The aim of this study was to comprehensively evaluate contemporary tibial component designs against global tibial anatomy. We hypothesized that anatomically designed tibial components offer increased morphological fit to the resected proximal tibia with increased alignment accuracy compared to symmetric and asymmetric designs.Using a multi-ethnic bone dataset, six contemporary tibial component designs were investigated, including anatomic, asymmetric, and symmetric design types. Investigations included (1) measurement of component conformity to the resected tibia using a comprehensive set of size and shape metrics; (2) assessment of component coverage on the resected tibia while ensuring clinically acceptable levels of rotation and overhang; and (3) evaluation of the incidence and severity of component downsizing due to adherence to rotational alignment and overhang requirements, and the associated compromise in tibial coverage. Differences in coverage were statistically compared across designs and ethnicities, as well as between placements with or without enforcement of proper rotational alignment.Compared to non-anatomic designs investigated, the anatomic design exhibited better conformity to resected tibial morphology in size and shape, higher tibial coverage (92 % compared to 85–87 %), more cortical support , lower incidence of downsizing (3 % compared to 39–60 %), and less compromise of tibial coverage (0.5 % compared to 4–6 %) when enforcing proper rotational alignment.The anatomic design demonstrated meaningful increase in tibial coverage with accurate rotational alignment compared to symmetric and asymmetric designs, suggesting its potential for less intra-operative compromises and improved performance.III. Though total knee arthroplasty (TKA) is a largely successful procedure , 16, 31,Several morphological assessments concluded that contemporary tibial component designs do not fit global population equally well , 34, 37.To date, all the studies on component coverage relied on manual component implantation , 25, 32,n = 316) and Caucasian (n = 163) ethnicities and spanning a wide range of patient statures, were used in this study , at 5°–analysis ) Fig. 1n = 316 aPersona™ The Personalized Knee System ; an asymmetric Design B: Natural-Knee® II System ; and four symmetric designs: (1) Design C: Vanguard® Complete Knee System ; (2) Design D: Triathlon® Knee System ; (3) Design E: Sigma® Knee Solutions ; and (4) Design F: NexGen® Complete Knee Solution . All the available sizes in each component design were used in the analysis.Six contemporary TKA tibial component design families were evaluated in this study Table , includiY) was defined as the line connecting the medial third of the tubercle and the center of the posterior cruciate ligament (PCL) attachment site, projected onto the resection plane [A two-dimensional coordinate system was constructed on each resected tibial surface for aligning the resection contours and performing the morphological measurements Fig. . The neuon plane . The medsize and shape : Areas enclosed by profile or bounding box : For overall resected plateau (plateau aspect ratio), the ratio was defined as the AP/ML ratio of the bounding box. For each compartment (compartment aspect ratio), the ratio was defined as the ratio between the AP dimension of the compartment and the ML width.Boxiness (shape): Boxiness was defined as the ratio between the area of the overall plateau or individual compartment and the area of the associated bounding box .Asymmetry (shape): Asymmetry metrics reflect the asymmetry between the medial and lateral compartments . AP asymmetry was defined as the ratio between medial and lateral AP dimensions; anterior radius asymmetry was defined as the ratio between medial and lateral anterior radii; and boxiness asymmetry was defined as the ratio between medial and lateral compartment boxiness.The metrics were regressed against ML width for each ethnicity. The regressions were compared with the same metrics measured from the six tibial component designs Fig. a to asseFive clinically relevant anatomic zones were identified on each resection contours as shown in Fig. Using the automated algorithm, the next two studies applied one or both of the two types of placement described below Fig. :Fig. 5A Placement maximizing coverage The largest component size was selected to maximize coverage without constraining rotation, provided ≤1 mm component overhang was achieved in zones 1–4.Placement with proper rotational alignment This placement repeated the procedure as in the placement maximizing coverage, but with the added constraint that rotational alignment had to be within ±5° of the neutral rotational alignment axis.Tibial coverage from placement with proper rotational alignment was correlated with tibial component size (ML width) for each design and compared between Asian and Caucasian population groups. For cases in which a compromise on either rotation or overhang was required , the component design family was identified as “no suitable tibial component fit” for that bone.Following the above methods, downsizing of the tibial component was identified if the predicted component size when rotational accuracy  was enforced was smaller than the size which provided maximum coverage (placement maximizing coverage). In order to assess the impact of downsizing on cortical support for the tibial component, the distance from the profile of the component to the exterior cortex in anatomic zones 1–4 was calculated for both placements. Specifically, twenty points were evenly sampled on the component profile from the region that fell in each anatomic zone. The shortest distance from the sampled point to the resection contour was calculated through projecting the point to the resection contour, which represent the profile of the exterior cortex of the bone. The cortical distances for each sampled profile point were then averaged across all the bones, with smaller averages indicating better cortical support around that point on the component. The degree of mal-alignment for placement maximizing coverage and the incidence of downsizing were compared across designs.Indian CT data Sant Parmanand Hospital, New Delhi, India.Japanese CT data PS Clinic, Fukuoka, Japan.Korean CT data Department of Radiology, Asan Medical Center, Seoul, South Korea.Chinese CT data The Affiliated Hospital of Medial College Qingdao University, Qingdao, Shandong, China.The Asian CT scans in this study were collected from live patients. Each CT data collection has been approved by the institution to which the study principle investigators were primarily affiliated. The following listed the names of the institutions that granted the approval:n = 92, Korean/Chinese group) can provide a power of 0.80 to detect a 1 % difference in coverage between two groups. Therefore, the sample size used was determined to be sufficient as sub-percentage difference in tibial coverage was deemed to be clinical irrelevant.A power analysis was performed for the sample size selection . Assuming a 3 % standard deviations in tibial coverage , the smap = 0.05.The arithmetic mean and standard deviation of the coverage measurements were determined. One-way analyses of variance (ANOVAs) were performed to compare the tibial coverage from placement with proper rotational alignment between ethnicities and designs, and between the two types of placement across designs. Distribution of the distance from the profile of the component to the exterior cortex in each anatomic zone was calculated and compared between the two placements for each design and across designs. The null hypothesis was that all the ethnic, design, or placement group means are equal; the level of significance was defined at size, the LAP of all six designs generally agreed well with the three ethnicities investigated  of the anatomic design were consistently closer to the anatomy than the other designs . Most bones without suitable component fit were Asian (1–5 % bones for Designs B–F) and only presented in Design F for Caucasian (1 %)  and to Japanese in Designs C and D (p ≤ 0.01)  Fig. , with thComponent internal rotation of more than 5° was required on 39–60 % of bones in the non-anatomic designs in order to maximize coverage, leading to component downsizing (sometimes multiple times). Furthermore, 30 % of the bones were internally rotated beyond 10°, and 2–11 % of the bones required downsizing of 2 or more sizes. In contrast for the anatomic design, only 3 % of the bones required a single downsize caused by small mal-rotations (≤10°)   . No differences in cortical support were found in other anatomic zones (2–4) between the two placements (n.s.).Across designs, in order to ensure proper rotational alignment, average coverage decreased from 90–93 to 85–92 % Figs. a. In the01) Fig. a and pos01) Fig. b, c. In The most important finding of the present study is that the anatomic tibial component design offers improved component fit compared to the non-anatomic designs investigated. It exhibited the closest morphological match to the size and shape of the resected tibia, provided the highest and most consistent tibial coverage, and resulted in the least compromise across the multi-ethnic dataset studied. Specifically, for the anatomic design, by properly leveraging anatomic data within component design, the accuracy of rotational alignment is facilitated, not impeded, by maximizing tibial coverage and minimizing overhang. In contrast, the surgical compromise required for the non-anatomic designs may be attributed primarily to the morphological mismatches between their profiles and the resected tibia, such as smaller medial compartment and overlooked or insufficient asymmetry in the anterior radius. To ensure accurate alignment, symmetric designs that have a smaller medial compartment have an increased tendency to overhang in the posterolateral region of the resected tibia . MismatcIt is worth noting that for the non-anatomic designs, although smaller (ML) component sizes were used for the dataset compared to those for the anatomic design Fig. , there sThere are several limitations to this study. First, although the resection parameters used for design comparison were representative of a typical TKA resection, design-specific tibial resections were not investigated. Second, ideal tibial resection was employed ; however, clinical variability in resection parameters has been reported in previous studies , 28. ThiAlthough all the measurements performed in this study were based on fully automated computer simulation, the expected resolution of the results is impacted by several aspects of the data pre-processing: (1) Accuracy of the automatic annotations of the landmarks depends on the resolution of the CT data, which had sub-millimeter accuracy ; (2) approval of automatically defined landmarks by experienced users introduces some level of inter- and intra-user variability, though this has been shown to be at sub-millimeter level  ; and (3)Good morphological fit between the tibial component and the resected tibial anatomy is an important factor for long-term success in TKA. This study comprehensively evaluated contemporary tibial component designs. Here, the term “good fit” not only means a close match in the basic dimensions or aspect ratios with the resected tibia, but also the design appropriately accommodates balancing of tibial coverage with accuracy in rotational alignment for proper kinematics and minimal overhang to avoid soft tissue impingement. The clinical implications of the observations in the present study revealed that the anatomic design allows for increased accuracy in rotational alignment of the tibial component with better proximal tibial coverage. Accurate rotational alignment of the tibial component improves knee kinematics and patella tracking, while better proximal tibial coverage influences fixation and reduces peripheral soft tissue impingement. These features influence the ultimate clinical outcome of TKA. The results here further revealed the current gap in the morphological fit of symmetric and asymmetric tibial component designs, demonstrating that one or more of the key placement objectives  often need to be compromised in many contemporary designs.Assessment of contemporary tibial component profiles using morphological measurements as well as simulated component sizing and placement indicates that anatomic designs most effectively balance the competing surgical goals of tibial coverage, proper rotation, and minimal component overhang.


