A1 and A2A adenosine receptors play a protective role to reduce prevalence of autoimmunity following tissue damage.

Adenosine is a potent modulator that has a tremendous effect on the immune system. Adenosine affects T cell activity, and is necessary in maintaining the T helper/regulatory T cell (Treg ) ratio. Adenosine signalling is also involved in activating neutrophils and the formation of neutrophil extracellular traps (NETs), which has been linked to autoimmune disorders. Therefore, adenosine, through its receptors, is extremely important in maintaining homeostasis and involved in the development of autoimmune diseases. In this study, we aim to evaluate the role of adenosine A1 and A2A receptors in involvement of autoimmune diseases. We studied adenosine regulation by NETosis in vitro, and used two murine models of autoimmune diseases: type I diabetes mellitus (T1DM) induced by low-dose streptozotocin and pristane-induced systemic lupus erythematosus (SLE). We have found that A1 R enhances and A2A R suppresses NETosis. In addition, in both models, A1 R-knock-out (KO) mice were predisposed to the development of autoimmunity. In the SLE model in wild-type (WT) mice we observed a decline of A1 R mRNA levels 6 h after pristane injection that was parallel to lymphocyte reduction. Following pristane, 43% of A1 R-KO mice suffered from lupus-like disease while WT mice remained without any sign of disease at 36 weeks. In WT mice, at 10 days A2A R mRNA levels were significantly higher compared to A1R-KO mice. Similar to SLE, in the T1DM model the presence of A1 R and A2A R was protective. Our data suggest that, in autoimmune diseases, the acute elimination of lymphocytes and reduction of DNA release due to NETosis depends upon A1 R desensitization and long-term suppression of A2A R.

The impact of gender on early scientific publication and long-term career advancement in Israeli medical school graduates.

BACKGROUND: Many medical schools and residency programs incorporate research projects into their curriculum, however most remain unpublished. Little is known on the long-term effect of early-career publication, especially in female graduates. METHODS: We collected data on physicians 15-20 years after graduation (representing a mid-career point), and analysed data on early publication, publication volume and impact according to graduates' gender and professional characteristics. Physicians were divided into those who never published, early-publishers (EP) who published within 2 years of graduation and late-publishers (LP). We analysed and compared the demographics, publication volume, publication quality as well as current mid-career position. RESULTS: Of 532 physicians, 185 were EP (34.8%), 220 were LP (41.3%), 127 (23.9%) never published, 491 (92.2%) became specialists and 122 (22.3%) achieved managerial position. Of the 405 who published, the average number of publications was 20.3 ± 33.0, and median (IQR) 9(19). H-index was significantly higher in EP, males, surgical specialists, and those holding a managerial position. Male gender was associated with higher publication rate (OR = 1.742; 95% CI 1.193-2.544; P = 0.004). Using quantile regression, female gender was negatively associated with the number of publications in Q50-Q95. Surgical specialty and managerial position were positively associated with publications in Q25 to Q75 and early publication in Q25 and Q75. CONCLUSIONS: We found a strong association between EP and the number, impact, and quality of publications throughout their academic career. This study illuminates the need for further investigations into the causes of gender discrepancies. We should invest in support programs encouraging early high quality research projects for young physicians and female graduates.

Chronic drug treatment among hemodialysis patients: a qualitative study of patients, nursing and medical staff attitudes and approaches.

