the survival study, mice were observed twice daily, at 7 am and 6 pm; any mice found dead were removed from the appropriate cages. The identifying number of each dead mouse was recorded along with the date and time of death, and kidneys were removed and processed for histological examination as detailed above.Quantitative data from timed sacrifices  were evaluated by two-way analysis of variance, considering the genotype and age of each mouse at sacrifice as independent variables. The significance of inter-group variation was evaluated by Bonferroni's t - statistic. Data were pooled from two independent experiments, because three-way analysis of variance disclosed that there was no significant difference between the two replicate experiments.Overall survival was measured from the date of birth to date of death, and censored at the date of last follow-up for survivors or at the date of sacrifice.  The differences among groups were tested using the log rank test, and the Cox proportional hazards model ,52 was ulpr : MRL/MpJ-Tnfrsf6lpr; dsDNA: double-stranded DNA; SNP: single-nucleotide polymorphism;MRL/The authors declare that they have no competing interests.in vitro experiments. JWS harvested tissues and assisted with in vitro experiments. XD performed backcrossing and genotyping of mice and in vitro experiments. QL performed backcrossing and genotyping of mice and in vitro experiments. DS performed in vitro experiments. MK performed in vitro experiments relating to antibody elution from kidneys. JRS developed the defective γ3 heavy chain allele and bred the original knockout line. PF performed statistical analyses and plotted the survival curves. CP designed and supervised in vitro experiments relating to antibody elution from kidneys and wrote the manuscript. SE designed, performed, and interpreted experiments relating to kidney histopathology and wrote the manuscript.All authors read and approved the final manuscript. NG contributed to developing the defective γ3 heavy chain allele, conceived and designed the studies, interpreted the data, and wrote the manuscript. ML harvested tissues and assisted with In the manuscript titled "IgG3 Deficiency Extends Lifespan and Attenuates Progression of Glomerulonephritis in MRL/lpr Mice", Greenspan et al., have studied the role of IgG3 antibody in the autoimmunity associated with MRL/lpr mice. Although IgG3 antibodies are implicated in autoimmunity, their definitive role in causing immunopathology is unclear. These authors have generated IgG3 deficient mice and have shown that the loss of IgG3 protects mice against glomerulonephritis-associated morbidity and mortality. The authors deserve credit for the well-controlled experiments using MRL/lpr mice with all three genotypes derived from the same litters of crosses between heterozygotes. They also deserve credit for presenting their data clearly. The authors have shown that although IgG3 deficient MRL/lpr mice suffer renal damage, the process of progression to end stage renal disease is retarded or arrested, thereby increasing their survival, compared to normal MRL/lpr mice. While these evidence imply that IgG3 antibodies play a pathogenic role in MRL/lpr mice, they are not conclusive. To further strengthen the studies in elucidating the role of IgG3, they should experimentally demonstrate that passive transfer of IgG3 antibodies derived from MRL/lpr mice or injection of hybridomas secreting IgG3 antibodies causes pathology or at least decreases the survival in IgG3 deficient mice.Minor concerns and questions:• It is known that IgG3-deficient mice in BALB/c background are more susceptibleto pneumococcal infection than wild-type BALB/c mice. In this study, did the authors observe any infection by commensal bacteria or any evidence of inflammatory bowel disease in IgG3 deficient mice?• In Figure The manuscript can be accepted for publication in "Biology Direct" provided that the authors perform the suggested experiment.The experiments requested by the reviewer were done many years ago and reported in multiple publications, two of which are cited as references 13 and 14 in our manuscript.In the present study, we did not investigate infections or the possible presence of inflammatory bowel disease. Such matters are beyond the scope of the present study.Antibodies of the IgG3 subclass are previously known to play a role in the pathogenesis of the spontaneous glomerulonephritis in MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mice. However, as put forward by the authors, the rationale of the current study is to provide a direct and a more definitive evidence that IgG3 antibodies are important in the spontaneous