burnetii genome in multiple copies has ensured detection of the bacteria. Designed PCR provided evidence of two C. burnetii-positive serum samples, no. 37 from Zemné and no. 47 from Vinice. Although in the course of testing we ended up with several unreadable results, all positive samples were reliably and repeatedly detected, and underwent PCR detection in duplicate.

Non-bacteria-positive, for example non-rickettsial, non-template controls, gave negative results in all runs performed. Furthermore, the clinical picture of the patients (A. Nyitray, unpublished data) endorses our results. Regardless of the applied method, we did not detect any case of Rickettsia mongolotimonae infection, which is known to cause lymphangitis-associated selleck products rickettsiosis (Fournier et al., 2005), nor did we find Rickettsia felis. However, reports of human infection with R. felis are AZD2014 rare (Renvoise et al., 2009). Similarly, some of Bartonella species used in this study remain undetected, for example Ba. henselae (Marseille), Bartonella alsatica, Bartonella vinsonii ssp. berkhoffii, Bartonella ‘weissi’, and Ba. vinsonii ssp. Arupensis. No infection

with human granulocytic ehrlichiosis (HGE) Anaplasma, or D. massiliensis was confirmed either. The use of two complementary methods, IFA and PCR, allowed us to show Rickettsia, Borrelia, Bartonella, Coxiella and Franciscella as possible sources of human infections in Slovakia. Not all serologically detected cases could be confirmed with PCR (Table 3). We are aware of certain limits of the PCR with a single template assay, as the number of organisms found in the

blood can be quite low. Detection limits for amplification of 47-kDa gltA and ompB gene targets of certain rickettsial strains are known be 2, 1 and 1 μL−1 in single template format, respectively (Paris et al., 2008). As few as seven copies of the 16S rRNA gene of R. helvetica could be detected in 200 μL of serum sample in another study (Choi et al., 2005). However, the use of two complementary tests, IFA and PCR, enabled the bacteria to be verified. Five of 16 rickettsial cases detected by IFA were confirmed by PCR. Rickettsiae have been detected in Slovakia previously (Rehacek et al., 1975; Kovacova et al., 2006), and R. slovaca (Sekeyova et al., 1998), R. helvetica (Spitalska et al., 2008) and R. raoultii (Boldis et al., 2008) Sclareol are ‘domestic’ and are frequently neglected by the local medical community. On the other hand, R. conorii serum reactivity in IFA (not confirmed with PCR) is questionable. This agent has never before been identified in Slovakia due to a missing corresponding tick vector (Rhipicephalus sanguineus). Rickettsiae need specific invertebrates as vectors or hosts (ticks, lice and fleas). Thus, together with other detected bacterial agents (Subramanian et al., 2011) they are probably one of the most important causes of systemic febrile illness in Europe (Parola & Raoult, 2001; Chmielewski et al.

Glomerular NEP levels were correlated inversely with glomerulosclerosis and proteinuria measured at the time of biopsy. Tubular NEP levels were associated inversely with interstitial fibrosis. Incubation of proximal tubular cells with MPA led to a dose- and time-dependent increase of NEP gene expression. For the first time, these data suggested that MPA treatment may modulate this enzyme directly,

contributing to the slow-down of the chronic glomerular progression and tubulointerstitial damage [105]. Additionally, a few other studies using in vitro and animal models have identified, using specifically designed microarray platforms, some genes PD0325901 research buy with putative relevance to efficacy and toxicity of immunosuppressive drugs used in nephrology. However, none of the results obtained by these studies has been confirmed in a clinical setting. Interestingly, high-throughput genomic screening technologies have been used to identify biomarkers associated with immunological tolerance in renal transplant patients. It is well known that long-term allograft survival requires lifelong immunosuppression, but recipients

rarely display spontaneous ‘operational tolerance’ with stable graft function in the absence of immunosuppression [106]. The lack of biological markers of this phenomenon precludes identification Navitoclax cost of potentially tolerant patients in which immunosuppression could be tapered or interrupted early. Therefore, the objective of all these