BACKGROUND: Dialysis patients have a high pill burden, increasing their care complexity. A previous study in our institution's dialysis unit found notable discrepancies between medication prescriptions, purchases and patient reports of medication use: overall adherence to medication was 57%, on average; staff reported patients took 3.1 more medication types than actual purchases; concordance of patient purchases and nurse reports was found in 5.7 out of 23.6 months of patient follow-up. We sought to investigate patients and staff concepts and attitudes regarding medication care and to understand better the previously identified inconsistencies. METHODS: We performed a qualitative research based on the grounded theory approach, using semi-structured, in-depth, interviews with patients and staff from the same dialysis unit studied previously, at the Hadassah Medical Center, Jerusalem, Israel. RESULTS: Though all respondents described a seemingly synchronized system of care, repeated questioning revealed that staff distrust patient medication reports. Patients, on their part, felt that their monitoring and supervision were bothersome and belittling. Along with patients, nurses and physicians, we identified a "fourth" factor, which influences medication care - the laboratory tests. They serve both as biological parameters of health, but also as parameters of patient adherence to the prescribed medication regimens. CONCLUSIONS: Participant responses did not clearly resonate with previous findings from the quantitative study. The central role of laboratory tests should be carefully considered by the staff when interacting with patients. An interaction process, less adversarial, centering on the patient attitudes to medication care, might establish better communication, better cooperation and better patient outcomes.

Vitamin D levels, vitamin D supplementation, and prognosis in patients with chronic kidney disease
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BACKGROUND: Vitamin D (Vit D) deficiency plays a central role in the pathogenesis of chronic kidney disease (CKD) complications, both skeletal and nonskeletal. The purpose of this study was to examine whether 25(OH)D levels and supplementation with oral cholecalciferol (Vitamin D3 (Vit D3)) are associated with morbidity and mortality among patients with significant CKD. METHODS: CKD patients attending the nephrology clinic at Shaare Zedek Medical Center between July 1, 2008 and January 31, 2012, tested at least twice for 25(OH)D levels, were enrolled. Primary endpoints included death, end-stage renal disease (ESRD) requiring start of dialysis, a rise of at least 50% in serum creatinine, or composite endpoints of the above. RESULTS: A total of 516 patients were studied, of whom 178, 257, and 81 patients had baseline vitamin D levels < 5 ng/mL, 15 - 30 ng/mL, and > 30 ng/mL, respectively. We found an association between baseline 25(OH)D level below 15 ng/mL and renal outcomes (start of dialysis or a rise of at least 50% in serum creatinine) in both crude and multivariate analyses (hazard ratio (HR) 3.17, 95% CI 1.12 - 8.94). Vit D3 supplementation demonstrated beneficial effects on combined renal outcomes and death in univariate analyses (p = 0.02). Moreover, an increment of 10 ng/mL in 25(OH)D levels was associated with a 25% reduction in mortality (HR 0.755 (95% CI 0.54 - 1.00), in crude but not adjusted analyses. CONCLUSIONS: Significant Vit D deficiency in CKD can serve as a biological marker indicating patients in whom adverse renal outcomes can be anticipated. Moreover, Vit D3 supplementation and rise of serum 25(OH)D levels may have beneficial influence on hard renal outcomes.
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Summary adherence estimates do not portray the true incongruity between drug intake, nurse documentation and physicians' orders.

BACKGROUND: Hemodialysis patients (HD) need to adhere to a complex medication regimen. Because their daily pill burden is one of the highest reported, poor compliance is a major cause of therapeutic failure. The primary aim of this study was to define patterns of medication prescription, intake and documentation among HD patients. METHODS: HD patients treated between 2007 and 2009 and assigned to the largest health service provider in Israel were randomly selected. Drug practices were abstracted from their records and compared to electronic pharmacy data. The discrepancy between drug intake reports and the actual purchase was measured to estimate adherence. Drug purchase, intake report and physician order were plotted in complementing diagrams to appreciate consistency and discrepancy patterns. RESULTS: The study included full analysis of 75 patients. The mean overall drug adherence was 56.7% (95% CI 53.6-59.9%), varying among drug families and over time. Often, there was a systematic disengagement between the nurses' documentation and the actual patient purchase. Specifically, we observed either different quantities of medication use, improper documentation of a non-purchased drug, drug purchase without nurse documentation and futile physician attempts to modify prescriptions of unpurchased medication. We found a high rate of physician order turbulence for active vitamin D and calcium. CONCLUSIONS: Drug prescription, documentation and adherence are incongruent and their mismatches are diverse. Summary estimates do not divulge the extent of these disparities. These system-wide communication failures compromise patient care. Strategies to promote system reconciliation and reasonable medication prescription are in need.

