SL/PS1M146L mice or in primates. Numerous transgenic mice with various plaque typologies have been developed to mimic cerebral amyloidosis and comparison of plaque detection between animal models and humans with new imaging methods is a recurrent concern. Here, we investigated detection of amyloid plaques by Gd-stained MRI in five mouse models of amyloidosis  presenting with compact, diffuse and intracellular plaques as well as in post mortem human-AD brains. The brains were then evaluated by histology to investigate the impact of size, compactness, and iron load of amyloid plaques on their detection by MRI. We show that Gd-stained MRI allows detection of compact amyloid plaques as small as 25 µm, independently of their iron load, in mice as well as in human-AD brains.Gadolinium (Gd)-stained MRI is based on Gd contrast agent (CA) administration into the brain parenchyma. The strong signal increase induced by Gd CA can be converted into resolution enhancement to record microscopic MR images. Moreover, inhomogeneous distribution of the Gd CA in the brain improves the contrast between different tissues and provides new contrasts in MR images. Gd-stained MRI detects amyloid plaques, one of the microscopic lesions of Alzheimer’s disease (AD), in APP Even if their role in AD onset is still debated, they appear as an effective biomarker of its preclinical stages. Currently, the clinical detection of amyloid plaques is based on positron emission tomography (PET) imaging with three radioactive agents recently approved by the Food and Drug Administration (FDA)2. However, the low spatial resolution of PET does not allow the visualization of individual plaques, and in animals, PET studies have provided controversial results4. For example, some studies successfully detected amyloid progression in APP235 and 5xFAD6 mice while others failed to detect signal changes related to amyloidosis8. Other imaging modalities, such as optical imaging9 or two-photon imaging after craniotomy10, have also been developed to detect amyloid plaques in animals. As with PET, optical imaging is too low-resolution to identify individual plaques. Two-photon imaging, however, can reveal individual amyloid plaques at very high resolution (1 μm)10 though the field of view of the technique is limited and does not allow recording of images from the whole brain11.Amyloid plaques are one of the earliest hallmarks of Alzheimer’s disease (AD), occurring up to 20 years before clinical diagnosis15 and in humans17. This approach is limited by a low sensitivity threshold and is strongly dependent on the iron load of the plaques which locally shortens relaxation times19. Also, the possibility to detect plaques by MRI in human tissues on the basis of their endogenous contrast is still disputed21. Thus, MR contrast agents seem to be required to facilitate amyloid plaque detection. The first option is to use MR contrast agents specifically targeting amyloid plaques, modulating their MR signal and so increasing their contrast with the brain parenchyma25. The second option uses non-targeted gadolinium (Gd) contrast agents such as gadoterate meglumine  that is administered in cerebral ventricles after stereotaxic injection26 or intravenously in association with a non-invasive and safe permeation of the blood-brain barrier using ultrasound27. With this method, called Gd-stained MRI, once the contrast agent has reached the brain, amyloid plaques appear as black spots since the hydrophilic Gd-contrast agent increases the signal of tissues surrounding the plaques but do not access their hydrophobic core28. As the volume of brain tissue is high compared to the volume of plaques, these agents induce a high signal increase in the brain. This can be converted into resolution enhancement to record high resolution images. Several in vivo studies in mice have shown that this method reveals amyloid plaques that otherwise cannot be detected by non-enhanced MRI27. Recently, it was used to characterize longitudinally the efficacy of an anti-amyloid immunotherapy29. Gd-stained MRI has also been used to detect amyloid plaques in primates30 and to detect prion plaques in post mortem brain samples from Creutzfeldt-Jakob patients31, but it has never been used to label amyloid plaques in human-AD brains.Continuous efforts are ongoing to implement amyloid plaque detection by high-resolution magnetic resonance imaging (MRI). MRI-based monitoring of amyloid plaques can be divided into three research fields. Some studies are based on the endogenous contrast of the plaques, in both mouse models of amyloidosisSL/PS1M146L, APP/PS1dE9, APP23, APPSwDI, 3xTg). Vascular abnormalities often co-exist with amyloid plaques in mouse models of amyloidosis as in AD patients32. Aβ peptide may accumulate into the vessel wall of cerebral arteries leading to cerebral amyloid angiopathy (CAA) and microhemorrhages. We also examined the capacity of Gd-stained MRI to detect these lesions. Finally, we explored the capacity of Gd-stained MRI to detect amyloid plaques in post mortem human-AD brains. The brain samples were then evaluated by histology to assess the impact of size, compactness, and iron load of amyloid plaques on their detection by MRI. Amyloid plaques from APPSL/PS1M146L, APP/PS1dE9 and human-AD brains had the most similar histological characteristics and could be detected by Gd-stained MRI. Also, we found that the key features associated to amyloid plaque detection by Gd-stained MRI are their size, compactness but not their iron load.Numerous models of amyloidosis with different plaque typologies are used for preclinical investigations. The choice of transgenic mouse model, as well as the stage of Aβ pathology, significantly contributes to the outcome of preclinical studies. Here, we investigated the extent to which Gd-stained MRI allows detection of different types of amyloid lesions including compact, diffuse and intracellular amyloid deposits in five mouse models of amyloidosis  and C57Bl/6 amyloid-free control animals. In vivo MRI was acquired at a resolution of 29 × 29 × 117 µm3 after intracerebroventricular injection of gadoterate meglumine. Ex vivo MRI was recorded at a resolution of 25 × 25 × 100 µm3 after incubation of the brains in a Gd solution. In vivo and ex vivo MRI without Gd-staining were also performed in APPSL/PS1M146L mice. Following ex vivo MRI acquisitions, brains were processed by histology to label amyloid plaques or iron . We found a significant correlation between the hypointense spots load and the amyloid load /PS1M146L mice39 while Meadowcroft et al. reported a very reduced iron load in the plaques of APPS(i.e. Tg2576)/PS1A246E mice18. In humans, some studies reported a close relationship between amyloid plaques and iron depositions18 while some others suggested high or low iron levels in amyloid plaques even within the same subject40. In fact, the relationship between amyloid plaques and iron depositions in humans is still not consensual41. Our study supports the presence of iron in some but not all amyloid plaques. Moreover, the shape of the iron deposits is highly variable with focal deposits in mice, while in humans we detected punctate iron accumulations in the entire plaques as well as ramified accumulations surrounding the plaques.Our histological evaluation showed that APPSL/PS1M146L, APP/PS1dE9 or APP23 with detection thresholds of 36 and 30 µm for in vivo and ex vivo images, respectively, which corresponds approximately to 1.2 times the voxel size. This threshold can explain the poor detection achieved in the 3xTg mice. In this strain, only the largest plaques could be detected, while most of the amyloid deposits that were intracellular and smaller than 20 µm were not seen by MRI. In human-AD brains, the minimum visible plaque size was similar to that in mice (i.e. 25 µm for human-AD and 30 µm for mice).One of the main purposes of our study was to compare MRI and histological characteristics of amyloid plaques in different mouse strains to investigate the critical parameters required for Gd-stained detection of amyloid plaques. The size of amyloid plaques is an important factor that influences their MR detection. Amyloid plaque detection was more efficient in the strains having the largest plaques as observed in APPi.e. APPSwDI or 3xTg mice). The mechanism of contrast enhancement is assumed to be due to the hydrophobic property of amyloid plaques that limits the penetration of the contrast agent within them. Because this hydrophobicity is related to the concentration of the amyloid peptide, diffuse deposits are probably less prone to be revealed by Gd-stained MRI than compact plaques. Interestingly, previous studies have shown that diffuse deposits are not detected by MRI without contrast agents in mice42 or in human brains43. Thus, it seems that Gd-stained MRI does not improve the ability to detect diffuse plaques as compared to contrast-agent free MRI.Our study also showed that the compactness of amyloid plaques seemingly impacts their detection by the Gd-staining procedure. Amyloid plaques were detected in models with compact plaques, but not in models with large diffuse Aβ deposits /PS1A246E mice and APPS(i.e. Tg2576)/PS1M146L), plaques that do not contain iron can be detected42. However, in most in vivo studies without contrast agent, iron is considered as critical for plaque detection39. Since iron accumulation in plaques is age-dependent and variable according to brain region, one of the advantages of Gd-stained MRI is to allow plaque detection without depending on iron accumulation which is a covariate related to aging and not to plaque load.Finally, our study showed that iron accumulation is not necessary for plaque detection after Gd-staining. Indeed, in mice, 33% of the amyloid plaques detected with Gd-stained MRI contained little or no iron. Also, in human-AD brain samples, although most of the plaques detected by Gd-stained MRI contained iron deposits, some plaques devoid of iron were detected. In 32. These lesions are hypointense on MR images35, and could thus be misinterpreted as amyloid plaques on Gd-stained MR images. We showed that large microbleeds are distinguishable from amyloid plaques based on their superior size. Small microbleeds are the only lesions that could be confounded with amyloid plaques. Their number is however low compared to that of plaques, and exclusion of these lesions is always possible by recording T2* or susceptibility-weighted MR images before Gd-staining. Regarding CAA, we observed that blood vessels appeared as hypointense structures by in vivo MRI owing to the paramagnetic properties of blood, independently of the presence of CAA. On post mortem MR images, the hypointense nature of blood vessels could reflect the presence of Aβ peptide in the vessel wall but also a small amount of blood trapped in the vessels. Gd-stained MRI is thus not appropriate to detect amyloid angiopathy.We then examined the capacity of Gd-stained MRI to detect microhemorrhages and CAA. Microhemorrhages are often associated with aging and amyloid pathology in mice and in humans26. This method can thus be used to study amyloid biology or anti-amyloid therapies29. Here, we showed that among the models used in our