A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis

Background

Stomatocytoses are a group of inherited autosomal dominant hemolytic anemias and include overhydrated hereditary stomatocytosis, dehydrated hereditary stomatocytosis, hereditary cryohydrocytosis and familial pseudohyperkalemia.

Design and Methods

We report a novel variant of hereditary stomatocytosis due to a de novo band 3 mutation (p. G796R-band3 CEINGE) associated with a dyserythropoietic phenotype. Band 3 genomic analysis, measurement at of hematologic parameters and red cell indices and morphological analysis of bone marrow were carried out. We then evaluated the red cell membrane permeability and ion transport systems by functional studies of the patient’s erythrocytes and Xenopus oocytes transfected with mutated band 3. We analyzed the red cell membrane tyrosine phosphorylation profile and the membrane association of the tyrosine kinases Syk and Lyn from the Src-family-kinase group, since the activity of the membrane cation transport pathways is related to cyclic phosphorylation-dephosphorylation events.

Results

The patient showed mild hemolytic anemia with circulating stomatocytes together with signs of dyserythropoiesis. Her red cells displayed increased Na⁺ content with decreased K⁺content and abnormal membrane cation transport activities. Functional characterization of band 3 CEINGE in Xenopus oocytes showed that the mutated band 3 is converted from being an anion exchanger (Cl⁻, HCO₃⁻) to being a cation pathway for Na⁺ and K⁺. Increased tyrosine phosphorylation of some red cell membrane proteins was observed in diseased erythrocytes. Syk and Lyn membrane association was increased in the patient’s red cells compared to in normal controls, indicating perturbation of phospho-signaling pathways involved in cell volume regulation events.

Conclusions

Band 3 CEINGE alters function from that of anion exchange to cation transport, affects the membrane tyrosine phosphorylation profile, in particular of band 3 and stomatin, and its presence during red cell development likely contributes to dyserythropiesis.     

CD8+ T-cell immunity against cancer-testis antigens develops following allogeneic stem cell transplantation and reveals a potential mechanism for the graft-versus-leukemia effect

Background

Allogeneic stem cell transplantation is associated with a powerful ‘graft-versus-leukemia’ effect that is generally considered to result from an alloreactive T-cell immune response. However, disease remission can also be observed after syngeneic transplantation and we investigated whether a T-cell immune response to cancer-testis antigens can be detected in patients in the post-transplant period.

Design and Methods

The T-cell immune response against cancer-testis antigens was studied in a cohort of 41 patients who underwent allogeneic stem cell transplantation for the management of acute myeloid leukemia or multiple myeloma. The cytokine secretion assay was combined with magnetic selection to allow detection of an interferon-γ-secreting T-cell response to a panel of cancer-testis antigen peptides.

Results

A cancer-testis antigen-specific CD8⁺ T-cell immune response was observed in the peripheral blood of five patients with an average magnitude of 0.045% of the CD8⁺ T-cell repertoire. Four of these patients had undergone reduced intensity conditioning transplantation with alemtuzumab for the treatment of acute myeloid leukemia and three remain in long-term remission. T-cell immunity was focused against peptides derived from MAGE proteins and was markedly increased within the bone marrow.

Conclusions

Functional cancer-testis antigen-specific CD8⁺ T-cell immune responses develop in the early period following reduced intensity allogeneic stem cell transplantation and are preferentially localized to bone marrow. These immune responses are likely to contribute to the cellular basis of the graft-versus-leukemia effect.     

Mammalian target of rapamycin activity is required for expansion of CD34+ hematopoietic progenitor cells

Background

The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic syndromes, suggesting that correct regulation of mammalian target of rapamycin is critical for normal hematopoiesis.

Design and Methods

An ex vivo granulocyte differentiation system was utilized to investigate the role of mammalian target of rapamycin in the regulation of myelopoiesis.

Results

Inhibition of mammalian target of rapamycin activity, with the pharmacological inhibitor rapamycin, dramatically reduced hematopoietic progenitor expansion, without altering levels of apoptosis or maturation. Moreover, analysis of distinct hematopoietic progenitor populations revealed that rapamycin treatment inhibited the expansion potential of committed CD34⁺ lineage-positive progenitors, but did not affect early hematopoietic progenitors. Further examinations showed that these effects of rapamycin on progenitor expansion might involve differential regulation of protein kinase B and mammalian target of rapamycin signaling.