This study was carried out to understand the Ca2+-antagonistic effect of ginsenoside Ro (G-Ro), an oleanane-type saponin in Panax ginseng. G-Ro, without affecting leakage of lactate dehydrogenase, dose-dependently inhibited thrombin-induced platelet aggregation, and the half maximal inhibitory concentration was approximately 155 μM. G-Ro inhibited strongly thrombin-elevated [Ca2+]i, which was strongly increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. G-Ro increased the level of cAMP and subsequently elevated the phosphorylation of inositol 1, 4, 5-triphosphate receptor I (IP3RI) (Ser1756) to inhibit [Ca2+]i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP3RI (Ser1756) by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS. In addition, G-Ro inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa), indicating inhibition of Ca2+ influx across plasma membrane. We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser1756) phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa) to decrease thrombin-elevated [Ca2+]i, which contributes to inhibition of ATP and serotonin release, and p-selectin expression. These results indicate that G-Ro in Panax ginseng is a beneficial novel Ca2+-antagonistic compound and may prevent platelet aggregation-mediated thrombotic disease.Intracellular Ca Elevation of [Ca2+]i by agonists is dependent on the mobilization from intracellular Ca2+ stores (endoplasmic reticulum or dense tubular system) and the influx of extracellular Ca2+ across plasma membrane (PM) i) from dense tubular system (DTS) by binding to IP3 receptor type I (IP3RI). The increased [Ca2+]i activates both the Ca2+/calmodulin-dependent phosphorylation of myosin light chain (20 kDa) and the DG-dependent phosphorylation of pleckstrin (47 kDa) to induce granule secretion  and platelet aggregation i mobilization i [2+]i via cAMP-dependent IP3RI phosphorylation and dephosphorylation of ERK in human platelets. This study provides novel information for antiplatelet effects of G-Ro in ginseng.Ginseng, the root oftoxicity , 22. In toxicity , anticoatoxicity , endothetoxicity , and inhtoxicity . In our 2 (TXA2) . It is r2) [2+]i . However3-receptor type I, antiphosphor-IP3-receptor type I (Ser1756), anti-ERK (1/2), antiphosphor-ERK (1/2), anti-rabbit IgG-horseradish peroxidase conjugate (HRP), and lysis buffer were obtained from Cell Signaling . Anti-β-actin was obtained from Santa Cruz Biotechnology . Mouse monoclonal to CD62P (p-selectin) antibody was purchased from Biolegend . Polyvinylidene difluoride (PVDF) membrane was from GE Healthcare . Enhanced chemiluminescence solution (ECL) was from GE Healthcare .Ginsenoside Ro G-Ro,  was obta3, 0.36 mM NaH2PO4, 5.5 mM glucose, and 1 mM Na2EDTA, pH 6.5). The washed platelets were then resuspended in suspension buffer  to a final concentration of 5 × 108/mL. All of the above procedures were carried out at 25°C to avoid platelet aggregation from any effect of low temperature. The Korea National Institute for Bioethics Policy Public Institutional Review Board  approved these experiments (PIRB12-072).Human platelet-rich plasma (PRP) anticoagulated with acid-citrate-dextrose solution  was obtained from Korean Red Cross Blood Center . PRP was centrifuged for 10 min at 125 g to remove a few red blood cells and white blood cells and was centrifuged for 10 min at 1,300 g to obtain the platelet pellets. The platelets were washed twice with washing buffer  were preincubated for 3 min at 37°C in the presence of 2 mM exogenous CaCl2 with or without substances and then stimulated with thrombin (0.05 U/mL) for 5 min. Aggregation was monitored using an aggregometer  at a constant stirring speed of 1,000 rpm. Each aggregation rate was calculated as an increase in light transmission. The suspension buffer was used as the reference (transmission 0). G-Ro was dissolved in platelet suspension buffer (pH 6.9).Human washed platelets (108/mL) were incubated for 5 min at 37°C with various concentrations of G-Ro and then centrifuged at room temperature for 2 min at 12,000 g. The supernatant was measured by LDH assay kit  at an optical density of 490 nm. LDH leakage is expressed as the percentage of the total enzyme activity in platelets completely lysed with 0.1% Triton X-100.Human platelet cytotoxicity was determined by the leakage of lactate dehydrogenase (LDH) from cytosol. Human washed platelets  for 5 min for evaluation of [Ca2+]i. Fura 2 fluorescence was measured with a spectrofluorometer  with an excitation wavelength that was changed every 0.5 sec from 340 to 380 nm; the emission wavelength was set at 510 nm. The [Ca2+]i values were calculated using the method of Grynkiewicz i  and 300 μM of G-Ro-reduced [Ca2+]i level, which is to observe an apparent elevation of [Ca2+]i by their inhibitors. As shown in μM) dose-dependently increased the [Ca2+]i level as compared with those  by G-Ro  in the thrombin-induced platelet aggregation (μM) increased G-Ro-decreased  [Ca2+]i levels to 103.6% and 85.8%, respectively. But Rp-8-Br-cGMPS (250 μM) increased G-Ro-decreased  [Ca2+]i levels to 26.4% and 36.9%, respectively  dose-dependently increased thrombin-attenuated cAMP level. With regard to cGMP, thrombin did not change cGMP level as compared with that of basal level; on the contrary, cGMP appears to be decreased by G-Ro (Because it was confirmed that inhibition of [Ca by G-Ro .μM) dose-dependently increased phosphorylation [IP3R (Ser1756)] of IP3RI (Ser1756) and the ratio of p-IP3RI (Ser1756) to IP3RI in thrombin-induced human platelet aggregation (3RI (Ser1756) phosphorylation by G-Ro was resulted from cAMP/PKA-pathway or cGMP/PKG-pathway, we investigated an apparent inhibitory effect of their inhibitors on IP3RI (Ser1756) phosphorylation by 300 μM of G-Ro that potently stimulates the phosphorylation of IP3RI (Ser1756). As shown in 3RI (Ser1756) in thrombin-induced platelet aggregation. PKG inhibitor Rp-8-Br-cGMPS also decreased G-Ro-induced p-IP3RI (Ser1756) (G-Ro (50 to 300 Ser1756) , Lane 3.2+ mobilized from DTS (ER) is involved in phosphorylation of PI3K and ERK to influx extracellular Ca2+ i from Ca2+ store DTS via IP3RI. Depletion of the intracellular Ca2+ store by IP3 is known to connect the influx of extracellular Ca2+, which is stimulated by PI3K and ERK [2+]i, it is not unknown if G-Ro inhibited the production of IP3 via inhibition of phospholipase Cβ. The Ca2+-antagonistic reaction by cAMP and cGMP is mediated by PKA/IP3RI and PKG/IP3RI phosphorylation pathways, respectively. Even if G-Ro would not inhibit PLC-β activity and IP3 production in thrombin-activated human platelets, if G-Ro that elevates the level of cAMP stimulates IP3RI (Ser1756) phosphorylation in thrombin-activated human platelets, this is a clear evidence that G-Ro is involved in inhibition of [Ca2+]i mobilization from DTS via cAMP/PKA/IP3RI (Ser1756) phosphorylation pathway. In this report, we confirmed that G-Ro inhibited [Ca2+]i mobilization via IP3RI (Ser1756) phosphorylation by cAMP/PKA, which is supported from the result that cAMP inhibitor Rp-8-Br-cAMPS inhibited G-Ro-elevated phosphorylation of IP3RI (Ser1756) in thrombin-induced human platelet aggregation; if not so, A-kinase inhibitor Rp-8-Br-cAMPS would not increase G-Ro-decreased [Ca2+]i mobilization in thrombin-induced human platelet aggregation. It is well established that PI3K and ERK are involved in influx of extracellular Ca2+ across PM. Because Fura 2-loaded washed platelets were stimulated by thrombin in the presence of CaCl2 (2 mM), thrombin-evoked [Ca2+]i resulted from the Ca2+ mobilization from DTS by IP3 and the Ca2+ influx across PM by PI3K or ERK. Because G-Ro activated the phosphorylation of IP3RI (Ser1756) and the dephosphorylation of ERK 2 (42 kDa), the inhibition of thrombin-elevated [Ca2+]i by G-Ro is thought to result from both phosphorylation of IP3RI (Ser1756) and dephosphorylation of ERK 2 (42 kDa) by G-Ro. It is known that IP3 induces serotonin release from platelet dense body, which means that IP3 is involved in serotonin release by elevating [Ca2+]i via IP3RI [3RI (Ser1756).Thrombin-elevated [Ca and ERK –18. Evenia IP3RI . This re2+]i to phosphorylate Ca2+/calmodulin-dependent myosin light chain (20 kDa), which is involved in granule secretion such as ATP and serotonin [2+]i level [2+]i level by stimulating the cAMP-dependent phosphorylation of IP3RI (Ser1756) and dephosphorylation of ERK 2 (42 kDa). Therefore, it is thought that the inhibition of ATP and serotonin secretion by G-Ro resulted from the inhibition of IP3RI-mediated [Ca2+]i mobilization, and ERK 2-mediated (42 kDa) Ca2+ influx.A lot of agonists such as collagen, thrombin, and ADP increase i level ; howeverα-granule i level, which contributed to attenuating the release of ATP, serotonin, and p-selectin. In addition, G-Ro could be clinically applied to the prevention of platelet-mediated thrombosis. Therefore, it is thought that G-Ro may represent a useful tool in the therapy and prevention of vascular diseases associated with platelet aggregation.In conclusion, the most important result of this study is that G-Ro cAMP-dependently phosphorylates IP


The mutation replaces an evolutionarily-conserved aspartic acid with glycine at position 244 (p.D244G) of CASQ1, the main sarcoplasmic reticulum (SR) Ca2+ binding and storage protein localized at the terminal cisternae of skeletal muscle cells. Here, immunocytochemical analysis of myotubes, differentiated from muscle-derived primary myoblasts, shows that sarcoplasmic vacuolar aggregations positive for CASQ1 are significantly larger in CASQ1-mutated cells than control cells. A strong co-immuno staining of both RyR1 and CASQ1 was also noted in the vacuoles of myotubes and muscle biopsies derived from patients. Electrophysiological recordings and sarcoplasmic Ca2+ measurements provide evidence for less Ca2+ release from the SR of mutated myotubes when compared to that of controls. These findings further clarify the pathogenic nature of the p.D244G variant and point out defects in sarcoplasmic Ca2+ homeostasis as a mechanism underlying this human disease, which could be distinctly classified as “CASQ1-couplonopathy”.An autosomal dominant protein aggregate myopathy, characterized by high plasma creatine kinase and  To support this, CASQ1 ablation in mice results in reduced levels of SR Ca2+ release and in altered contractile properties . Ou. Ou2+ cotructure . Lewis a2+ diffusion towards the open Ca2+ release channels by altering the local concentration of diffusible Ca2+ ions, their one-dimensional directionality or CASQ1-RyR allosteric interactions, overall reducing RyR-mediated Ca2+ release from the SR as documented in this study. It has been shown that CASQ1 inhibits RyR channels, depending on its phosphorylation status and whether it is bound to RyR directly or indirectly via anchoring proteins [The D244G-induced disruption in CASQ1 polymerization may also affect Caproteins ,17, all 2+, the SK current makes a useful, albeit indirect, functional assessment tool for investigating mechanisms that underlie SR calcium dysfunction. The reduced SK tail-current densities measured from patient myotubes is functional proof that the mutation results in a physiologically significant reduction in Ca2+ release from the SR. A more direct evidence lies in the results from the Ca2+ signal recordings where myotube depolarization evoked Ca2+ signals of lower amplitude than those evoked in control myotubes. Also the exposure to increasingly elevated levels of caffeine, an RyR agonist, resulted in significantly lower Ca2+ transients in patient myotubes. Consistently, the SK tail-current activation results, both from depolarization- and caffeine-induced Ca2+ signals, indicate that the CASQ1’s p.D244G mutation disrupts normal muscle function by compromising SR Ca2+ release via RyR channels. Noteworthy, caffeine has been widely used to deplete the SR of Ca2+ [2+ transients is commonly taken as an index of the SR Ca2+ load [i.e., the amount of Ca2+ sequestered by mutated CASQ1), is consistent with data showing that the D244G protein possesses a lower binding capacity in vitro, and that the aggregates are less able to bind Ca2+ [That in this study, patient-derived myotubes had large CASQ1 aggregates, greater in area than that in myotubes derived from control subjects and similar to that observed from patient muscle biopsies, make myotubes differentiated from biopsied primary myoblasts an appropriate model to investigate the pathogenic mechanisms underlying this CASQ1-couplonopathy. Having basic electrophysiological properties comparable to healthy myotubes is consistent with the clinically mild phenotype of the disease and further strengthens the usefulness of this model. Activated by and dependent on intracellular Ca of Ca2+ , and thea2+ load . Our obsind Ca2+ .2+ homeostasis within muscle fibers as a possible mechanism underlying muscle cramping, weakness and fatigue. With a much clearer understanding of the D244G mutation and its effect on altering sarcoplasmic Ca2+ release, the major challenges to be tackled in the near future would be a better understanding of the mechanisms leading to D244G-induced cell toxicity and designing possible therapeutic interventions for this newly identified CASQ1-couplonopathy.Overall, the evidence provided here further implicates D244G substitution as pathogenic and points out defects in Ca