study, APPSL/PS1M146L, APP/PS1dE9 and APP23 are the models of choice to apply Gd-stained MRI and amyloid plaque detection in these models provides similar results to those obtained with human brain samples.In this article, we also showed for the first time that Gd-stained MRI is able to detect amyloid plaques in human-AD brain tissues. Several significant technical barriers must be solved for this method to become suitable for use in living human subjects, including the ability to administer the contrast agent in the brain as well as resolution and imaging time considerations. The aim of this article is not to propose immediate solutions for these issues and we acknowledge that today the application of Gd-stained MRI as a routine method can be envisioned only in animal models. Gd-stained MRI can be repeated over the lifetime of animals and individual plaques can be followed-up longitudinallySL/PS1M146L, APP/PS1dE9 or APP23 mice independently of their iron load, and suggest that detection of amyloid plaques by Gd-stained MRI in APPSL/PS1M146L and APP/PS1dE9 is the most similar to that in human-AD brains.In conclusion, this study clearly highlights differences among amyloid plaques found in different mouse models of amyloidosis, and provides a better understanding of the origin of contrast induced by amyloid plaques in Gd-stained MRI. We also showed that Gd-stained MRI can be used to detect amyloid lesions in models with large compact amyloid plaques such as APPSL/PS1M146L (n = 6), APP/PS1dE9 (n = 6) and APP23 (n = 4)), diffuse amyloid plaques (APPSwDI (n = 6)) and intracellular amyloid deposits (3xTg (n = 7)). C57Bl/6 amyloid-free mice (n = 2) were used as controls. In vivo and ex vivo Gd-stained MR images from brains of these animals were recorded to detect amyloid plaques. Two APPSL/PS1M146L were also imaged in vivo and ex vivo before Gd-staining. Ages of the animals were selected to image the youngest animals with already well-established lesions  and the oldest animals available in our colony . A cohort of 75 week-old APPSL/PS1M146L mice (n = 5) was further evaluated to study microhemorrhage detection. Their brains were studied before and after Gd-staining. An overview of the selected strains is presented below.We selected male mice from five transgenic strains presenting with compact amyloid plaques (APPSL/PS1M146L mice co-express human APP with Swedish double mutation (KM670/671NL) and London mutation (V717I) and human presenilin 1 (PS1) with M146L mutation under the control of a neuron-specific Thy1 promoter. This leads to a 3-fold higher expression of the human APP transgene than endogenous murine APP. These mice develop dense-cored amyloid plaques that reach a significant level in the neocortex and in the hippocampus at ~26 weeks44. These plaques have already been widely evaluated with Gd-stained MRI29 and they can be detected in ~26-week-old animals26. These mice were imaged from 24 to 78 weeks .APPdE9 mice co-express human APP with the Swedish double mutation (KM670/671NL) and human PS1 deleted in exon 9 under the control of the mouse prion protein promoter. Significant amyloid plaque burden is seen in the hippocampus and cortex by 36 weeks45 and increases with age46. These mice develop amyloid plaques with a dense core, surrounded by dystrophic neurites45. These mice were imaged from 35 to 126 weeks (n = 6).APP/PS147. APP23 mice develop a significant amyloidosis between 24 and 56 weeks48. Then, dense-cored amyloid plaques increase in size and number with age, mainly in the neocortex and the hippocampus47. These mice were imaged from 39 to 77 weeks (n = 4).APP23 mice express human APP with the Swedish double mutation (KM670/671NL) driven by the mouse Thy1.2 promoter allowing a neuron-specific expression of the transgene. This leads to a 7-fold higher expression of the human mutated APP than the endogenous murine APPSwDI mice express human APP containing three mutations: Swedish (KM670/671NL), Dutch (E693Q) and Iowa (D694N), under the control of the mouse Thy1 promoter. These mice display mainly diffuse amyloid plaques and a significant number of plaques is reached at 52 weeks49. These mice were imaged from 52 to 114 weeks (n = 6).APPKM670/671NL, MAPTP301L, and PSEN1M146V) to comparable levels in the same brain regions. Consequently, they display both amyloid and neurofibrillary tangle pathologies. Amyloidosis starts at 12 weeks in the neocortex and at 24 weeks in the hippocampus, before neurofibrillary tangle formation. At this age, amyloid is mainly intraneuronal. Extracellular amyloid deposits become readily evident at 52 weeks50. These mice were imaged from 44 to 112 weeks (n = 7).3xTg mice express three mutated transgenes (APPC57Bl/6 are amyloid-free mice used as controls. These mice were imaged at 48 and 79 weeks (n = 2).All animal experiments were conducted in accordance with the European Communities Council Directive (2010/63/UE). Animal care was in accordance with institutional guidelines and experimental procedures were approved by local ethics committees .post mortem brain samples from the cerebral cortex and adjacent white matter of three AD patients were obtained from the Gie-Neuro-CEB brain bank. This brain bank is run by a consortium of patients associations including France Alzheimer, with the support of Fondation Plan Alzheimer and IHU A-ICM. All methods using human brains were carried out in accordance with French guidelines and regulations. The informed consent forms were signed by either the patients themselves or their next of kin in their name, in accordance with French bioethical laws. The Brain Bank GIE NeuroCEB has been declared at the Ministry of Higher Education and Research and has received approval to distribute samples (agreement AC-2013-1887).Human 51. Blunt Hamilton syringes were used to inject gadoterate meglumine  into the lateral ventricles at coordinate −1.75 mm relative to the surface of the dura mater. A total volume of 1 µL (0.5 mmol/mL) was injected into each side at a rate of 0.1 µL/minute. Upon completion of the injections, needles were not moved for 10 minutes to allow the diffusion of the contrast agent. Then, needles were slowly withdrawn to minimize any outflow from pressure release and the skin was then sutured back.Animals were anesthetized with a mixture of isoflurane (1–2%) and air (1 L/min). After their heads were shaved, the mice were placed on a stereotaxic frame using ear bars and a tooth bar to secure them. A heating pad maintained physiological temperature throughout the procedure. After a midline incision of the skin, the coordinates of the bregma were recorded for anterior-posterior (A/P) and lateral (L) references. The skull was bilaterally perforated with a Dremel at coordinates A/P −0.2 mm and L ± 1 mm, according to a stereotaxic atlasIn vivo MRI was performed with a 7T spectrometer  interfaced with a console running VnmrJ 3.2. The spectrometer was equipped with a rodent gradient insert of 700 mT/m. A birdcage coil  and a mouse brain surface coil  were used for emission and reception, respectively. A high-resolution 3D-Gradient Echo sequence was used to achieve a resolution of 29 × 29 × 117 µm3  = 50 ms, echo time (TE) = 13 ms, flip angle = 20°, number of averages (Nex) = 2, bandwidth = 25 kHz, acquisition time = 1 h 49 min)26. All the MR images were recorded starting at 60 minutes after administration of the Gd contrast agent. During MRI experiments, animals were anesthetized with a mixture of isoflurane (0.75–1.5%) and carbogen (95% O2–5% CO2). Their breathing rate and their body temperature was monitored. Carbogen was used to reduce the signal coming from circulating blood52.in vivo MRI experiments using a high dose of sodium pentobarbital (100 mg/kg) and then fixed with a transcardiac perfusion of 4% paraformaldehyde (PFA). The brains were then removed, immersed in 4% PFA overnight at 4 °C, and preserved in PBS 0.1 M at 4 °C until ex vivo MRI experiments.All animals were sacrificed after ® diluted to 2.5 mM in PBS) for 48 hours before MR experiments. Then, they were placed in a tight plastic tube filled with an aprotonic perfluorocarbon-based fluid  that provides a black background. A high-resolution 3D-Gradient Echo sequence was used to achieve a resolution of 25x25 × 100 µm3 .Brains were incubated in a Gd solution .To detect microhemorrhages, Brains were cryoprotected in 30% PBS-sucrose solution for 72 hours, cut into 40 µm thick coronal sections on a freezing microtome and mounted on slides . Sections were stained for β-amyloid deposits (BAM10 immunohistochemistry and Congo red staining) and for iron deposits (Perls-DAB staining).2O2). Then, they were pretreated with 0.2% octylphenol ethylene oxide condensate . After this pretreatment, they were incubated with an anti-amyloid primary antibody  for 48 hours and then with a secondary antibody  for 1 hour. Before revelation , the reaction was amplified for 1 hour with a biotin-avidin complex 53. For Congo red staining, sections were pretreated with 1% NaOH in 80% EthOH saturated with NaCl for 30 min. Then, they were again immersed in the same solution saturated with Congo red for 30 min. For Perls’ staining, endogenous peroxidases were first inactivated by immersion in a methanol/H2O2 solution. Then, sections were stained with 2% potassium ferrocyanide  and 2% hydrogen chloride for 20 min. Iron staining was finally intensified using DAB (1 g/l), Tris (0.2 M) and 30% H2O2 for 20 min54.For BAM10 immunohistochemistry, sections were first rinsed in PBS 0.1 M and then in 30% hydrogen peroxide  in hydrochloric acid (20%). After 3 washings in distilled water, they were counterstained for 5 minutes in a filtered nuclear fast red solution .55. This plugin enables manual rotation of the 3D MR volume in any direction. We identified typical landmarks such as layers of the hippocampus, blood vessels or amyloid plaques on histological sections. The 3D MR images were then manually rotated until we could identify these landmarks in the MR images. The minimum plaque size resolvable by in vivo or ex vivo MRI was established by measuring plaque diameters on the registered BAM10 stained sections employing ZEN lite 2012 analysis software . Freehand boundaries were drawn around the plaques and their diameter estimated from the average length of the major and minor axes from the resulting ellipsoid18.MR images were manually registered to histological sections using the “3D/Volume viewer” plugin from ImageJSL/PS1M146L, APP/PS1dE9, and APP23 mice. Only cortical amyloid plaques with a diameter ≥36 µm (which corresponds to the minimum plaque size resolvable in vivo) were considered. A total number of 369 amyloid plaques detected on histological sections were classified into four categories. 1. Iron-positive plaques detected by MRI, 2. Iron-negative plaques detected by MRI, 3. Iron-positive plaques not detected by MRI, 4. Iron-negative plaques not detected by MRI.The properties of the plaques detected by MRI were determined after registration between MRI and histological sections double-stained for Aβ and iron in APPin vivo MR images and histological sections of APPSL/PS1M146L (n = 5), APP/PS1dE9 (n = 5), and APP23 (n = 4) mice. For MR images, cortical amyloid load was calculated by using a method similar to that reported by Jack et al.39. A total of 32 regions of interest (ROIs) per animal were analyzed for in vivo and ex vivo samples: 8 slices equally spaced along the rostro-caudal axis with 4 circular ROIs (diameter ~900 µm) drawn in the cortex on each of these slices (2 in each hemisphere). Hypointense spots were manually counted in each ROI, excluding hypointense elements that could be tracked over more than 2 adjacent slices, or that had a tube-like shape, suggesting that they were blood vessels. The area of each hypointense spot was measured in each ROI. Plaque load was determined as the ratio of the total area of hypointense spots over the area of the ROI.Amyloid load was quantified from For histological sections, amyloid and iron load were quantified by the same method used for quantification of amyloid load on MR images after digitization of amyloid and iron-stained sections with a Zeiss Axio Scan.Z1  whole slide imaging microscope at a lateral resolution of 0.5 μm.Supplementary Figures