renal disease of MRL/lpr mice, using mice with genetic deficiency in the capacity to synthesize IgG3 antibodies. In order to validate the direct pathological role of IgG3, the authors must perform the passive transfer of the antibodies in their mouse model. I agree that it has been previously shown that passive transfer of IgG3 monoclonal antibodies can elicit renal pathology analogous to the damage that develops spontaneously in MRL/lpr mice. However as authors argued, the serum levels of such antibodies required for those effects may surpass those that are physiologically relevant in the spontaneous disease. Moreover those experiments were not performed in mice that were specifically defective in IgG3 subclass antibodies in MRL/lpr background. Therefore, the authors must demonstrate a direct role of this subclass of antibodies in the pathology in mice defective in IgG3 subclass antibodies in MRL/lpr background, which was not done in the previous studies, cited as references 13 and 14. It is necessary to show that such a passive transfer of physiological levels of IgG3 antibodies reverses and reduces the longer life span, observed in the IgG3-deficient MRL/lpr mice in their model. The manuscript can be accepted for publication in "Biology Direct" provided that the authors perform this experiment.lpr mouse model, such experiments are not guaranteed to recreate the in vivo conditions that obtain during the evolution of spontaneous glomerulonephritis. It would take substantial effort to produce the relevant reagents , define the necessary experimental parameters , and carry out the experiments. These efforts potentially represent an amount of work sufficient for another manuscript. Furthermore, neither of the other two reviewers commented on the need for passive transfer experiments to justify the publication of the present manuscript. Therefore, we respectfully disagree with the reviewer on this point.While we agree with reviewer 1 that it would potentially be useful to perform passive transfer experiments with IgG3 antibodies in the IgG3-deficient MRL/The aim of the study was to provide direct evidence that IgG3 isotype antibodies are critical in the glomerulitis that develops in MRL/lpr mice. The method was to backcross a 'defective' gamma3 heavy chain into MRL/lpr mice and observe serum anti-dsDNA antibodies particularly of the IgG3 isotype; kidney lesions; IgG and IgG3 eluted from the kidneys; kidney function and survival. The mice with the 'defective' gamma3 (termed -/-) demonstrated a slight decrease in general serum IgG anti-dsDNA with essentially no IgG3; less IgG and much less IgG3 eluted from the kidneys; modified histologic kidney pathology; and delayed death compared to wild type (termed +/+) and heterozygous mice (termed +/-).I have several problems with the conclusions one may draw from these results:1. The so-called -/- mice are transgenic for a defective gamma3 heavy chain; they are not gene knockouts, and, in fact, IgG3 was eluted from their kidneys. Therefore, they do produce at least some IgG3 antibody. So the observations and results cannot be attributed to the absence of IgG3 antibodies.2. We are not given any details about the actual defect in the transgene, or how expression of the defective gene might have affected expression of the wild type gene that was left, or ideas about the possible role of IgG3 in the recognition of dsDNA, or the likely relationship between the mouse IgG3 isotype and the equivalent human IgG isotype (IgG2?) and their possible roles in SLE in humans vs mice.3. The mice with the defective IgG3 heavy chain gene died, albeit later than their counterparts; was there any effect of the transgene on the general lymphoproliferative disease of the mice? Did the transgene affect only the kidneys? What actually killed the mice? I enjoyed reading the paper; I found it to be thought provoking and raised the issue of how a single gene product might affect the pathophysiology of a complex immune system and organ system disease.The original gamma 3 heavy chain-deficient mice were obtained by homologous recombination of the targeting vector at the gamma 3 heavy chain locus (ref. 30 in the manuscript). The SNP analysis, included for reviewers only, strongly suggests that the defective gamma 3 heavy chain allele inserted as intended eliminating the wild-type allele and making the genetically-manipulated mice true knockouts.H1. This gene structure was designed to be unexpressed.The targeting vector, as described in ref. 30, contained a neomycin-resistance gene just upstream of the sequence coding for CThe Western blot analysis of sera from mice possessing the defective