The incidence and prognosis of renal dysfunction following cardioversion of atrial fibrillation.

OBJECTIVE: Limited data are available regarding the incidence and clinical impact of renal dysfunction following cardioversion of atrial fibrillation. The objective of this study was to assess the incidence and implications of renal dysfunction following cardioversion of atrial fibrillation. METHODS: We conducted a nested case-control study to determine the incidence, timing, risk factors and outcome of atrial fibrillation cardioversion associated with renal dysfunction (AFCARD) in a tertiary medical center. Consecutive patients undergoing direct current cardioversion (DCCV) for atrial fibrillation in our institution during 2008-2009 with measurements of creatinine before and following cardioversion were included. AFCARD was defined as a rise in serum creatinine greater than 25% from baseline within a week following DCCV. RESULTS: One hundred and twelve patients were included in the study, of whom 19 (17%) developed AFCARD. One patient required hemodialysis. Patients with AFCARD had a higher incidence of advanced heart failure, diabetes mellitus and were more frequently treated with digoxin and enoxaparin. Patients with AFCARD had a significantly decreased survival rate at 1 year (63 vs. 92%; p < 0.001). CONCLUSIONS: AFCARD is relatively common and is associated with increased mortality. These findings suggest a role for close surveillance of renal function following DCCV.

The MONitoring Dialysis Outcomes (MONDO) initiative.

BACKGROUND: Systematic collection and analysis of global hemodialysis patient data may help to improve patient outcomes. METHODS: The MONitoring Dialysis Outcomes (MONDO) initiative comprises data from eight dialysis providers worldwide. Data are combined into one repository. Extensive procedures are employed to merge data across countries and providers. RESULTS: The MONDO database comprises longitudinal data of currently 128,000 hemodialysis patients from 26 countries on five continents. Here we report data from 62,345 incident hemodialysis patients. We found lower catheter rates in South-East Asia and Australia, lower hemoglobin levels in South-East Asia, and a higher prevalence of diabetes in North America. Longitudinal analyses suggest that there is a decline in interdialytic weight gain and serum phosphorus and an increasing neutrophil-to-lymphocyte ratio before death in all regions studied. CONCLUSIONS: While organizationally lean and low-cost, MONDO is the largest global dialysis database initiative to date, with a particular focus on high longitudinal data density and geographical diversity.

Monitoring dialysis outcomes across the world--the MONDO Global Database Consortium.

BACKGROUND/AIMS: Dialysis providers frequently collect detailed longitudinal and standardized patient data, providing valuable registries of routine care. However, even large organizations are restricted to certain regions, limiting their ability to separate effects of local practice from the pathophysiology shared by most dialysis patients. To overcome this limitation, the MONDO (MONitoring Dialysis Outcomes) research consortium has created a platform for the joint analysis of data from almost 200,000 dialysis patients worldwide. METHODS: We examined design and operation of MONDO as well as its methodology with respect to patient inclusion, descriptive data and other study parameters. RESULTS: MONDO partners contribute primary databases of anonymized patient data and collaboratively analyze populations across national and regional boundaries. To that end, datasets from different electronic health record systems are converted into a uniform structure. Patients are enrolled without systematic exclusions into open cohorts representing the diversity of patients. A large number of patient level treatment and outcome data is recorded frequently and can be analyzed with little delay. Detailed variable definitions are used to determine if a parameter can be studied in a subset or all databases. CONCLUSION: MONDO has created a large repository of validated dialysis data, expanding the opportunities for outcome studies in dialysis patients. The density of longitudinal information facilitates in particular trend analysis. Limitations include the paucity of uniform definitions and standards regarding descriptive information (e.g. comorbidities), which limits the identification of patient subsets. Through its global outreach, depth, breadth and size, MONDO advances the observational study of dialysis patients and care.