Pediatric tuberculous meningitis is a highly morbid, often fatal disease. Its prompt diagnosis and treatment saves lives, in fact delays in the initiation of therapy have been associated with high mortality rates.Mycobacterium tuberculosis were negative, while a non classical method based on intracellular cytokine flow cytometry response of CD4 cells in cerebral spinal fluid helped us to address the diagnosis, that was subsequently confirmed by a nested polymerase chain reaction amplifying a 123 base pair fragment of the M. tuberculosis DNA.This is a case of an Italian child who was diagnosed with tuberculous meningitis after a history of a month of headache, fatigue and weight loss. Cerebrospinal fluid analysis revealed a lymphocytic pleocytosis with predominance and decreased glucose concentration. Microscopy and conventional diagnostic tests to identify M. tuberculosis DNA. An early diagnosis is required in order to promptly initiate a therapy and to increase the patient’s survival.We diagnosed tuberculous meningitis at an early stage through an innovative immunological approach, supported by a nested polymerase chain reaction for detection of The online version of this article (doi:10.1186/s13104-017-2444-9) contains supplementary material, which is available to authorized users. Mycobacterium (M.) tuberculosis have a low sensitivity [Tuberculous meningitis (TBM) represents roughly 1% of all cases of TB and it is associated with high mortality and residual neurologic sequelae, even with adequate treatment. In endemic countries the highest incidence of TBM is reported in children aged 2–4 years [sitivity , 4. Alsositivity . An addisitivity , even ifsitivity .We present a case study of an Italian child with TBM who was hospitalized in the Pediatric Infectious diseases department of Umberto I° Hospital in Rome, and in whom the early diagnosis, based on immunological flow cytometry test and molecular assay, allowed us to start an early treatment, fundamental requirement to increase patient’s survival.Neisseria meningitis A-C-Y-W135 Ag, Streptococcus pneumoniae Ag) were negative; cultures for common pathogens and M. tuberculosis, quantitative polymerase chain reaction (PCR) to detect herpetic viruses  were negative. The GeneXpert MTB/RIF assay (Cepheid) was also negative for CSF. In order to investigate other CNS diseases we performed the Link index [A 9-year-old Italian girl developed headache, fatigue and weight loss of about 6 kg 1 month prior the admission to our hospital. One week before admission, because of the occurrence of dizziness and vomiting, she was hospitalized to the Neurology Department of another hospital in Rome, where the computed tomography (CT) scan of head and audiovestibular exams were normal. The patient was discharged with a diagnosis of “Symptomatic paroxysmal vertigo, migraine without aura and acute gastroenteritis”. Few days after the discharge, she reported history of sleepiness, persistence of migraine and fever. Thereafter, she was admitted to the Pediatric Emergency Department of our hospital where she had normal blood tests and a normal brain magnetic resonance imaging (MRI). The MRI is shown in Fig. nk index  and oligMtb-specific CD4+ in peripheral blood and we proposed an immune-based approach, which could improve the identification at single time point of subjects with no TB infection or patients having active or latent TB. A receiver operating characteristic (ROC) analysis was performed to calculate optimal cut-off values for both activated CD4+ T cells and polyfunctional CD4+ T cells in order to discriminate infected (active TB and LTBI) from uninfected patients and active from latent stage. We performed a ROC analysis, and a cut-off >0.45% for activated CD4+ T cells was found as the value allowing the best combination of sensitivity  and specificity  to differentiate Mtb-infected patients (active TB and LTBI) from healthy controls [In order to improve the diagnosis of TB, we performed an alternative immunological method based on multifunctional T cells, which has been suggested in recent years as a new tool for the discrimination between active TB and latent TB infection (LTBI) . In a prAlthough this study involved pulmonary TB, we tried to apply our algorithm to TB meningitis. The same method to stain peripheral blood was used to stain CSF. CSF (0.5 mL) was added to the 3 tubes of QFT-IT containing respectively, saline solution (negative control), phytohaemagglutinin (positive control), and TB antigens . A costimulation with 5 μL/mL anti-CD28 plus anti-CD49d  and 10 μg/mL BrefeldinA (Sigma-Aldrich) was added in all tubes [M. tuberculosis in CSF was negative. Considering the polyfunctionality of T CD4+ cells, those simultaneously producing IFN-γ+ , IL-2+ and TNF-α+ in WB were 0.01% and in CSF 0.40% of total CD4+ cells  (0.94%) and in CSF 2.86%) and using the cut-off (>0.45%) we scored the patient as a positive subject TB infected. Worthy of note, at this time the TST was negative, Quantiferon TB-gold was undetermined and a second PCR for .86% and Moreover we carried out on the CSF extracted from the LP in the first day of hospitalization, the l ADA level which amounted to 18.31 UI/L, higher than the cut-off 10 .Two days later, due to persistent clinical symptoms and uncertainty in the tests of coordination, a second brain MRI was performed with a report of hypertensive hydrocephalus and a diffuse leptomeningeal enhancement of the basal cistern, in particular of the interpeduncular cistern, attributable to infectious-inflammatory alterations. The brain MRI is shown in Fig. Based on the clinical, radiological findings and CSF results  TBM was considered on admission day 3 and therapy was started with antituberculous drugs with isoniazid (H) 300 mg/day plus ethambutol (E) 500 mg/day plus rifampicin (R) 400 mg/day and pyrazinamide (Z) 1000 mg/day, as well as intravenous steroids (dexamethasone 4 mg every 8 h).Analysis of CSF obtained from LP and encephalic drainage in the following days revealed a different protein concentration, with higher level of protein in CSF extracted from LP compared to CSF extracted by ventricular drain Table .Table 1SM. tuberculosis DNA was performed using CSF with a positive result, while the The GeneXpert MTB/RIF assay was negative.Renal and liver function, bilirubin and uric acid levels were monitored regularly. Serial CSF values are shown in Table The family screening was performed and only the father was found TST positive with normal chest radiography and chest CT scan.Once the patient’s condition stabilized, the HERZ regimen was continued and dexamethasone was gradually decreased. However, on admission day 27, because of an acute onset of aphasia with right hemiplegia, an emergency brain MRI was performed Fig. d. The fiOn admission day 74, there were no further episodes of fever, the neurological examination was normal with total disappearance of hemiplegia, the facial asymmetry and the double vision, thanks to neurological rehabilitation. The patient was discharged in good condition without significant neurological sequelae besides the presence of a slight disorder of attention and concentration and with recommendation to continue the treatment with HERZ regimen and aspirin. She was followed up at the outpatient clinic and a follow-up MRI of brain is shown on the Fig. We report a case of TBM complicated with hydrocephalus and cerebral vasculitis tuberculosis (TVC), which benefited from early diagnosis, based on unconventional methods, and an early TB treatment increasing the chance of a favourable outcome.M. tuberculosis and a recent large childhood study documented neurological disability in about 75% of survivors, despite an exceptionally low mortality rate of only 13% and a treatment with antituberculosis chemotherapy [TBM remains the most lethal form of otherapy .Early diagnosis and treatment of TBM is the single most important factor determining outcome –15. AlthThe diagnosis of TBM is difficult because of unspecific symptoms and signs.There are some evidences that a combination of clinical and simple laboratory data might help in the diagnosis . CertainM. tuberculosis from the CSF is dependent upon meticulous microscopy and culture of a large volume (>5 mL) of CSF [In our case report neither the microscopy, to detect acid-fast bacilli in the CSF, nor culture resulted positive since TBM is a paucibacillary form of tuberculosis so they were unhelpful in making diagnosis. The likelihood of seeing or culturing ) of CSF  that is ) of CSF .In the last decades nucleic acid-based amplification (NAA) tests have emerged as potentially important tools for diagnosing TBM, unfortunately they have a high specificity (97–99%) but low sensitivity (46–66%) . In our To increase the chance to detect TB infection we used whole blood QFT-G-IT  to detect the response to stimuli with specific TB antigens. Our child obtained an IGRA on whole blood indeterminate, in accordance with Vidhate and colleagues  who founWe used unconventional diagnostic method to address the diagnosis. We measured the adenosine deaminase activity (ADA), an enzyme that is widely distributed in tissues and body fluids, that has been used in the diagnosis of pleural, meningeal and pericardial TB . VariousM. tuberculosis infection with a high local immune specificity. In CSF the increased proportion of CD4 cells producing cytokines  could reflect the high number of mononuclear cells localised to infected sites, typical of TBM. A similar phenomenon was described in pleural [Furthermore we performed an innovative assay to detect the CD4 intracellular response in whole blood and CSF based on the premise that mononuclear cells localised to infected sites produce more cytokines than peripheral blood mononuclear cells (PBMC) , which h pleural  and alve pleural  in thoraIn our study the use of non conventional methods to detect TBM, such as intracellular staining of peripheral blood and of CSF followed by the detection of ADA activity and a in house nested PCR, helped us in the early diagnosis and treatment of TBM, despite the consecutive negativity of classical diagnostic tests. A prospective study with a large number of children is needed to confirm this isolate observation.