gamma 3 heavy chain allele in both ref. 27 and the present manuscript support the inference that if there is any IgG3 antibody in the sera of these mice it is at a concentration below the level of detection of this assay. In addition, in ref. 27 it was shown by ELISPOT analysis that there were no spleen cells spontaneously secreting IgG3 (sensitivity approximately one cell per million) in mice of the BALB/c background that were genotyped as gamma 3 -/-. In contrast, spleens from wild-type BALB/c mice contained numerous cells secreting IgG3.et al. :3134-8. PubMed PMID: 12626570) have described IgG2 dimers that form due to disulfide bonds between Fc regions of otherwise separate IgG monomers.Mouse IgG3 and human IgG2 are not fully 'equivalent,' although both isotypes are associated with responses to polysaccharide immunogens. Human IgG2 has not been described to dynamically self-associate non-covalently like murine IgG3, but Yoo The manuscript by Greenspan et al. describes that, when an inactivated IgG3 locus is introgressed in MLR/lpr mice those become partially protected against glomerulomephritis.On the positive side, I found the observations reported convincing, and those could clearly be relevant to deciphering the pathogenesis of kidney damage in lupus, and other antibody-mediated diseases.On the other hand, although the overall quality of the English was more than adequate, I also found that many aspects of the work were very poorly presented, and this was particularly true for the material and methods and the results sections. I would therefore strongly suggest that many modifications should be made to the manuscript before it is published. Below, I have provided a non exhaustive list of suggestions, but I would also advise the authors to seek advice from willing colleagues inside and outside of their immediate field of study to help them improve the overall structure and contents of their manuscript.http://www.informatics.jax.org/external/festing/mouse/docs/MRL.shtml)1) Nowhere in the title or abstract does it say that MRL/lpr mice are a commonly used model for SLE. In fact, I would also suggest adding a bit more info on these mice in the introduction, such as that provided at the Jackson (2) In the abstract, I suggest replacing the sentence "gamma3 -/- mice exhibited minimal titers of these auto-antibodies " by "gamma3 -/- mice exhibited baseline levels of these auto-antibodies"; Indeed, 'minimal titers' suggests that there are some IgG3 still present, and according to McLay et al. 2002, the IgG3 KO mice have a complete absence of IgG3 synthesis. Any signal detected should be due either to background noise, or cross reactivity of the reagents on other isotypes.3) In the introduction, in the sentence "In terms of IgG subclasses, there is evidence implicating both IgG2a and IgG3 antibodies in the pathogenesis of renal disease " you sho4) further down, I would change 'Subsequent reports revealed that many IgG3 antibodies self-associate in antigen-independent -20' to '5) In the section on genotypes of mice in the M&M, there should be a much more thorough description of the IgG3 +/- genotyping protocol, with a reference to panel A of Figure 6) In the section entitled, Measure of auto-antibodies specific for double-stranded (ds) DNA and g-actinin, I am not exactly sure of what the purpose of the passage of dsDNA through a 0,45 micron filter is. It is possibly to sterilize it, and/or to shear the DNA, but certainly not to purify it !7) In the section "Elution of antibodies from kidneys": the buffer(s) used, and the precise conditions should be specified . Also, I suggest changing 'The total eluted IgG or IgG3 in each group was determined ...' to 'The amounts of eluted IgG or IgG3 in each group were determined ...'. On this same subject, in the results section, the amounts of antibodies eluted are expressed as concentrations, which really does not mean anything if one does not know the final volume used for elution. I would strongly suggest expressing those as total amounts of antibody per kidney, or per mouse.8) Evaluation of renal structure: Upon sacrifice (at specific ages selected to provide insight into ( not to) the progression9) I suggest that the first paragraph of the results really belongs in the M&M section. The layout of Figure 10) On Figure 11) In the text describing Figure 12) In the section on eluted antibodies: as mentioned earlier, these need to be expressed as total amounts (in micrograms) per kidney or mouse. You also need to describe how the ratios between total IgG and IgG3 were established. The most disturbing thing to me, however, was that these data were not presented as a