Parasitological, molecular, and epidemiological investigation of Trypanosoma evansi infection among dromedary camels in Balochistan province.

Surra is a zoonotic disease caused by Trypanosoma evansi (T. evansi), which affects a wide variety of animals worldwide. The disease has a severe impact on the productivity, health, and working capacity of camels and causes mortality and extensive economic losses if not diagnosed early. This is the first comprehensive report on the prevalence of T. evansi infection in dromedaries in Balochistan province. In the present study, 393 blood samples (indigenous, n = 240; imported, n=153) were collected from one-humped camels (Camelus dromedarius) and were tested by molecular methods to estimate the prevalence of T. evansi in three districts (Pishin, Nushki, and Lasbella) of Balochistan province. The overall prevalence of T. evansi among examined camel samples was 28.24% (95% confidence interval (CI): 24.02-32.89%). The risk of T. evansi infection in adult camels (> 10 years) is higher than that in young ones (odd-ration (OR) = 2.7; 95% CI: 1.3357-5.3164%). Moreover, male camels were six times more likely to get an infection than female camels. The detection of T. evansi infection in camels sampled in summer and spring was 3.12- and 5.10-fold higher, respectively, than in camels sampled in winter. In conclusion, our findings showed a high rate of T. evansi infection in camels from the three districts. Our study emphasizes the need for a strict surveillance program and risk assessment studies as prerequisites for control measures.

Biosynthesis and characterization of zinc oxide nanoparticles using Nigella sativa against coccidiosis in commercial poultry.

Coccidiosis causes huge economic losses worldwide. Current study evaluated the effect of biosynthesized Zinc oxide nanoparticles (ZnONPs) using Nigella sativa, on Eimeria tenella infected broilers. Scanning electron microscopy showed spherical ZnONPs with 50-100 nm diameter, Fourier transforms infrared spectroscopy revealed the functional groups involved in the reduction of zinc acetate dihydrate to ZnONPs, UV-vis spectroscopy showed a peak at 354 nm, and Zeta potential exhibited stability at - 30 mV. A total of 150, a day-old broiler chicks were divided into 5 equal groups. Control negative: uninfected and untreated; Control positive: Infected and untreated; 3rd, 4th and 5th group were infected orally with 5 × 104 sporulated oocysts of Eimeria tenella and treated with 60 mg/kg ZnONPs, 1% Nigella sativa seeds and amprolium 125 ppm, respectively. ZnONPs significantly (p < 0.05) improved the growth performance in the infected birds and decreased the oocyst shedding and anti-coccidial index. A significant (p < 0.05) decrease in the level of aspartate transferase and alanine transferase, whereas, a significantly higher amount of antioxidants like catalase and superoxide dismutase in ZnONPs treated group was observed. Pro-inflammatory cytokines like IL-2 and TNF-α were significantly decreased by ZnONPs (p < 0.05). In conclusion, biogenic ZnONPs with Nigella sativa might have enhanced anticoccidial, antioxidant, and anti-inflammatory effects with improved growth performance.

Protective Effects of Dexmedetomidine Infusion on Genotoxic Potential of Isoflurane in Patients Undergoing Emergency Surgery.

Background: Isoflurane (ISO) has been extensively uses in general anesthesia and reported to cause deoxyribonucleic acid (DNA) damage in prolonged surgical procedures. Dexmedetomidine (DEX) is an adrenergic agonist and having antioxidant activity that may reduce the genotoxic potential (DNA damage) and oxidative stress induced by ISO in patients undergoing major neurosurgical procedures. Methods and Findings. Twenty-four patients of ASA (American Society of Anesthesiologists) classes I and II were randomly divided into two groups (n = 12). Group A patients received ISO, while group B patients received DEX infusion for maintenance of anesthesia. Venous blood samples were collected at different time intervals and used to evaluate the oxidative stress marker malondialdehyde (MDA) and endogenous antioxidants superoxide dismutases (SOD) and catalases (CAT). A single-cell gel electrophoresis (SCGE)-comet assay was used to investigate the genotoxic potential of ISO. Conclusion: Increased level of antioxidants and decreased value of MDA and genetic damage index were seen in group B (P < 0.001) in a time-dependent manner. Genetic damage was highest at point T 2 (0.77 vs. 1.37), and continued to decrease till T 3 (0.42 vs. 1.19), with respect to negative controls or baseline values following DEX infusion. Significantly, higher level of MDA was recorded in serum of group A (P < 0.001) as compared to group B (1.60 ± 0.33 vs. 0.03 ± 0.001). Enzymatic activities of CAT and SOD were significantly higher in group B than group A (10.11 ± 2.18 vs. 5.71 ± 0.33), (1.04 ± 0.05 vs. 0.95 ± 0.01), respectively. It may play a contributing role in daily anesthesia practice and improve the toxic effects on patients as well as anesthesia personnel. Trial Registration. Ethical Committee of Post Graduate Medical Institute (PGMI), Lahore General Hospital approved the use of humans in this study vide human subject application number ANS-6466 dated February 04, 2019. Furthermore, as the clinical trials required registration from an appropriate registry approved by World Health Organization (WHO), this trail also retrospectively registered at Thai Clinical Trials Registry (an approved WHO registry for clinical trials registration) under reference ID TCTR20211230001 on December 30, 2021.

