WITHDRAWN: Proposal for Initiative of Evidence-based Treatment of COVID-19 Patients with Worsening Hypoxia.

Ahead of Print article withdrawn by publisher.

Noninvasive Imaging of Activated Complement in Ischemia-Reperfusion Injury Post-Cardiac Transplant.

Ischemia-reperfusion injury (IRI) is inevitable in solid organ transplantation, due to the transplanted organ being ischemic for prolonged periods prior to transplantation followed by reperfusion. The complement molecule C3 is present in the circulation and is also synthesized by tissue parenchyma in early response to IRI and the final stable fragment of activated C3, C3d, can be detected on injured tissue for several days post-IRI. Complement activation post-IRI was monitored noninvasively by single photon emission computed tomography (SPECT) and CT using (99m) Tc-recombinant complement receptor 2 ((99m) Tc-rCR2) in murine models of cardiac transplantation following the induction of IRI and compared to (99m) Tc-rCR2 in C3(-/-) mice or with the irrelevant protein (99m) Tc-prostate-specific membrane antigen antibody fragment (PSMA). Significant uptake with (99m) Tc-rCR2 was observed as compared to C3(-/-) or (99m) Tc-PSMA. In addition, the transplanted heart to muscle ratio of (99m) Tc-rCR2 was significantly higher than (99m) Tc-PSMA or C3(-/-) . The results were confirmed by histology and autoradiography. (99m) Tc-rCR2 can be used for noninvasive detection of activated complement and in future may be used to quantify the severity of transplant damage due to complement activation postreperfusion.

Bimodal Imaging of Inflammation with SPECT/CT and MRI Using Iodine-125 Labeled VCAM-1 Targeting Microparticle Conjugates.

Upregulation of cell adhesion molecules on endothelial cells is a hallmark of inflammation and an early feature of several neurological conditions. Here, we describe bimodal in vivo imaging of this inflammatory event in the brain using functionalized micron-sized particles of iron oxide. The particles were conjugated to anti-VCAM-1 antibodies and subsequently labeled with iodine-125. Radiolabeling of the antibody-coated particles was straightforward and proceeded in high radiochemical yields using commercially available iodination tubes. The corresponding contrast agent was evaluated in a rat model of cerebral inflammation based on intracerebral injection of tumor necrosis factor alpha and a rat model of status epilepticus. Biodistribution studies and phosphorimaging of cryosections were used to verify in vivo imaging data obtained with single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). The contrast agent showed rapid and highly localized binding to the vasculature of inflamed brain tissue, and was effectively cleared from the blood pool within 2 min postinjection. Overall, the pattern of hypointensities observed with MRI was in good agreement with the distribution of the contrast agent as determined with SPECT and phosphorimaging; however, conspicuous differences in the signal intensities were observed. The results demonstrate that radiolabeled micron-sized particles of iron oxide enable multimodal in vivo imaging with MRI and nuclear techniques, and highlight the value of validating different imaging methods against one another.

Basic implications on three pathways associated with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts between the host and virus and govern induction, resulting in multiorgan impacts. Its pathophysiology involves the followings: 1) the angiotensin-converting enzyme (ACE2) and Toll-like receptor (TLR) pathways: 2) the neuropilin (NRP) pathway: 3) the spike protein pathway. Therefore, it is necessary to block the pathological course with modulating innate lymphoid cells against diverse corona variants in the future.

Benefits of Using Dapsone in Patients Hospitalized with COVID-19.

Since the start of the SARS-CoV-2 pandemic, refractory and relentless hypoxia as a consequence of exuberant lung inflammation and parenchymal damage remains the main cause of death. We have earlier reported results of the addition of dapsone in this population to the standard of care. We now report a further chart review of discharge outcomes among patients hospitalized for COVID-19. The 2 × 2 table analysis showed a lower risk of death or discharge to LTAC (Long term acute care) (RR = 0.52, 95% CI: 0.32 to 0.84) and a higher chance of discharge home (RR = 2.7, 95% CI: 1.2 to 5.9) among patients receiving dapsone compared to those receiving the usual standard of care. A larger, blinded randomized trial should be carried out urgently to determine if dapsone indeed improves outcomes in COVID-19.