Hypoxia causes remodeling and contractile responses in both pulmonary artery (PA) and pulmonary vein (PV). Here we explore the effect of hypoxia on PV and pulmonary venous smooth muscle cells (PVSMCs).2 for 21 days. Rat distal PVSMCs were isolated and cultured for in vitro experiments. The fura-2 based fluorescence calcium imaging was used to measure the basal intracellular Ca2+ concentration ([Ca2+]i) and store-operated Ca2+ entry (SOCE). Quantitative RT-PCR and western blotting were performed to measure the expression of mRNA and levels of canonical transient receptor potential (TRPC) protein respectively.Chronic hypoxic pulmonary hypertension (CHPH) model was established by exposing rats to 10% O2+]i and SOCE in both freshly dissociated and serum cultured distal PVSMCs. Moreover, hypoxia increased TRPC6 expression at mRNA and protein levels in both cultured PVSMCs exposed to prolonged hypoxia  and distal PV isolated from CHPH rats. Hypoxia also enhanced proliferation and migration of rat distal PVSMCs.Hypoxia increased the basal [Ca2+]i in PVSMCs. This enhancement is potentially correlated with the increased expression of TRPC6. Hypoxia triggered intracellular calcium contributes to promoted proliferation and migration of PVSMCs.Hypoxia induces elevation of SOCE in distal PVSMCs, leading to enhancement of basal [Ca In the pulmonary vasculature, exposure to hypoxia is associated with vasoconstriction and vasculature remodeling which contributes to pulmonary hypertension Pulmonary vessels consist of three longitudinal vascular segments: the arteries, microvessels, and veins. In earlier studies, pulmonary arteries attracted much attention while pulmonary veins (PV) were seen simply as conduit vessels causing them to be largely overlooked. Over the past few decades, increasing evidence indicates important role of PV in regulation of pulmonary circulation 2+ entry (SOCE) and presence of TRPC1, 6 in rat distal Pulmonary vein smooth muscles (PVSMCs) 2) can elevates basal [Ca2+]i and triggers SOCE in PVSMCs 2+]i and SOCE is still unknown. Therefore, in this study, we aim to determine whether chronic hypoxia affects the SOCE and TRPCs expression in rat distal PV, as well as the proliferation and migration of PVSMCs. Our work could enrich our understanding of the mechanism underlying the hypoxic pulmonary vein contraction and remodeling.It was reported that hypoxia could lead to contraction and remodeling of the pulmonary vein. However, the underlying mechanism remains unclear. Our recent report described the occurrence of store-operated Ca2+ HBSS (20 µM CaCl2) at room temperature, and 23 minutes in reduced-Ca2+ HBSS containing collagenase, papain, Bovine Serum Albumin (BSA), and dithiothreitol at 37°C to disperse PVSMCs into a single-cell suspension. HBSS solution was then added into digestive solution and the tube was centrifuged for 5 minutes at 300 g. The supernatant was poured out and the cells pellet were washed with HBSS and centrifuged for 5 minutes at 300 g again. Then the cells were resuspended in Smooth Muscle Basal Medium  which contained 0.3% serum for 2 days, and then cultured in Smooth Muscle Growth Medium-2  which contained 5% serum for 5–6 days in incubator with humid atmosphere of 5% CO2 -95% air at 37°C. Cells were growth arrested in Basal Smooth Muscle Medium with 0.3% serum for 12–24 hours before treatment.The Animal experiment protocols were approved by the Animal Care and Use Committee of Guangzhou Medicine University and the Animal Care and Use Committee of the Johns Hopkins Medical Institution. All surgery was performed under anesthesia with sodium pentobarbital (65 mg/kg i.p.), and all efforts were made to minimize animal suffering. The method of isolation and culture of PVSMCs is as following our previous description with some modifications Right ventricular pressure and right ventricular hypertrophy were measured using the method we described previously The method of PVSMCs immunostaining was performed as previously described. In a word, cells grown on 25 mm coverslips were serum free starved for 12–24 hours, fixed with ethanol, and then incubated with mouse monoclonal antibody for smooth muscle α-actin . The cells were then probed with Cy3-linked goat anti-mouse secondary antibody . Nucleuses were stained with YO-PRO-1 dimeric cyanine dye . The coverslips were then fixed on slides with FluoroGuard Antifade . Cells were examined under a laser-scanning confocal microscope with a Zeiss Plan-Neofluor 40 oil-immersion objective .2 to maintain 10% O2. The successful development of the CHPH model was evaluated by measuring the weight ratio of the RV to the LV plus the septum [RV/(LV+S)], right ventricular systolic pressure (RVSP) and Hematocrit value.Rats were randomized into a chronic hypoxic group and a normoxic control group. Rats in the chronic hypoxia group were exposed to hypoxic chamber for up to 21 days. The chamber was continuously flushed with a mixture of room air and NAGCCTCTTGACAAACGAGGA, R:ACCTGACATCTGTCCGAACC);TRPC6;18S. The procedure of real-time PCR consisted of three steps including a hot start at 95°C for 15 minutes, 40 cycles with each containing 94°C for 15 seconds, 57.5°C for 20 seconds, and 72°C for 20 seconds, and melting curves performed at 95°C for 1 minute, 55°C for 1 minute, and 80 repeats of increments of 0.5°C. Detection threshold cycle (CT) values were measured by iCyclerIQ software. Relative concentration of each transcript was calculated by using classic the Phaffl method. Data were presented as a ratio of TRPC to 18S in the same sample.The total RNA of rat distal PV tissue and PVSMCs was extracted by using TRIzol reagent  and RNeasy kit  as previously described Samples of PV tissue or PVSMCs were homogenized or lysed in buffer containing 62.5 mM Tris·HCl (pH 6.8), 2% SDS, 10% glycerol, 5% protease inhibitor cocktail, 1 mM EDTA, and 200 M 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride. Protein concentration was measured by using the BCA Protein Assay Kit (Bio-Rad) with BSA as a standard . The homogenized proteins were mixed with 150 mM dithiothreitol, heated at 95°C for 3 minutes, and resolved by 10% SDS-PAGE. These separated proteins were then transferred to polyvinylidene difluoride membranes  and incubated with rabbit polyclonal antibodies against TRPC1 (Sigma), TRPC6 (Sigma), STIM1  or Orai1 (Alomone labs) for overnight or mouse monoclonal antibody that was specific for α-actin (Sigma) for 1 hour. Secondary antibodies were probed with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG  for 1 hour and then detected using an enhanced chemical luminescence system .2+ measurement experiments were accomplished as previously described 2, 0.57 mM MgSO4, 1.18 mM KH2PO4, 25 mM NaHCO3 and 10 mM glucose. During the experiments, temperature of coverslip was kept at 37°C and the solution was continuously equilibrated with 16% O2 and 5% CO2. The tubing and a manifold to an inline heat exchanger  were used to warm the perfusate again just before it entered the polycarbonate chamber. The chamber platform was controlled at 37°C by a dual-channel heater controller . Data were collected by using InCyte II software . Intracellular Ca2+ concentration was estimated from the ratio of fura-2 fluorescence emitted at 510 nm after excitation at 340 nm to that after excitation at 380 nm (F340/F380). For measurement of VDCC, the concentration KCL was increased to 60 mM, and NaCl was decreased to 62.7 mM.Ca2+ restoration and Mn2+ quenching were used to assess SOCE. As described above, coverslips with PVSMCs growing on were perfused for 10–15 min with Ca2+-free KRB solution containing 5 µM nifedipine, 10 µM Cyclopiazonic acid (CPA), and 1 mM EGTA. First, we measured the basal intracellular Ca2+ concentration at 0.2 minute intervals and SOCE was measured from the increase in intracellular Ca2+ that was caused by restoration of extracellular Ca2+. Second, we measured fura-2 fluorescence excited at 360 nm at 0.5 minute intervals before and after the addition of MnCl2 (200 M) which act as Ca2+ surrogate and reduces Fura-2 fluorescence on binding to the dye to the perfusate. Since fluorescence excited at 360 nm was the same for Ca2+-bound and Ca2+-free fura-2, changes in fluorescence can be thought to be caused by Mn2+ alone. SOCE was evaluated by the rate at which fura-2 fluorescence was quenched by Mn2+.Ca3 cells/well, cultured under normoxia or hypoxia (4% O2) condition for 60 hours and labeled with Brdu for 24 hours. The cells were then fixed, blocked, probed with anti-Brdu, developed with TMB substrate, and stopped with sulphuric acid. The optical density of wells was measured using a microplate reader (Bio-Rad) at 450 nm.The proliferation assay of PVSMCs was performed by the Cell Proliferation Biotrak ELISA Kit , according to the operation manual. In short PVSMCs were seeded in 96 well plates in SMBM  at a density of 4×105 cells/insert) and incubated under normoxia and hypoxia (4% O2) condition for 24 hours. Cells were then fixed using 95% freezing-cold ethanol for 10 minutes, stained with Brilliant blue R Staining Solution for 5 minutes, and washed with PBS for three times. After staining, these cells were imaged under microscope at five fixed positions of the Transwell membrane and counted to represent the total cell number. The cells on the upper surface of the membrane inserts were then gently wiped off by using cotton swabs. Images for the migrated on the lower surface of membrane were taken at the same fields as indicated above for all PASMCs. Cell migration rates were measured as the counts ratio of the migrated cells to the total cells on each membrane.As we reported before 2 at 37°C. The final concentration of each siRNA was 1000 ng/ml.Small interfering RNA (siRNA) targeted to STIM1 (siSTIM1) and Orai1 (siOrail1) were designed and synthesized by Dharmacon  and Shanghai GenePharma respectively. Primary cultured cells at 50–70% confluence were transfected with siSTIM1 or siOrai1, and nontargeting control siRNA using transfection vehicle  as carrier for 4 hours in serum-free SMBM in 5% COUnless otherwise specified, all materials and drugs were obtained from Sigma. Fura-2 AM (Invitrogen) was prepared before the experiment as a 2.5 mM stock solution in 20% DMSO containing 20% pluronic F-127 (Invitrogen). Stock solutions of nifedipine and CPA were both made in DMSO at 30 mM.2+ concentration, the number of cells in each experiment was from 25 to 30. Statistical analyses were performed using variance (ANOVA) or Student's t-test. When P<0.05, comparison differences were considered significant.Data is represented as means ± SE; n is the number of experiments, which equals the number of animals providing veins or PVSMCs. When fura-2 fluorescence was measured for calculating intracellular Ca2+]i in the cultured PVSMCs and all cells in visual fields exhibited clear increased [Ca2+]i  successfully developed CHPH. As shown in Rats exposed to chronic hypoxia  and nomoxic control rats were prepared for basal [Ca2+]i measurement, respectively. Cells from CHPH rats showed significantly increased basal [Ca2+]i (190±31 nM) compared with that from controls (100±17 nM) as shown in 2, 60 hours) or normoxia. Basal [Ca2+]i in the hypoxic PVSMCs  was significantly greater than that in nomoxic cells  than that observed in normoxic control cells , indicating enhanced SOCE in the hypoxic PVSMCs.We assessed SOCE in two ways. By measuring both intracellular Ca2+]i caused by restoration of extracellular Ca2+ can be affected by factors other than SOCE such as changes in membrane potential, we also measured the rate at which Mn2+ quenched Fura-2 fluorescence. As shown in 2+-free Krebs containing 200 µM Mn2+. Mn2+ quenching, expressed as the percentage decrease in F360 at 10 min in hypoxia exposed PVSMCs at 40.1±7.1% (n = 4 experiments in 95 cells) was significantly enhanced compared with 22.1±6.1% (n = 4 experiments in 108 cells) in normoxia control cells. It suggests that extracellular Ca2+ entry through store-operated Ca2+ channels (SOCCs) in hypoxic PVSMCs is enhanced when stores are depleted.Considering that the increase in i (Δ[Ca2+]i) and SOCE were measured in each respective treated cell. Results showed that the Δ[Ca2+]i was significantly reduced in STIM1 knockdown group compared with that in NT siRNA and control