figure. Is this because this was done only once or twice (which would explain why there is not deviation provided)? If it is the case, you should really repeat this a few more times instead of providing data in this shoddy format which is both difficult to apprehend, and does not correspond to data which had been documented sufficiently thoroughly.13) Regarding figure plots, I would strongly encourage you to convert them to a smaller format with white backgrounds. Figures 14) I would like to express my very strong disagreement (not to say outrage) regarding the following sentences: ""Serum creatinine in both the +/+ and +/- mice at 26 weeks was significantly  higher than the level in the same genotype at 18 weeks, whereas the difference in serum creatinine between 18 and 26 week old -/- mice was not significant (t = 2.2). These data indicate that there was progressive renal insufficiency (and presumably nephron loss) in the mice capable of producing IgG3, but not in -/- mice.". Indeed, just because a difference does not reach statistical significance, it does not mean that there is no difference. I would thus suggest the following wording: "Serum creatinine in both the +/+ and +/- mice at 26 weeks was significantly  higher than the level in the same genotype at 18 weeks, whereas the difference in serum creatinine between 18 and 26 week old -/- mice was less pronounced and did not reach statistical significance (t = 2.2). These data thus suggest that there was progressive renal insufficiency (and presumably nephron loss) in the mice capable of producing IgG3, but that this was attenuated in -/- mice."15) I found the whole section on histopathology extremely difficult to read and understand. Indeed, whereas, in other sections of the manuscript a clear effort is made to be didactic for a non specialist reader , I found that this section was incredibly difficult to decipher. I would actually suggest pooling Figures 16) Regarding Figure 17) In the discussion, the idea about an IgG3 rheumatoid factor is interesting, but it would be helpful to define clearly what a rheumatoid factor is (i.e. an antibody that can bind to the Fc portion of other antibodies).18) Further down, replace "with the notion that elimination of IgG3 antibodies attenuates the irreversible renal damage associated with the MRL/lpr mouse strain." by "with the notion that the absence of IgG3 antibodies attenuates the irreversible renal damage associated with the MRL/lpr mouse strain."lpr strain has been widely studied as a model of human systemic lupus erythematosus.1) The abstract now notes that the MRL/2) The requested wording change in the abstract has been implemented.3) The relevant sentence (p. 4) now indicates that the reference to Ig3 and IgG2a antibodies applies to mice. Humans do not have a subclass referred to as "IgG2a."4) The relevant passage  has been modified to read as follows: "Subsequent reports revealed that a notable characteristic of many IgG3 antibodies is to self-associate in antigen-independent -20 or an5) In the M&M section on "Genotypes of mice," reference is now made to Figure 6) Passage through the 0.45 micron filter is to remove the single stranded DNA, thus enriching for dsDNA. This is a standard method which has been used in all of our (CP) previous relevant publications, and it has never been questioned.7) The wording changes requested have been implemented in the M&M section on the methods for elution of IgG antibodies from kidneys. As requested, details of the methods have also been inserted.8) The requested revision in the text has been implemented in the M&M section on "Evaluation of renal structure."9) For the gel photograph in Figure lpr mice of all three γ3 genotypes . Similar results were obtained in a repeat experiment with spleen cells from BALB/c γ3 -/- mice.10) We have reduced Figure 11) The requested revision has been made . In addition, we have modified the wording of the final sentence of the section to read: "The greater concentration of the IgG3 auto-antibodies in the IgG3 producing mice could contribute to any observed γ3 genotype-associated differences in renal function, renal histopathology, and survival (see below)."12) See response to comment 7.13) We acknowledge the reviewer's comment but do not believe it is necessary to consolidate the figures or create a figure for the limited kidney elution data.14) We have implemented requested change in wording in paragraph 7 of Results.15) As suggested, the first paragraph of the section has been significantly revised.16) As suggested, we have eliminated Figure 17) A description of rheumatoid factors has been inserted, as requested, in paragraph 