studies was to identify markers able to identify a tolerant population clearly. Several genes have been suggested as potentially useful predictors of tolerance, including genes involved in immune quiescence, apoptosis and memory T cell response [107]. However, further validation and prospective clinical trials using these selective biological elements are needed. Microarray studies have been performed to identify specific genomic FAD fingerprints modulated during acute [108] and chronic [109,110] dialysis therapy. Interestingly, several genes were de-regulated in CKD patients undergoing these renal replacement treatments. Among the genes selected were those encoding for several chemokines with proinflammatory and chemotactic activity [e.g. interleukin (IL)-8, chemokine (C-C motif) receptor 7 (CCR7), tumour necrosis factor (TNF)-α, chemokine (C-X-C motif) receptor 4 (CXCR4)], key regulators of oxidative stress [e.g. v-rel reticuloendotheliosis viral oncogene homologue A (RELA) and glutathione synthetase (GSS)] and those implicated in the mitochondrial oxidative phosphorylation system (e.g. ATP5O, COX6C, COX7C, NDUFS5, NDUFA6, UQCRH, NDUFA1, ATP5J, UQCRB, NDUFB1 and ATP5I).

aeruginosa motility might theoretically result in spreading

of the infection in the body, which needs to be clarified in our next study. Ginseng is one of the traditional Chinese medicines that is widely used not only in China, Korea and Japan but also in the rest of SAHA HDAC order the world, including Europe and North America. It is well accepted in China that ginseng is a tonic medicine with multiple modulating effects on different organ systems in the human body (Huang, 1993). Our previous animal studies showed that ginseng has therapeutic effects on chronic P. aeruginosa lung infections by inducing a TH1-dominated immune response. The present results suggest that ginseng can disturb the development of P. aeruginosa biofilm and cause dissolution of mature biofilms, most likely by activating the motility of P. aeruginosa. Apparently, ginseng is a potentially promising remedy for the treatment of P. aeruginosa biofilm infections. “
“The sensitivity of influenza

rapid diagnostic tests (IRDTs) currently available in Japan for various influenza virus strains, including human H7N9 and H5N1 isolates, were compared and it was found that all of the IRDTs examined detected these viruses; however, their detection sensitivities differed. “
“Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA Pyrophosphorylated metabolites of isoprenoid-biosynthesis (phosphoantigens, PAgs) activate Vγ9Vδ2 T cells during infections and trigger antitumor activity. This activation depends on expression of butyrophilin 3 A1 (BTN3A1) by antigen-presenting BTK inhibitor cells. This report defines the minimal

genetic requirements for activation of Vγ9Vδ2 T cells by PAgs and mAb 20.1. We compared PAg-presentation by BTN3A1-transduced CHO hamster cells with that of CHO cells containing the complete human chromosome 6 (Chr6). BTN3A1 expression alone was sufficient for activation of Vγ9Vδ2 T-cell receptor transductants by mAb 20.1., while activation by PAgs also required the presence of Chr6. We take this finding as evidence that gene(s) on Chr6 in addition to BTN3A1 are mandatory for PAg-mediated activation of Vγ9Vδ2 T cells. This observation is important for the design of animal models for PAg-mediated immune responses and Branched chain aminotransferase provokes speculations about the analogy between genes controlling PAg presentation and MHC-localized genes controlling peptide-antigen presentation. Vγ9Vδ2 T cells are activated by phosphorylated antigens (PAgs) such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) which is found in many microbes and plants [1] and also by cells with an elevated level of isopentenyl pyrophosphate. Elevated levels of isopentenyl pyrophosphate are found in some tumors [2] or in cells after inhibition of expression or function of the isopentenyl pyrophosphate-consuming enzyme farnesyl pyrophosphate synthase (FPPS) by inhibitory RNA [3], aminobisphonates (e.g. zoledronate) [3, 4] or alkylamines (e.g. sec-butylamine) [5].

At the completion of the

experiments, blood was harvested by cardiac puncture with a heparinized syringe and the animal was killed. Blood was assessed for lactate concentration, leukocyte count, and hematocrit X-396 supplier using standard assays in the clinical hematology laboratory of Hamilton Health Sciences Corporation, McMaster site. Purified human AGP was radiolabeled using 125I by the Iodogen method [12] and injected into C57BL/6 mice either intravenously or intraperitoneally, using a dose of 3.3 × 106 counts per minute in 0.1 mL of normal saline; the acid-precipitable radioactivity in plasma samples obtained by sampling from the tail vein was followed over time, and reported as a percentage of the total injected radiolabeled AGP dose as previously described [39, 2]. All values are reported as the mean ± the SEM. Data were analyzed using GraphPad