Community-based serum chloride abnormalities predict mortality risk.

INTRODUCTION: This population-based study aimed to investigate the prognostic value of ambulatory serum chloride abnormalities, often ignored by physicians. METHODS: The study population included all non-hospitalized adult patients, insured by "Clalit" Health Services in Israel's southern district, who underwent at least 3 serum chloride tests in community-based clinics during 2005-2016. For each patient, each period with low (≤97 mmol/l), high (≥107 mmol/l) or normal chloride levels were recorded. A Cox proportional hazards model was used to estimate the mortality risk of hypochloremia and hyperchloremia periods. RESULTS: 664,253 serum chloride tests from 105,655 subjects were analyzed. During a median follow up of 10.8 years, 11,694 patients died. Hypochloremia (≤ 97 mmol/l) was independently associated with elevated all-cause mortality risk after adjusting for age, co-morbidities, hyponatremia and eGFR (HR 2.41, 95%CI 2.16-2.69, p<0.001). Crude hyperchloremia (≥107 mmol/L) was not associated with all-cause mortality (HR 1.03, 95%CI 0.98-1.09 p = 0.231); as opposed to hyperchloremia ≥108 mmol/l (HR 1.14, 95%CI 1.06-1.21 p<0.001). Secondary analysis revealed a dose-dependent elevated mortality risk for chloride levels of 105 mmol/l and below, well within the "normal" range. CONCLUSION: In the outpatient setting, hypochloremia is independently associated with an increased mortality risk. This risk is dose-dependent where the lower the chloride level, the higher is the risk.

Can Dialysis Patients Identify and Diagnose Pulmonary Congestion Using Self-Lung Ultrasound?

BACKGROUND: With the recent developments in automated tools, smaller and cheaper machines for lung ultrasound (LUS) are leading us toward the potential to conduct POCUS tele-guidance for the early detection of pulmonary congestion. This study aims to evaluate the feasibility and accuracy of a self-lung ultrasound study conducted by hemodialysis (HD) patients to detect pulmonary congestion, with and without artificial intelligence (AI)-based automatic tools. METHODS: This prospective pilot study was conducted between November 2020 and September 2021. Nineteen chronic HD patients were enrolled in the Soroka University Medical Center (SUMC) Dialysis Clinic. First, we examined the patient's ability to obtain a self-lung US. Then, we used interrater reliability (IRR) to compare the self-detection results reported by the patients to the observation of POCUS experts and an ultrasound (US) machine with an AI-based automatic B-line counting tool. All the videos were reviewed by a specialist blinded to the performer. We examined their agreement degree using the weighted Cohen's kappa (Kw) index. RESULTS: A total of 19 patients were included in our analysis. We found moderate to substantial agreement between the POCUS expert review and the automatic counting both when the patient performed the LUS (Kw = 0.49 [95% CI: 0.05-0.93]) and when the researcher performed it (Kw = 0.67 [95% CI: 0.67-0.67]). Patients were able to place the probe in the correct position and present a lung image well even weeks from the teaching session, but did not show good abilities in correctly saving or counting B-lines compared to an expert or an automatic counting tool. CONCLUSIONS: Our results suggest that LUS self-monitoring for pulmonary congestion can be a reliable option if the patient's count is combined with an AI application for the B-line count. This study provides insight into the possibility of utilizing home US devices to detect pulmonary congestion, enabling patients to have a more active role in their health care.

Admission of kidney patients to a closed staff nephrology department results in a better short-term survival.