groups . There was no significant difference in Δ[Ca2+]i between NT siRNA treated and control cells  (Specific siRNA against STIM1 (siSTIM1) was synthesized and non-targeted siRNA (siNT) was used as a control. PVSMCs were were incubated with siSTIM1 or NT siRNA, control cells were treated with transfection vehicle alone. The specificity and knockdown efficiency of siSTIM1 was detected using Western blotting. As shown in ol cells . Consist P<0.01) .P<0.01). Additionally, SOCE was also measured using Mn2+ quenching measurement, which revealed that siOrai1 transfection significantly decreased the Mn2+ quenching compared with NT siRNA treated group   was synthesized and non-targeted siRNA (siNT) was used as a control. Cultured PVSMCs were incubated with siSTIM1 or NT siRNA, control cells were treated with transfection vehicle alone. The specificity and knockdown efficiency of siOrai1 was detected using Western blotting. As shown in  P<0.01) .in vitro showed that hypoxia increased proliferation and migration of PVSMCs. Rat distal pulmonary vein smooth muscle was demonstrated to exhibit a similar response to hypoxia in compare to pulmonary artery smooth muscle.In this study, we investigated the effect of hypoxia on SOCE and TRPCs expression in rat distal pulmonary vein smooth muscle. The proliferation and migration of rat distal PVSMCs exposed to hypoxia were also detected. Our results revealed that hypoxia increased SOCE and enhanced TRPC6 expression in rat distal pulmonary vein smooth muscle. Experiments with rat distal PVSMCs Previous studies of mechanisms underlying CHPH development mainly focused on the effects of hypoxia on pulmonary arteries and PASMCs. It was found that pulmonary arterial vasoconstriction and structural remodeling were two major links during CHPH development. However, based on the pathophysiology of pulmonary circulation, when oxygen partial pressure in pulmonary circulation decreases, hypoxemia first affected pulmonary vein. Thus, exploring the mechanism underlying CHPH development from the PV side is necessary and meaningful. For investigating the function of pulmonary vein, sufficient and purified PVSMCs are required. In the past, we developed an efficient method to isolate and culture PVSMCs from rat distal pulmonary veins 2+ concentration to hypoxia highlighted smooth muscle cell proliferation and migration, we found that both freshly isolated PVSMCs from CHPH rats and cultured PVSMCs exposed to prolonged hypoxia exhibited elevated intracellular Ca2+ concentration than cells from normoxic rats. These findings are similar to those observed in PA and PASMCs from different species in the past, suggesting that basal Ca2+ concentration increase in PV is pivotal during CHPH development.In the past decades, an increasing number of studies reported that similar to pulmonary artery, hypoxia can also induce remodeling in pulmonary vein 2+ concentration can be achieved mainly by: the Ca2+ release from sarcoplasmic reticulum, Ca2+ influx from extracellular fluid through voltage-dependent calcium channels (VDCCs), receptor-operated Ca2+ channels (ROCCs), or SOCCs 2+ influx via VDCCs. We calculated the SOCE as the increase in intracellular Ca2+ following the restoration of extracellular Ca2+ and as the decreased rate of Mn2+ quenching in PVSMCs. We found prolonged hypoxia could enhance the SOCE in PVSMCs, which was similar to that in PASMCs. SOCC is known to be mainly composed of the classical TRPC family members, among which TRPC1 and TRPC6 have been found to be expressed in the rat pulmonary vein and PVSMCs at both mRNA and protein levels Based on previous knowledge, the enhanced intracellular Ca2+ store depletion and mitogen-mediated PASMCs proliferation 2+]i signaling pathway.TRPC6 is usually considered to be a key component of ROCCs, which could be activated by diacylglycerol (DAG) and other signaling messengers involved in the G protein signal pathway 2+ concentration in various cell types, including PASMCs 2+]i, enhanced SOCE and triggered proliferation and migration in PVSMCs. These results suggest that although similar enhanced intracellular Ca2+ concentration takes place in both PA and PV, the detail mechanism seems different. These results could potentially explain why PV exhibits less degree of remodeling than PA, and why PVSMCs exhibit smaller increase in SOCE and basal [Ca2+]i than PASMCs, when exposed to hypoxia. Moreover, STIM1 protein has been identified to act as Ca2+ sensor and is located in the internal Ca2+ stores. After emptying the stores STIM molecule aggregate to activate Orai channels which are responsible for SOCE. In PASMCs, it is well accepted that stimuli inducing ER depletion leads to STIM1 translocation to the plasma membrane, interacts with and activates Orai and TRPC channels to mediate SOCE. However, whether the similar machinery is also present in PVSMCs remains unknown. In this study, by using specific siRNA knockdown strategy, we performed additional experiments to demonstrate that like PASMCs, in PVSMCs, STIM1 and Orai1, also contribute to and largely mediate SOCE, respectively. Under this condition, in hypoxic PVSMCs, the increased TRPC6 expression is likely responsible for hypoxia-elevated intracellular calcium homeostasis.Unlike to TRPC6, TRPC1 is mostly determined as a primary component of SOCCs, which provides a pathway for SOCE, thus participating in the regulation of intracellular Ca2+ has been reported to be an important determinant 2+]i and SOCE in hypoxic PVSMCs, we further confirmed that hypoxia also elevated the proliferation and migration of PVSMCs, suggesting a similar pattern to that in hypoxic PASMCs.It is well known that hypoxia induced pulmonary vascular remodeling is directly associated with the PA smooth muscle cell proliferation and migration ex vivo freshly isolated and in vitro cultured rat distal PVSMCs, leading to elevated basal [Ca2+]i enhancement in PVSMC. This enhancement was mainly dependent on the increased expression of TRPC6. Hypoxia increased basal [Ca2+]i triggered proliferation and migration of PVSMCs, and led to PV structural remodeling which contributed to pulmonary circulation peripheral resistance enhancement.In conclusion, we initially demonstrated that hypoxia induced enhanced SOCE in both


We confirmed that in HEK cells expressing PTH1R, acute stimulation with PTH(1-34) potentiated carbachol-evoked Ca2+ release. This was mediated by locally delivered cyclic AMP (cAMP), but unaffected by inhibition of protein kinase A (PKA), exchange proteins activated by cAMP, cAMP phosphodiesterases (PDEs) or substantial inhibition of adenylyl cyclase. Sustained stimulation with PTH(1-34) causes internalization of PTH1R–adenylyl cyclase signalling complexes, but the consequences for delivery of cAMP to IP3R within cAMP signalling junctions are unknown. Here, we show that sustained stimulation with PTH(1-34) or with PTH analogues that do not evoke receptor internalization reduced the potentiated Ca2+ signals and attenuated carbachol-evoked increases in cytosolic IP3. Similar results were obtained after sustained stimulation with NKH477 to directly activate adenylyl cyclase, or with the membrane-permeant analogue of cAMP, 8-Br-cAMP. These responses were independent of PKA and unaffected by substantial inhibition of adenylyl cyclase. During prolonged stimulation with PTH(1-34), hyperactive cAMP signalling junctions, within which cAMP is delivered directly and at saturating concentrations to its targets, mediate sensitization of IP3R and a more slowly developing inhibition of IP3 accumulation.Parathyroid hormone (PTH) stimulates adenylyl cyclase through type 1 PTH receptors (PTH Many ef (GPCRs) . PTH rec[Ca2+]c) . The N-t+]c)  (3), and hence Ca2+ release from intracellular stores. Typically, such Ca2+ signals are evoked by higher concentrations of PTH than are required for stimulation of adenylyl cyclase . Association of PTH1R with the scaffold proteins, Na+/H+ exchange regulatory factors-1 and 2 (NHERF-1 and 2), both of which are expressed in HEK cells  allows it to activate the small G protein Rap2B, which then stimulates PLCε  , which c cyclase . Furthercoupling  , PTH(1-34) stimulates adenylyl cyclase. The cAMP produced directly sensitizes IP3 receptors (IP3Rs) to the IP3 produced when receptors, like endogenous M3 muscarinic receptors, stimulate PLC. Hence, concentrations of PTH(1-34) that do not alone evoke increases in [Ca2+]c potentiate the Ca2+ signals evoked by carbachol, which activates muscarinic receptors  when PTH1R signalling pathways might be reconfigured. We show that sustained stimulation with PTH leads to diminished potentiation of carbachol-evoked Ca2+ signals. This does not require internalization of PTH1R. We provide evidence that the hyperactive cAMP signalling junctions that mediate sensitization of IP3R by PTH also cause inhibition of IP3 formation during sustained stimulation. Our results suggest that delivery of cAMP to its targets within signalling junctions allows rapid potentiation of IP3R activity followed by a more slowly developing inhibition of IP3 accumulation.We have shown that in HEK cells stably expressing human PTHeceptors . This poed IP3Rs . We descl muscle , where r2+ release potentiated the Ca2+ signals evoked by carbachol .In HEK-PR1 cells, concentrations of PTH(1-34) that did not alone stimulate Caarbachol  . We do not detect stimulation of PLC by PTH(1-34) in HEK-PR1 cells  was indistinguishable from PTH(1-34) in stimulating adenylyl cyclase and PLC (1PTH(1-34) were as effective as PTH(1-34) in stimulating adenylyl cyclase, but they failed to activate PLC . Selective activation of adenylyl cyclase by PTH(2-38) and Tyr1PTH(1-34) is consistent with evidence that N-terminal modifications of PTH attenuate coupling to PLC (At the highest concentrations used (>300 nM), PTH(1-34) alone evoked small (<40 nM) increases in [Ca2+]c   mimicked PTH(1-34) in both stimulating adenylyl cyclase and potentiating carbachol-evoked Ca2+ signals . Furthermore, the relationship between the change in intracellular cAMP concentration and the potentiated Ca2+ signals was indistinguishable for the three analogues  or Tyr1PTH(1-34)  to potentiate carbachol-evoked Ca2+ signals  than was cAMP accumulation . The Ca2+ signals evoked by very high concentrations of PTH(1-34) probably result from stimulation of PLC. Our inability to detect IP3 formation under these conditions  and Tyrnalogues . HoweverTH(1-34) . These r signals   did not mimic the effects of PTH(1-34) or 8-Br-cAMP on carbachol-evoked Ca2+ signals. That conclusion is strengthened by results with a new membrane-permeant antagonist of EPAC1 and EPAC2  (ESI-09)  on carbachol-evoked Ca2+ signals or the small Ca2+ signals directly evoked by high concentrations of PTH(1-34) . It was impracticable to use higher concentrations of ESI-09 or more prolonged treatments because they directly inhibited carbachol-evoked Ca2+ release . Others have also recently reported non-specific effects of ESI-09 .We have previously provided evidence that the effects of PTH(1-34) on carbachol-evoked Caor EPACs . The lat(ESI-09) . ESI-09 f ESI-09 . A compe cyclase . However2+ signals by PTH(1-34) or the direct effects of high concentrations of PTH(1-34) on Ca2+ signals. The latter, with evidence that some analogues of PTH stimulate adenylyl cyclase without directly evoking Ca2+ signals  stimulates delivery of cAMP to IPunctions . This, texes see , prompte2+ signals by ∼50%, while increasing the sensitivity to PTH(1-34) by almost 10-fold . These effects were apparent after 15–30 min, and were not further increased by extending the incubation with PTH(1-34) to 60 min. The inhibition (∼50%) was similar whether maximal or submaximal carbachol concentrations were used to evoke the Ca2+ signals. Analyses of single cells showed that the reduced maximal response after prolonged incubation with PTH(1-34) was due to diminished Ca2+ signals within individual cells rather than to fewer cells responding  nor PTH(2-38) directly stimulated Ca2+ release from intracellular stores . Furthermore, addition of ionomycin to cells in Ca2+-free HEPES-buffered saline (HBS) to assess the Ca2+ contents of the stores after incubation with PTH(1-34) showed that the increase in [Ca2+]c evoked by ionomycin was unaffected by acute or sustained stimulation with PTH(1-34) (2+ indicator because restoration of extracellular Ca2+ after ionomycin evoked a much larger increase in fluo 4 fluorescence (2+ content of the stores after stimulation with PTH(1-34) and carbachol, showed that more Ca2+ remained within the stores of cells stimulated with carbachol after prolonged treatment with PTH(1-34)  than after brief treatment  (2+ from intracellular stores. We conclude that sustained stimulation with PTH(1-34) reduces the maximal potentiation of carbachol-evoked Ca2+ signals without