InStat version 3.01 statistical analysis software (GraphPad Software, Inc., San Diego, CA, USA). For multiple comparisons, data were analyzed using ANOVA with Tukey’s post-test, if the data sets met conditions of normal distribution and similarity of standard deviations, and non-parametric ANOVA (Kruskal–Wallis) with Dunn’s post-test if they did not. For comparisons of two groups, a non-paired, two-tailed Student’s t-test was used for parametric analysis if these conditions were met and the Mann–Whitney test was used if they were not. Statistical significance was set at p < 0.05 in all cases. In all experiments, whether involving endotoxemia or CLP, all animals were alive and active four hours post-LPS or CLP, when subjected to anesthesia in Nivolumab preparation for intravital microscopy; in addition, none died under Cediranib (AZD2171) anesthetic cover prior to the point in the protocol at which euthanasia was planned. As shown in Table 1, there were no significant differences among groups of mice in the endotoxemia experiments in either hematocrit or lactate levels, suggesting that not only did the mice have similar intravascular fluid status but that they were also well resuscitated. A similar

situation was found with respect among groups of mice in the CLP experiments (see Table 2). Administration of LPS significantly reduced circulating leukocyte counts, irrespective of whether saline, AGP, or HAS were employed as the resuscitation fluid (see Figure 1A). Leukocyte counts were reduced to 23 ± 8% of levels seen in sham-treated mice by LPS treatment with saline resuscitation, and to 18 ± 8% and 13 ± 4%, respectively, in LPS-treated mice resuscitated with AGP or HAS, respectively (mean ± SD). These reductions were highly statistically significant with reference to their respective sham values but did not differ significantly among the three resuscitation fluid groups. As shown in Figure 2A, the leukopenia associated with CLP was less marked than that associated with endotoxemia; reductions in leukocyte counts of 50–60% were observed, relative to sham-treated mice, for both saline- and AGP-treated mice.

To test whether CD4+CD25+ T cells constitutively express FasL, freshly isolated CD4+CD25+ T cells and CD4+CD25− T cells were tested for co-expression of FoxP3 and FasL by flow cytometry. The majority of cells expressing FasL were detected within the FoxP3+ population of CD4+CD25+ T cells check details (Fig. 2B, 10.1±2.8% of CD4+CD25+ T cells co-expressed FoxP3

and FasL, n=3 samples tested), while CD4+CD25− T cells did not express FoxP3 and only a small population of these cells (1.9±0.1%, n=3) expressed FasL. The results indicate a population of CD4+CD25+FoxP3+ cells that constitutively expresses FasL. To test the potential killing of hapten-presenting DC by regulatory CD4+CD25+ T cells through Fas–FasL interactions, DC purified from FITC-sensitized mice were cultured with CD4+CD25+ T cells purified from skin-draining LN of naïve mice. Following 4 h of culture, DC were gated as a CD11c+ cell population (Fig. 3A, gate R2) and analyzed for apoptosis by staining with Annexin-V. First, FITC+ and FITC− DC were gated as described above in Fig. 2A and tested for Annexin-V staining after culture with CD4+CD25+ T cells. To normalize the intensity of Annexin-V staining, the autofluorescence of unstained DC was subtracted from the MFI of each DC population stained with Annexin-V. The

intensity of the Annexin-V staining was Torin 1 price significantly increased in the FITC+ DC population when compared with FITC− DC (MFI=113.0±3.3 for FITC+versus MFI=71.6±2.9 for FITC− DC, n=3, p<0.01). While both FITC+ and FITC− DC populations contained Annexin-V-positive cells, the percentages of these cells were significantly increased in the FITC+ DC population (Fig. 3A, 80.7±2.6% versus 52.3±5.1%, n=3, p<0.01). The considerable proportion of Annexin-V-positive cells within the FITC− DC population is likely due to the spontaneous death of DC in vitro, which has been reported in studies using similar cultures of DC alone or with CD4+ T cells 2. Overall, the results indicated the increased death of hapten-presenting DC during culture with CD4+CD25+ T cells in comparison to the death of non-presenting DC under the same culture conditions.

These results correlated with our in vivo studies indicating that the numbers of Reverse transcriptase FITC+, but not FITC−, DC significantly increased in the priming site when CD4+CD25+ T cells were attenuated by anti-CD25 mAb. While apoptosis of FITC+ DC was increased when these DC were cultured with CD4+CD25+ T cells, we did not detect this increase after DC co-culture with CD4+CD25− T cells (Fig. 3B). Furthermore, addition of anti-FasL mAb to the co-cultures of DC and CD4+CD25+ T cells resulted in a significant decrease of FITC+ DC apoptosis (Fig. 3B, *p<0.05), while this FasL blockade had no effect on the death of FITC− DC. This inhibitory effect was dose-dependent as lower concentrations of anti-FasL mAb resulted in less inhibition of DC apoptosis mediated by CD4+CD25+ T cells (data not shown).