BACKGROUND: The outcome of patients with chronic kidney disease (CKD) and acute kidney injury (AKI) is often dismal and measures to ameliorate their course are scarce. When admitted to the hospital, kidney patients are often hospitalized in general Medicine wards rather than in a specialized Nephrology department. In the current study, we compared the outcome of two cohorts of kidney patients (CKD and AKI) admitted either to general open-staff (with rotating physicians) Medicine wards or to a closed-staff (non-rotating Nephrologists) Nephrology ward. METHODS: In this population-based retrospective cohort study, we enrolled 352 CKD patients and 382 AKI patients admitted to either Nephrology or General Medicine wards. Short-term (< = 90 days) and long-term (>90 days) outcomes were recorded for survival, renal outcomes, cardiovascular outcomes, and dialysis complications. Multivariate analysis was performed using logistic regression and negative binomial regression adjusting to potential sociodemographic confounders as well as to a propensity score based on the association of all medical background variables to the admitted ward, to mitigate the potential admittance bias to each ward. RESULTS: One hundred and seventy-one CKD patients (48.6%) were admitted to the Nephrology ward and 181 (51.4%) were admitted to general Medicine wards. For AKI, 180 (47.1%) and 202 (52.9%) were admitted to Nephrology and general Medicine wards, respectively. Baseline age, comorbidities and the degree of renal dysfunction differed between the groups. Using propensity score analysis, a significantly reduced mortality rate was observed for kidney patients admitted to the Nephrology ward vs. general Medicine in short term mortality (but not long-term mortality) among both CKD patients admitted (OR = 0.28, CI = 0.14-0.58, p = 0.001), and AKI patients (or = 0.25, CI = 0.12-0.48, p< 0.001). Nephrology ward admission resulted in higher rates of renal replacement therapy (RRT), both during the first hospitalization and thereafter. CONCLUSIONS: Thus, a simple measure of admission to a specialized Nephrology department may improve kidney patient outcome, thereby potentially affecting future health care planning.

Blockade of transforming growth factor-beta1 accelerates lymphatic regeneration during wound repair.

Lymphedema is a complication of cancer treatment occurring in approximately 50% of patients who undergo lymph node resection. Despite its prevalence, the etiology of this disorder remains unknown. In this study, we determined the effect of soft tissue fibrosis on lymphatic function and the role of transforming growth factor (TGF)-ß1 in the regulation of this response. We determined TGF-ß expression patterns in matched biopsy specimens collected from lymphedematous and normal limbs of patients with secondary lymphedema. To determine the role of TGF-ß in regulating tissue fibrosis, we used a mouse model of lymphedema and inhibited TGF-ß function either systemically with a monoclonal antibody or locally by using a soluble, defective TGF-ß receptor. Lymphedematous tissue demonstrated a nearly threefold increase in the number of cells that stained for TGF-ß1. TGF-ß inhibition markedly decreased tissue fibrosis, increased lymphangiogenesis, and improved lymphatic function compared with controls. In addition, inhibition of TGF-ß not only decreased TGF-ß expression in lymphedematous tissues, but also diminished inflammation, migration of T-helper type 2 (Th2) cells, and expression of profibrotic Th2 cytokines. Similarly, systemic depletion of T-cells markedly decreased TGF-ß expression in tail tissues. Inhibition of TGF-ß function promoted lymphatic regeneration, decreased tissue fibrosis, decreased chronic inflammation and Th2 cell migration, and improved lymphatic function. The use of these strategies may represent a novel means of preventing lymphedema after lymph node resection.

Adipose-derived stem cells promote lymphangiogenesis in response to VEGF-C stimulation or TGF-ß1 inhibition.