affecting the Ca2+ content of the stores.Varying the duration of the incubation with PTH(1-34) before addition of carbachol established that sustained exposure to PTH(1-34) reduced the maximal amplitude of the carbachol-evoked Casponding . The dimr stores , but resTH(1-34) . The indrescence . Using s P<0.05) . This ag3 concentration evoked by a submaximal concentration of carbachol (30 µM) were measured in single HEK-PR1 cells using a Förster resonance energy transfer (FRET)-based IP3 sensor. Cells were first stimulated with 1 mM carbachol  to identify responsive cells (3 evoked by carbachol (3.The effects of acute and sustained stimulation with PTH(1-34) on the changes in cytosolic IPve cells . After wve cells , and it ve cells . These rve cells . This isve cells . Howeverarbachol . We conc1R to assess whether endocytosis of functional adenylyl cyclase signalling pathways contributes to the sustained effects of PTH(1-34) on carbachol-evoked Ca2+ signals. PTH(1-34) evokes receptor internalization and sustained signalling from endosomal adenylyl cyclase, PTH(2-38) does not evoke receptor internalization, Tyr1PTH(1-34) is a weak partial agonist for receptor internalization and PTHrP(1-36) evokes receptor internalization but no persistent adenylyl cyclase signalling . The acute and sustained effects of each analogue on carbachol-evoked Ca2+ signals were similar to those evoked by PTH(1-34) . For each PTH analogue, the maximal amplitude of the Ca2+ signal evoked by carbachol was smaller after sustained stimulation, despite each causing intracellular levels of cAMP to be greater after stimulation for 30 min relative to 1 min . Although PTHrP(1-36) mimicked the effects of PTH(1-34) in potentiating carbachol-evoked Ca2+ signals, it stimulated less cAMP accumulation. This is unexpected because others have suggested that PTHrP(1-36) . This suggests that a component of the increased sensitivity of the Ca2+ signals is probably due to the increased sensitivity of adenylyl cyclase activation to PTH during prolonged stimulation. The more important point for the present work is that, for all the PTH analogues, sustained stimulation causes greater accumulation of cAMP, but lesser potentiation of carbachol-evoked Ca2+ signals. Collectively, these results suggest that internalization of functional PTH1R signalling complexes is unlikely to be responsible for the sustained effects of PTH on carbachol-evoked Ca2+ signals. We therefore assessed the effects of more directly evoking sustained elevations in intracellular cAMP concentration on carbachol-evoked Ca2+ signals.We used PTH analogues that differ in their abilities to evoke internalization of PTHrP(1-36) , PTHrP(1rP(1-36)  are as epression . We have2+ signals . However, the relationship between intracellular cAMP and Δ[Ca2+]c is different for PTH(1-34), PTHrP(1-36) and NKH477  or NKH477. This indicates that IP3R cannot be responding to a uniformly delivered global increase in cytosolic cAMP.Brief stimulation (1–5 min) with 8-Br-cAMP, PTH(1-34) or NKH477, a soluble analogue of forskolin that directly activates adenylyl cyclase , caused  signals  and thei signals . Becauseous work  by confid NKH477 : the eff2+ signals, and again the maximal effects of each were non-additive . For both PTH(1-34) and NKH477, prolonged stimulation reduced the apparent effectiveness of cAMP in potentiating carbachol-evoked Ca2+ signals . There was also no effect of H89 on the amount of cAMP produced after stimulation with PTH(1-34) for 1 min or 60 min. For cells treated with H89, amounts of intracellular cAMP detected after stimulation with 3 µM PTH(1-34) for 1 min or 60 min were 95±0.1% and 98±0.2% (mean±s.e.m.) of those detected in matched control cells (n = 3) . We conclude that sustained elevations of intracellular cAMP, whether evoked by activation of PTH1R or directly, attenuate the potentiation of carbachol-evoked Ca2+ signals. Neither the potentiation of Ca2+ signals by cAMP nor the diminished response after sustained elevation of cAMP requires activation of PKA.Sustained exposure to PTH(1-34), NKH477 or 8-Br-cAMP caused similar decreases in the maximal potentiation of carbachol-evoked Ca signals . Neither signals  . The reduced effectiveness of cAMP with increased duration of exposure is clear from comparison of the relationships between cAMP and potentiated Ca2+ signals for cells stimulated acutely or chronically with PTH(1-34) or NKH477  was unaffected by substantially inhibiting cAMP formation  or degradation (by inhibiting cyclic nucleotide PDEs with IBMX), although both treatments had the expected effects on intracellular cAMP . Supplementary material Fig. S3E demonstrates that if the cAMP that regulates IP3R were uniformly distributed, the observed 60–70% inhibition of adenylyl cyclase by SQ/DDA would cause a detectable inhibition of the effects of PTH(1-34) on carbachol-evoked Ca2+ signals. The lack of effect of SQ/DDA on Ca2+ responses is not, therefore, a limitation of our methods. Similar results were obtained when NKH477 was used to acutely stimulate adenylyl cyclase. SQ/DDA and IBMX had the expected effects on intracellular concentrations of cAMP , despite substantial effects on intracellular concentrations of cAMP, suggests that cAMP is locally delivered at super-saturating concentrations to IP3Rs (Acute (1 min) potentiation of carbachol-evoked Ca of cAMP , but the signals . These rto IP3Rs . We propto IP3Rs .2+ signals . Similar effects were observed after sustained stimulation with NKH477: cAMP accumulation was substantially inhibited by SQ/DDA without affecting the concentration-dependent effects of NKH477 on carbachol-evoked Ca2+ signals  for 60 min, cAMP formation was reduced by ∼70% after inhibition of adenylyl cyclase by SQ/DDA, but there was no significant effect on the potentiation of carbachol-evoked Ca signals . These r2+ signals to PTH(1-34) and NKH477 without affecting the maximal amplitude of the increase in [Ca2+]c . The latter remained smaller than the increase observed after acute stimulation, demonstrating that even massive increases in intracellular cAMP concentration cannot surmount the attenuation of potentiated Ca2+ signals after sustained stimulation with PTH. As with all other analyses, inhibition of PKA (with H89) had no effect on the potentiation of carbachol-evoked Ca2+ signals by PTH(1-34) in the presence of IBMX , re-affirming that PKA is not involved in the potentiation of carbachol-evoked Ca2+ signals.IBMX massively increased the amount of cAMP produced after sustained stimulation with PTH(1-34) or NKH477. In parallel analyses, IBMX significantly increased the sensitivity of carbachol-evoked Ca2+ signals by PTH(1-34) alone or with IBMX , it partially reversed the increase in sensitivity to PTH(1-34) during sustained stimulation with PTH(1-34) and IBMX  confirm the role of cAMP in mediating the effect. Sustained stimulation (60 min) with PTH(1-34) in the presence of IBMX generated levels of intracellular cAMP that were 22-fold greater than those evoked by acute (1 min) stimulation . Although SQ/DDA substantially inhibited adenylyl cyclase, the amount of intracellular cAMP in cells stimulated with PTH(1-34) for 60 min with IBMX remained substantially greater than during acute stimulation . These results suggest that when the global intracellular cAMP concentration is massively increased, it achieves levels that can sensitize the Ca2+ signals evoked by carbachol without the need for cAMP signalling junctions. Under these conditions, cAMP will sensitize both junctional IP3R and extra-junctional IP3R. It would be expected that recruitment of the latter would be attenuated by inhibition of adenylyl cyclase, whereas junctional signalling would be unaffected  or to direct stimulation of adenylyl cyclase (with NKH477) were insensitive to inhibition of either adenylyl cyclase (with SQ/DDA) or cyclic nucleotide PDEs (with IBMX), although each inhibitor had the expected effect on global concentrations of intracellular cAMP . This, together with the inconsistent relationship between intracellular cAMP and Ca2+ signals for different analogues of PTH and direct stimulation of adenylyl cyclase  then result from recruitment of these digital junctions, rather than from graded activity within individual junctions  requires local communication between adenylyl cyclase and IP3Rs motivated our analysis of sustained responses to PTH(1-34) during which functional adenylyl cyclase signalling pathways are internalized (see PTH(1-34) potentiates carbachol-evoked Ca of IP3R . The potproteins . Despite cyclase , establiunctions   was increased . The diminished Ca2+ responses were not due to fewer cells responding or to loss of Ca2+ from intracellular stores   is mediated by a sustained increase in cytosolic cAMP that does not require PKA or internalization of PTH1R signalling complexes.Sustained stimulation with PTH(1-34) potentiated carbachol-evoked Cancreased ; Fig. 4.r stores , and ther stores . Acute aTH(1-34) . Collect and Akt , are unl3R that occur during sustained activation of adenylyl cyclase. We conclude, and despite evidence that sustained stimulation with PTH(1-34) evokes internalization of functional adenylyl cyclase signalling complexes  in the presence of IBMX caused the global concentration of intracellular cAMP to increase to levels sufficient to sensitize IP3R without the usual need for junctional delivery of cAMP. This was evident from the increased sensitivity to PTH(1-34) and NKH477 after sustained stimulation in the presence of IBMX, and its partial reversal by inhibition of adenylyl cyclase with SQ/DDA . Although the global increase in cAMP increased the sensitivity to PTH(1-34) and NKH477, it had no effect on the maximal response, which remained smaller than that evoked by acute stimulation. This demonstrates that ineffective delivery of cAMP to IP3R during sustained stimulation does not cause the diminished potentiation of carbachol-evoked Ca2+ signals. Instead, sustained increases in intracellular cAMP reduce the accumulation of cytosolic IP3 after carbachol stimulation  are mediated by cAMP (The insensitivity of the sustained responses to PTH(1-34) and NKH477 to substantial inhibition of adenylyl cyclase  suggestsomplexes , that PTh SQ/DDA . Our demmulation . We have by cAMP –8, indep by cAMP  and depe by cAMP .1R stimulates adenylyl cyclase and locally delivers cAMP at supersaturating concentrations to associated IP3Rs, thereby increasing their sensitivity to IP3 and so potentiating the Ca2+ signals evoked by carbachol  on Ca2+ signals and the longer term attenuation of the response without need for activation of PKA.We conclude that PTH(1-34) through PTHarbachol . This juN-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89) and 8-Br-cAMP were from R&D Systems . 2′,5′-dideoxyadenosine (DDA), N,N-dimethyl--5-(acetyloxy)-3-ethenyldodecahydro-10,10b-dihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-1H-naphthopyran-6-yl ester β-alanine hydrochloride (NKH477) and 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) were from Merck Biosciences . -adenine was from Perkin Elmer . 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was from Molekula . Carbamylcholine chloride  and 3-isobutyl-1-methylxanthine (IBMX) were from Sigma-Aldrich . Ionomycin was from Apollo Scientific . Cell culture media, G-418, fluo 4AM and fura 2AM were from Life Technologies . 3-[5-(tert-butyl)isoxazol-3-yl]-2-[2-(3-chlorophenyl)hydrazono]-3-oxopropanenitril (ESI-09) was from Biolog Life Science Institute . All PTH analogues were human forms and supplied by either Bachem  or, for PTHrP(1-36), custom-synthesized by Selleckchem . Sequences of the analogues used are listed in supplementary material Table S1.5 PTH1R/cell) were cultured as described previously  was used to record fluorescence at intervals of 1.44 s  (F) was calibrated to [Ca2+]c from: [Ca2+]c = KD(F−Fmin)/(Fmax−F), where KD is the equilibrium dissociation constant of fluo 4 for Ca2+ (345 nM); Fmin and Fmax were measured from cells treated with Triton X-100  in the presence of BAPTA (10 mM) or CaCl2 (10 mM).HEK-PR1 cells (∼10eviously . Measureescribed . Briefly>525 nm) . Fluores2+]c, near-confluent cultures of HEK-PR1 cells were grown on poly-l-lysine-coated round coverslips (22-mm diameter) and loaded with fura 2 by incubation with fura 2AM  in HBS. The medium was removed and cells were incubated for a further 45 min in HBS at 20°C before single-cell imaging using an Olympus IX71 inverted fluorescence microscope. Cells were alternately excited at 5-s intervals with light (340 nm and 380 nm) from a Xe-arc lamp and monochromator, while collecting emitted light at 510 nm using a Luca EMCCD camera  and MetaFluor software . Autofluorescence was determined at the end of each experiment by addition of ionomycin (1 µM) and MnCl2 (10 mM) and subtracted from measurements before computing fluorescence ratios (R = F340/F380). These were calibrated to [Ca2+]c from:KD for fura 2 is 224 nM, Rb and Rf are the fluorescence ratios for fura 2 with and without Ca2+ bound, and Fb and Ff are the fluorescence recorded at 380 nM with and without Ca2+.For single-cell measurements of c. HEK-PR1 cells were grown in 24-well plates until ∼90% confluent, [3H]adenine (2 µCi per well) was then added to the culture medium. After 2 h at 37°C in 5% CO2, the medium was removed, cells were washed with HBS, and used for experiments in HBS at 20°C. Because many cells extrude cAMP into the extracellular medium  at its N-terminus and enhanced yellow fluorescent protein (YFP) at its C-terminus  using Lipofectamine LTX reagent with PLUS reagent, according to the manufacturer's instructions . Cells were imaged after 48 h. An Olympus IX71 inverted fluorescence microscope with a 40× objective and a 440 nm/520 nm dual band-pass dichroic mirror was used to record fluorescence from widefield images after excitation at 440 nm (to excite CFP). A Luca EMCCD camera  was used to collect emitted fluorescence simultaneously at 1-s intervals from YFP (520–550 nm) and CFP (455–485 nm) using a Cairn Optosplit 2 image-splitter fitted with a 495-nm dichroic mirror. After correction for background fluorescence (determined from cytosolic areas of non-transfected cells), FRET ratios are presented as the ratio of CFP emission to YFP emission, so that the ratio increases (decreased FRET) after IP3 binding. The transfection efficiency was ∼65%, and 52±2%  of transfected cells responded to carbachol (1 mM) with discernible FRET changes; only these responsive cells were included in analyses of the effects of PTH(1-34).A FRET sensor based on the IPof IP3R1  was usedeviously . The sen