Conclusions

Together, these results indicate that mammalian target of rapamycin activity is essential for expansion of CD34⁺ hematopoietic progenitor cells during myelopoiesis. Modulation of the mammalian target of rapamycin pathway may be of benefit in the design of new therapies to control hematologic malignancies.     

High levels of CD34+CD38low/-CD123+ blasts are predictive of an adverse outcome in acute myeloid leukemia: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study

Background

Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34⁺CD38^low/− compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34⁺CD38^low/−CD123⁺ phenotype.

Design and Methods

The percentage of CD34⁺CD38^low/−CD123⁺ cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses.

Results

A proportion of CD34⁺CD38^low/−CD123⁺ cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34⁺CD38^low/−CD123⁺ higher than 15% retained significance with an odds ratio of 0.33 (0.1–0.97; P=0.044). A greater than 1% population of CD34⁺CD38^low/−CD123⁺ cells negatively affected disease-free survival (0.9 versus 4.7 years; P<0.0001) and overall survival (1.25 years versus median not reached; P<0.0001). A greater than 1% population of CD34⁺CD38^low/−CD123⁺ cells retained prognostic significance for both parameters after multivariate analysis.

Conclusions

The percentage of CD34⁺CD38^low/−CD123⁺ leukemic cells at diagnosis was significantly correlated with response to treatment and survival. This prognostic marker might be easily adopted in clinical practice to rapidly identify patients at risk of treatment failure.     

Changes in peripheral CD19+Foxp3+ and CD19+TGFβ+ regulatory B cell populations in rheumatoid arthritis patients with interstitial lung disease

Objective

Although the role of regulatory B cells (Bregs) in rheumatic disease has gained increasing attention, two lesser-known Breg subsets that express either Foxp3 or transforming growth factor beta (TGFβ) are rarely examined in studies of rheumatic disease. This study investigates the association between the relative proportions of CD19⁺Foxp3⁺ and CD19⁺TGFβ⁺ Bregs, and clinical indicators of disease severity in rheumatoid arthritis (RA) patients with or without interstitial lung disease (ILD).

Methods

A total of 31 RA patients (14 with and 17 without ILD) and 26 healthy control subjects were included. All subjects did not have other autoimmune disease except RA, tumor, active infection, or a history of related drug administration. Peripheral blood mononuclear cells (PBMCs) were isolated and analyzed by flow cytometry (FCM). The relationship between the relative proportions of CD19⁺Foxp3⁺ and CD19⁺TGFβ⁺ Bregs and their associations to RA and ILD incidence, as well as disease severity assessed by common clinical indicators, were then examined.

Results

Our analyses revealed RA patients had significantly lower proportions of peripheral CD19⁺Foxp3⁺ and CD19⁺TGFβ⁺ Bregs as compared to healthy controls. While no association was observed between CD19⁺Foxp3⁺ Bregs and ILD incidence, patients with ILD had a substantially lower percentage of CD19⁺TGFβ⁺ Bregs compared to RA patients without ILD. In addition, CD19⁺Foxp3⁺ Bregs were negatively correlated with erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels in RA patients, whereas CD19⁺TGFβ⁺ Bregs were only correlated with CRP in RA patients with ILD. Furthermore, there was a negative association between CD19⁺Foxp3⁺ Bregs and disease severity scores, which was not found in analyses with CD19⁺TGFβ⁺ Bregs.

Conclusions

The proportions CD19⁺Foxp3⁺ and CD19⁺TGFβ⁺ Bregs were significantly decreased in RA patients, particularly in those with ILD complications, suggesting that Breg phenotypes may have different functions in the pathogenesis of RA and ILD.     

Impact of constitutional polymorphisms in VCAM1 and CD44 on CD34+ cell collection yield after administration of granulocyte colony-stimulating factor to healthy donors

Background

The number of CD34⁺ cells mobilized from bone marrow to peripheral blood after administration of granulocyte colony-stimulating factor varies greatly among healthy donors. This fact might be explained, at least in part, by constitutional differences in genes involved in the interactions tethering CD34⁺ cells to the bone marrow.

Design and Methods

We analyzed genetic characteristics associated with CD34⁺ cell mobilization in 112 healthy individuals receiving granulocyte colony-stimulating factor (filgrastim; 10 μg/kg; 5 days).