AIMS: Recent studies have demonstrated that augmentation of lymphangiogenesis and tissue engineering hold promise as a treatment for lymphedema. The purpose of this study was to determine whether adipose-derived stem cells (ASCs) can be used in lymphatic tissue-engineering by altering the balance between pro- and anti-lymphangiogenic cytokines. MATERIALS & METHODS: ASCs were harvested and cultured in media with or without recombinant VEGF-C for 48 h. ASCs were then implanted in mice using Matrigel plugs. Additional groups of animals were implanted with ASCs transfected with a dominant-negative TGF-ß1 receptor-II adenovirus with or without VEGF-C stimulation, since TGF-ß1 has been shown to have potent antilymphangiogenic effects. Lymphangiogenesis, lymphatic differentiation and cellular proliferation were assessed. RESULTS: Stimulation of ASCs with VEGF-C in vitro significantly increased expression of VEGF-A, VEGF-C and Prox-1. ASCs stimulated with VEGF-C prior to implantation induced a significant (threefold increase) lymphangiogenic response as compared with control groups (unstimulated ASCs or empty Matrigel plugs; p < 0.01). This effect was significantly potentiated when TGF-ß1 signaling was inhibited using the dominant-negative TGF-ß1 receptor-II virus (4.5-fold increase; p < 0.01). Stimulation of ASCs with VEGF-C resulted in a marked increase in the number of donor ASCs (twofold; p < 0.01) and increased the number of proliferating cells (sevenfold; p < 0.01) surrounding the Matrigel. ASCs stimulated with VEGF-C expressed podoplanin, a lymphangiogenic cell marker, whereas unstimulated cells did not. CONCLUSION: Short-term stimulation of ASCs with VEGF-C results in increased expression of VEGF-A, VEGF-C and Prox-1 in vitro and is associated with a marked increase lymphangiogenic response after in vivo implantation. This lymphangiogenic response is significantly potentiated by blocking TGF-ß1 function. Furthermore, stimulation of ASCs with VEGF-C markedly increases cellular proliferation and cellular survival after in vivo implantation and stimulated cells express podoplanin, a lymphangiogenic cell marker.

Renal Tubular Acidosis Type I with Prominent Hypokalemia and Nephrolithiasis as a Presentation of Sjögren's/Systemic Lupus Erythematosus Disease.

Female patient, suffering from nephrolithiasis, at the age of 32 was admitted for renal colic caused by a stone obstructing UP junction with left hydronephrosis. Nephrostomy was placed, resulting in brisk diuresis. Severe metabolic acidosis with normal anion gap and urine pH of 6.5 was noted. Potassium level dropped to extremely low level (1.6 mEq/L), causing muscle paralysis and respiratory failure, necessitating mechanical ventilation. The patient was treated by potassium chloride infusion, followed by correction of severe metabolic acidosis by sodium bicarbonate. Diagnosis of distal type renal tubular acidosis type I (dRTA) was made based on normal anion gap metabolic acidosis, alkaline urine, hypokalemia, and nephrolithiasis. Five years later, the patient presented with severe hypoxia, lung opacities, and bronchiolitis obliterans organizing pneumonia which was confirmed by bronchoscopy with lung tissue biopsy. Concurrently, the patient presented with dry mouth, pruritus, skin rash with hypocomplementemia, elevated anti-DNA, anti-Ro, and anti-SmAb. Diagnosis of overlap Sjögren's/systemic lupus erythematosus disease was done and treatment by hydroxychloroquine, prednisone, and azathioprine was started. Possible presence of Sjögren's syndrome should be considered in adult patients with unexplained dRTA.

Cannabis is associated with blood pressure reduction in older adults - A 24-hours ambulatory blood pressure monitoring study.

BACKGROUND: Medical cannabis use is increasing rapidly in the past several years, with older adults being the fastest growing group. Nevertheless, the evidence for cardiovascular safety of cannabis use is scarce. The aim of this study was to assess the effect of cannabis on blood pressure, heart rate, and metabolic parameters in older adults with hypertension. METHODS: We conducted a prospective study of patients aged 60 years or more with hypertension and a new prescription of cannabis. We have performed the following assessments: 24-hours ambulatory blood pressure monitoring, ECG, blood tests, and anthropometric measurements prior to the initiation of cannabis therapy and 3 months afterward. The primary outcome was change in mean 24-h blood pressure at 3 months. RESULTS: Twenty-six patients with a mean age of 70.42 ± 5.37 years, 53.8% females completed the study. At 3 months follow-up, the mean 24-hours systolic and diastolic blood pressures were reduced by 5.0 mmHg and 4.5 mmHg, respectively (p<0.001 for both). The nadir for the blood pressure and heart rate was achieved at 3 hours post-administration. The proportion of normal dippers changed from 27.3% before treatment to 45.5% afterward. No significant changes were seen in the different metabolic parameters assessed by blood tests, anthropometric measurements, or ECG exam. CONCLUSION: amongst older adults with hypertension, cannabis treatment for 3 months was associated with a reduction in 24-hours systolic and diastolic blood pressure values with a nadir at 3 hours after cannabis administration.