AbstractAcute myeloblastic leukemia accounts for approximately 20% of acute leukemias in children. The days the microscope represented the main tool in the diagnosis and classification of Acute Myeloblastic Leukemia seem to be very far. This review summarizes the current diagnosis of this malignancy, where the morphological, cytochemical, immunophenotyping, cytogenetic and molecular characterization represents the basement of a risk group related therapy. Acute myeloblastic leukemia (AML) which is also known as acute non-lymphoblastic leukemia, acute myeloid leukemia or acute granulocytic leukemia contains a heterogeneous group of hematologic neoplasia with different biological and clinical connotations, characterized by a blocking event in maturation and the proliferation of precursor myeloid cells.1]. If AML is the most common form of leukemia in the adult, in children it is a rare disease, representing 15-20% of the acute leukemias in pediatric age, with a peak of incidence in the neonatal period and a relatively constant incidence during childhood, increasing during the teenage period . The angiogenesis represents the process of forming new vessels from the preexistent vessels. The angiogenesis and the angiogenic factors have also proved to be important in the hematological malignancies not only in solid tumors. AML is associated with a growth of the vascularization in the bone marrow but also with a growth of the level of angiogenic factors such as VEGF, the fibroblastic growth factor, the derived growth factor of the platelets, the epidermal growth factor, TNF-alpha and beta, the hepatocyte growth factor (HGF), the angiogenic, angiopoietin-1 in the case of hematologic malignancies, most of these angiogenic factors seem to be secreted by hematopoietic neoplastic cells, assuring the growth and proliferation of the leukemic cells in an autocrine manner. VEGF and bFGF – the most powerful inductors of angiogenesis – are synthesized in leukemic cells but also in fibroblasts, immune cells, osteoclasts, platelets and megakaryocytes. Keith et al. showed that the gene expression of these factors is regulated in the bone marrow [ From a pathogenic point of view AML is divided in primary forms (AML de novo) and secondary forms which developed consecutively to a myelodysplasia syndrome or related to the exposure to leukaemogenic agents (therapy related AML).  Although in the last decades, amazing progresses have been made regarding the discovery of the elements of pathogenesis in acute myeloblastic leukemia in children, there are still some questions that did not find an answer, such as: Which are the oncogenes that are important in AML? Do all the malignant cells have the same properties? Which is the role of the medullary microenvironment in AML?  Diagnosis work-up  • Anamnesis • Clinical examination + anthropometric indices  • Hemoleucogram + leukocyte formula, peripheral blood smear  • The examination of the bone marrow  immunophenotyping, cytogenesis, molecular analysis)  • Lumbar puncture, if the patient is symptomatic  • Biochemical profile  • aPTT, PT, fibrinogen  • Rx thorax  • Cardiac evaluation  before the anthracycline treatment • Infectious status  • Sanguine group, Rh  • HLA typification, in patients who are possible candidates to hematopoietic stem cells transplant (HSCT) Diagnosis classification systems  Acute leukemias differentiate according to the type of proliferative cellular type, clinical manifestations, evolution and therapeutic answer; as a result, the possibility of diagnosing various types of diseases is crucial.  The first stage, which is mandatory, and which remains the cornerstone in establishing a certain diagnosis in acute myeloblastic leukemia but also in the exclusion of non-hematological proliferations and the lymphoproliferative diseases is represented by the cytomorphological examination.  The morphological analysis presupposes the use of blood smears obtained by marrow aspiration puncture and peripheral blood smears, colored in May-Grunwald-Giemsa. In most of the cases, the marrow preparation is hypercellular, with 80-100% atypical myeloblasts, but for the positive diagnosis a percent of 30% atypical myeloblasts is enough (according to FAB classification) or 20% atypical myeloblasts (according to WHO).  In cases of leukopenia, as well as in cases of uncertainty, the repetition of the aspiration puncture or the realizing of the bone biopsy puncture is indicated. The cytological framing using the French-American-British (FAB) system, considered the most complete morphological classification system, becomes more exact if it is completed by the immunohistochemistry criteria. Although this classification system represented the most important step in AML classification in children, the correlation with the prognostic indicators of survival and the answer to the treatment is limited.  FAB Classification System admits 8 subtypes of acute myeloblastic leukemia:  M0 – Undifferentiated AML – The blast population is made up of great myeloblasts, agranular which can sometimes be confused with the LAL-L2 ones. The diagnosis cannot be certainly established in optic microscopy but only based on demonstrating the presence of cytoplasmic granulations in electronic microscopy, of expressing some antigens belonging to the myeloid line by the blast cells, as well as expressing the absence of antigens specific to the lymphoid line.  M1 – AML without maturation – Blast population - 80-90% of the non-erythroid cells – is made up of large myeloblasts with rare azurophilic granulations. Auer corpuses are present in 50% of the cases. The diagnosis is positive if more than 3-5% of the cells react positively to MPO or Sudan black. The morphological study alone cannot differentiate this subtype of AML from LAL.  M2 – AML with maturation – 30-80% of the non-erythroid cells are very well differentiated myeloblasts characterized by a rich cytoplasm and a various number of azurophilic granulations. Auer corpuses are present in 65% of the cases. In 10% of the cells, the maturation exceeds the blast stage. The reaction for MPO is intensely positive, just like the one for Sudan black.  M3 – Promyelocytic acute leukemia – The myeloid population is dominated by the presence of pathologic promyelocytes, large cells with round nucleus, abundant cytoplasm, numerous great azurophilic granulations, with Auer corpuses placed “in bundles", sometimes masking the nucleus. The myeloperoxidase reaction is very intense.  M3V – Promyelocytic acute leukemia, microgranular version  M4 – Myelomonocytic acute leukemia – Precursors of the granulocytic and monocytic lines are present in the marrow in varying proportions, each line representing more than 20%, but not more than 80% of the nucleated cells. The myeloblasts contain Auer corpuses. The monoblasts are bigger than the myeloblasts, have a round nucleus, obvious nucleoli, abundant cytoplasm which contains azurophilic granulations and chromatin which is delicately dispersed. There is a high number of monocytes and promonocytes in the peripheral blood. The reaction for MPO and esterases completes the certitude diagnosis. M4eoz – Myelomonocytic acute leukemia with eozinophils – it has the same criteria as M4, but additionally, abnormal eozinophils with eozinophils and dysplastic basophilic granules are present.  M5 – Monoblastic acute leukemia – 80% of the non-erythroid cells are represented by monoblasts, promonocytes and monocytes. The reaction for MPO is negative, the reaction for nonspecific esterase is intensely positive.  M6 – Acute erythroleukemia (Di Guglielmo Leukemia) – The erythrocyte precursors represent over 50% of the medullary nucleate cells, with megaloblastic modifications and nuclear anomalies just like the ones in myelodysplasia. Over 20% of the nucleate cells are myeloblasts. The Auer corpuses can be met in 2/3 of the cases. From the cytochemical point of view, the erythroblasts are positive for the PAS reaction and esterase.  M7 – Megakaryoblastic myeloid leukemia (megakaryocytic) – numerous megakaryoblasts in different differentiation stages  - undifferentiated smaller forms, nucleus with dense chromatin, hardly visible nucleoli, reduced cytoplasm. - differentiated bigger forms, with delicate granular chromatin, abundant cytoplasm with azurophilic granulations.  The medullary product contains numerous reticulin fibers, which often makes the marrow puncture difficult to obtain. The diagnosis is specified by the cytochemical, immunophenotypical (the presence of specific markers) and ultrastructural (the presence of platelet chromatin peroxidase) examinations.  This subtype is frequently met in children with trisomy 21.  Histochemical analysis  The colors which are usually used – myeloperoxidase (MPO), periodical acid-Schiff (PAS), Sudan black B and esterases – find their utility in differentiating various subtypes of AML but also in differentiating AML from LAL in cases in which flow-cytometry studies offer doubtful results.  The immunophenotypical analysis allows the differentiation of AML from LAL through flow-cytometry, according to the antigenic markers expressed at the surface or in the cytoplasm of the blasts and identifies different subtypes correspondent to the stages of cellular differentiation, markers which are reunited under the name of CD (cluster of differentiation-antigen). There are no antigens with absolute specificity or even more, there are some antigens that are characteristic to LAL, which can be also expressed in AML. The immunophenotypical characterization presupposes the use of some very large panels, which will include myeloid lineage markers , markers associated to line B  and markers associated to line T . HLA-DR (Human Leukocyte Antigen) testing proves its utility in identifying the LAM-M3 cases, whose positivity is rarely met in this subtype. Another practical application is the association of LAM-M7 with CD41, CD42 and CD61. The chromosomal and molecular biology analysis  Starting from the fact that the chromosomal and molecular biology particularities offer the most important prognostic information before the starting of the treatment, although the investigations are expensive, the purpose is that these modern methods are part of the basic diagnosis.  Molecular characterization: The biological material (minimum 5x10^6 cells) is processed in order to obtain the RNA. A molecular screening is done for AML1-ETO and CBFβ-MYH11 from the results of t and inv(16)/t, anomalies which have an important prognosis value. The criteria used in describing the cytogenetic clone and the karyotype follow the recommendations of the International Nomenclature System of Human Cytogenetics, the characterization of a normal chromosome needing the analysis of minimum 20 cells. What seems to be of great interest is the presence of t  responsible for the fusion of PML-RARα genes which are characteristic to the AML-M3 morphological form. In AML in children, mutations of the tyrosine kinase receptor are described; FLT3-ITD – identified in 16,5% of the cases – implies a negative prognosis of the disease.  