S2c). FcγRIIIB was expressed by a smaller percentage of CD4+ T cells (Fig. S2). The examination of three independent fields of cells expanded using anti-CD3 and anti-CD28 showed that a total of 49% of cells expressed FcγRIIIA, 27% expressed FcγRIIIB and 22% stained for MRs. Treatment of the cells with TCC, ICs purified from SLE patients (SLE–ICs) or TCC together with ICs did not alter the

protein pattern of immunoprecipitates Ribociclib clinical trial generated using anti-FcγRIIIA/B (Fig. S7). Western analysis of immunoprecipitates obtained using monoclonal anti-FcγRIIIA/B from naive CD4+ T (CD45RA+) cells showed protein bands migrating at the molecular weights of 26–29 kD that correspond to a previously reported molecular mass for FcγRIIIA and B

(Fig. S6) [29]. In naive CD4+ T cells, an additional band at approximately 34 kD was also observed (Fig. S6). The FcγRIIIA consists of 254 amino acids with a predicted molecular mass of 29 kD (Accession no. P08637-1) and FcRIIIB consists of 233 amino acids with a predicted molecular mass of 26 kD (Accession no. P75015-1). In addition to the light and heavy chains of immnoglobulins, faint protein bands at 72, 98 and 130 kD were also observed. These proteins were also observed in the immunoprecipitates prepared from Jurkat cells. Jurkat cells are used traditionally to study T cell activation (Fig. S6). To further confirm the presence of FcγRIIIA/B in the CD4+ T cells, we analysed the presence of RNA Montelukast Sodium transcripts by RT–PCR. The RT–PCR analysis of the total RNA isolated from both check details peripheral CD4+ T cells and naive CD4+ T cells using a primer set designed from the gene ID NM_001127596·1 (FCGRA) and a second primer set published recently [27] showed the presence of appropriate-sized products for the FcγRIII gene. These FcγRIII transcripts were

also amplified from the total leucocyte RNA. Negative controls without the template RNA did not show the PCR amplification product. Both CD4+ T cells (not shown) and naive CD4+ T cells showed transcripts for the FcγRIIIA/B gene. Jurkat cells also demonstrated these RNA transcripts (Fig. 4). The sequencing of PCR-amplified cDNA confirmed it to be the FcγRIIIA/B gene product. The staining pattern of FcRγ chain in T cells showed them to be present in microclusters, a pattern that is observed for TCR signalling proteins in activated CD4+ T cells (Fig. 3a). The treatment of cells with purified ICs triggered the microclusters to move towards one side of the cell due to capping (Fig. 3a). The presence of TCC during IC treatment further enhanced staining for the FcRγ chain. We observed that the ICs and TCC treatment triggered migration of these receptors into MRs (Figs 5 and S5). We have observed previously that the assembly of non-lytic C5b-9 using purified C5b-6, C7, C8 and C9 labelled with AlexaFluor® 594 trigger MR aggregation beneath C5b-9 deposits (Fig. S4). In quiescent cells, both FcγRIIIB and the FcγRIIIA were not observed in the MRs.

Several studies have shown that autoantibodies are heavily

mutated and back mutation of mutated human V genes to the germline sequences resulted in a loss of antigen binding [20–22]. However, other reports did not support these findings [23–25]. Some studies have shown a low rate of somatic mutation in autoantibodies of patients with SS [17, 26, 27]. In another study, an increased rate (19.6%) of unmutated clones was reported in the parotid gland specimen from a patient with SS [18]. In addition, VH gene analyses of non-Hodgkin lymphomas in patients with SS have shown that neoplastic B cell populations are often unmutated [14–28]. Our finding that B cells infiltrating inflammatory lesions of patients with SS possess less mutated VH genes is in line with these observations and supports the hypothesis KPT-330 that some germline or less mutated genes may play a role in the development of this autoimmune disease. Moreover, Selleck IWR 1 autoantibodies encoded by such genes fail to be deleted in patients with SS. IgG4-related sclerosing sialadenitis is a chronic inflammatory disorder characterized by a dense infiltration of IgG4-positive plasma cells. As treatment with steroids is very effective, an autoimmune mechanism is highly implicated in the aetiology of IgG4-related sclerosing sialadenitis. In this study, we