4 of the Discussion.18) We have replaced "elimination" with "the absence" in paragraph 5 of the Discussion.The manuscript by Greenspan et al. describes that, when an inactivated IgG3 locus is introgressed in MLR/lpr mice those become partially protected against glomerulomephritis. I found the observations reported convincing, and those could clearly be relevant to deciphering the pathogenesis of kidney damage in lupus, and other antibody-mediated diseases.Compared to the initial version, I find that the modifications introduced following the suggestions of the three referees have resulted in a very significant improvement of the manuscript.I do, however, remain thoroughly unsatisfied with the aspects dealing with elution of immunoglobulins from kidneys, for the following reasons:- The authors only provide the concentrations of antibodies recovered, but not the total volumes of those eluates. There is not even an indication that the same volumes were used for all samples. What should really be provided, and compared, are amounts of antibodies per kidney (or per mg of tissue).- From the way the data is presented in the text, I have come to suspect very strongly that these data were obtained only once, with each value corresponding to one eluate obtained from the pooled half-kidneys in each of the cohorts. I can understand that this may be an unavoidable limitation of the approach, but this should have been made very clear, and justified, in the text.- In this paragraph, the authors state that "< 12% of the IgG in eluates from -/- mice was IgG3.", suggesting that there are some IgG3 to be found in IgG3 KO mice. According to McLay et al. 2002 (ref 27), however, the IgG3 KO mice have a complete absence of IgG3 synthesis. Any signal detected would thus be due either to background noise, or cross reactivity of the reagents on other isotypes.- And what to say of the +/- mice, for which 'IgG3 represented ~10% of the total IgG eluted', i.e. roughly the same levels as those from IgG3 KO mice?- All those considerations are particularly relevant since, contrary to what the authors state, the differences in total IgG concentrations between +/+ and -/- mice are actually rather minor: 16.8 vs 6 μg/ml at 18 weeks, and 86 vs 66 μg/ml at 21 weeks. From what I can make out from the data in the form in which it is provided, the IgG deposits are mostly of the IgG3 subtype in +/+ mice, and not so much less, but of other subtypes in -/- mice, and very surprisingly also in +/- mice. This last point is completely brushed over by the authors. This observation in itself underlines the need for those experiments to be repeated on additional mice, or cohorts of mice, before those results can be considered as sound and reliable.I was also sorry to see that the authors had chosen to ignore my suggestions on two much less important points:- In the paragraph of the M&M section about anti dsDNA ELISA, I thought that it would be helpful to clarify that the purpose of passing the DNA through a 0.45 micron filter was to eliminate single stranded DNA.- I also suggested that the layout of figures should be modified. As they are now, I suspect that those figures will become extremely difficult to read once they have been reduced to printing sizes, and will become completely useless to people if printed in black and white (as well as for color-blind people).We now explicitly state in the relevant section of Materials and Methods that the kidney elutions were only performed once. As for the methodology, the identical volumes of fluid were used for eluting each kidney. We believe that the apparent IgG3 concentrations eluted from the γ3 -/- kidneys most likely arose from the crossreactivity of the detection reagent for the ELISA.As requested, we have added the rationale for the filtration of the DNA to the section on the ELISA for autoantibodies specific for dsDNA in Materials and Methods.Although we appreciate the reviewer's substantial efforts in reviewing the manuscript and take seriously his suggestions , we are not convinced that it will be a net benefit to modify the figures.