Results

Genetic variants in VCAM1 and in CD44 were associated with the number of CD34⁺ cells in peripheral blood after granulocyte colony-stimulating factor administration (P=0.02 and P=0.04, respectively), with the quantity of CD34⁺ cells ×10⁶/kg of donor (4.6 versus 6.3; P<0.001 and 7 versus 5.6; P=0.025, respectively), and with total CD34⁺ cells ×10⁶ (355 versus 495; P=0.002 and 522 versus 422; P=0.012, respectively) in the first apheresis. Of note, granulocyte colony-stimulating factor administration was associated with complete disappearance of VCAM1 mRNA expression in peripheral blood. Moreover, genetic variants in granulocyte colony-stimulating factor receptor (CSF3R) and in CXCL12 were associated with a lower and higher number of granulocyte colony-stimulating factor-mobilized CD34⁺ cells/μL in peripheral blood (81 versus 106; P=0.002 and 165 versus 98; P=0.02, respectively) and a genetic variant in CXCR4 was associated with a lower quantity of CD34⁺ cells ×10⁶/kg of donor and total CD34⁺ cells ×10⁶ (5.3 versus 6.7; P=0.02 and 399 versus 533; P=0.01, respectively).

Conclusions

In conclusion, genetic variability in molecules involved in migration and homing of CD34⁺ cells influences the degree of mobilization of these cells.     

Identifying factors associated with changes in CD4+ count in HIV-infected adults in Saskatoon,Saskatchewan

OBJECTIVE:

To assess the impact of clinical and social factors unique to HIV-infected adults in Saskatoon, Saskatchewan, regarding the rate of CD4⁺ count change, and to identify factors associated with a risk of CD4⁺ count decline.

METHODS:

A retrospective longitudinal cohort study from medical chart reviews at two clinics was conducted in Saskatoon. Univariate and multivariate linear mixed effects models were used to assess the impact of selected factors on CD4⁺ count change.

RESULTS:

Four hundred eleven HIV-infected patients were identified from January 1, 2003 to November 30, 2011. Two hundred eighteen (53%) were male, mean (± SD) age was 35.6 ±10.1 years, 257 (70.8%) were First Nations or Métis, 312 (80.2%) were hepatitis C virus (HCV) coinfected and 300 (73.3%) had a history of injection drug use (IDU). In univariate models, age, ethnicity, HCV, IDU, antiretroviral therapy and social assistance were significant. Using ethnicity, HCV and IDU, three multivariate models (models 1, 2, 3) were built due to high correlation. First Nations or Métis ethnicity, HCV coinfection and a history of IDU were associated with significantly lower CD4⁺ counts in multivariate models. Older age and social assistance were associated with significantly lower CD4⁺ counts in models 1 and 3. Age was marginally significant in model 2 (P=0.055). Not prescribed antiretroviral therapy was associated with a significantly negative CD4⁺ count slope in all multivariate models.

CONCLUSION:

The unique epidemiology of this HIV-infected population may be contributing to CD4⁺ count change. Increased attention and resources focused on this high-risk population are needed to prevent disease progression and to improve overall health and quality of life.     

Three-year data from the XIENCE V® INDIA study: Safety and efficacy of XIENCE V® in 1000 real world Indian patients

Background

Cardiovascular disease in Asia has reached epidemic proportions in recent years. Use of drug eluting stents in Asians has rapidly expanded with varying penetration rates across different countries. The XIENCE V^® INDIA Study included ‘real world’ patients who underwent XIENCE V^® stent implantation to assess short and intermediate term outcomes in Indian patients with diverse risk factors.

Objective

To evaluate 3-year clinical outcomes in a cohort of ‘real world’ Indian patients with CAD being treated with XIENCE V^® Everolimus Eluting Coronary Stent System.

Methods

1000 patients were enrolled from 18 sites in India between June 2008 and March 2009. Patients were included if their index procedures were completed using only XIENCE V^®. There were no clinical or angiographic exclusions. An independent Clinical Events Committee adjudicated all endpoint-related events. The primary endpoint was stent thrombosis rate annually through to 3 years as defined by the Academic Research Consortium criteria. The co-primary endpoint was the composite rate of cardiac death and myocardial infarction at 1 year.

Results

At 1-year the primary endpoint of definite/probable stent thrombosis rate was 0.51%. No additional very late stent thrombosis was reported through a 3-year follow up. The composite endpoint of cardiac death and any myocardial infarction was 1.9%, 2.7% and 3.1% at 1, 2 and 3 years respectively.