WHO Classification , which was first put to practice in 2002 and revised in 2008 has been the most complex until presently and significantly differs from the FAB system through the following:  • the reduction of the percent of marrow blasts which differentiate the myelodysplastic syndrome from AML, from 30% to 20%.  • the inclusion of cytogenetic criteria or the equivalent molecular anomalies in the classification.  • the inclusion of the cases preceded by myelodysplastic/chronic myeloproliferative syndromes in the classification.  • the inclusion of multilineage dysplasia with or without precedent marrow affecting in the classification.  • the inclusion of cases secondary to precedent cytotoxic therapy in the classification.  • the introduction of new morphologic subtypes.  Defining the stages of the disease  ∎ Start: The AML diagnosis is defined according to WHO criteria – the presence of myeloid blasts in the marrow aspirate in a percent of at least 20% of the nucleate cells.  AML de novo: is the term that determines the form of the disease met in children who did not receive any treatment, except for the treatments that mark the symptoms of the disease.  AML secondary to a treatment (t-AML) or to any other affection.  ∎ Extramedullary disease:  It is well known that acute myeloblastic leukemia can invade the non-hematopoietic tissue . Among the most important ones, the following should be mentioned:  1. SNC Affection – presupposes the presence of over 5 leucocytes/mmc of cephalorachidian liquid (CRL). If below 5 blast cells/mmc are present, the exploration must be repeated. The clinical neurological manifestations  which can be the expression of central nervous system involvement cannot be neglected.  2. Granulocytic sarcoma – presupposes the proliferation of myeloid cells at the level of the soft tissues without the involvement of the bone marrow, which can be identified through morphological methods. These forms need different diagnosis procedures and another therapeutic attitude.  ∎ Complete remission (CR) is defined by:  ∘ the presence of <5% myeloblasts in BM, in optical microscopy, in the conditions of a marrow cellularity of over 20% and at least 1500 circullant neutrophils with a duration of at least 4 weeks. The minimum residual disease (MRD/BRM) is defined as a cell mass of undetectable leukemic cells through usual morphological techniques. The immunological and molecular genetics techniques promise the evaluation of a more accurate minimum residual disease. What is studied is whether the action of defining CR through BRM is superior to defining it through optical microscopy.  ∘ lack of any symptom / sign connected to the disease  ∘ absence of the extramedullary disease and the SNC infiltration detected in the clinical examination and the CRL investigation.  ∎Partial remission: The presence of 5-20% myeloblasts in the blood marrow obtained by medullary aspiration at the end of an induction stage.  ∎Recurrent AML: The reappearance of the disease after obtaining a complete remission.  ∎Refractive AML: Failing to obtain a complete remission at the end of the induction stage, after applying the chemotherapeutical treatment according to current protocols (20% blast cells).  ∎CRL Remission: The patients with SNC involvement in the beginning stage are considered in CRL remission if 2 consecutive lumbar punctures indicate 5 leukocytes/mmc and do not show any blast cells as a result of centrifugation.  In conclusion, the remarkable progresses that have been obtained in the last couple of years in AML in children by adopting an individualized therapy, and which are adapted to risk groups, have as a base a set of diagnostic procedures. Although the classification of the patients on risk groups is influenced by many clinical and laboratory factors, the genetic modifications that represent the basis of pathogenesis are prominent in most of the classifications and are indispensible together with the immunophenotypical characterization in the current AML diagnosis in children.


There is limited information on a special subtype of Acute myeloid leukemia (AML) characterized by >20% myeloblasts and >20% abnormal promyelocytes in bone marrow and peripheral blood.The objective of the present investigation was to explore the clinical and laboratory features of seven patients with AML-M2/M3.We retrospectively assessed cell morphology, cytochemistry, immunophenotype, cytogenetics, and clinical features of seven patients with this rare subtype of AML.1.All seven cases had thrombocytopenia, coagulation abnormalities, >20% myeloblasts and abnormal promyelocytes. The PML/RARα fusion gene was present in six patients and two patients presented a mixed PML/RARα and AML1/ETO genotype. Five cases achieved CR and two cases did not achieve remission and one case transform into AML-M2 after CRThe clinical and laboratory features of seven patients with AML-M2/M3 are demonstrated in the present study, providing information on the FAB sub-classification. Acute myeloid leukemia (AML) is characterized by clonal expansion of myeloid blasts in bone marrow (BM), peripheral blood (PB), or other tissues. In the World Health Organization (WHO) scheme (2008), a myeloid neoplasm with 20% or more myeloblasts in the PB or BM is considered to be AML. Acute myeloid leukemia with the gene rearrangement t 8; 21) ; (AML1/ETO), which is also referred to as the RUNX1 gene, and AML with the rearrangement t   (PML/RARα) are considered distinct AML subtypes. In the French-American-British (FAB) classification scheme for AML, the criteria for AML-M2 is 30% to 89% myeloblasts, >10% promyelocytes and neutrophilic myelocyte, and <20% monocytes [ , CD117(+++), TdT(-), CD34(-), L26(-), UCHL-1(+),CD3(-), CD79a partial(+), Fe(-), and reticular fiber(-) , CD20 partial (+), CD79а partial (+), CD3 partial (+), some CD138 (+), CD4 (-), CD5 (-), Ag (+), and Fe (-).In BM smears, the myeloid lineage was hyperactive, with 39.5% myeloblasts and 49% abnormal promyelocytes.Results of BM biopsy were consistent with AML: MPO(+), CD117(+), LCA(+), Ki-67 approximately 20%(+), CD3(-), CD34(-),TdT(-), CK(-), Ag(++), and Fe(-).Bone marrow smears revealed 42.5% myeloblasts and 30% abnormal promyelocytes. Cytochemistry indicated 99% MPO (+) cells. Most cells also appeared AS-DCE (+). The immunophenotype was MPO (+), CD117 (+), LCA (+), about 20% Ki-67(+), CD3 (-), CD34 (-), TdT (-), CK (-), Ag (++), and Fe (-). Cytogenetic analysis indicated the presence of the PML/RARа fusion gene, S type. Immunophenotyping by FCM revealed that 76.4% of BM karyocytes were abnormal . Most cells were weakly CD45 (+), while 30.9% were CD13 (+) 11.6% CD15 (+), 95.5% CD33 (+), 96.5% CD56 (+), 16.9% CD64 (+), 78.4% CD117 (+), and 84.3% were positive for the cytoplasmic antigen of cMPO. Cells were negative for antigens of the lymphocytic lineage , 6.5% CD15 (+), 0.2% CD14/CD11b (+), and 3.5% HLA-DR/CD13 (+). No antigens of the lymphocytic lineage were detected.Bone marrow smears revealed 49% myeloblasts and 13% abnormal promyelocytes, while the cells in the PB smear were 14% myeloblasts and 13% promyelocytes. The BM cells harbored both the PML/RARа fusion gene, S type, and the AML1/ETO fusion gene. Immunophenotyping by FCM revealed two groups of blast cells. The SSC lower cell group was approximately 44.9% of the total and this population was 13.5% CD11b(+), 90.8% CD13(+), 40.3% CD15(+), 82.6% CD33(+), 62.3% CD34(+), 12.4% CD56(+), 40.3% CD64(+), 88.0% CD117(+), 85.9% HLA-DR (+), and 97.8% cMPO(+), while no antigens of other lineages were expressed. The SSC lower cell group was approximately 48.3% of the total and this population was 46.6% CD11b(+), 94.9% CD13(+), 17.3% CD14(+), 95.3% CD15(+), 83.0% CD33(+), 26.1%(+) CD56, 70.4% CD64(+), 43.8% CD117(+), 35.0% HLA-DR(+), and 98.9% cMPO(+). Antigens of other lineages were not expressed  and AS-DCE(+) fractions, results of immunophenotyping by flow cytometry, and results of cytogenetic analysis for all seven case patients are summarized in Tables Five of the six cases were treated by standard chemotherapy ,18 daun.Table 5When newly diagnosis, 85.7% of case patients (6/7) had fever, 71.4% (5/7) had sternum pressing pain, hemorrhagic symptoms, and anemia, 28.6% (2/7) presented with hepatomegaly or splenomegaly, 100% had leukocytosis or leukopenia, and thrombocytopenia, 28.6% showed reduced hemocytes , 100% had coagulation abnormalities, and 85.7% had elevated serum LDH. All patients had >20% myeloblasts and >20% abnormal promyelocytes, of which 71.4% had >30% BM myeloblasts and abnormal promyelocytes. All six patients tested were MPO (+). Six patients were PML/RARа positive and two of these patients harbored both the AML1/ETO and PML/RARа fusion genes. Immunophenotyping (Table The FAB classification scheme (1976) divided AML into subtypes based on the morphology of BM or PB cells, while the current World Health Organization (WHO) classification system incorporates morphology, cytogenetics, molecular genetics, and immunological markers for the diagnosis of AML subtypes ,5,8. AcuAccording to the diagnostic criteria for AML, AML-M2 is characterized by significantly increased myeloblasts, while AML-M3 is associated with abnormal promyelocytes. However, six of our cases meet the diagnostic criteria for both AML-M2 (>30% myeloblasts) and AML-M3 . We also observed two patients with both the AML1/ETO and the PML/RARα fusion gene. Such cases cannot be classified as AML-M2 or AML-M3, so we suggest a new subtype of AML, AML-M2/M3 ,8,15-17.The diagnosis of AML-M2/M3 requires all available information, including morphology, cytochemistry, immunophenotyping, genetics, and clinical features . In partIn BM and PB smears, all cases showed a simultaneous increase in myeloblasts and abnormal promyelocytes. Some promyelocytes contained dust-like or sparsely distributed granules. Promyelocyte nuclei were kidney-shape or even bi-lobed with inconspicuous to prominent nucleoli. Some promyelocytes also contained Aure rods. These morphological characteristics are distinct from the typical abnormal promyelocytic cells observed in AML-M3 -3.Immunophenotyping showed that the SSCs expressed CD9, CD11b, CD13, CD15, CD33, CD34, CD38, CD45, CD56, CD64, CD117, and HLA-DR, antigens characteristic of both AML-M2 and AML-M3. Three cases expressed BM cells strongly positive for CD9 and four cases exhibited cells strongly positive for CD13 and CD33. Two of 6 cases exhibited cells with CD117 expression. In addition, CD34, cMPO, CD117, HLA-DR, CD11b, CD15, CD38, CD56, and CD64 were expressed in some cases. However, it is difficult to distinguished AML-M2 from AML-M3 according to immunophenotype -27.It is uncertain whether the two cases with both fusion genes harbor two genetic rearrangements within the same clone or two independent clones with different rearrangements. Unfortunately, as this was a retrospective study, no further analysis was possible and there was no information on how the various FACS and molecular assays were performed. Further studies are needed to examine the frequency of this double genetic rearrangement in AML-M2/M3.Many of the clinical characteristics of AML-M2/M3 are common to other subtypes of leukemia, including anemia, fever, hemorrhage, and infiltration. However, this group of patients was prone to DIC, cerebral hemorrhage, and other serious complications, with high mortality rates. Moreover, patients showed a trend for recurrence and transformation into AML-M2 . AlthougAt present, there are no guidelines for the treatment of this new subtype of AML. All of the patients were treated with ATRA, ATO, or CRINT combined with the DA regimen and five achieved complete temporary remission. Our initial observations suggest that hematopoietic stem cell transplantation may be beneficial for long-term survival. Therefore, the significance of the present study was that the patients with AML could be treated with ATRA, ATO, or CRINT combined with the DA regimen, and hematopoietic stem cell transplantation might be beneficial for long-term survival, possibly providing data for the guidelines for the FAB sub-classification and treatment of this new subtype of AML.In the present study, we identified a novel entity of AML according to FAB classification and examined its clinical characteristics. Although the “gray-zone” entity of each of the FAB sub-classification has been recognized since the first proposal of FAB classification in 1976, unfortunately, this issue was not endowed with clear solution. Later, in a “proposed revised criteria for the classification of AML” by the FAB cooperative group in 1985 , the potIn conclusion, the clinical and laboratory features of seven patients with AML-M2/M3 are demonstrated in the present study, providing information on the FAB sub-classification.