Introduction. The management of pelvic tumors is a challenge for orthopaedic oncologists due to the complex anatomy of the pelvis and the need to have extensive exposure. Various reconstructive techniques have been proposed with poor functional results and a high percentage of complications. Our purpose is to determine the functional results and the rate of complications of iliac stem prosthesis for acetabular defects following resections for periacetabular tumors. Materials and Methods. Between 1999 and 2012, 45 patients underwent pelvic resections for periacetabular bone tumors followed by reconstruction with stem cup prosthesis. The most common diagnosis was CS , followed by OS  and metastasis (3 cases). In 33 cases, this implant was associated with massive bone allografts. Minimum follow-up required to evaluate functional outcome was 2 years. We classified pelvic resections according to Enneking and Dunham's classification and we used MSTS  score to evaluate functional outcomes. Results and Discussion. Sixteen patients died of their disease, three were lost to follow-up, four are alive with disease, and twenty-two are alive with no evidence of disease. Fifteen patients had local recurrence. Sixteen patients had bone or lung metastasis. We have had 6 infections, 2 aseptic loosening, and 2 cases of hip dislocation. Iliac sovracetabular osteotomy was fused in all cases at 10 months from surgery. Functional results were good or excellent in 25 of 31 patients with long-term follow-up (77%), with a percentage similar to that reported in the literature. Conclusion. The use of iliac stem prosthesis is a simple reconstructive technique that reduces operative times and risk of infection. It allows having good results and low rate of complications, but it should be performed in selected cases and centres of reference. The management of pelvic tumors is a challenge for orthopaedic oncologists. Since 1980, when Enneking and Dunham [This surgery is characterized by long operative times and consequently high number of complications, as reported by several authors –10.Various reconstructive techniques have been proposed, such as iliofemoral coaptation and ischiofemoral arthrodesis, with poor functional results. The use of saddle or custom-made prosthesis can improve hip function in major resections but is characterized by high number of complications.The use of large pelvic allograft is the most common technique to restore acetabular defects, but this procedure may be associated with high rate of complications, such as infections, nonunions, and fractures.We believe that the use of iliac stem prosthesis, alone or in association with pelvic allograft, for acetabular defects following resections for periacetabular tumors simplifies reconstructive techniques and consequently can reduce the complications.This technique can be used in a wide variety of patients, including children and patients who have received chemotherapy.Our purpose is to determine the functional results and the rate of major complications of this type of reconstructions.Between 1999 and 2012, 45 patients underwent pelvic resections for periacetabular bone tumors followed by reconstruction with stem cup prosthesis . In 33 cGeneral data and oncological outcomes are recorded . Twenty-The patients underwent staging exams according to Enneking's stage system . The mosAll patients received intravenous antibiotic therapy preoperatively and for 3 weeks after surgery. When the tumor was located in the periacetabular region, an ilioinguinal surgical approach was used with an anterolateral and ileofemoral extension in order to obtain a good view of the neurovascular structures and a good internal and external exposure of the pelvis and hip joint. When the tumor was located in the proximal femur, an extended ileo-femoral approach was performed.We classified pelvic resections according to Enneking and Dunham's classification  Figure .We performed 13 extra-articular femoral resections, removing the entire hip en bloc, when the tumor was located in the proximal femur and involved the joint and 32 intra-articular acetabular resections when the tumor was located in the pelvis, all of them involving P2 area. The most common type of resection was P2-P3 (14 cases) followed by P2 (12 cases). The mean operative time was 5.9 hours (range 4–10 hours). Complications and functional outcome are reported .Reconstructions were performed all with iliac stem prosthesis  and in 3In partial P2 resection, if residual bone stock was sufficient to obtain a stable implant, only stem cup prosthesis was used.In P2 or P2-P3 resections we used CISP: stem prosthesis cemented in the allograft and press-fit in the residual iliac host bone .In P1-P2 resections, the stem cap prosthesis was completely cemented .In cases of CISP is very important to obtain a good matching between graft and host bone. Moreover, to improve the donor-to-host bone contact, a transiliac screw is added Figures  and 8. EHemovac drains were removed at a mean of 4-5 days after surgery. Partial weight bearing was started at 6 months and total weight bearing was begun at 9–12 months. Hip cast was used for the first 3 months.Patients had follow-ups with X-ray of the pelvis and