Conclusion

Despite the high risk population of coronary artery disease, the use of XIENCE V^® in ''real world'' Indian patients was associated with very low clinical event rates upto three years of follow up.     

Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro

Background

In the bone marrow mesenchymal stromal cells and osteoblasts form functional niches for hematopoietic stem and progenitor cells. This microenvironment can be partially mimicked using in vitro co-culture systems. In this study, we examined the oxygen tension in three distinct compartments in a co-culture system of purified CD34⁺ cells and mesenchymal stromal cells with regard to different spatial localizations.

Design and Methods

Hypoxic cells in the co-culture were visualized by pimonidazole staining. Hematopoietic cell distribution, and functional and phenotypic characteristics were analyzed by flow cytometry. The secretion of vascular endothelial growth factor and stromal-derived factor-1 by mesenchymal stromal cells in low oxygen co-cultures was determined by an enzyme-linked immunosorbent assay. The effect of co-culture medium on the hematopoietic cell migration potential was tested in a transwell assay.

Results

In co-cultures under atmospheric oxygen tension, regions of low oxygen tension could be detected beneath the feeder layer in which a reservoir of phenotypically more primitive hematopoietic cells is located in vitro. In low oxygen co-culture, the adhesion of hematopoietic cells to the feeder layer was decreased, whereas hematopoietic cell transmigration beneath mesenchymal stromal cells was favored. Increased vascular endothelial growth factor-A secretion by mesenchymal stromal cells under low oxygen conditions, which increased the permeability of the monolayer, was responsible for this effect. Furthermore, vascular endothelial growth factor-A expression in low oxygen mesenchymal stromal cells was induced via hypoxia-inducible factor signaling. However, stromal cell-derived factor-1 secretion by mesenchymal stromal cells was down-regulated under low oxygen conditions in a hypoxia-inducible factor-independent manner.

Conclusions

We demonstrate for the first time that differences in oxygen tension cause selective modification of hematopoietic cell and mesenchymal stromal cell interactions in a co-culture system, thus confirming that oxygen tension plays a critical role in the interaction between hematopoietic cells and the niche environment.     

Cytotoxic molecule-positive classical Hodgkin's lymphoma: a clinicopathological comparison with cytotoxic molecule-positive peripheral T-cell lymphoma of not otherwise specified type

Background

Classical Hodgkin’s lymphoma is characterized by Hodgkin and Reed Sternberg cells, which are of B-cell origin in many cases. We recently highlighted the adverse prognostic significance of cytotoxic molecule expression in patients with classical Hodgkin’s lymphoma. However, the clinical characteristics of cytotoxic molecule-positive classical Hodgkin’s lymphoma remain controversial.

Design and Methods

We investigated the clinicopathological profiles of 32 patients with cytotoxic molecule-positive Hodgkin’s lymphoma, comprising 23 with nodular sclerosis and 9 with mixed cellularity, and compared these profiles with those of 55 patients with cytotoxic molecule-positive nodal peripheral T-cell lymphoma, not otherwise specified and 439 patients with cytotoxic molecule-negative Hodgkin’s lymphoma.

Results

The patients with cytotoxic molecule-positive Hodgkin’s lymphoma consisted of 20 men and 12 women with a median age of 50 years (range, 19 to 81). All these patients had lymphadenopathy at presentation, and 14 showed mediastinal involvement. Physical findings included hepatomegaly and splenomegaly in six patients each. Four patients had a bulky mass, and nine showed stage IV disease. The tumor cells of patients with cytotoxic molecule-positive Hodgkin’s lymphoma had a prototypic immunophenotype of CD15⁺ CD30⁺ CD45RO⁻ fascin⁺, with positivity for Epstein-Barr virus in 39% of cases. All patients were negative for Pax5. In comparison with patients with cytotoxic molecule-positive nodal peripheral T-cell lymphomas, not otherwise specified, patients with cytotoxic-positive Hodgkin’s lymphoma had relatively mild clinical symptoms, similar to those of patients with cytotoxic molecule-negative Hodgkin’s lymphoma. Regarding prognosis, the survival of patients with cytotoxic molecule-positive Hodgkin’s lymphoma was worse than that of patients with cytotoxic molecule-negative Hodgkin’s lymphoma (P=0.0003) but better than that of patients with cytotoxic molecule-positive peripheral T-cell lymphomas, not otherwise specified (P=0.002).