Survival outcomes for patients with nasopharyngeal carcinoma in non-endemic region in the UK treated with intensity modulated based radiotherapy 65 Gy in 30 fractions ± weekly cisplatin chemotherapy.

Background: Nasopharyngeal carcinoma (NPC) is rare in the UK. The aim of the current study was to investigate survival outcomes for patients with NPC treated with (chemo)radiotherapy using 65 Gy in 30 fractions in a non-endemic region. Materials and methods: All consecutive 62 patients with histology proven non-metastatic nasopharyngeal carcinoma diagnosed between January 2009 to June 2019 were included in this retrospective analysis. Results: Median age was 59 years (range:19-81). The majority of patients had stage III disease (66.1%). Induction chemotherapy was given in 21% of patients and 82.3% of patients received concomitant systemic therapy. All patients were treated with 65 Gy in 30 fractions. There was disease recurrence in 17.4% patients. The 5-year disease-free, disease-specific and overall survival were 81.9%, 79.2% and 76.4%, respectively. On univariate analysis, disease recurrence was associated with N-stage (p = 0.047) and overall stage group (p = 0.023). Conclusion: To the best of authors' knowledge, this is the first report of the use of 65 Gy in 30 fractions of radiotherapy ± weekly cisplatin chemotherapy in NPC in a real-world setting. Our results are comparable to that from other non-endemic regions of the world using different dose fractionation of (chemo)radiotherapy. Future randomised control trials are warranted to compare various dose fractionations in these settings.

Molecular and Morphological Characterization of Eimeria crandallis Isolated from Deer (Cervidae) in Different Captive Animals.

Coccidiosis is a protozoan disease that is characterized by diffuse diarrhea, dehydration, emaciation accompanied by moderate morbidity and mild mortality in animals and birds. The current study targeted the molecular characterization of Eimeria isolates in captive deer from different localities in Lahore. The host species was the Cervidae family, such as Hog deer (Axis porcinus) and Punjab urial (Ovis aries vignei). The Eimeria crandallis was isolated from zoo animals. The DNA was extracted from oocysts and amplified by using reported oligonucleotide primers that exhibited the 809 bp product. These were analyzed by using the small subunit 18S rRNA gene-based evolutionary relationship with 36 other Eimeria species reported in caprine, cervinae, bovines, avians, and rodents. Light microscopic examination exhibited 3.29% (7/213) Eimeria-positive fecal samples with morphological features, including sub-spherical forms, the presence of micropyle with polar cap, and oocysts diameters (µm) ranging from 24.32 ± 1.61 to 18.94 ± 1.51. The phylogenetic tree constitutes four distinct clusters with relatively higher values. The evolutionary network showed that sequences were clustered in the monophyletic group of Eimeria species reported in caprine and cervinae. The nucleotide and amino acid sequence similarity matrix analysis exhibited 99.5-99.9% identity of the study isolates with Eimeria crandallis (AF336339). This study provides relevant baseline data to develop strategic control measures for coccidiosis in zoo animals. However, further investigations are required to place the hog deer and Punjab urial-derived E. crandallis into the caprine-originated cluster.

Modified QuEChERS Method for Extracting Thiamethoxam and Imidacloprid from Stingless Bees: Development, Application, and Green Metrics.