CT of the chest every 3 months during the first 3 years, then every 6 months during the fourth and fifth year, and yearly thereafter. Radiographic evaluation was assessed by two authors (DD and MD), in order to evaluate signs of nonunion, resorption of the graft, failure, or migration of the prosthetic components. Union of the graft to the host bone was said to have occurred if there was no visible osteotomy line at the junction sites or if greater than or equal to 75% of cortical thickness was fused on follow-up radiographs, according to the ISOLS radiological implants evaluation system . MarginsFunctional evaluation was performed at the most recent follow-up examination and using MSTS score system .Patients'  and prosThe study was approved by the Institutional Ethics Committee and patients were informed about the proposed surgical procedure and alternative procedures, and informed consent was obtained in each case.Sixteen patients died of their disease, three were lost to follow-up, four are alive with disease, and twenty-two are alive with no evidence of disease.Fifteen patients had local recurrence (33%): margins were wide only in 9 of them. Ten were CS. All of them were extracompartimental, stage IB, or IIB.Six of them were treated with hindquarter amputation, three with chemotherapy and radiotherapy, three with excision of the local recurrence, and one with resection and reconstruction with saddle prosthesis. One refused hindquarter amputation; one was lost to follow-up. Sixteen patients had bone or lung metastasis.The probability to have the prosthesis still in place was 60% at medium follow-up of 5 years .We have had 6 infections (13%) that required in case 15 and 44 allograft and prosthesis removal , in 1 case only allograft removal (case 11—replaced with cement spacer), in 1 case surgical debridement (case 36), and in two cases hindquarter amputation (case 22 and 26). Three of them were patients who underwent chemotherapy for osteosarcoma.Case 11 was a IIB OS that received chemotherapy and extrarticular femoral resection (H1-H2-P2) and reconstruction with allograft prosthetic composite . At last follow-up (99 months) he has no evidence of disease and poor function. Case 15 was a wide resection (P1-P2-P3) for a central CHS staged IB. A composite reconstruction was performed, but he developed lung metastasis and at last follow-up (75 months) he is alive with disease. Case 22 was a central CHS staged IB and treated with P2 resection and reconstruction with CISP. At last follow-up (112 months) she has no evidence of disease. Case 26 was a central CHS staged IB and treated with P2-P3 resection and reconstruction with CISP. She was lost to follow-up at 5 months. Case 36 was a IIB angiosarcoma who underwent P2-P3 resection and reconstruction with CISP. She was lost to follow-up at 38 months. Case 44 was a IIB OS that underwent P1-P2-P3 resection and reconstruction with CISP. At last follow-up she has a poor function.We have had 2 aseptic loosenings (case 1 and 9), due, respectively, to allograft fracture during operation and wrong positioning of the stem cup prosthesis.Case 1 was a P2 resection and was treated with revision of the implant . At last follow up (144 months) he has no evidence of disease and good function.Case 9 was a P2 resection reconstructed only with stem cup prosthesis and underwent revision of the prosthesis. At 127 months from surgery she has no evidence of disease and fair function.We report 2 cases of hip dislocation (case 12 and 27) both treated with revision surgery using longer head prosthesis and an artificial ligament between iliopubic branch and intertrochanteric area of the femur. Case 12 was a P2 resection and it has no evidence of disease at last follow-up (84 months) and has an excellent function. Case 27 was a radio-induced OS treated with P2-P3 resection and CSIP. She developed local recurrence, but she refused amputation.Among minor complications, we report 2 cases of sciatic popliteal nerve palsy, partially recuperated, 2 cases of delay healing of wound surgery, 3 vein thrombosis, and 1 intolerance to sacroiliac screw which was removed.Iliac sovracetabular osteotomy was fused in all cases at 10 months from surgery, while we report a frequent nonunion of anterior pelvic ring that has not affected implant stability .Three of the patients evaluated with long follow-up had a resorption at the prosthesis-host bone interface, with a migration of the implant <20 mm in 2 of them. Anyone underwent revision surgery.Functional results were good or excellent in 25 of 31 patients with long-term follow-up (77%). Sixteen of them were partial P2 resections or P2-P3 resections