Conclusions

Cytotoxic molecule-positive Hodgkin’s lymphoma is characterized by an unfavorable prognosis, even if its clinicopathological features are within the boundaries of classical Hodgkin’s lymphoma. More effective chemotherapy for cytotoxic molecule-positive Hodgkin’s lymphoma is clearly required.     

A functional dynamic scoring model to elucidate the significance of post-induction interim fluorine-18-fluorodeoxyglucose positron emission tomography findings in patients with Hodgkin��s lymphoma

Background

The findings of interim fluorine-18-fluorodeoxyglucose positron emission tomography (FDG-PET/CT) predict progression-free survival of patients with Hodgkin’s lymphoma. Historically, the assessment was based on a static all-or-none scoring system. However, the clinical significance of any positivity in interim FDG-PET/CT has not been defined.

Design and Methods

Ninety-six patients with Hodgkin’s lymphoma who underwent interim FDG-PET/CT were evaluated using dynamic and visual scores, employing mediastinal or liver blood pool uptake as a comparator. FDG-PET/CT was prospectively defined as positive if any abnormal F¹⁸FDG uptake was present. In a retrospective analysis dynamic score 0 indicated resolution of all disease sites; score 1 defined a single residual focus; score 2 denoted a reduction in the number of foci; score 3 defined a reduction in intensity with no reduction in number; and score 4 indicated no change in the number and intensity of foci or appearance of new foci.

Results

The dynamic visual score review reduced the number of positive interim studies from 24 to 6 if a score of 2 or less was considered negative, with significantly better specificity (96%) as compared to static visual scores (78%–86%). The 5-year progression-free survival and overall survival rates in patients who had a negative dynamic score were 92% and 97%, respectively; the corresponding figures for patients with positive results were 50% and 67%.

Conclusions

A dynamic visual score may be a better indicator for tailoring therapy than static visual scoring.     

Ion-binding properties of a K+ channel selectivity filter in different conformations

K⁺ channels are membrane proteins that selectively conduct K⁺ ions across lipid bilayers. Many voltage-gated K⁺ (K_V) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K⁺ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to K_V channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na⁺ or low [K⁺]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K⁺ ion binding to channels with an inactivated or conductive selectivity filter is different from K⁺ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.K⁺ channels are found in all three domains of life, where they selectively conduct K⁺ ions across cell membranes. Specific stimuli trigger the activation of K⁺ channels, which results in a hinged movement of the inner helix bundle (1–7). This opening on the intracellular side of the membrane initiates ion conduction across the membrane by allowing ions to enter into the channel. After a period, many channels spontaneously inactivate to attenuate the response (8–17). The inactivation process is a timer that terminates the flow of ions in the presence of an activator to help shape the response of the system. Two dominant types of inactivation have been characterized in voltage-dependent channels: N-type and C-type (18). N-type inactivation is fast and involves an N-terminal positively charged “ball” physically plugging the pore of the channel when the membrane is depolarized. C-type inactivation, on the other hand, is a slower process involving a conformational change in the selectivity filter that is initiated by a functional link between the intracellular gate and the selectivity filter (10, 19).Several experimental observations indicate a role for the selectivity filter in C-type inactivation. First, mutations in and around the selectivity filter can alter the kinetics of inactivation (20–23). Second, increasing concentrations of extracellular K⁺ ions decrease the rate of inactivation, as if the ions are stabilizing the conductive conformation of the channel to prevent a conformational change in the selectivity filter (14, 16, 17, 22). Finally, a loss of selectivity of K⁺ over Na⁺ has been observed during the inactivation process in Shaker channels, suggesting a role for the selectivity filter (24, 25). Together, these data indicate that channels in their inactivated and conductive conformations interact with K⁺ ions differently, and suggest that C-type inactivation involves a conformational change in the selectivity filter. Although several structures of K⁺ channels in their conductive state have been solved using X-ray crystallography, there is at present no universally accepted model for the C-type inactivated channel (1, 3–5, 9, 19, 26–28) (Fig. 1B).Open in a separate windowFig. 1.Macroscopic recordings and structural models of KcsA K⁺ channel. (A) Macroscopic currents of WT KcsA obtained by a pH jump from pH 8 to pH 4 reveal channel inactivation. Two models representing the conformation of the channel are shown below. (B) Conductive [Left, Protein Data Bank (PDB) ID code 1K4C] and constricted (Right, PDB ID code 1K4D) conformations of the selectivity filter are shown as sticks, and the ion-binding sites are indicated with green spheres. The thermodynamic properties of the conductive, constricted, and inactivated (Middle) conformations are the subject of this study.Inactivation in the K⁺ channel from Streptomyces lividians (KcsA) has many of the same functional properties of C-type inactivation, which has made it a model to understand its structural features (20). KcsA channels transition from their closed to open gate upon changing the intracellular pH from high to low (Fig. 1A). The rapid flux of ions through the channel is then attenuated by channel inactivation, where most open WT channels are not conducting, suggesting that crystal structures of open KcsA channels would reveal the inactivated channel. In some crystal structures of truncated WT KcsA solved with an open gate, the selectivity filter appears in the constricted conformation, similar to the conformation observed in structures of the KcsA channel determined in the presence of only Na⁺ ions or low concentrations of K⁺ ions (3, 10, 29, 30) (Fig. 1B). Solid-state and solution NMR also indicate that the selectivity filter of the KcsA channel is in the constricted conformation when the cytoplasmic gate is open (31–33).However, a recently published study shows that even when the constricted conformation of KcsA’s selectivity filter is prevented by a nonnatural amino acid substitution, the channel inactivates like WT channels, suggesting the constricted filter does not correspond to the functionally observed inactivation in KcsA (28). In this study, we use isothermal titration calorimetry (ITC) to quantify the ion-binding properties of WT and mutant KcsA K⁺ channels with their selectivity filters in different conformations and EPR spectroscopy to determine the conformation of the channels’ intracellular gates. A comparison of these ion-binding properties leads us to conclude that the conductive and inactivated filters are energetically more similar to each other than the constricted and inactivated filters.     