Atherosclerosis is a common inflammatory disease. Stem cell and endothelial progenitor cell treatments can improve cardiac function after myocardial infarction. Granulocyte colony-stimulating factor (G-CSF) is a mobilisation agent, mobilising stem cells from the bone marrow to circulation in the blood. G-CSF may constitute a treatment of atherosclerosis. We have conducted meta-analysis to evaluate the current evidence for the effect of G-CSF on the progression of atherosclerosis in animal models and to provide reference for preclinical experiments and future human clinical trials of atherosclerosis treatment. We searched several databases and conducted a meta-analysis across seven articles using a random-effect model. All statistical analyses were performed using Review Manager Version 5.2 and Stata 12.0.P = 0.006). No significant differences in total serum cholesterol (P = 0.54) and triglyceride levels (P = 0.95) were noted in G-CSF treatment groups compared with controls. Multivariable metaregression analysis revealed that the animal type  and frequency of G-CSF administration  impacted the atherosclerotic lesion area changes. We found that G-CSF therapy was associated with reduced atherosclerotic lesion area (weighted mean difference (WMD): 7.29%; 95% confidence interval (CI): 2.06–12.52%;  The meta-analysis suggested that G-CSF treatment might inhibit the progression of atherosclerosis in animal models. Atherosclerosis is a common disease with serious implications for the human health. It is the main pathological basis of ischemic cardiovascular disease, including coronary heart disease, cerebrovascular disease, and thromboembolic disease . AtherosGranulocyte colony-stimulating factor (G-CSF), a prototypical mobilising agent, induces hematopoietic stem/progenitor cell mobilisation , 6 and sAnimal models comprise a valuable tool for preclinical studies, informing the development of treatment approaches for human diseases. A number of animal experiments have been carried out to study the effect of G-CSF on the progression of atherosclerosis. However, the potential of G-CSF treatment remains controversial. The basic function of G-CSF is to stimulate the proliferation of neutrophil progenitor cells ; furtherhttps://www.ncbi.nlm.nih.gov/pubmed), EMBASE (https://www.elsevier.com), the Chinese BioMedical Literature , and China National Knowledge Infrastructure , up to July 4, 2016. The following keywords and their combinations were used: “Granulocyte colony-stimulating factor or G-CSF” and “atheroscleroses or atherogenesis or atherosclerosis”.We searched the following databases: PubMed (Two reviewers (MLL and LJP) independently qualified all studies. Qualified studies had to satisfy the following experimental criteria: (1) randomised controlled trial of an arterial atherosclerosis animal model; (2) animal model without vascular injury or artery narrowing rings; (3) injection G-CSF as the only experimental intervention measure; and (4) evaluation of the degree of atherosclerosis  as the final results. Principal criteria for the exclusion of studies were as follows: (1) irrelevant topic, duplicate, review, discussion, and comment; (2) no appropriate data; and (3) data partly published in another journal.All data from the qualified studies were independently extracted by the two reviewers. The following data types were extracted: basic characteristics of the animal model atherosclerotic lesion area, total serum cholesterol levels, total serum triglyceride levels, G-CSF dose, total number of injections, and the time point of atherosclerotic lesion area ratio measurements. If required, data were estimated from graphic elements provided in the qualified studies .P < 0.1). Multivariable metaregression analysis was used to determine the factors that underpin the heterogeneity, followed by subgroup analysis. Multivariable metaregression analysis was performed with the following factors that could lead to heterogeneity: animal type ; route of delivery ; G-CSF dose (≤100 μg/kg/d or >100 μg/kg/d); the total number of injections (≤20 or >20); the time point of atherosclerotic lesion area measurement . Subgroup analyses were performed for the same factors as above. Continuous variables were expressed as weighted mean differences (WMDs) with 95% confidence intervals (CIs) between the control groups and G-CSF-treated animals. P < 0.05 was deemed statistical significant.Our main indicator of treatment success was the difference of mean atherosclerotic lesion areas between experimental and control groups. We analysed the data using a random-effect model, but heterogeneity test revealed significant heterogeneity  and Stata 12.0.Articles in English (496) and in Chinese (56) were retrieved for this study from databases specified in  Z = 2.73; P = 0.006), with significant heterogeneity  and the frequency of G-CSF administration  were significantly associated with the reduction in atherosclerotic lesion area.Pooled analysis revealed that the atherosclerotic lesion area was 7.29% after G-CSF injection and was significantly lower than in the control group  was 8.42% lower in the G-CSF treatment group than in the control group. In mouse model studies, the atherosclerotic lesion area was 5.78% (95% CI: −1.64–13.21%) smaller in the G-CSF group relative to the control group. In studies using intraperitoneal G-CSF injection  and intravenous injection , the lesion areas were lower compared with a hypodermic injection treatment . After administration of high doses of G-CSF (>100 μg/kg/d), the atherosclerotic lesion area was 7.67% smaller in the G-CSF group than in the matched control group (95% CI: −19.85–35.19%). After administration of low doses of G-CSF (≤100 μg/kg/d), the atherosclerotic lesion area was significantly smaller  than in controls. Multiple injections of G-CSF (>20) resulted in a greater reduction of the atherosclerotic lesion area  in the experimental group compared with a matched control group. The atherosclerotic lesion area after ≤20 G-CSF injections was 2.55% (95% CI: −2.56–7.66%) smaller in the experimental group than in a matched control group.The results of subgroup analyses are shown in P = 0.03). The atherosclerotic lesion area measured at 8 or 9 weeks  and ≤6 weeks since the beginning of treatment  was lower in the experimental group compared with matched controls.The atherosclerotic lesion area ratio measured >12 weeks since the commencement of G-CSF treatment was significantly lower in the G-CSF group compared with a matched control group .G-CSF regulates proliferation, survival, and differentiation of hematopoietic stem cells/hematopoietic progenitor cells. It is one of the hematopoietic growth factor family members . PreviouAtherosclerosis is a type of inflammatory disease . AtherosIn the present meta-analysis, the atherosclerotic lesion area ratios, total cholesterol, and triglyceride levels were analysed. G-CSF might inhibit the atherosclerotic process since it can effectively reduce the area of atherosclerotic lesions. However, no significant differences were reported for the total cholesterol and triglyceride levels between the experimental and control groups. We hypothesized that the effect of G-CSF on the size of atherosclerotic lesions might be associated with its ability to mobilise stem cells rather than with its effect on the total serum levels of the above compounds. Tousoulis et al.  showed tG-CSF had been shown to increase the number of neutrophils in the circulation by promoting the differentiation of bone marrow progenitor cells into neutrophils and accelerating the maturation and release rate of neutrophils , 38. G-CThe effect of G-CSF on atherosclerotic plaque stability remains unresolved. Plaque vulnerability was characterized by a large necrotic lipid core, thin fibrous cap, and high content of macrophage . In the μg/kg/d) treatment on atherosclerosis progression was not different from high-dose G-CSF treatment (>100 μg/kg/d). But clinical data revealed that low-dose G-CSF treatment is safe in patients with stroke or acute myocardial infarction (<10 μg/kg/d) [μg/kg/d in patients with acute ischemic stroke [Furthermore, our findings revealed that the effect of G-CSF treatment on atherosclerosis progression was significantly affected by the type of animal model and the frequency of G-CSF administration, with rabbit models and multiple administration >20) resulting in a more pronounced decrease of atherosclerotic lesion area ratio. It is likely that the effect of G-CSF on large blood vessels (rabbit) is greater than on small blood vessel (mouse). This result may also be related to the difference between species. The results of subgroup analyses indicated that the effect of low-dose G-CSF (≤100 μg/kg/d) , 49 and c stroke . In addi resultinc stroke . TherefoThe Reviewing Animal Trials Systematically (RATS) Group  suggesteWe here adopted multiple regression analysis to evaluate the effect of various factors on the progression of atherosclerosis in animal models. We discovered that the type of animal model and the frequency of G-CSF administration impact the G-CSF treatment efficacy. Keeping in mind that the analysis was based on published data rather than on accessed individual data, our meta-analysis effectively summarises the available data from animal experiments, drawing conclusions about the efficacy of G-CSF therapy and providing a reference for future clinical treatment of atherosclerosis.


