Anti-Tumor Activity of a miR-199-dependent Oncolytic Adenovirus

Peer reviewe

Phenotypic Characterization and Antibiotic Resistance Patterns of Extended-Spectrum β-Lactamase- and AmpC β-Lactamase-Producing Gram-Negative Bacteria in a Referral Hospital, Saudi Arabia

Background. Emergence of pathogenic bacteria carrying β-lactamase-resistant determinants has become a major health problem in the hospital setting. The study aimed to determine antibiotic-resistant patterns and frequency of extended-spectrum β-lactamase- (ESBL-) producing Gram-negative bacteria (GNB) and AmpC β-lactamase-producing GNB. Methodology. A prospective cross-sectional study was conducted during a period from September 2017 to August 2018 at King Abdullah Hospital, Bisha Province, Saudi Arabia. GNB (n = 311) were recovered from patients’ clinical specimens including sputum, urine, wound pus, blood, tracheal aspirates and high vaginal swabs, umbilical discharge, eye discharge, and cerebrospinal fluids. Isolates were identified by the Phoenix identification system. Antimicrobial susceptibility was tested by the Kirby–Bauer disk procedure. Phenotypic characterization of ESBLs and AmpC β-lactamases was performed utilizing the double-disk synergy test and inhibitor-based method, respectively. Associations with outcome measures were determined by simple descriptive statistics and a chi-square test. Results. Out of 311 GNB isolates, the frequency of ESBL and AmpC β-lactamase producers was 84 (27%) and 101 (32.5%), respectively. Klebsiella pneumoniae and Escherichia coli were common ESBL producers. AmpC β-lactamases predominate among Acinetobacter spp. and Pseudomonas aeruginosa. Coproduction of ESBLs and AmpC β-lactamases was found in 36 (11.6%) isolates, with very close relative frequencies among K. pneumoniae, Acinetobacter spp., and P. aeruginosa. β-Lactamase producers were predominantly found in the surgical department (56.5%) and ICUs (44.2%). ESBL producers revealed high resistance for cefuroxime (96.4%), cefotaxime (92.9%), and trimethoprim/sulfamethoxazole (90.5%). The resistance rates were significantly higher among ESBL producers than nonproducers for cephalosporins (p<0.001), amoxicillin/clavulanate (p<0.001), piperacillin/tazobactam (p=0.010), nitrofurantoin (p=0.027), aztreonam (p<0.001), ciprofloxacin (p=0.002), and trimethoprim/sulfamethoxazole (p<0.001). Significantly higher (p<0.05) resistance rates were observed among AmpC β-lactamase producers than nonproducers for all tested antibiotics. Conclusions. This finding showed a high prevalence of ESBL- and AmpC β-lactamase-producing GNB in our hospital. Quality control practice and routine detection of β-lactamase producers before deciding on antibiotic therapy are advocated

Enteroparasitosis infections among renal transplant recipients in Khartoum state, Sudan 2012–2013

Abstract Objectives Renal transplantation procedure markedly increased over the past few decades. The risk of harboring parasitic diseases may affect transplant recipients during life expectancy. We aimed in this study to determine the enteroparasitosis frequency among renal transplant recipients in Khartoum state, Sudan. A case–control hospital-based study performed between November 2012 and May 2013, on 300 renal transplant recipients attending Sudanese Kidney Association hospital in Khartoum state, Sudan, along with 300 normal healthy individuals matching the case in age and sex. Stool samples were collected for parasitological studies. Results Out of the 300 renal transplant recipients: 242 (80.7%) were males mean age 43 ± 11.28 and 58 (19.3%) were females mean age 41 ± 13.41. Intestinal parasitic infection was observed in 118 participants and the overall frequency was 19.7%; of which 64 were cases (21.3%) and 54 (18.0%) were controls. Eight different species of intestinal parasites were identified; Entamoeba histolytica/dispar (7.5%), Entamoeba coli (6.5%), Giardia lambelia (3.2%), Cryptosporidium parvum (1.2%), Ascaris lumbricoides (0.6%), Enterobius vermicularis (0.3%), (0.2%) for each of Strongyloides stercoralis and Hymenolepis nana

miR-199a-3p Modulates MTOR and PAK4 Pathways and Inhibits Tumor Growth in a Hepatocellular Carcinoma Transgenic Mouse Model