Herpesvirus saimiri-based endothelin-converting enzyme-1 shRNA expression decreases prostate cancer cell invasion and migration.

The zinc metalloprotease, endothelin-converting enzyme-1 (ECE-1), which converts the mitogenic peptide endothelin-1 (ET-1) from its biologically inactive precursor big-ET-1, is commonly upregulated in prostate cancer (PC) cells. Consequently, we have sought to suppress ECE-1 expression by using RNAi as a potentially novel therapeutic approach. Therefore, a synthetic 64-nt short-hairpin RNA (shRNA), designed to target the ECE-1 gene, was expressed in an Herpesvirus saimiri (HVS)-based delivery vector. ECE-1 expression in cells transduced with the vector was examined by real-time PCR and Western blotting. The effects of ECE-1 knockdown on PC cell migration and invasion were studied using a scratch assay and Matrigel invasion. These studies, in vitro and ex vivo, demonstrated that the HVS-shRNA viruses could infect and silence ECE-1 expression effectively in human PC cells. Furthermore, it was observed that ECE-1 knockdown in either stromal cells or epithelial cells could significantly reduce invasion of PC-3 cells in coculture by 33 and 31%, respectively. In addition, suppressed migration was also observed in HVS-ECE-1 shRNA-infected PC-3 cells compared to uninfected and HVS-GFP-infected control cell cultures. These findings highlight the potential tumor-suppressing effect of ECE-1 knockdown in cancer cells and novel strategies for future therapeutic developments in advanced PC.

SPECT/CT lymphoscintigraphy of heterotopic cardiac grafts reveals novel sites of lymphatic drainage and T cell priming.

The normal function of lymphatic vessels is to facilitate the trafficking of antigen presenting cells to draining lymph nodes where they evoke an immune response. Donor lymphatic vessels are not connected to that of recipients' during organ transplantation. The pathophysiology of this disruption has received little attention. Murine heterotopic cardiac transplantation has been used extensively in transplantation research. Following vascularized organ transplantation, the main site of allosensitization is thought to be in the spleen of the recipient as a result of migration of donor passenger leukocytes via blood. Here, using Single Photon Emission Computed Tomography/Computerized Tomography (SPECT/CT) lymphoscintigraphy, we studied the pattern of lymphatic flow from mouse heterotopic abdominal cardiac grafts and identified mediastinal lymph nodes as the draining nodes for the donor graft. Staining with HY tetramer after transplantation of HY mismatched heart grafts and ELISPOT following allogeneic grafts to detect donor specific T cells revealed them as important sites for allosensitization. Our data indicates that mediastinal lymph nodes play a crucial role in the alloimmune response in this model, and should be used for ex vivo and adoptive transfer studies after transplantation in addition to the spleen.

Osteoporosis prophylaxis in patients receiving chronic glucocorticoid therapy.

BACKGROUND AND OBJECTIVES: Glucocorticoid-induced osteoporosis (GIOP) is the most common form of secondary osteoporosis, yet few patients receive proper measures to prevent its development. We retrospectively searched prescription records to determine if patients receiving oral prednisolone were receiving prophylaxis or treatment for osteopenia and osteoporosis. METHODS: Patients who were prescribed > or =7.5 milligrams of prednisolone for 6 months or longer during a 6- month period were identified through the prescription monitoring system. Demographic and clinical data were extracted from the patient records, and dual energy x-ray absorptiometry (DEXA) scans were retrieved, when available. Use of oral calcium, vitamin D and anti-resorptives was recorded. RESULTS: One hundred males and 65 females were receiving oral prednisolone for a mean (SD) duration of 40.4 (29.9) months in males and 41.2 (36.4) months in females. Twenty-one females (12.7%) and 5 (3%) males had bone mineral density measured by DEXA. Of those, 10 (47.6%) females and 3 (50%) males were osteoporotic and 11(52.4%) females and 2 (40%) males were osteopenic. Calcium and vitamin D were prescribed to the majority of patients (60% to 80%), but none were prescribed antiresorptive/anabolic therapy. CONCLUSIONS: Patients in this study were neither investigated properly nor treated according to the minimum recommendations for the management of GIOP. Physician awareness about the prevention and treatment of GIOP should be a priority for the local health care system.