Lactobacillus plantarum (L. plantarum) strains in alleviation of cadmium (Cd) toxicity in vivo and demonstrated that the observed protection largely depended on the tolerance of the strains to Cd-induced stress. It was also observed that there were significant intra-species differences in Cd tolerance of L. plantarum strains. In this study, we investigated the mechanism of Cd induced stress response of L. plantarum strains using the isobaric tags for relative and absolute quantitation (iTRAQ) based comparative proteomics. L. plantarum CCFM8610 (strongly resistant to Cd) and L. plantarum CCFM191 (sensitive to Cd) were selected as target strains, and their proteomic profiles in the presence and absence of Cd exposure were compared. We propose that the underlying mechanism of the exceptional Cd tolerance of CCFM8610 may be attributed to the following: (a) a specific energy-conservation survival mode; (b) mild induction of its cellular defense and repair system; (c) an enhanced biosynthesis of hydrophobic amino acids in response to Cd; (d) inherent superior Cd binding ability and effective cell wall biosynthesis ability; (e) a tight regulation on ion transport; (f) several key proteins, including prophage P2b protein 18, CadA, mntA and lp_3327.Our previous study confirmed the protective potential of  Cd has also been classified as a potent human carcinogen by the International Agency for Research on Cancer4. Chelation therapies, the most commonly used treatments for heavy metal toxicity, have a number of safety and efficacy concerns and as yet none of them have been approved for clinical use against Cd poisoning in humans6. The development of dietary supplements against Cd toxicity represents a potential alternative strategy.Cadmium (Cd) is a representative non-essential element and known as an environmental hazard to human health. This heavy metal can contaminate the food chain and cause cumulative toxic effects in the liver, kidney, bone and the reproductive systems of humans7. Among these LAB, Lactobacillus plantarum (L. plantarum) strains have been widely used in the food industry as probiotics and functional food supplements8. Our previous studies have demonstrated that L. plantarum CCFM8610 can sequester Cd in the intestines of the host, which in turn promotes fecal Cd excretion and reduction in Cd accumulation in tissues, indicating that this strain can be considered a dietary supplement for the prevention and alleviation of Cd toxicity11. It was further noted that CCFM8610 offers significantly better protection than some other L. plantarum strains in vivo11, and the protective effects were dependent on bacterial cell viability9.Lactic acid bacteria (LAB) are members of commensal inhabitants of the human intestinal microbiota that can confer health benefits on the host14. As L. plantarum cells contact with Cd directly in the intestines of the host, the protective effects of the strains against Cd toxicity largely depend on their abilities to thrive after Cd exposure. Therefore, understanding the mechanism of Cd tolerance of L. plantarum strains is essential for the development of probiotic based strategy against Cd toxicity. Our previous work showed that the minimum inhibitory concentration (MIC) of L. plantarum CCFM8610 (tested on Cd-containing agar plates) is more than 1000 mg/L, while some other L. plantarum strains have MIC values 10 to 20 times lower15. A bacterium is considered tolerant to Cd if its MIC value exceeds 100 mg/L16 or 112.4 mg/L of Cd17. Based on this definition, L. plantarum CCFM8610 can be considered a highly Cd-resistant strain. This may partly explain the protection of the host against Cd toxicity in vivo offered by this strain. Therefore, it is of interest to understand the resistance mechanism of L. plantarum strains against Cd exposure, and to explore why CCFM8610 is highly tolerant to Cd.Cd exposure, even at low levels, can disrupt cell wall synthesis, respiratory chain function and metal ion homeostasis in microorganisms, which in turn causes oxidative stress, DNA damage and energy metabolism dysfunctions, inducing extreme cellular toxic effectsPseudomonas spp., Escherichia coli, Bacillus subtilis and Saccharomyces cerevisia20. The related metabolomic pathways and key proteins identified include sulfur assimilation/glutathione synthesis pathway, metallothionein, oxidoreductase and ion transport proteins. However, comprehensive heavy metal tolerance mechanisms have not yet been well defined in LAB strains. To our knowledge, the Cd resistance modes have only been studied in Streptococcus thermophiles and Lactococcus lactis, with a focus on two Cd resistance-associated genes, CadA and CadC24. Numbers of reports also indicated the presence of these genes in some strains of L. plantarum species26, but the Cd stress response network in these strains is yet to be elucidated.The physiological bases of heavy metal tolerance have been well investigated in environmental and industrial microorganisms such as 29. The most commonly used approaches for proteomic analysis are gel-based methods, such as two-dimensional gel electrophoresis (2-DE) and two-dimensional difference gel electrophoresis (2-D DIGE). However, these methods were reported to have limitations in sensibility, reproducibility, and proteome coverage31. Isobaric tags for relative and absolute quantitation (iTRAQ) is a labelling approach that allows reliable quantitative description of differentially regulated proteins in complex systems. The main advantage is to have the possibility to analyze several samples together, with biological and technical replicates (4 or 8 plex). The subsequent use of high resolution mass spectrometry analyses provides accurate relative ratio between protein concentrations, present in the different samples31. Therefore, this approach was selected in this study.Proteomics has been reported to be efficient to provide global physiological profiles of bacteria in protein levelL. plantarum species were investigated by using iTRAQ based proteomic approach. L. plantarum strains CCFM8610 (strongly resistant to Cd) and CCFM191 (sensitive to Cd) were selected for comparative proteomic analysis based on their differing Cd tolerant phenotype. The comparative proteomic profiles between non-stimulating and Cd-exposed conditions were compared in the two strains. The proteomic results were further confirmed by RT-qPCR and by the measurement of several biological properties of the bacterial cells in response to Cd exposure.In this study, the Cd-tolerance related key proteins and pathways within the L. plantarum strains were cultured in MRS broth containing different concentrations of Cd, and the relative growth rates were determined , was selected as a Cd-sensitive strain for the comparative proteomic analysis with CCFM8610. As shown in Fig. 34.Twenty ed Table . While i1592 and 1527 proteins were detected and identified in CCFM8610  and CCFM191 , respectively Fig. , and theIntra-species difference of inherent proteomic profiles between Cd resistant and sensitive strains was evaluated, so as to pinpoint the proteins that might be implicated in the Cd stress response process. Total of 206 proteins with fold change more than 1.5 were selected as differentially expressed proteins  by Cd stress were categorized into biological processes including global stress response, transportation, lipid, amino acid and pyrimidine metabolism and cell wall biosynthesis. For CCFM191, the abundances of 111 proteins were markedly changed after Cd exposure   and some well-studied Cd-tolerant microorganisms36, Cd tolerance related proteins Cd-/zinc-/cobalt-transporting ATPase (lp_3327), Cd-transporting P-type ATPase (CadA) and Cd-/manganese-transporting P-type ATPase (mntA) were further analyzed by RT-qPCR assay, as the information of these low-abundance membrane proteins is easily to be lost during proteomic analysis. Carbamoyl-phosphate synthase, pyrimidine-specific, large chain (pyrAB), Carbamoyl-phosphate synthase, pyrimidine-specific, small chain (pyrAA), D-alanine-poly (phosphoribitol) ligase subunit 2-1 (dltC1), D-alanine-poly(phosphoribitol) ligase subunit 2-2 (dltC2), Transcription regulator of CopAB ATPases (copR), Prophage P2a protein 13 (lp_2444), Nucleotide-binding protein, universal stress protein UspA family (lp_2993), DNA-directed DNA polymerase III subunit epsilon (lp_0811) and Cold shock protein 1 (CspP), were randomly selected for RT-qPCR assay to confirm the reliability of proteomic results. For this reason, the proteins in the same operon are preferred, such as pyrAA and pyrAB, and dltC1 and dltC2. The mRNA expressions of Cd tolerance-related proteins, CadA, mntA and lp_3327, showed clear up-regulation in CCFM8610 after Cd exposure, while these genes (lp_3327 and mntA) were down-regulated or remained unchanged (CadA) in CCFM191 after Cd exposure  levels of two strains before and after Cd exposure Fig. . The resL. plantarum strains, we examined the proteomic profiles of CCFM8610 (strongly resistant to Cd) and CCFM191 (sensitive to Cd) in non-stimulating conditions as well as after Cd exposure using the iTRAQ approach. The results revealed that L. plantarum CCFM8610 displayed a complex biological network to tackle Cd stress, which may be related to carbohydrate, purine and pyrimidine metabolism, global stress responses, lipid and amino acid metabolism, metal binding properties, cell wall biosynthesis and transporters of the bacterial cell  and the four citrate lyases  were in lower abundance in CCFM8610. This corresponded to the lower citrate concentration in this strain (unpublished data). We also observed reduced abundance of 11 proteins involved in the PTS system in CCFM8610, further indicating repression of carbohydrate metabolism even in the absence of Cd stress.The comparative proteomic profiles between the two strains in the absence of Cd treatment showed that CCFM8610 has lower abundance of 24 proteins and higher abundance of only 2 proteins associated with carbohydrate metabolism  to tellurite exposure indicated that even in the absence of tellurite, T5 cells displayed a “poised” status with altered intracellular levels of glutathione and branched-chain amino acids, along with increased resistance to other toxic metals and metabolic inhibitors37. Such a mechanism was believed as an inherent tolerance of the mutant strain primed for tellurite exposure, which further supports our hypothesis.The differentially expressed proteomic profiles in the two strains after Cd exposure also confirmed this self-protection mechanism of CCFM8610. No significant changes in the proteins involved in carbohydrate metabolism were found in CCFM8610 after Cd stress Table , indicat39. The superior Cd tolerance of CCFM8610 may be in part due to its ability to maintain a steady physiological status, including purine and pyrimidine metabolism, after Cd exposure, which can be further validated in the following analysis of its global stress responses.Very limited effect on pyrimidine metabolism-related proteins was observed after Cd exposure in CCFM8610. PurA, an important enzyme in the de novo pathway of purine nucleotide biosynthesis, was down-regulated in CCFM8610 after Cd exposure. NrdE, lp_0811 and dnaE, three catalytic proteins involved in DNA replication, were also changed by Cd stress. In contrast, nine purine and pyrimidine metabolism-related proteins were altered during the Cd stress responses of CCFM191 Table , most ofP. brassicacearum19.Induction of global stress responses is an important strategy for bacteria to endure harsh environmental conditions. The comparative proteomic profiles between native and Cd-treated CCFM8610 showed that Cd stress caused fluctuations of only two proteins that are involved in the universal stress response, lp_2993 (up-regulated) and hsp1 (down-regulated) Table . The for40, was also found after environmental stress in other microorganisms, such as L. reuteri41, L. acidophilus42, and Oenococcus oeni43.In contrast, CCFM191 showed more marked changes with 16 significantly altered proteins related to stress response after Cd shock Table . These pIn non-stimulating conditions, CCFM191 showed higher abundance (compared to CCFM8610) of two proteins (mutS2 and recU) involved in DNA repair, four proteins  related to protein repair, two proteins (lp_2952 and lp_2616) related to bacteriocin immunity, and one protein (hsp1) involved in stress response. In addition, a marked distinction was observed between CCFM8610 and CCFM191 in 23 oxidoreductases, indicating that these two strains display different oxidative stress status even in the absence of Cd exposure.44. Looking at the different nucleic acid metabolism–related and global stress response-related proteomic profiles between these two strains in non-stimulating conditions, as well as in response to Cd exposure, we conclude that compared with CCFM191, CCFM8610 is able to respond to environmental stress with weaker induction of the cellular defense and repair system. The intracellular ROS level of CCFM8610 was significantly lower than that of CCFM191 both in the presence and absence of Cd stress, which further supported our conclusion  was in a higher abundance in CCFM8610 than that in CCFM191. This protein is responsible for the methylation reaction that translates preexisting 47. Our in vitro assays demonstrated a significant up-regulation of hydrophobic amino acids and surface hydrophobicity in both strains  and two acyl carrier proteins (acpA1 and acpA2) were up-regulated in both CCFM8610 and CCFM191 after Cd exposure Tables  and S3.  