An in-vitro tumour microenvironment model using adhesion to type I collagen reveals Akt-dependent radiation resistance in renal cancer cells.

BACKGROUND: Renal cell carcinoma (RCC) is considered resistant to ionizing radiation. Recently, the extracellular matrix (ECM) has been shown to play a role in both drug resistance and radiation resistance (RR). While fibronectin has been extensively investigated in the context of RR, the role of type I collagen [col(I)], a principal constituent of the ECM in tumour metastases, in RR of RCC is unknown. METHODS: RCC cell adhesion to matrix was studied via pre-coating a variety of ECM glycoproteins onto plates. Cancer cell apoptosis and cell cycle were evaluated with flow cytometry using annexin V and propidium iodide stains, respectively. Activation of cellular survival signalling was analysed with western blots, and specific molecular inhibitors were correspondingly employed to block signalling. Hypoxia (<1%) was induced via N(2)/CO(2) gas flow in a specialized chamber. RESULTS: While adherence to col(I) enhanced RCC cell proliferation in general, col(I) and fibronectin, but not fibrinogen, could confer specific anti-apoptotic RR to RCC cells. The radioprotective effect of col(I) was maintained during both hypoxia/reoxygenation and normoxia conditions. In contrast to intact col(I), micronized col(I), lacking the natural fibrillar structure, was not radioprotective. The effect of col(I) in RCC cells is mediated via attenuation of apoptosis rather than cell cycle redistribution, involving the PI3 kinase/Akt pathway but not the MAP kinase pathway. CONCLUSIONS: Adherence to col(I) appears to be a relevant environmental cue enhancing RR in RCC cells, Akt dependently. Our results support inhibition of the PI3-kinase/Akt pathway as a radiosensitizing approach.

Herpes simplex virus type 1 preferentially targets human colon carcinoma: role of extracellular matrix.

Viral therapy of cancer (viral oncolysis) is dependent on selective destruction of the tumor tissue compared with healthy tissues. Several factors, including receptor expression, extracellular components, and intracellular mechanisms, may influence viral oncolysis. In the present work, we studied the potential oncolytic activity of herpes simplex virus type 1 (HSV-1), using an organ culture system derived from colon carcinoma and healthy colon tissues of mouse and human origin. HSV-1 infected normal colons ex vivo at a very low efficiency, in contrast to high-efficiency infection of colon carcinoma tissue. In contrast, adenoviral and lentiviral vectors infected both tissues equally well. To investigate the mechanisms underlying the preferential affinity of HSV-1 for the carcinoma tissue, intracellular and extracellular factors were investigated. Two extracellular components, collagen and mucin molecules, were found to restrict HSV-1 infectivity in the healthy colon. The mucin layer of the healthy colon binds to HSV-1 and thereby blocks viral interaction with the epithelial cells of the tissue. In contrast, colon carcinomas express small amounts of collagen and mucin molecules and are thus permissive to HSV-1 infection. In agreement with the ex vivo system, HSV-1 injected into a mouse colon carcinoma in vivo significantly reduced the volume of the tumor. In conclusion, we describe a novel mechanism of viral selectivity for malignant tissues that is based on variance of the extracellular matrix between tumor and healthy tissues. These insights may facilitate new approaches to the application of HSV-1 as an oncolytic virus.

A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus.