In the present study, a method for the determination of residues of the neonicotinoid insecticides thiamethoxam and imidacloprid in the stingless bee Melipona scutellaris Latreille (1811) was optimized through a factorial design, tested using green metrics, and then applied to exposed bees. It combines the extraction with a modified quick, easy, cheap, effective, rugged, and safe method and the determination by liquid chromatography-tandem mass spectrometry analysis. Different parameters such as the mass of the sample, dispersive sorbents, and elution solvents were assessed. Method validation parameters were checked and include sensitivity, specificity, and linearity. The limit of quantification of 0.0025 µg g-1 was obtained for both insecticides, where accuracy was 94%-100% with satisfactory intraday and interday precisions (relative standard deviation <10%). The qualified method was applied to orally and topically exposed bee samples, and the results indicated that it is suitable for the determination and quantification of neonicotinoid pesticide residues in this species. Moreover, green analytical metrics like the National Environmental Methods Index, Eco Scale score, high-performance liquid chromatography with an environmental assessment tool (HPLC-EAT), waste generation, and amount of sample were compared with methods described in the literature involving neonicotinoid analysis in honeybees. As a result, the present study displayed the highest Eco Scale score and HPLC-EAT score and the second smallest amount of sample and waste generated. Thus, the method meets green analytical metrics more than other methods. In this sense, besides the application, the multicriteria decision analysis tool employed suggests that this is a good option as a green analytical method. Environ Toxicol Chem 2022;41:2365-2374. © 2022 SETAC.

High-throughput sequencing of Fasciola spp. shows co-infection and intermediate forms in Balochistan, but only Fasciola gigantica in the Punjab province of Pakistan.

Fasciola gigantica and Fasciola hepatica are digenetic trematodes causing fasciolosis in ruminants. The host and geographical distribution of both Fasciola species are influenced by environmental and climatic conditions favouring survival and development of free-living stages and intermediate hosts, and livestock management practices. The aim of the present study was to describe the host distribution of the two Fasciola species in buffalo, cattle, goats, and sheep in the Balochistan and Punjab provinces of Pakistan. 359 flukes were collected from a total of 32 livers from the four livestock species. Deep amplicon sequencing of the internal transcribed spacer region 2 of ribosomal DNA (rDNA ITS-2) and mitochondrial nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND-1) loci confirmed co-infection of F. hepatica and F. gigantica in Balochistan and single species F. gigantica infection in Punjab. In Balochistan, co-infections and hybrids of both Fasciola species were identified in cattle, with more F. hepatica detected than F. gigantica. However, F. hepatica was the only species identified in goats, and F. gigantica was the only species identified in buffalo. In Punjab, all flukes were confirmed as F. gigantica in each of the four livestock species. Overall, the results indicate differences in the host and geographical distribution of F. gigantica and F. hepatica, and provide useful knowledge for the development of control strategies for livestock and humans.

Epidemiological and Clinical Characteristics of COVID-19: A Retrospective Multi-Center Study in Pakistan.

The emergence of a pathogen responsible for a mysterious respiratory disease was identified in China and later called a novel coronavirus. This disease was named COVID-19. The present study seeks to determine the epidemiological and clinical characteristics of COVID-19 in Pakistan. This report will exhibit a linkage between epidemiology and clinical aspects which in turn can be helpful to prevent the transmission of the virus in Pakistan. A retrospective, multiple center study was performed by collecting the data from patients' with their demographics, epidemiological status, history of co-morbid conditions, and clinical manifestations of the disease. The data was collected from 31 public-sector and 2 private hospitals across Pakistan by on-field healthcare workers. A Chi-square test was applied to assess the relationship between categorical data entries. A total of 194 medical records were examined. The median age of these patients was found to be 34 years. A total of 53.6% active cases were present including 41.2% males and 12.4% females till the end of the study. Adults accounted for most of the cases (94.3%) of COVID-19. Fever (86.60%), cough (85.05%), fatigue (36.60%), dyspnea (24.74%), and gastrointestinal discomfort (10.31%) were among the most frequently reported signs and symptoms by the patients. However, 4.12% of the total patient population remained asymptomatic. The median duration of hospital stay was found to be 14 (0-19) days. The earliest source of the spread of the virus may be linked to the foreigners traveling to Pakistan. Spread among men was more as compared to women. A few cases were found to be positive, due to the direct contact with pets or livestock. Hypertension (7.73%), diabetes (4.64%), cardiovascular conditions (2.58%) were the most common co-morbidities. The percentage mortality was 2.50% with the highest mortality among elders.