reconstructed only with iliac stem prosthesis.Surgical management of periacetabular tumors is a challenge for orthopaedic surgeons because of the complex anatomy of the pelvis and because there are important anatomical structures to preserve. Pelvic continuity and durable hip function are very difficult to achieve.Several reconstructive options have been described to restore pelvic ring continuity, such as saddle prosthesis, custom-made prosthesis, and the use of allografts, alone or in association with prosthesis (composite reconstruction).Saddle prosthesis was first described by Nieder in 1979 to fill wide acetabular defects in revision hip surgery. Then, they were used to reconstruct pelvic anatomy following resections for periacetabular tumors. But several studies , 14, 15 One of the first studies dealing with custom-made prosthesis was performed by Abudu et al.  in 1997.Customized prosthesis is made based on preoperative imaging data, which may not reflect the situation after resection if tumor-related or mechanical problems are discovered at surgery and require more extensive resection than planned. Conversely, pelvic allografts are larger than needed and are cut to size at the end of the resection step .The most important advantages of composite reconstructions are the possibility to restore any bone defect by fitting the graft during surgery as well as osteointegration, revascularization, and partial replacement with host bone and the possibility to suture soft tissues to the graft, providing better results by developing a biologic union with the recipient bone. The most important disadvantage is the high infection rate, reported between 10 and 50% in the literature. Other disadvantages are fracture, non-union, the risk of viral disease transmission, and the necessity to have a musculoskeletal tissue bank.Few records are reported in the literature about the use and results of pelvic allografts. In 1996, Ozaki et al.  presenteIn 2000, Yoshida et al.  describeDelloye et al.  describeMore recently, Donati et al.  analyzedOur purpose in this study was to determine implant survival and functional outcomes of these patients.The most important limitation of our study is the presence of many uncontrolled variables, such as tumor grade and extent, the treatment, patient age, and reconstructive procedure, which have a confounding effect on dependent variables such as prosthesis survival. Moreover, there is the difficulty to compare our results with those of other authors because of differences in tumor type, extent of resection, and reconstruction techniques.We observed 6 infections (13%), a rate similar to other series. All of them occurred in patients who underwent composite reconstruction with massive allografts and 3 of them in patients who underwent chemotherapy for OS. Other series, like for example, Ozaki's one , reporteThe use of rifampin solution to thaw the allografts is a factor influencing our low rate of infections, but other factors must be searched to explain it. According to us, the most important factor reducing infections is the low operative time (mean time 5.9 hours), due to an easy surgical technique that avoids the use of long plates and in selected cases the use of the stem cup prosthesis without allograft, such as when resection line can spare part of the acetabular roof and posterior wall.We reported 2 aseptic loosening of the implant, due to wrong positioning of the prosthesis during surgery (that leads to an improper usury of polyethylene) and to an intraoperative fracture of the allograft.Two dislocations are reported: both cases were not treated with artificial ligament. They were revised with longer head prosthesis and an artificial ligament between iliopubic branch and intertrocanteric area of the femur.We had 15 local recurrences (30%). Among these patients 6 had inadequate margins and 8 were high-grade sarcomas. Ten were CS and all were extracompartimental, stage IB, or IIB. This high rate of local recurrence can be justified by different variables, such as the inadequate margins of resection in 6 of them (Donati et al.  have demOur functional results are similar to those reported in the literature, between 56% and 75%.The use of a stemmed acetabular cup, alone or in association with a massive bone allograft, seems to be a valid alternative and a simple reconstruction technique. This type of reconstruction avoids complex osteosynthesis, such as contoured plates, therefore reducing significantly the operative time and early complication rates.It allows having good results and low rate of complications, but it should be performed in selected cases and centres of reference.