showed that VH fragments of IgG4-related sclerosing sialadenitis and SS cases shared a common characteristics, a high rate of unmutated VH clones probably derived from the VH3 family. This finding suggests that an autoimmune mechanism similar to that of SS may also be responsible to the development of IgG4-related sclerosing sialadenitis. In conclusion, we studied VH usage and VH

somatic hypermutation in SS and IgG4-related sclerosing sialadenitis using sialolithiasis tissues as a control. The VH fragments, especially those of the VH3 family, were often unmutated when compared with those of the sialolithiasis cases. This finding will provide insight into the pathogenesis of SS and IgG4-related sclerosing sialadenitis. H. S., T. J., K. S, and H. I. designed research; H.S., F.O., and S.M. performed research; H.S., F. O., S. M., and H.I. analyzed data; and H. S. and H.I. wrote the paper. Authors thank Dr Hitoshi Miyachi, Aichi-Gakuin University School of Dentistry, for his valuable advice and Mr Takeo Sirolimus nmr Sakakibara for his technical assistance. H. Sakuma and F. Okumura contributed equally to the study and should both be regarded as first authors. This study has no conflicts of interest. Data S1 Sequence analysis of Sjogren’s syndrome cases. Data S2 Sequence analysis of IgG4-related sclerosing sialadenitis cases. Data S3 Sequence analysis of sialolithiasis cases. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

In the motor cortex, loss of Betz cells was also confirmed. Synaptophysin immunostaining of the lumbosacral cord also revealed decreased expression outside the old lesions, excluding the posterior horn. Interestingly, decreased expression of synaptophysin was also evident in the cervical anterior horns, where no old lesions were observed. No Bunina bodies, TDP-43 inclusions, or Golgi fragmentation were found. Neurogenic atrophy was evident in the iliopsoas and scalenus muscles, and inclusion body myositis-like changes were also observed in these muscles and the tongue. Was it possible to have diagnosed this patient as having ALS? We consider that

the features in this case may have represented the pathology of long-standing and/or fatal PPS itself, and not ALS. “
“We describe a 78-year-old Japanese woman with early-stage progressive supranuclear palsy (PSP). She had a 3-week Veliparib ic50 history of postural instability and gait disturbance. On examination, upper vertical gaze palsy, akinesia, hyperreflexia with pathological reflexes, hesitation, and postural instability were observed. Rigidity and resting tremors were not apparent. Brain MRI revealed atrophy of the frontotemporal see more lobes and dilatation of the third ventricle. A month later, she died of cerebral infarction. The total duration

of her clinical course was approximately 2 months. The brain weighed 1180 g after fixation. Macroscopically, mild atrophy of the frontal lobes and mild depigmentation Lonafarnib of the substantia nigra were observed. The conspicuous findings included degeneration confined to the subthalamic nucleus and substantia nigra and widespread but infrequent tau-positive neurofibrillary tangles/pretangles and glial fibrillary tangles (tuft-shaped astrocytes, coiled bodies and argyrophilic threads)

in the brain. It has been reported that the most affected areas in PSP are the globus pallidus, subthalamic nucleus and substantia nigra. We suggest that degeneration in PSP would start with involvement of the substantia nigra and subthalamic nucleus. “
“Y. Liu, X. Zhang, Y. Liang, H. Yu, X. Chen, T. Zheng, B. Zheng, L. Wang, L. Zhao, C. Shi and S. Zhao (2011) Neuropathology and Applied Neurobiology37, 395–405 Targeting X box-binding protein-1 (XBP1) enhances sensitivity of glioma cells to oxidative stress Aims: Reactive oxygen species (ROS) and oxidative stress are tightly linked with cancers including gliomas. We previously reported the protective role of X box-binding protein-1 (XBP1) against oxidative stress in both mouse embryofibroblasts and human Hela cells. This study was to investigate XBP1-mediated protection against oxidative stress in the treatment of gliomas. Materials and methods: XBP1 expression levels were knocked down by siRNA transfection in the U251MG cell line.