Disposition of cyadox in domesticated cats following oral, intramuscular, and intravenous administration.

Cyadox (CYX) is a synthetic antibacterial agent of quinoxaline with much lower toxic effects. A safety criterion of CYX for clinical use was established by studying the pharmacokinetics and metabolism of CYX after oral (PO), intramuscular (IM), and intravenous (IV) administration. CYX was administered in six domesticated cats (three males and three females) by PO (40 mg/kg.b.w.), IM (10 mg/kg.b.w.), and IV (10 mg/kg.b.w.) routes in a crossover pattern. Highly sensitive liquid chromatography with ultraviolet detection (HPLC-UV) method was developed for detection of CYX and its metabolites present in plasma, urine, and feces. The bioavailability of CYX after PO and IM routes was 4.37% and 84.4%. The area under curves (AUC), mean resident time (MRT), and clearance (CL) of CYX and its metabolites revealed that CYX quickly metabolized into its metabolites. The total recovery of CYX and its main metabolites was >60% after each route. PO delivery suggesting first pass effect in cats that might make this route suitable for intestinal infection and IM injection could be better choice for systemic infections. Less ability of glucuronidation did not show any impact on CYX metabolism. The findings of present study provide detailed information for evaluation of CYX.

Characterization of the Complete Mitochondrial Genome of a Whipworm Trichuris skrjabini (Nematoda: Trichuridae).

The complete mitochondrial (mt) genome of Trichuris skrjabini has been determined in the current study and subsequently compared with closely related species by phylogenetic analysis based on concatenated datasets of mt amino acid sequences. The whole mt genome of T. skrjabini is circular and 14,011 bp in length. It consists of a total of 37 genes including 13 protein coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNAs) genes, and two non-coding regions. The gene arrangement and contents were consistent with other members of the Trichuridae family including Trichuris suis, Trichuris trichiura, Trichuris ovis, and Trichuris discolor. Phylogenetic analysis based on concatenated datasets of amino acids of the 12 PCGs predicted the distinctiveness of Trichuris skrjabini as compared to other members of the Trichuridae family. Overall, our study supports the hypothesis that T. skrjabini is a distinct species. The provision of molecular data of whole mt genome of T. skrjabini delivers novel genetic markers for future studies of diagnostics, systematics, population genetics, and molecular epidemiology of T. skrjabini.

Advances in engineered trans-acting regulatory RNAs and their application in bacterial genome engineering.

Small noncoding RNAs, a large class of ancient posttranscriptional regulators, are increasingly recognized and utilized as key modulators of gene expression in a broad range of microorganisms. Owing to their small molecular size and the central role of Watson-Crick base pairing in defining their interactions, structure and function, numerous diverse types of trans-acting RNA regulators that are functional at the DNA, mRNA and protein levels have been experimentally characterized. It has become increasingly clear that most small RNAs play critical regulatory roles in many processes and are, therefore, considered to be powerful tools for genetic engineering and synthetic biology. The trans-acting regulatory RNAs accelerate this ability to establish potential framework for genetic engineering and genome-scale engineering, which allows RNA structure characterization, easier to design and model compared to DNA or protein-based systems. In this review, we summarize recent advances in engineered trans-acting regulatory RNAs that are used in bacterial genome-scale engineering and in novel cellular capabilities as well as their implementation in wide range of biotechnological, biological and medical applications.

Seroprevalence and Risk Factors for Theileria equi Infection in Equines from Khyber Pakhtunkhwa Province, Pakistan.

BACKGROUND: Theileria equi is a tick borne protozoan parasite which causes piroplasmosis among equines worldwide. The present study was aimed to determine seroprevalence of T. equi in donkeys, horses, and mules from two equine populated districts (Peshawar and Charsadda) of Khyber Pakhtunkhwa (KPK), Pakistan. METHODS: A total of 393 equine (195 horses, 194 donkeys and 4 mules) serum samples were collected from five and four randomly selected localities in Charsadda (n = 193) and Peshawar (n = 200), respectively. The presence of antibodies to T. equi was determined using a commercially available competitive enzyme-linked immunosorbent assay. RESULTS: An overall seroprevalence of 38.2% (n=150) was observed among all the tested animals suggesting a higher seropositivity among equids belonging to Charsada (50.3%) as compared to Peshawar (27.5%). Binary logistic regression analysis revealed that being a donkey (OR 2.94), having tick infestation (OR 4.32), history of voiding red (i.e., blood containing) urine (OR 3.97) and anemia (OR 2.1) were the factors significantly associated with the seroprevalence of T. equi. For animals with higher anti-T. equi antibody titers, a strong association of seroprevalence for T. equi was recorded with species, age, sex, tick infestation, anemia and history of hematuria. CONCLUSION: The present study indicates a high level of exposure of working equids to T. equi in KPK region, Pakistan. Future studies should focus on tick vector identification and other factors responsible for spread of the disease.