Design, manufacture, and assay of the efficacy of siRNAs for gene silencing.

Small interfering RNAs (siRNAs) have been widely exploited for nucleotide-sequence-specific posttranscriptional gene silencing, as a tool to investigate gene function in eukaryotes, and they hold promise as potential therapeutic agents. Conventionally designed siRNAs are 21-mers with symmetric 2-nt 3' overhangs that mimic intermediates (microRNAs or miRNAs) of the natural processing of longer dsRNA (double-stranded RNA). siRNAs are sequences with full complementarity to their target mRNA and can be generated by either chemical synthesis or processing of shRNAs (short hairpin RNAs) transcribed from DNA vectors. To minimize off-target effects, any homology to nontarget mRNA can be verified using the expressed sequence tag (EST) database for the relevant organism. Here, we provide a practical guide and an overview to the design and selection of effective and specific siRNAs.

Safety evaluation through genotoxicity and apoptotic markers following RISUG® induced contraception and its reversal in male rabbits.

The safety evaluation following vas occlusion with RISUG® and its reversal with DMSO and NaHCO3, using genotoxicity tests and apoptotic marker assays, was carried out in rabbits. Animals were divided into groups of sham operated control, vas occlusion with RISUG® for 3 & 12 months, reversal with DMSO and NaHCO3 after 3 & 12 months, respectively. Minimum incidences of micronuclei in erythrocytes and frequency of aberrant chromosomes were observed. Caspase-3 and TUNEL positive cells in testis and cauda epididymis sections were observed within control limits. Comet assay in leukocytes and testicular cells revealed damaged cell range at the control level. DNA damage in cauda epididymal spermatozoa was observed between 2-3 % by in vitro study and annexin V assay indicated a significant enhancement (p < 0.05) of positive cells in 3 months vas occlusion group. In conclusion, RISUG® induced occlusion and its reversal has not been correlated with any toxicity.

Expression and localization of endothelin-converting enzyme-1 in human prostate cancer.

Endothelin (ET)-1 can influence cancer invasion and metastasis by exerting an autocrine (epithelial) or paracrine (stromal) influence on growth. ET-1 is generated from big ET-1 by endothelin-converting enzyme (ECE)-1, which has four recognized isoforms, ECE-1a, ECE-1b, ECE-1c, and ECE-1d, differing only in their amino-terminal regions. This study investigated the expression and localization of the ECE-1 isoforms in prostate cancer (PC). The epithelial cell lines used were androgen-sensitive LNCaP, androgen-independent PC-3 and Du145, and nonmalignant transformed PNT1-a, PNT2-C2, and P4E6 prostate cells. Primary cells derived from malignant and benign tissue from radical prostatectomies were also exploited. Previously, we reported increased ECE-1 expression in androgen-independent PC cell lines, as compared with androgen-sensitive cells. Our present data show that transcripts for all ECE-1 isoforms were present in all epithelial cell lines analyzed. However, only the ECE-1c protein was detectable in PC-3, Du145, PNT2-C2, and PNT1-a cells. ECE-1c localized to both the cell surface and intracellular compartments in individual cell lines. In primary stromal cells, all individual ECE-1 isoforms were expressed at the mRNA level, with the exception of ECE-1a. ECE-1b and ECE-1c protein levels were higher in malignant stromal cells, as compared with benign cells. In stroma, ECE-1c protein was localized to the cell surface, with filamentous immunoreactivity throughout the cell, whereas ECE-1b immunoreactivity was punctate throughout the cytoplasm. The upregulation of the ECE-1c isoform in PC cell lines is being investigated further.