BACKGROUND: Construction of conditionally-replicative Adenovirus (CRAd) is complex and time-consuming. While homologous recombination (HR) using a two-plasmid system in bacteria is commonly used to generate CRAds, alternative methods may be required when HR fails. Previously, in vitro ligation has been suggested to facilitate construction of E1/E3-deleted, replication-incompetent Ad vectors. However, in vitro ligation has only rarely been used to generate CRAds and may be a complex procedure for molecular biologists who are not experts in the field. METHODS AND RESULTS: A modified in vitro ligation approach was developed to construct a double-targeted, E1B55k-deleted CRAd. The method allowed the incorporation of a tumor-specific promoter, e.g. the heat-shock protein 70 (hsp70) promoter, upstream of E1a, deletion of the E1B55k gene, and HR-free cloning of the recombined E1Delta55k gene into the Ad genome. The genetic structure of the CRAd was confirmed using restriction analysis and PCR. The replication rate of the hsp70E1Delta55k CRAd was 1.5-2% of Ad without E1Delta55k deletion. CONCLUSION: A 3-step cloning approach can generate a double-targeted, E1B55k-deleted CRAd using a straight-forward, modified in vitro ligation procedure.

Analysis of adenoviral attachment to human platelets.

BACKGROUND: Systemic adenoviral (Ad) vector administration is associated with thrombocytopenia. Recently, Ad interaction with mouse platelets emerged as a key player determining liver uptake and platelet clearance. However, whether Ad can activate platelets is controversial. Thus, in vitro analysis of Ad attachment to platelets is of interest. METHODS: We developed a direct flow cytometry assay to specifically detect Ad particles adherent to human platelets. The method was pre-validated in nucleated cells. Blocking assays were employed to specifically inhibit Ad attachment to platelets. Platelet activation was analyzed using annexin v flow cytometry. RESULTS: We found in vitro that Ad binding to human platelets is synergistically enhanced by the combination of platelet activation by thrombin and MnCl2 supplementation. Of note, Ad binding could activate human platelets. Platelets bound Ad displaying an RGD ligand in the fiber knob more efficiently than unmodified Ad. In contrast to a previous report, CAR expression was not detected on human platelets. Integrins appear to mediate Ad binding to platelets, at least partially. Finally, alphaIIbbeta3-deficient platelets from a patient with Glanzmann thrombasthenia could bind Ad 5-fold more efficiently than normal platelets. CONCLUSION: The flow cytometry methodology developed herein allows the quantitative measurement of Ad attachment to platelets and may provide a useful in vitro approach to investigate Ad interaction with platelets.

Risk Factors Before Dialysis Predominate as Mortality Predictors in Diabetic Maintenance Dialysis patients.

Diabetic patients undergoing maintenance dialysis (MD) have a particularly high mortality rate. Many of the risk factors for mortality have been identified in diabetics who die before reaching end stage renal disease (ESRD), i.e. before dialysis (BD). In addition, many risk factors for mortality have been identified in diabetics after dialysis onset (AD). However, whether in the BD period there are long-term risk factors for AD mortality in diabetics is unknown. We therefore investigated a new concept, i.e. that clinical and biochemical risk factors during the BD stage affect long-term AD mortality. We performed a population based retrospective cohort study, in diabetic CKD patients in a single center in south Israel who initiated MD between the years 2003 and 2015. Clinical and biochemical data 12 months BD and 6 months AD were collected and evaluated for association with mortality AD using Cox's proportional-hazards model. BD parameters that were found to be significant were adjusted for significant parameters AD, thus generating a "combined" regression model in order to isolate the contribution of BD factors on long term mortality. Six hundred and fifty two diabetic MD patients were included in the final analysis. Four independent BD parameters were found in the multivariate model to significantly predict AD mortality: age, BMI (inversely), pulse pressure (U-shaped) and cardiovascular comorbidity. AD independent risk factors for mortality were age, BMI (inversely) and albumin (inversely). Of note, BD factors remained dominantly significant even after additionally adjusting for AD factors. No association was found between either BD HbA1C levels or BD proteinuria and AD mortality. In diabetics who reach ESRD, BD parameters can predict long term AD mortality. Thus, some of the factors affecting the poor survival of diabetic MD patients appear to begin already in the BD period.