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Prognosis is poor, and therapeutic options are limited. MicroRNAs (miRNAs) have emerged as potential therapeutic molecules against cancer. Here, we investigated the therapeutic efficacy of miR-199a-3p, an miRNA highly expressed in normal liver and downregulated in virtually all HCCs. The therapeutic value of miR-199a-3p mimic molecules was assayed in the TG221 mouse, a transgenic model highly predisposed to the development of liver cancer. Administration of miR-199a-3p mimics in the TG221 transgenic mouse showing liver cancer led to a significant reduction of number and size of tumor nodules compared to control animals. In vivo delivery confirmed protein downregulation of the miR-199a-3p direct targets, mechanistic target of rapamycin (MTOR) and p21 activated kinase 4 (PAK4), ultimately leading to the repression of FOXM1. Remarkably, the anti-tumor activity of miR-199a-3p mimics was comparable to that obtained with sorafenib. These results suggested that miR-199a-3p may be considered a promising HCC therapeutic option

Phenotypic and Genotypic Analysis of Multidrug-Resistant Mycobacterium tuberculosis Isolates from Sudanese Patients

Background. Currently, mutations in rpoB, KatG, and rrs genes and inhA promoter were considered to be involved in conferring resistance to rifampicin, isoniazid, and streptomycin in Mycobacterium tuberculosis (MTB). Objective. The aims of this study were to detect the prevalence of first-line tuberculosis (TB) drug resistance among a group of previously treated and newly detected TB patients, to determine the association between prevalence of multidrug resistance (MDR) and demographic information (age and sex), to explain genes correlated with MDR Mycobacterium tuberculosis, and to characterize MTB via 16S ribosomal RNA (16S rRNA) analysis. Methods. A hundred MTB isolates from Sudanese pulmonary TB patients were included in the study. The proportional method of drug susceptibility test was carried out on Löwenstein-Jensen media. Multiplex PCR of rpoB and KatG genes and inhA promoter was conducted; then rrs genes were amplified by conventional PCR and were sequenced. The sequences of the PCR product were compared with known rrs gene sequences in the GenBank database by multiple sequence alignment tools. Result. The prevalence of MDR was 14.7% among old cases and 5.3% among newly diagnosed cases. Conclusion. Mutations in rrs could be considered as a diagnostic marker

Anti-Tumor Activity of a miR-199-dependent Oncolytic Adenovirus

none15siThe down-regulation of miR-199 occurs in nearly all primary hepatocellular carcinomas (HCCs) and HCC cell lines in comparison with normal liver. We exploited this miR-199 differential expression to develop a conditionally replication-competent oncolytic adenovirus, Ad-199T, and achieve tumor-specific viral expression and replication. To this aim, we introduced four copies of miR-199 target sites within the 3' UTR of E1A gene, essential for viral replication. As consequence, E1A expression from Ad-199T virus was tightly regulated both at RNA and protein levels in HCC derived cell lines, and replication controlled by the level of miR-199 expression. Various approaches were used to asses in vivo properties of Ad-199T. Ad-199T replication was inhibited in normal, miR-199 positive, liver parenchyma, thus resulting in reduced hepatotoxicity. Conversely, the intrahepatic delivery of Ad-199T in newborn mice led to virus replication and fast removal of implanted HepG2 liver cancer cells. The ability of Ad-199T to control tumor growth was also shown in a subcutaneous xenograft model in nude mice and in HCCs arising in immune-competent mice. In summary, we developed a novel oncolytic adenovirus, Ad-199T, which could demonstrate a therapeutic potential against liver cancer without causing significant hepatotoxicityopenCallegari E; Elamin BK; D'Abundo L; Falzoni S; Donvito G; Moshiri F; Milazzo M; Altavilla G; Giacomelli L; Fornari F; Hemminki A; Di Virgilio F; Gramantieri L; Negrini M; Sabbioni SCallegari E; Elamin BK; D'Abundo L; Falzoni S; Donvito G; Moshiri F; Milazzo M; Altavilla G; Giacomelli L; Fornari F; Hemminki A; Di Virgilio F; Gramantieri L; Negrini M; Sabbioni