Plasmodium falciparum population structure in Sudan post artemisinin-based combination therapy.

Over the past decade, Sudan has stepped up malaria control backed by WHO, and this has resulted in significant reduction in parasite rate, malaria morbidity and mortality. The present study analyzed Plasmodium falciparum parasites in four geographical separated areas, to examine whether the success in malaria control following the use of artemisinin-based combination therapy (ACT) has disrupted the population structure and evolution of the parasite. We examined 319 P. falciparum isolates collected between October 2009 and October 2012 in four different areas in Sudan (Jazira [central Sudan], Southern Darfur [western Sudan], Upper Nile [southern Sudan] and Kasala [eastern Sudan]). Twelve microsatellites were analyzed for allelic diversity, multi-locus haplotype and inter-population differentiation. Level of diversity was compared to that detected for three of the above microsatellites among P. falciparum parasites in central and eastern Sudan in 1999, prior to introduction of ACT. Diversity at each locus (unbiased heterozygosity [H]) was high in all areas (Jazira, H=0.67), (Southern Darfur, H=0.71), (Upper Nile, H=0.71), and (Kasala, H=0.63). Microsatellites were distributed widely and private alleles, detected in a single population, were rare. The extent of diversity in the above sites was similar to that seen, in 1999, in central (Khartoum, H=0.73) and eastern Sudan (Gedaref, H=0.75). Significant Linkage disequilibrium (LD) was observed between the microsatellites in all populations. Pairwise FST analysis revealed that parasites in the four areas could be considered as one population. However, the parasites in Sudan clustered away from parasites in West Africa and the Arabian Peninsula. Despite marked reduction in malaria risk in Sudan, the extent of diversity and parasite genetic structure are indicative of a large population size. Further considerable reduction in transmission would be needed before fragmented sub-population can be seen. In addition, the large divergence of P. falciparum in Sudan from West Africa and Arabian Peninsula populations may result from differential evolutionary pressures acting at the population level, which shall be considered in eradication plans.

Significance of fractional exhaled nitric oxide measurements in detecting primary ciliary dyskinesia in Saudi children.

OBJECTIVE: To examine the usefulness of fractional exhaled nitric oxide (FENO) measurements in detecting primary ciliary dyskinesia (PCD) in children. METHODS: This observational study was conducted at the Department of Pediatrics and Physiology, College of Medicine, King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia from January 2011 to December 2011. The study population consisted of 22 children with symptoms suggestive of PCD and the diagnosis was confirmed by ciliary biopsy. Using the American Thoracic Society guidelines, measurements of FENO were performed in 22 subjects with proven PCD biopsies and in 11 healthy age-matched subjects. RESULTS: No significant differences were found on the basis of age or ventilatory function tests between the PCD patients and control groups. Fractional exhaled nitric oxide values were significantly lower in children with PCD (6.19+/-1.43) compared to control group (17.00+/-6.30) (CI: -14.854 to -5.927, p<0.0001). Rhinorrhea was seen in 7 (31.8%), recurrent acute otitis media in 16 (72.7%), chronic otitis media in 5 (22.7%), recurrent sinusitis in 5 (22.7%), chronic productive cough in 8 (36.4%), bronchospasm in 11 (50%), and dextrocardia in 3 (13.6%) subjects. There was no correlation between age, FENO, and ventilatory function parameters. CONCLUSION: The measurement of FENO appears to be a useful tool for screening children for PCD. It can complement other tests such as nasal biopsy and electron microscopy studies.

Medication administration errors in Eastern Saudi Arabia.