Enhanced L-type calcium currents in cardiomyocytes from transgenic rats overexpressing SERCA2a

BACKGROUND:

Previous research reported that transgenic rats overexpressing the sarco(endo)plasmic reticulum Ca²⁺-ATPase SERCA2a exhibit improved contractile function of the myocardium. Furthermore, impaired Ca²⁺ uptake and reduced relaxation rates in rats with diabetic cardiomyopathy were partially rescued by transgenic expression of SERCA2a in the heart.

OBJECTIVE:

To explore whether enhanced Ca²⁺ cycling in the cardiomyocytes of SERCA2a transgenic rats is associated with changes in L-type Ca²⁺ (I_Ca-L) currents.

METHODS:

The patch-clamp technique was used to measure whole-cell currents in cardiomyocytes from transgenic rats overexpressing SERCA2a and from wild-type (nontransgenic) animals.

RESULTS:

The amplitudes of I_Ca-L currents at depolarizing pulses ranging from −45 mV to 0 mV (350 ms duration, 1 Hz) were significantly higher in cardiomyocytes of SERCA2a transgenic rats than in nontransgenic rats (1985±48 pA [n=32] versus 1612±55 pA [n=28], respectively). The inactivation kinetics of I_Ca-L showed subtle differences with increased tau fast and tau slow decay constants in cardiomyocytes of SERCA2a transgenic animals. Beta-adrenergic stimulation with 50 nM isoproterenol reduced tau fast and tau slow decay constants in cardiomyocytes of transgenic rats to values that were not significantly different from those in normal cardiomyocytes. Furthermore, isoproterenol enhanced I_Ca-L currents 3.2-fold and 2.3-fold in cardiomyocytes with and without the SERCA2a transgene, respectively, and this effect was abolished by buffering intracellular Ca²⁺ with BAPTA.

CONCLUSIONS:

These findings indicate that enhanced Ca²⁺ cycling in the hearts of SERCA2a transgenic rats, both under normal conditions and during beta-adrenergic stimulation, involves changes in I_Ca-L currents. Modified I_Ca-L kinetics may contribute, to some extent, to the improved contractile function of the myocardium of transgenic rats.     

Glyburide prevents isoflurane’s reducing effects on hydroxyl radical formation in the postischemic reperfused rat heart

BACKGROUND:

The role of K_ATP channels in isoflurane’s reducing effects on oxygen free radical formation are not well known.

OBJECTIVES:

To investigate whether glyburide, an ATP-regulated potassium (K_ATP) channel blocker, abolishes isoflurane-induced cardioprotective effects and whether it affects hydroxyl radical formation in the postischemic reperfused heart.