miR-199 controls Ad-199T replication <i>in vitro</i>.

<p>(<b>A</b>-<b>B</b>) To asses viral replication in absence and in presence of miR-199, HepG2 and HepG2/199 cell lines were infected with Ad-199T or Ad-Control adenoviruses. A progressive accumulation of viral DNA, indicated as fold change copies of Adeno DNA referred the lower level of Adenovirus DNA copies, indicates that active viral replication is efficiently occurring in both cell lines infected with Ad-Control virus. A progressive accumulation of viral DNA in the HepG2 cells infected with Ad-199T indicates that active viral replication is efficiently occurring in this cell line; on the contrary, in HepG2/199 cells infected with Ad-199T the viral DNA did not increase over time, confirming that the virus replication was inhibited by the presence of miR-199. P-values of the comparisons are shown.</p

Description of the conditionally replication-competent oncolytic Adenovirus.

<p>(<b>A</b>) Schematic illustration of Adenovirus type 5 genome: the viral early and late genes and the Inverted Terminal Repeat (ITR) are indicated. (<b>B</b>) TheAd-Control viral genome encloses the E1A/E1B genes and the IRES-EGFP expression cassette. (<b>C</b>) The Ad-199T viral genome contains a specific sequence complementary to microRNA 199a-3p (miR-199 target) in the 3’ untranslated region of the E1A gene. (<b>D</b>) The miR-199 target sequence is made of 4 copies of a 22bp DNA segment complementary to the mature miRNA.</p

Ad-199T replicates in tumor cells <i>in vivo</i>.

<p>(<b>A</b>) 2x10⁶ HepLuc cells were intra-hepatically implanted in B6D2 wild type mice at 3 days of birth and examined at the In Vivo Imaging System (IVIS) two hours after cells implantation. After 24 hours, three experimental groups, consisting of six mice each, were defined: one was injected intra-hepatically with 1x10⁸ I.U. of the Ad-199T virus; one was infected with 1x10⁸ I.U. of the Ad-Control virus; one was not infected (no virus). Mice were monitored at the IVIS at 24h (<b>B</b>), 48h (<b>C</b>) and 72h (<b>D</b>) after virus inoculation. Reduction in pseudo-color images, representing bioluminescence intensity, indicates that the implanted tumor cells were eliminated, likely due to the active viral replication by either Ad-Control or Ad-199T viruses. (<b>E</b>) A quantitative photon analysis of the region-of-interest showed a highly significant decrease (p value <0.05) of luminescence in mice infected with Ad-Control and Ad-199T viruses versus uninfected animals.</p

Differential replication of Ad-199T in normal liver versus tumor cells.

<p>2x10⁶ HepLuc cells were intra-hepatically implanted in six B6D2 wild type mice 3 days after birth. After 24 hours, the animals were injected intra-hepatically with 1x10⁸ I.U. of the Ad-199T virus. Mice were sacrificed at 24h and 48h after the treatment and livers as well as tumor cells masses were collected. Genomic DNA extracted both from normal liver and tumor masses was analyzed by analytical PCR (<b>A</b>) and quantitative PCR as fold change copies of Adeno DNA referred to the lower level of Adenovirus DNA copies (<b>B</b>). The results show a significant suppression of Ad-199T replication in normal liver (NL) compared to tumors (Tumor). P-values of the comparisons are shown.</p