OBJECTIVE: To assess the prevalence and characteristics of medication errors (ME) in patients admitted to King Fahd University Hospital, Alkhobar, Kingdom of Saudi Arabia. METHODS: Medication errors are documented by the nurses and physicians standard reporting forms (Hospital Based Incident Report). The study was carried out in King Fahd University Hospital, Alkhobar, Kingdom of Saudi Arabia and all the incident reports were collected during the period from January 2008 to December 2009. The incident reports were analyzed for age, gender, nationality, nursing unit, and time where ME was reported. The data were analyzed and the statistical significance differences between groups were determined by Student's t-test, and p-values of <0.05 using confidence interval of 95% were considered significant. RESULTS: There were 38 ME reported for the study period. The youngest patient was 5 days and the oldest 70 years. There were 31 Saudis, and 7 non-Saudi patients involved. The most common error was missed medication, which was seen in 15 (39.5%) patients. Over 15 (39.5%) of errors occurred in 2 units (pediatric medicine, and obstetrics and gynecology). Nineteen (50%) of the errors occurred during the 3-11 pm shift. CONCLUSION: Our study shows that the prevalence of ME in our institution is low, in comparison with the world literature. This could be due to under reporting of the errors, and we believe that ME reporting should be made less punitive so that ME can be studied and preventive measures implemented.

Endothelin system in oral squamous carcinoma cells: specific siRNA targeting of ECE-1 blocks cell proliferation.

The present study focused on the endothelin axis in human oral squamous cell carcinoma (SCC) cells. We investigated the expression and distribution of endothelin-1 (ET-1), its receptors (endothelin-A receptor (ET(A)R) and endothelin-B receptor (ET(B)R)) and isoforms of its specific converting enzyme (ECE-1a, 1b, 1c) and the report on their relative influences on cell proliferation. We also investigated the effect of an ECE-specific inhibitor (ECE-i) and siRNA targeting of the ECE-1 gene on SCC cell proliferation. We observed the expression of ET-1, ET(A)R, ET(B)R and all endothelin-converting enzyme-1 (ECE-1) isoforms in oral SCC cells, but only the expression of ET-1, ET(B)R and ECE-1 was increased when compared to normal human epidermal keratinocytes. ET-1 alone stimulated proliferation of oral SCC cells. Antagonists of either ET(A)R or ET(B)R inhibited ET-1-mediated proliferation. Decreased ECE-1 expression after ECE siRNA treatment reduced SCC cell proliferation. Antiproliferative effects were also observed by inhibiting ECE activity with ECE-i. In conclusion, the present study demonstrates that modulation of the endothelin system in oral SCC cells might provide a novel therapeutic protocol for oral cancer.

Differential expression of neutral endopeptidase-24.11 (neprilysin) and endothelin-converting enzyme in human prostate cancer cell lines.

Neutral endopeptidase-24.11 (neprilysin; NEP/CD10) is a cell surface metallopeptidase expressed by prostatic epithelial cells that degrades various bioactive peptides including endothelin. Endothelin-converting enzyme (ECE), the key enzyme of endothelin biosynthesis, catalyses the final processing step in the pathway. Neuropeptide substrates of NEP, including endothelin, have been implicated in the growth of androgen-independent prostate cancer. We have surveyed the expression of NEP and ECE in a range of prostate cancer cell lines. Western analysis reveals that ECE-1 is expressed abundantly in all the malignant cell lines tested, except for LNCaP. In contrast, LNCaP cells express high levels of NEP, while NEP was not detected in PC-3, DU145 and other metastatic cell lines that were tested. Of the normal immortalized prostate epithelial cell lines, PNT1a shows equivalent amounts of NEP and ECE. PNT2-C2 shows poor NEP expression but an abundance of ECE. P4E6, by comparison, has low levels of both ECE and NEP. These differences in expression may render these cell lines useful in experimental models for future study. Benign prostatic hyperplasia primary epithelial cells express much higher levels of NEP than malignant primary epithelial cells, but neither show ECE expression. On the other hand, surrounding stromal cell populations have detectable ECE levels. An absence of ECE in malignant and benign prostatic hyperplasia cells of primary epithelial origin suggests an important role for stromal interaction and paracrine production of ECE within the host. The upregulation of ECE expression in metastatic cells in culture may be indicative of its role in metastatic progression. A differential profile of ECE and NEP could contribute to an abundance of mitogenic peptides aiding the progression of androgen-independent prostate cancer.