Figures  and S10,49. As Cd binds preferentially to sulfur ligand50, the up-regulation of sulfur amino acid metabolism may be a self-detoxification mechanism of CCFM191 against Cd exposure.For CCFM191, significant changes in amino acid metabolism were observed after Cd exposure Table . Two pro51. The higher abundance of fhuD, an iron chelatin, might improve the Cd-binding capacity of CCFM8610. Another protein with higher abundance in CCFM8610, hydrolase phosphohydrolase (gph1), can release free phosphate from lipids and precipitate Cd as CdHPO4 or other chemical forms onto the cell surface52. Consistent with these analyses, our in vitro adsorption assays in aqueous phase solution showed that CCFM8610 possesses significantly better Cd binding ability and sequesters a higher proportion of Cd in the cellular surface than CCFM191  biosynthesis, showed a 2.56-fold up-regulation in CCFM8610, which was higher than that in CCFM191  was observed to be down-regulated by 1.68-fold in CCFM8610 after Cd exposure, which could be regarded as a response of the strain to osmotic stress. The phosphoenolpyruvate-dependent PTS is a major carbohydrate transport system in LAB strains. Two PTS sugar transporters (rafP and pts35B) were down-regulated in CCFM8610 after Cd exposure, indicating the energy-conservation survival mode of this strain during Cd stress.Cd exposure inevitably induces osmotic stress in bacterial cells, which in turn leads to further cell damage. The tight regulation of metal import is one of the most basic mechanisms of metal homeostasisCompared with CCFM8610, CCFM191 showed extra changes in three carbohydrate transporters , five ABC transporters , one branched-chain amino acid transporter (livA) and two glycerol uptake facilitators (glpF4 and glpF3) after Cd exposure. These changes might indicate that in order to survive the Cd stress, the sensitive strain CCFM191 needs to shut down more carbohydrate transporters, optimize ABC transporters and increase the proportion of hydrophobic amino acids.56. Prophage-dependent thermo-resistance via plasmid integration has been reported in Staphylococcus aureus57. The phage genes have also been observed to be up-regulated in E. coli upon Cd stress12. These analyses highlighted the prophage P2b protein 18 as a potential key determinant in L. plantarum strains for the response to Cd stress. The acquisition of this stress resistance-related protein by CCFM8610 is a clear benefit to the strain.In the non-treated conditions, the abundance of prophage P2b protein 18 (lp_2463) in CCFM8610 was 6.88-fold higher than that in CCFM191, which was the second-highest fold change among all up-regulated proteins between the two strains. In addition, this prophage protein was observed to be the most significantly up-regulated protein (4.45-fold) in CCFM8610 after Cd exposure. In the Cd-sensitive strain CCFM191, prophage P2b protein 18 remained unaffected Table . In pathL. plantarum strains and some well-studied Cd-tolerant microorganisms58. Since the information of these membrane proteins is easily to be lost during proteomic analysis due to their low abundance and the insufficient extraction, the expressions of their genes were quantified by RT-qPCR  after Cd exposure in CCFM8610, while in CCFM191 it was only marginally down-regulated (0.85 fold). The Cd-resistant function of CadA is well established in S. aureus, L. monocytogenes and L. lactis60, the significantly enhanced efflux of Cd by the up-regulation of CadA might be a detoxification strategy of CCFM8610. The expression of mntA, a Cd2+-Mn2+ shared transporter, and lp_3327, a Cd-/zinc-/cobalt-transporting ATPase, were up-regulated 6.06-fold and 4.57-fold in CCFM8610 after Cd exposure, respectively. However, the expressions of these two proteins slightly decreased in CCFM191. Besides their metal transporting ability, mntA and lp_3327 have been reported to harbor multiple transmembrane domains that bind metal. Therefore, these proteins may also prevent Cd-induced cytotoxicity by Cd sequestration. This may also explain the difference of Cd tolerance between these two strains.Cd-tolerance-associated proteins, including CadA, mntA and lp_3327, are believed to be Cd transporters in the cell membranes of L. plantarum strains using comparative and functional proteomic analysis of L. plantarum CCFM8610 (strongly resistant to Cd) and L. plantarum CCFM191 (sensitive to Cd). The proteomic profiles in non-stimulating conditions and the altered proteomic profiles after Cd stress in both strains were explored. Of the total 1415 identified proteins, 206 were differentially expressed for the comparison of natural proteomic profiles of CCFM8610 and CCFM191, 27 were differently regulated in CCFM8610 after Cd exposure, and 111 were changed in CCFM191 in response to Cd stress. Both strains showed physiological alterations in energy metabolism, purine and pyrimidine metabolism, global stress responses, lipid and amino acid metabolism, metal binding properties, cell wall biosynthesis and transporters in response to Cd exposure. The underlying mechanism of the intra-species distinctions between CCFM8610 and CCFM191 on Cd tolerance can be attributed to the following aspects. (a) CCFM8610 possesses a specific energy-conservation survival mode, which can be regarded as an inherent “poised” physiological status primed for Cd exposure. (b) CCFM8610 can cope with environmental stress with mild induction of the cellular defense and repair system, which enables the strain to survive Cd exposure without drastic physiological response. (c) CCFM8610 induces the biosynthesis of hydrophobic amino acids that enhance surface hydrophobicity of the cell and prevent Cd-induced protein damage. (d) CCFM8610 has inherent superior Cd binding ability and effective cell wall structures, which promotes Cd sequestration on the surface of the cell, preventing the uptake of this toxic metal into the cytoplasm. (e) CCFM8610 exhibits a tight regulation on ion transport to withstand the Cd-induced osmotic stress. (f) Several key proteins, including prophage P2b protein 18, CadA, mntA and lp_3327, also play a potential role in Cd tolerance in CCFM8610. This study provides an overview of the Cd stress response network  and other chemicals were purchased from Shanghai Sinopharm Chemical Reagent Company (China). Trizol reagent was obtained from Ambion Life Technologies (USA). TAKARA RR047A kits were purchased from TAKARA BIO INC (China). iTaqTM Universal SYBR® Green Supermix was purchased from Bio-Rad (USA). iTRAQ reagents were purchased from Applied Biosystems (USA).Cadmium chloride . All strains were cultured in de Man, Rogosa and Sharpe (MRS) broth  at 37 °C for 18 h routinely.Twenty 61, twenty L. plantarum strains were inoculated (2% v/v) into MRS broth containing Cd2+ ranging from 0 mg/L to 50 mg/L and then incubated at 37 °C. The OD600 values were measured at different time points until the stationary phase of the strains. The relative growth rate of each strain was expressed as a percentage of that of the control culture (without Cd exposure) which was assigned a value of 100%61. The MIC value of each strain was determined as the lowest Cd concentration that completely inhibited the growth rate of the strains.As it is normally to incubate the bacteria in liquid medium for proteomic analysis34. The strains were also incubated in Cd-free media as a control.Based on the data of Cd tolerance assay, two strains with the widest difference in Cd tolerance values were selected for proteomics analysis. The strains were grown in the Cd containing MRS broth at 37 °C and the Cd concentration was set as the 1/2 MIC value of the sensitive strain (5 mg/L) as that was established previously600 = 6.0)61, the cells were harvested and washed twice with ice-cooled phosphate-buffered saline (PBS) solution and pelleted. The collected pellets were immediately stored at −80 °C and used for protein extraction. The cell protein was extracted as previously described with minor modifications62. The detailed procedure of protein extraction could be found in supplementary materials.At the early stationary phase  in each of four conditions is shown in Fig. The detailed procedures can be seen in the supplementary materials.L. plantarum WCFS1, based on our preliminary studies revealing that there is a utmost genome similarity between our CCFM8610 and WCFS1. The function of the identified proteins was annotated using Gene Ontology (GO) analysis, and the metabolomics pathway was analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The threshold for differentiated expressed proteins was set as P < 0.05 and fold change >1.5 or <−1.5.The raw data obtained from LC/LC−MS/MS analysis were processed using Thermo Proteome Discoverer software  and searched with Mascot  at the in-house server to perform database comparisons against 63. The primers listed in Table L. plantarum WCFS164. The L. plantarum 16S rRNA gene was used as an expression control with primers specific for L. plantarum strains, and each reaction was conducted in four duplicates65.The total RNA of the strains was extracted using Trizol reagent (Life Technologies). Reverse transcription was performed with TAKARA RR047A kits according to the instruction manual. The alterations of mRNA expressions were evaluated as previously reportedIntracellular metal accumulation, Cd binding, bacterial hydrophobicity, autoaggregation, scanning electron microscope (SEM), intracellular reactive oxygen species (ROS) production, cellular components involved in Cd binding, glucose consumption and hydrophobic amino acid production.The detailed procedures of these assays can be seen in the material supplementary materials.P value of <0.05 was considered to indicate statistical significance.Data were expressed as the mean ± standard error of the mean (SEM) for each group. Differences between groups were analyzed using one-way analysis of variance (ANOVA), followed by the Tukey post-hoc test. A Supplementary Info