Washout of the CDR antibodies partially recovered CB immunoreactivity, suggesting a transient structural change in CB calcium-binding site. We discovered that CDR2 and CB co-immunoprecipitate. Furthermore, the expression levels of voltage-gated calcium channel Cav2.1, protein kinase C gamma and calcium-dependent protease, calpain-2, were increased after CDR antibody internalization. Inhibition of these signaling pathways prevented or attenuated CDR antibody-induced CB and L7/Pcp-2 immunoreactivity loss, morphological changes and increased protein expression. These results signify that CDR antibody internalization causes dysregulation of cell calcium homeostasis. Hence, drugs that modulate these events may represent novel neuroprotective therapies that limit the damaging effects of CDR antibodies and prevent PC neurodegeneration.Paraneoplastic cerebellar degeneration (PCD) is characterized by loss of Purkinje cells (PCs) associated with progressive pancerebellar dysfunction in the presence of onconeural Yo antibodies. These antibodies recognize the cerebellar degeneration-related antigens CDR2 and CDR2L. Response to PCD therapy is disappointing due to limited understanding of the neuropathological mechanisms. Here, we report the pathological role of CDR antibodies on the calcium homeostasis in PCs. We developed an antibody-mediated PCD model based on co-incubation of cerebellar organotypic slice culture with human patient serum or rabbit CDR2 and CDR2L antibodies. The CDR antibody-induced pathology was investigated by high-resolution multiphoton imaging and biochemical analysis. Both human and rabbit CDR antibodies were rapidly internalized by PCs and led to reduced immunoreactivity of calbindin DThe online version of this article (doi:10.1007/s00401-014-1351-6) contains supplementary material, which is available to authorized users. Paraneoplastic cerebellar degeneration (PCD), caused by Purkinje cell (PC) death in the cerebellum, is a paraneoplastic neurological disorder with severe pancerebellar symptoms, such as ataxia, nystagmus, and dysarthria . Cross-rBrain, normal ovary tissue, and tumor tissue express CDR2 protein , 30, 70.2+ levels and Ca2+ flux via cytoplasmic Ca2+-binding protein calbindin D28K (CB) and the GoLoco domain protein, Purkinje cell-specific protein-2 (L7/Pcp-2), are essential for cellular and molecular mechanisms involved in neurotransmitter release, ion channel permeability, enzyme activity, and gene transcription ; 6 days; ***p < 0.0001; nE = 4; and rCDR [125 ng/mL]; ***p < 0.0001; 6 days; nE = 4; Fig. rCDR2 and rCDR2/2L internalization . rCDR2L internalization had no effect on PKCγ protein expression, while CB+ - and L7/Pcp-2+-PC loss was rescued by K252a co-treatment in the rCDR2L group. The antagonist had no effect on the CB+- or L7/Pcp-2+ count or PKCγ protein expression in the rIgG and untreated naive control.The postsynaptic Carization , 73. PKCrization . Missensrization , 60. We The results show that the CDR antibody-mediated pathology can be attenuated in the presence of VGCC, AMPA receptor, and PKC signaling inhibition.2+ levels and induce Ca2+-dependent proteases activity. To examine whether CDR internalization affects Ca2+-dependent proteases activity of calpain-1 and calpain-2, we performed Western blot analysis of rCDR-treated cOTSC lysates. Calpain-1 expression was not affected, but calpain-2 expression was significantly increased  (6 days) reduced the CB+-PC loss to ~45 %, as well as the CDR-induced pathology on the dendritic arborizations in the remaining PCs  treatment  (18 %) and hCDR2/2L+(PS3) (48 %) -induced L7/Pcp-2+-PC loss was partially rescued by 10 μM MDL28170  showed an improvement of the dendritic morphology and significant reduction of CB+-PC loss .Changes in VGCC, AMPA receptor, and PKC signaling can lead to elevated intracellular Ca2+ to the millimolar range, which thereby activate the Ca2+-dependent protease calpain 2.These data show that CDR internalization leads to an increase in intracellular unbound CahCDR2/2L+  and MAP kinase inhibitor U0126 (5 μM) did not prevent the CB+- or L7/Pcp-2+-PC immunoreactivity loss (CB: hCDR2/2L+(PS3): 70 ± 10 vs. 68 ± 7 % (5 μM U0126); p = 0.4268; L7/Pcp-2: hCDR2/2L+(PS4): 65 ± 3 vs. 64 ± 2 % (5 μM U0126), p = 0.5249; nE = 4; Fig. Activation of mitogen-activated protein (MAP) kinase signaling cascades are associated with PKC-dependent synaptic depression/declustering of glutamate receptors, inflammation, death receptor activation, apoptosis, and oxidative stress , 23, 35.hCDR2/2L.These data suggest that the MAP kinase signaling cascade is not involved in the CB or L7/Pcp-2 immunoreactivity reduction induced by CDR antibodies, also known as onconeuronal Yo antibodies, have attracted increasing interest due to their suggested pathological role in the progressive loss of PCs seen in PCD after binding to CDR2 and CDR2L antigens , 33, 70.OTSC can be used to study neurochemical, structural, and physiological changes linked to diseased in vivo brains , 44. HenhCDR and rCDR can cause similar PC pathology. By using rCDR we exclude cytotoxic T cell involvement, since the cOTSC was not exposed to related peptides which could activate brain-naive resident T cells [Here, we demonstrate that both  T cells . Therefo T cells . However T cells . Our CDR T cells , but the T cells  demonstr T cells .h/rCDR internalization changes PC physiology by targeting postsynaptic signaling factors and thereby modifies Ca2+ homeostasis. We described these factors along with related neurological diseases in Table S1. We found that the immunoreactivity of Ca2+ homeostasis regulator CB and VGCC modulator L7/Pcp-2 was reduced. These proteins are functionally linked, since CB shapes the post-tetanic potentiation induced by the Ca2+ influx through AMPAR and VGCC and can act as an activity-dependent sensor [2+-binding mechanism or protein structure, as seen in other neurodegenerative diseases [In Fig. t sensor , 57. Whet sensor , 37, 74.diseases , 50.Fig.2+-transient elevation which greatly influences Ca2+ homeostasis [2+ microdomain variations due to AMPAR, VGCC, or PKC activity play a crucial role [2+ influx [2+ influx in synaptic Ca2+ microdomains [2+ influx into PCs, which can activate cell death pathways by enhancing PKC and Ca2+-dependent protease activity [CB dysfunction or deletion can result in drastic fast intracellular Caeostasis . In PC sial role , 57, 68.+ influx , 57, 73.odomains . Inhibitactivity .rCDR-treated cOTSCs. We therefore hypothesize that CDR internalization enhances PKC activity, which: first, reduces L7/Pcp-2 expression; second, increases Cav2.1 expression; third, increases intracellular Ca2+, which activates Ca2+-dependent proteases such as calpain-1 and calpain-2 and thereby induces neuronal death.PKC is associated with apoptosis in ischemia and stroke , 29, 60.2+ concentrations. In Alzheimer’s, Huntington’s, and Parkinson’s disease, calpain over-activation or dysregulation triggers neuronal death by truncation of important synaptic substrates [2+ level above 0.250 mM [2+ transient by mediating over-activation of calpain-2 [Calpain-1 and calpain-2 are activated by different cellular Cabstrates , 53, 59.0.250 mM . Inhibit0.250 mM . We hypoalpain-2 . As calpalpain-2 , 23, 35.2+ homeostasis, which lead to the silencing of the PCs. Second, cytototoxic T cells and microglia mediate the clearing of diseased cells, as known from autopsy studies [PCD pathogenesis is largely unknown. We propose a two-step pathology mechanism. First, internalization of CDR antibodies modifies important regulatory factors of Ca studies .2+ homeostasis due to alterations of CB, VGCC, or PKC [2+ levels by inhibition, the VGCC–AMPAR–PKC signaling pathway  There is no pathology seen in hematoxylin and eosin stained, paraffin-embedded ultra thin sections of cOTSC after 6 days of hCDR2/2L+(PS3) serum treatment. Purkinje (PCL), molecular (MCL) and granule (GCL) cell layer, scale bars 50 μm. (b) Double immunostaining of CB (magenta) and cleaved “active” caspase-3 (green) in rIgG and rCDR2/2L-treated cOTSC after 6 days. Orthogonal view shows that cleaved caspase-3 positive apoptotic cells were scattered in the cerebellum in both rIgG control and rCDR2/2L treated group. All of these cells were small and labeling was not co-localized with CB+ PCs; scale bars 15 μm. Supplementary material 1 (TIFF 4816 kb)Fig. S2hCDR and rCDR internalization induces GoLoco domain protein L7/Pcp-2 immunoreactivity loss. (a) L7/Pcp-2 (green) positive PCs are reduced under hCDR2/2L serum from PCD patient 3 and 4 at day 6 (D6); scale bars 40 μm. (b) Internalization of hCDR2/2L+(PS3) and hCDR2/2L+(PS4) (1 μL/mL) reduced the L7/Pcp-2 positive PCs (L7/Pcp-2+) to two-third over time (samples taken: day 2 (D2), day 4 (D4), day 6 (D6); (nE = 3)). (c) rCDR internalization caused similar reduction pattern of L7/Pcp-2 (green) positive PCs after 6 days as seen for hCDR in (a), scale bars 40 μm. (d) rCDR2, rCDR2L and rCDR2/2L internalization led to ~70 % L7/Pcp-2+ PC loss with no difference between the groups (nE = 6). Data in mean ± SEM. Non-parametric two-tailed paired Mann–Whitney’s U test. *p < 0.05; **p < 0.01; ***p < 0.001. The percentage changes to the controls are summarized in Table 1. Supplementary material 2 (TIFF 5778 kb)Supplementary material 3 (DOC 59 kb)Supplementary material 4 (DOC 29 kb)Supplementary material 5 (DOC 51 kb)Below is the link to the electronic supplementary material.