5). A reduction

in p27kip levels permits resting B cells to transition from the G0/G1 to S phase [25]. In addition, siRNA for pro-IL-16 increased the activation of ERK1/2 and p38 MAP kinases and decreased that of JNK1/2 (Fig. 6). These results indicate that ERK and p38 MAP kinases are associated with the activation signalling pathway, while JNK inhibits B cell activation by inducing stress responses in this cell system. In earlier studies, we showed that the function of MHC class II molecules in resting B cells is not limited to their antigen-presenting Ferrostatin-1 supplier role. Rather, they are flexible receptors capable of triggering a variety of signalling pathways and regulating B cell function in a negative manner [6, 16, 17, 46]. In this study, we used a proteomics strategy to demonstrate that pro-IL-16 is associated with B cell proliferation through regulation

of Skp2 and p27kip as well as MAP kinases and NF-κB activation. In addition, impairment of cell growth by nuclear pro-IL-16, which had been shown in T lymphocytes, was observed in resting B cells. This is the first report of the role of pro-IL-16 in B cell function. We believe that further understanding of the mechanisms and pathways involved in MHC class II-mediated negative signalling involving pro-IL-16 will enable us to control B cell function and may yield therapeutic targets Fulvestrant mw for diseases associated with abnormal B cell function. This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0022168 and 2012-0008189). Drs. Y.-S. Jang and S.-H. Kim were supported by the research funds of Chonbuk National University in 2012. We would like to extend special thanks to Drs. Y.-J. Chung and Y.-J. Chang at the Center for University-Wide Research Facilities

of Chonbuk National University for helping with the mass spectrometry spot analysis. “
“Primary immunodeficiency diseases (PIDs) comprise a heterogeneous group of rare disorders. This study was devised in order to compare management of these diseases in the northern hemisphere, given Histone demethylase the variability of practice among clinicians in North America. The members of two international societies for clinical immunologists were asked about their management protocols in relation to their PID practice. An anonymous internet questionnaire, used previously for a survey of the American Academy of Allergy, Asthma and Immunology (AAAAI), was offered to all full members of the European Society for Immunodeficiency (ESID). The replies were analysed in three groups, according to the proportion of PID patients in the practice of each respondent; this resulted in two groups from North America and one from Europe.

UF heparin is infused into the arterial line and protamine into the venous line. Protamine is a basic protein

that binds heparin, forming a stable compound and eliminating its anticoagulant effect. Full neutralization see more of heparin can be achieved with a dose of 1 mg protamine/100 units heparin but protamine has a shorter half-life than heparin so there may be an increased risk of bleeding 2–4 h after dialysis. Most authors agree that the procedure can be technically challenging and has no significant advantage over ‘low-dose’ heparin regimens. Reactions to protamine are not uncommon and may be serious. As all forms of heparin are absolutely contraindicated in HIT Type II this form of regional anticoagulation cannot be used in that syndrome. Citrate binds ionized calcium and is a potent inhibitor of coagulation. Regional citrate regimens generally utilize iso-osmotic trisodium citrate or hypertonic trisodium citrate infusion RG7204 manufacturer into the arterial side of the dialysis circuit. This methodology avoids the use of heparin and limits anticoagulation to the dialysis circuit – effects which can

be used for routine dialysis,25 in patients at increased risk of bleeding26 or for dialysis anticoagulation in the stable phase of HIT Type II. The citrate–calcium complex is partially removed by the dialyser. The procedure may require, or be enhanced by, the use of calcium and magnesium-free dialysate. A low bicarbonate dialysate is also recommended to reduce the risk of alkalosis, especially in the setting of daily dialysis, as citrate is metabolized to bicarbonate. To neutralize the effect of citrate, calcium DNA Synthesis inhibitor chloride solution is infused into the venous return at a rate designed to correct ionized calcium levels to physiologic levels. Plasma

calcium must be measured frequently, e.g. second hourly, with prompt result turnaround. As commercial citrate for this purpose is not available in Australia, Ferrari et al. has recently published an approach with locally prepared citrate and point-of-care calcium testing.27 The procedure can be complex and high risk. There is a requirement for two infusion pumps and point of care calcium measurement. Either high or low calcium levels in the patient may risk severe acute complications. Hypertonic citrate may risk hypernatraemia. The metabolism of citrate generates a metabolic alkalosis. Nevertheless, the technique has been used with great success in some hands, with few bleeding complications and improved biocompatibility with reduced granulocyte activation and less deposition of blood components in the lines or on the dialyser.2 Simplified protocols have been proposed.28 This form of regional anticoagulation utilizes prostacyclin as a vasodilator and platelet aggregation inhibitor. It has a very short half-life of 3–5 min and is infused into the arterial line. Of importance prostacyclin is adsorbed onto polyacrylonitrile membranes.