ANIMALS AND METHODS:

Thirty-nine male Wistar rats were divided into four groups: group C (control, n=10), group I (isoflurane, n=9), group G (glyburide, n=10) and group GI (glyburide and isoflurane, n=10). The hearts were perfused as a Neely’s working heart model. Afterwards, global heart ischemia was induced for 15 min followed by reperfusion for 20 min. The formation of hydroxyl radicals in the coronary effluent and heart was measured with high performance liquid chromatography.

RESULTS:

Isoflurane alone and glyburide alone produced significant decreases in the duration of ventricular fibrillation during reperfusion (group C 452±345, group I 247±60, group G 261±135 s; P<0.05). In the presence of glyburide, isoflurane did not further decrease the duration of arrhythmia (group GI 230±48 s). Isoflurane reduced hydroxyl radical formation significantly in the coronary effluent during ischemia and reperfusion, but this was prevented by glyburide.

CONCLUSION:

The results suggest that isoflurane reduces hydroxyl radical formation, at least in part, through activation of K_ATP channels.     

Development of a Wilms�� tumor antigen-specific T-cell receptor for clinical trials: engineered patient��s T cells can eliminate autologous leukemia blasts in NOD/SCID mice

Background

The Wilms’ tumor antigen (WT1) is an attractive target for immunotherapy of leukemia. In the past, we isolated and characterized the specificity and function of a WT1-specific T-cell receptor. The goal of this translational study was to develop a safe and efficient WT1-T-cell receptor retroviral vector for an adoptive immunotherapy trial with engineered T cells.

Design and Methods

We generated a panel of retroviral constructs containing unmodified or codon-optimized WT1-T-cell receptor α and β genes, linked via internal ribosome entry sites or 2A sequences, with or without an additional inter-chain disulfide bond in the T-cell receptor constant domains. These constructs were functionally analyzed in vitro, and the best one was tested in an autologous primary leukemia model in vivo.

Results

We identified a WT1-T-cell receptor construct that showed optimal tetramer staining, antigen-specific cytokine production and killing activity when introduced into primary human T cells. Fresh CD34⁺ cells purified from a patient with leukemia were engrafted into NOD/SCID mice, followed by adoptive immunotherapy with patient’s autologous T cells transduced with the WT1-T-cell receptor. This therapeutic treatment evidently decreased leukemia engraftment in mice and resulted in a substantial improvement of leukemia-free survival.

Conclusions

This is the first report that patient’s T cells, engineered to express the WT1-T-cell receptor, can eliminate autologous leukemia progenitor cells in an in vivo model. This study provides a firm basis for the planned WT1-T-cell receptor gene therapy trial in leukemia patients.     

Low awareness but positive attitudes toward fecal transplantation in Ontario physicians

BACKGROUND:

Despite mounting evidence supporting fecal transplantation (FT) as a treatment for recurrent Clostridium difficile infection (CDI), adoption into clinical practice has been slow.

OBJECTIVE:

To determine the health literacy and attitudes of academic physicians in Toronto and infectious disease physicians in Ontario toward FT as a treatment for recurrent CDI, and to determine whether these are significant barriers to adoption.

METHODS:

Surveys were distributed to 253 general internists, infectious diseases specialists, gastroenterologists and family physicians.

RESULTS:

The response rate was 15%. More than 60% of physicians described themselves as being ‘not at all’ or ‘somewhat’ familiar with FT. Of the 76% of physicians who had never referred a patient for FT, the most common reason (50%) was lack of awareness of where to access the treatment. The ‘ick factor’ accounted for only 13% of reasons for not referring. No respondent believed that the procedure was too risky to consider.

CONCLUSION:

Despite general poor health literacy on FT, most physicians sampled share similar positive attitudes toward the treatment.     

Study of damage to red blood cells exposed to different doses of γ-ray irradiation

Background.

The aims of this research were to study alterations in the ultrastructure of red blood cells, the changes in concentrations of plasma electrolytes and the killing effect of lymphocytes in samples of blood exposed to different doses of γ-ray irradiation.

Materials and methods.

Blood samples were treated with different doses of γ-ray irradiation and then preserved for different periods. Specimens were prepared for standard electron microscopy and transmission electron microscopy. At the same time, changes in the concentrations of Na⁺, K⁺ and Cl⁻ and pH values in the plasma as well as Fas and FasL expression of lymphocytes before and after irradiation were determined.

Results.