Rana dybowskii) from metamorphosis to hibernation (18 weeks) and out-hibernation to hibernation (20 weeks) under the same environmental conditions. Brown frog growth was studied and mathematically modelled using various nonlinear, linear, and polynomial functions. The model input values were statistically evaluated using parameters such as the Akaike’s information criterion. The body weight/size ratio (Kwl) and Fulton’s condition factor (K) were used to compare the weight and size of groups of frogs during the growth period. The results showed that the third- and fourth-order polynomial models provided the most consistent predictions of body weight for age 1 and age 2 brown frogs, respectively. Both the Gompertz and third-order polynomial models yielded similarly adequate results for the body size of age 1 brown frogs, while the Janoschek model produced a similarly adequate result for the body size of age 2 brown frogs. The Brody and Janoschek models yielded the highest and lowest estimates of asymptotic weight, respectively, for the body weights of all frogs. The Kwl value of all frogs increased from 0.40 to 3.18. The K value of age 1 frogs decreased from 23.81 to 9.45 in the first four weeks. The K value of age 2 frogs remained close to 10. Graphically, a sigmoidal trend was observed for body weight and body size with increasing age. The results of this study will be useful not only for amphibian research but also for frog farming management strategies and decisions.Well-controlled development leads to uniform body size and a better growth rate; therefore, the ability to determine the growth rate of frogs and their period of sexual maturity is essential for producing healthy, high-quality descendant frogs. To establish a working model that can best predict the growth performance of frogs, the present study examined the growth of one-year-old and two-year-old brown frogs ( Growth is one of the most important features of animals and can be defined as an increase in body weight or body dimensions over time or with age . The groCurrently, studies of amphibian development are mostly based on years . SeveralMathematical modelling is used in livestock production to assist technicians and researchers in the development of animal breeding and nutrition programmes to improve the accuracy, profitability, and sustainability of zootechnical activities . The groRana dybowskii) is mainly distributed in north–eastern China, and it is an important species with both medicinal and economic value , the Durbin–Watson statistic (DW), the Bayesian information criterion (BIC), and Akaike’s information criterion (AIC). The use of several nonlinear, linear, and polynomial functions to model frog body weight and size data in this research reflects the most comprehensive study of amphibians to date.All frogs used in this study were handled in strict accordance with Northeast Agricultural University (NEAU) Institutional Animal Care and Use Committee (IACUC) protocols (IACUC#09-012) and tissues-of-opportunity waivers were approved by NEAU. Tissues-of-opportunity are defined as samples collected (1) during the course of another project with an approved IACUC protocol from another institution; (2) during normal veterinary care provided by appropriately permitted facilities; or (3) from free-ranging animals at appropriately permitted facilities. No endangered or protected species were harmed during this study.The experiments were carried out from April to October of 2010 at a farm located in Jiankou Town  owned by the Folin company in Jiamusi City, Heilongjiang Province, China. The area experiences a temperate continental monsoon climate with a long winter and a short summer, a frost-free season that typically lasts for 130 d, average yearly precipitation of approximately 510 mm, and an average temperature of 2.8 °C.The open-air breeding pens were enclosed by a sealed fence, a sprinkler was installed, and some low vegetation was planted. This simulated environment designed for rearing brown frogs resembled an actual forest habitat to ensure the necessary conditions for growth. Age 1 and age 2 brown frogs were reared separately in different pens, but all pens were maintained under the same conditions, including the air temperature, photoperiod, and air moisture as well as the construction materials and physical properties of the pen. The temperature and humidity were recorded every 2 h by temperature–humidity metres. The air humidity in the pens varied slightly but was maintained at approximately 60∼80% by spraying water into the air, and the ground-level humidity of the pens was controlled within a range of 25∼35%. During the trial, the average diurnal, maximum, and minimum temperatures recorded inside the facility were 18.73 ± 3.73 °C, 24.36 ± 3.40 °C, and 13.10 ± 2.29 °C, respectively. The temperature of the pens rarely exceeded 35 °C due to ventilation and spraying, thus preventing stress from rapid changes in the frogs’ body temperature.2 in size. The density of the age 1 brown frogs in the pens was 8/m2, and that of the age 2 brown frogs was 4/m2. During the experiments, the frogs and their behaviour were periodically examined. When the average temperature was above 10 °C and the frogs were observed to be moving, we provided food (Tenebrio molitor). The frogs were fed once a day at 09:00, and the feeding amount was approximately 4% of their mean body weight. Insects in the natural environment were also available for consumption. The age 2 frogs used in this experiment had been cultured the previous year (2009) using breeding methods consistent with this experiment (2010). In the fall of 2009, the groups of age 2 frogs were placed in separate wintering ponds to avoid errors in discriminating between the different ages. The initial body weights for the age 1 and age 2 brown frogs were 3.47 ± 0.74 and 0.48 ± 0.05 g, respectively.The age 1 and age 2 frogs were reared in three different breeding pens, each of which was approximately 80 mThe snout–vent lengths (SVLs) and body weights of 40 brown frogs were measured each week. Callipers with a precision of approximately 0.02 mm were used to measure the body sizes (SVLs) of the specimens, and their body weights were measured using electronic scales (accurate to 0.01 g). Age 1 brown frogs included frogs in the developmental stages from metamorphosis to out-hibernation in the second year, and age 2 brown frogs included frogs in the stages from out-hibernation in the second year to out-hibernation in the third year. Additionally, the one-year-old brown frogs completed their metamorphosis by 5th June and began eating between 10th June and 15th June. Brown frogs cannot eat after 1st October due to low temperatures, which terminates growth, so they must enter hibernation one month later. For age 2 brown frogs, the hibernation period ended on 15th May, and they began to feed from 25th May to 1st June before entering hibernation at the same time as the one-year-old brown frogs.Males and females were mix-reared in the first year because the brown frogs did not exhibit sexual dimorphism, but late in the second year, males, and females were reared separately.Frog growth was studied and mathematically modelled using various nonlinear, linear, and polynomial functions. The nonlinear mathematical models included the logistic, Gompertz, von Bertalanffy, Brody, Janoschek, and Richards models.These models contain several common parameters and can associate any biological meaning to each of them.W is the measurement value (g or cm); t is the number of experimental weeks; A is the body weight or length at maturity, B is an integration constant related to the initial weight of the animal, and C is the maturation rate. The value m is the parameter that gives shape to the curve by indicating where the inflection point occurs.Logistic Model. The following equation describes the Logistic  growth mGompertz Model. The following equation describes the Gompertz  growth mVon Bertalanffy Model. The following equation describes the Von Bertalanffy  growth mBrody Model. The following equation describes the Brody  growth mJanoschek Model. The following equation describes the Janoschek  growth mRichards Model. The following equation describes the Richards  growth mTo estimate body weight and body size at a certain age, first-, second-, third-, and fourth-order polynomial functions were fitted to the brown frog body weight and body size data.Polynomial Model. The following equation describes the Polynomial  growth mr is the second to the fourth order of fit; d0 is intercept; id is the regression coefficient.The value of R2adj). Furthermore, the residual standard deviation, or the RMSE, DW, AIC, and BIC were used.The models were assayed for prediction consistency and quality using an adjusted coefficient of determination ); the total sum of squares is denoted as SST; and the residual sum of squares is denoted by SSE. Additionally, n and p are the number of observations or data points and the number of parameters in the equation, respectively. The coefficient of determination is denoted by R2 and is a statistical measure of how well a regression line can approximate real data points.R2 value is a measure of the relationship between the general deviation around the average trend expressed by the growth curve model, and its value lies between 0 and 1. If the regression line of a model has a coefficient of determination of 1, it is considered a perfect approximation of the real data. Therefore, the predictions of a model are considered satisfactory if the coefficient of determination approaches unity.The importance of testing a regression line in this study to resolve polynomial models renders the determination coefficient a useful statistic. The n is the total number of observations (data points); and p is the number of parameters in the equation. The SSE is the squared sum of residuals, which is the squared sum of the individual differences between values estimated from the model and the observed values. The SSE determines the level of discrepancy between data and a model and is therefore a vital criterion for model selection.The root mean square deviation is a particularly generalized type of standard deviation that may occur among sets of observations and represents the sample standard variation of differences between the values predicted by a model and the observed values . IndividThe RMSE value is a major criterion for analysing the predictability and adequacy of the growth curve model of a desired function. Consequently, the growth curve of a function with the smallest error in its RMSE is considered the most adequate model.The DW is used in regression analyses to detect relationships between values separated from one another by a particular time lag; these relationships are generally referred to as autocorrelations. The DW was employed as discussed above, and the results are discussed below.te is the residual at time t, and te−1 is the residual at time t−1.The DW lies between the values of 0 and 4; a value close to 2 suggests no autocorrelation; a value trending towards 0 implies a positive autocorrelation; and a value trending towards 4 implies a negative autocorrelation . The DW Furthermore, AIC was calculated using the following expression :(11)\doAkaike’s information criterion is a good statistic for comparing models or functions of varying complexity due to its ability to modify the SSE for a different range of parametric inputs in a particular model. A smaller AIC value implies greater consistency between the data and the model. The basic use of AIC is in model selection, which has its foundation in information theory and is carried out by implicit comparison of several models. Notably, if all the contrasting models fit the data poorly, AIC selection is affected but not disrupted during usage.Meanwhile, the BIC , which iSimilar to AIC values, smaller BIC values from the above expression suggest better consistency between the data and the model.The model input variables (parameters) were estimated, and statistical analyses were conducted using R software version 2.14.2 . The nonKwl) was calculated as:W is the body weight (g), and L is the body length (cm).The body weight/size ratio (K) was calculated as follows:K is fatness (g/cm3); W is the weight (g); and L is the length (cm). Generally, larger fatness values indicate that the frog is in good shape  is interpreted as an estimate of initial body weights or body sizes, although parameter d1, which is defined as an average body weight or body size, changed over the course of a week. d1 values for the body weight of age 1 frogs, indicating imprecise estimates of this parameter.For the linear model, A is an estimate of asymptotic weight, which can be described as the mature or adult weight. The Brody and Janoschek models yielded the highest and lowest estimates of A, respectively, for the body weights of all frogs. The Janoschek growth model is similar to the Richards model in terms of the level of flexibility  dates were determined. The ages of the frogs were known from management of the frog farm. The above time points are pertinent to the intrinsic growth rate, which provides a better understanding of growth over weeks or the explicit relationship between growth and feeding.The artificial breeding technology used for frogs in this study is specific to in China . SimilarStudying frog growth and development is essential in the first two years of development  from metThe growth curve for frogs is sigmoidal, or S-shaped, from out-hibernation to hibernation, showing a slow rate of increase in the initial stage before accelerating at the metaphase and then slowing again at a later stage . From thThe most rapid rate of growth in body weight and size occurs in the first two years , and theAs shown in In brown frog breeding, growth requires a reasonable food supply and balanced nutrition, but the frogs often showed dietary deficiencies from the 4th to the 10th week after initiating feeding . TherefoEnvironmental factors, such as temperature (time constraints), obviously restrict growth, as occurs with the gradual decline in temperature before hibernation. The brown frog can experience hypothermia, which directly affects its metabolism , followeKnowledge regarding how parameters, such as the growth rate, differ among all developmental stages in frog breeding is important for formulating culture management strategies. Management must be appropriate for populations at different stages.R2adj values for the Richards growth functions were slightly higher than those of the remaining nonlinear models, which all had similar R2adj values; the Richards growth function also showed the lowest AIC values. The R2adj value measures linearity and is consequently a more suitable and appropriate factor for linear models.In this study, the logistic and Gompertz functions were used because of their ability to simply describe vital factors. Some of these simple descriptions include modelling the growth rate as a basic function of size, continuous growth, asymptotes, parameters with biological points, points of inflection, and sigmoidal trends. According to R2adj values suggest that the fourth-order polynomial model was more suitable. The estimated A and C parameter values are interpreted as mature body weight and the instantaneous relative growth rate, respectively.According to A linear model, unlike nonlinear models, lacks parameters with biological meaning , but nonObtaining frog descendants with rapid growth and sexual maturity rates  requiresR. dybowskii is a rare species among breeding frogs. It usually lives on land in a forest environment during its terrestrial stage, which differs from other frogs that exhibit a more typical, amphibious lifecycle.R. dybowskii, an amphibian that lives in terrestrial habitats, influencing the micro-climate factors of the captive environment was easy, which is unlike cultivation of other frogs or snakes and turtles that have complex lives both in water and on land. The rearing cycle of R. dybowskii was up to two- to three-years long, and the frog has the characteristics of ‘three adaptions and nine inadaptations’; the three adaptions are gloomy, dampness, and clean, and the nine inadaptations are high light, rain, dry, wind, high temperature, predators, dirty, noise, and off-flavour. These features reflect the high risk of frog culture due to complicated breeding technology and high morality. High-density breeding is the main factor affecting the occurrence of disease and high morality. The present study was conducted at a site within the natural distribution of R. dybowskii, and the density of the captive frogs was relatively low, which can largely prevent the occurrence of disease and ensure normal growth of the brown frog.For K, Kwl, and fatness of the cultured frogs were greater than those of wild frogs or frogs grown in a much more natural environment (Many factors affect the growth of frogs, including not only a captive environment but also the quality and quantity of food and health status . Culturiironment , suggestR2adj values than those of the other models. The results will be useful for amphibian research, and the growth curves will inform frog farm management strategies and decision-making regarding the culling of poor producers and the selection of highly productive animals. Furthermore, the relationship between growth characteristics and environmental changes has yet to be revealed.The growth of brown frogs was studied, and a mathematical model was developed using various nonlinear, linear, and polynomial functions. However, third- and fourth-order polynomial models provided the best fit for growth curves of body weight for age 1 and age 2 brown frogs, respectively. Both the Gompertz and third-order polynomial models yielded similarly adequate results for the body size of age 1 brown frogs, while the Janoschek model produced a similarly adequate result for the body size of two-year-old brown frogs due to lower RMSE, AIC, and BIC values and greater 10.7717/peerj.4587/supp-1Supplemental Information 1Click here for additional data file.


Kidney transplantation confers superior outcomes for patients with end stage kidney disease, and live donor kidneys associate with superior outcomes compared to deceased donor kidneys. Modern immunosuppression has improved rejection rates and transplant survival and, as a result, recurrence of glomerulonephritis has emerged as a major cause of allograft loss. However, many glomerulonephritides have significant genetic risk which may manifest through kidney intrinsic or systemic mechanisms. We hypothesise that heritable kidney intrinsic predisposition to glomerulonephritis will result in increased risk of glomerulonephritis recurrence in kidneys transplanted from genetically related donors.We investigated the effect of living related donation on rates of recurrence and subsequent graft outcomes in 7236 patient from 28 years of ANZDATA transplant registry data. Data were analysed in R, using Kaplan Meier Survival analysis and adjusted analyses performed using Cox Proportional Hazards methods. A competing risk model was also analysed.p < 0 · 001). In IgA nephropathy, transplantation from living related donor kidneys demonstrated a 10 year recurrence rate of 16 · 7% compared to 7 · 1% in living unrelated donors and 9 · 2% in deceased donors . In focal segmental glomerulosclerosis, risk of recurrence at 10 years was 14 · 6% in living related donors compared to 10 · 8% in living unrelated donors and 6 · 6% in deceased donors  for living related vs deceased donors. Primary glomerulonephritis death censored graft survival was superior for living donor grafts, related or unrelated, compared to deceased donor grafts.Glomerulonephritis recurrence rates were significantly higher in living related donor grafts compared to either living unrelated or deceased donor grafts (We identified a significant increase in the risk of glomerulonephritis recurrence in IgA Nephropathy and Focal Segmental Glomerulosclerosis in living related donors compared to a deceased donors. Kidney transplantation confers the best prognosis  for patiIt has been observed that kidneys transplanted from living donors have  both earWe have investigated the effect of living related donation on rates of GN recurrence and subsequent graft outcomes from 28 years of ANZDATA transplant registry data, comparing living related, living unrelated and deceased donors.The Australia and New Zealand Dialysis and Transplant Registry (ANZDATA) collects data on all renal transplants performed in Australia and New Zealand. Baseline clinical and demographic data are recorded with follow up data collected every 6 months from all transplant centres. Patient anonymity is assured with coding of data on entry. Recurrence of glomerulonephritis is recorded in the registry as the date of the relevant biopsy. Biopsy practice is defined by each centre according to its current clinical practice. This will necessarily have changed over the last 30 years with some centres moving to protocol biopsies. Biopsy indication is not available for this population currently. Data were extracted from ANZDATA for all renal allografts transplanted for patients with a primary GN between 1985 and 2013 within Australia and New Zealand. Transplants included first, second, or subsequent transplants. Primary end points were death censored graft loss (DCGS) or GN recurrence until December 2013.All statistical analyses were carried out in R . BaselinSixteen thousand twenty-three renal transplants were performed between 1985 and 2013 of which 7,236 (45 · 16%) were for biopsy-proven primary glomerulonephritis (GN). Characteristics of the patients are summarised in Table p < 0 · 001)  or living unrelated donor kidneys . A competing risk model was used to compare rates of recurrence in deceased and related donor allografts. Ten year recurrence rates were slightly reduced at 7.5% for deceased and 14.8% for related donors but they remained statistically significant. These are compared with standard survival analysis results in Table GN recurrence occurred in 424 10 · 5%) of all primary GN allografts. GN recurrence rates were significantly higher in living related donor grafts compared to either living unrelated or deceased donor grafts (0 · 5% ofp = 0 · 0005) or living unrelated donor kidneys  ) compared to 7 · 1% ) for living unrelated donors and 9 · 2% ) for deceased donors. In adjusted analysis, the risk of IGAN recurrence was significantly increased for living related donor kidneys compared to either deceased donor kidneys  and not with living unrelated donor kidneys . There was no difference in the rates of recurrence free survival based on donor category among patients with MN (p = 0 · 2) or MCGN (p = 0 · 6)  compared to 10 · 8% among living unrelated donors  and 6 · 6% in deceased donors . However, on adjusted analysis, the risk of FSGS recurrence in living related donor kidneys was significantly increased compared only with deceased donor kidneys  and this survival advantage was maintained in living related donor grafts  was superior for live donor grafts compared to graft survival in deceased donor grafts   Fig. . In cont02) Fig. . In FSGS02) Fig. .Fig. 4DeWe examined graft survival from time of recurrence Fig. . There wWe tested whether increased recurrence in IGAN and FSGS related to shared HLA antigens. The risk of recurrence was not significantly increased by in zero-mismatched grafts Fig. . This suOur study demonstrates an increased risk of primary GN recurrence within renal allografts from living related donors, mainly in patients with IGAN and FSGS. Increased risk with live donation has been reported in a number of studies of primary GN, and specifically in IGAN –21, FSGSCurrent studies of the effect of donor status on recurrence and outcome are unclear. A meta-analysis of the effect of donor status reported a higher risk of IgA recurrence in living related grafts but a non-significant trend in the risk of graft loss . The ERAThe mechanism of increased GN recurrence risk in living related grafts is unclear. This study implicates genetic risks for IGAN and FSGS manifesting in a kidney-intrinsic manner. However, it remains unclear if recurrence of IGAN is , 35 or iDespite the elevated risk of GN recurrence demonstrated in grafts from living related donors, death-censored graft survival was still superior for patients with IGAN compared to deceased donors, and no difference was observed in death-censored graft survival in MN, FSGS or MCGN. Our study shows that, for those with a primary diagnosis of IGAN or FSGS, the benefit of live allograft donation is maintained regardless of the elevated risk of GN recurrence. This benefit does not appear to be present for patients with membranous or MCGN. Therefore, increased recurrence rates do not outweigh the superior outcomes associated with living donation, in agreement with previous studies , 19, 26.Whilst this is a large, well-characterised study population with 28 years follow-up, we acknowledge the limitations inherent in registry analysis. Despite these limitations, this study represents the most complete and well-populated registry analysis to date.The increasing use of protocol biopsies post transplantation has the potential to alter the results of this analysis. The significance of histological evidence of recurrence in the absence of clinical evidence of glomerulonephritis is unclear. It may be argued that to adequately document the incidence and prevalence of post-transplant GN, a protocol biopsy regimen should be mandatory. Under those circumstances, it is highly likely that the GN recurrence rate would rise, but the overall impact on graft survival may fall. Whilst this might increase scientific rigour, it remains to be shown that such a policy can improve outcomes, both for recurrent GN and graft survival.In this Registry analysis of 7,236 patients with primary glomerulonephritis who received renal allografts between 1985 and 2013 we identified a significant increase in the risk of GN recurrence in patients suffering from IGAN and FSGS where donation occurs from a living related donor compared to a deceased donor. Despite this elevated risk of recurrent disease, the survival advantage of living related donation is maintained. We conclude that there is no reason to avoid living related donation in recipients with primary GN despite the elevated risks. Potential recipients should, however, be informed of the increased risk of disease recurrence when receiving an organ from a relative.