The proportions of reversibly and irreversibly transformed cells, for example, echinocytes, sphero-echinocytes, and degenerated forms, increased with increasing doses of irradiation and storage period, while the number of discocyte shaped red blood cells decreased. The change in K⁺ concentration was greater than that of Na+ or Cl⁻ after irradiation and was dosage-dependent. Plasma pH was influenced by different doses of radiation and storage time. After exposure to ¹³⁷Cs γ-irradiation, the expression of both Fas and FasL in lymphocytes differed significantly from that in the control group: the expression was positively correlated with irradiation dose (r=0.95, 0.96), but no significant difference in the Fas/FasL ratio was observed (P>0.05).

Discussion.

We conclude that the ultrastructure of red blood cells is not changed obviously by irradiation with some doses of γ-rays and various periods of storage. However, irradiation does have some dose-dependent and time-dependent adverse effects on the erythrocytes.     

Barriers to the recognition and reporting of occupational asthma by Canadian pulmonologists

BACKGROUND:

Occupational asthma is a common, but probably under-recognized problem.

OBJECTIVE:

To identify the factors that suggest work-related asthma when a pulmonologist encounters an adult patient with new-onset asthma, and to identify the barriers to recognizing and reporting such cases.

METHODS:

A postal questionnaire was sent to all pulmonologists in Canada. The questionnaire asked participants to respond to several questions about recognizing, diagnosing and reporting occupational asthma. Answers were scored using visual analogue scales.

RESULTS:

A total of 201 eligible responses were received from 458 pulmonologists. Pulmonologists identified that the most important factor in initially considering the role of work in occupational asthma was having seen others affected at the same workplace, or exposed to the same agent. Important perceived barriers to considering a diagnosis of occupational asthma were physicians’ low awareness, lack of knowledge and time. The most important barriers to reporting cases were the pulmonologists’ perceived patient concerns regarding job security and income. Quebec pulmonologists generally perceived barriers to recognizing and reporting occupational asthma to be less important, and believed that the use of specific inhalation challenge was more important in considering a diagnosis.

CONCLUSIONS:

Pulmonologists most readily recognized occupational asthma caused by a substance or process that they previously encountered as a possible cause of asthma. Time constraints and knowledge may hamper their ability to recognize occupational asthma. Concerns regarding the effect of the diagnosis on the patient’s job and income may discourage reporting.     

Differences in Patient–Provider Communication for Hispanic Compared to Non-Hispanic White Patients in HIV Care

Background

Hispanic Americans with HIV/AIDS experience lower quality care and worse outcomes than non-Hispanic whites. While deficits in patient–provider communication may contribute to these disparities, no studies to date have used audio recordings to examine the communication patterns of Hispanic vs. non-Hispanic white patients with their health care providers.

Objective

To explore differences in patient–provider communication for English-speaking, HIV-infected Hispanic and non-Hispanic white patients.

Design

Cross-sectional analysis.

Setting

Two HIV care sites in the United States (New York and Portland) participating in the Enhancing Communication and HIV Outcomes (ECHO) study.

Subjects

Nineteen HIV providers and 113 of their patients.

Measurements

Patient interviews, provider questionnaires, and audio-recorded, routine, patient–provider encounters coded with the Roter Interaction Analysis System (RIAS).

Results

Providers were mostly non-Hispanic white (68%) and female (63%). Patients were Hispanic (51%), and non-Hispanic white (49%); 20% were female. Visits with Hispanic patients were less patient-centered (0.75 vs. 0.90, p = 0.009), with less psychosocial talk (80 vs. 118 statements, p < 0.001). This pattern was consistent among Hispanics who spoke English very well and those with less English proficiency. There was no association between patient race/ethnicity and visit length, patients’ or providers’ emotional tone, or the total number of patient or provider statements categorized as socioemotional, question-asking, information-giving, or patient activating. Hispanic patients gave higher ratings than whites (AOR 3.05 Hispanic vs. white highest rating of providers’ interpersonal style, 95% CI 1.20-7.74).

Conclusion

In this exploratory study, we found less psychosocial talk in patient–provider encounters with Hispanic compared to white patients. The fact that Hispanic patients rated their visits more positively than whites raises the possibility that these differences in patient–provider interactions may reflect differences in patient preferences and communication style rather than “deficits” in communication. If these findings are replicated in future studies, efforts should be undertaken to understand the reasons underlying them and their impact on the quality and equity of care.KEY WORDS: HIV/AIDS, patient–physician relations, patient–physician communication, health disparities