Metabolism and toxicity of arsenicals in mammals.

Arsenic (As) is a metalloid usually found in organic and inorganic forms with different oxidation states, while inorganic form (arsenite As-III and arsenate As-v) is considered to be more hazardous as compared to organic form (methylarsonate and dimethylarsinate), with mild or no toxicity in mammals. Due to an increasing trend to using arsenicals as growth promoters or for treatment purposes, the understanding of metabolism and toxicity of As gets vital importance. Its toxicity is mainly depends on oxi-reduction states (As-III or As-v) and the level of methylation during the metabolism process. Currently, the exact metabolic pathways of As have yet to be confirmed in humans and food producing animals. Oxidative methylation and glutathione conjugation is believed to be major pathways of As metabolism. Oxidative methylation is based on conversion of Arsenite in to mono-methylarsonic acid and di-methylarsenic acid in mammals. It has been confirmed that As is only methylated in the presence of glutathione or thiol compounds, suggesting that As is being methylated in trivalent states. Subsequently, non-conjugated trivalent arsenicals are highly reactive with thiol which converts the trivalent arsenicals in to less toxic pentavalent forms. The glutathione conjugate stability of As is the most important factor for determining the toxicity. It can lead to DNA damage by alerting enzyme profile and production of reactive oxygen and nitrogen species which causes the oxidative stress. Moreover, As causes immune-dysfunction by hindering cellular and humeral immune response. The present review discussed different metabolic pathways and toxic outcomes of arsenicals in mammals which will be helpful in health risk assessment and its impact on biological world.

The complete mitochondrial genome sequence of Eimeria innocua (Eimeriidae, Coccidia, Apicomplexa).

The complete mitochondrial genome of Eimeria innocua KR strain (Eimeriidae, Coccidia, Apicomplexa) was sequenced. This coccidium infects turkeys (Meleagris gallopavo), Bobwhite quails (Colinus virginianus), and Grey partridges (Perdix perdix). Genome organization and gene contents were comparable with other Eimeria spp. infecting galliform birds. The circular-mapping mt genome of E. innocua is 6247 bp in length with three protein-coding genes (cox1, cox3, and cytb), 19 gene fragments encoding large subunit (LSU) rRNA and 14 gene fragments encoding small subunit (SSU) rRNA. Like other Apicomplexa, no tRNA was encoded. The mitochondrial genome of E. innocua confirms its close phylogenetic affinities to Eimeria dispersa.

Synthesis and Evaluation of Estradiol Derivatives as Anti-Breast Cancer Agents.

3-N-alkyloxyestradiol derivatives were synthesized, characterized and tested for activity in MCF-7 human breast cancer cells. Among the compounds, the diisopropyl and piperidinyl derivatives were found to be more active than 4-hydroxytamoxifen (HO-Tam), the active metabolite of tamoxifen based upon IC(50) values. The IC(50)s were correlated with structures using molecular modeling.

Safety and pharmacokinetic analysis of methotrexate administered directly into the fourth ventricle in a piglet model.

We have developed a piglet model to assess chemotherapy administration directly into the fourth ventricle as a potential treatment for medulloblastoma and other malignant posterior fossa tumors. The objective of this study was to assess safety and pharmacokinetics after methotrexate infusions into the fourth ventricle. Catheters were inserted into the fourth ventricle and lumbar cistern in five piglets. Two milligrams of Methotrexate (MTX) was infused into the fourth ventricle on five consecutive days. Safety was assessed by neurological examination, 4.7 T MRI, and post-mortem pathological analysis. MTX levels in serum and cerebrospinal fluid (CSF) were measured, and area under the concentration-time curve (AUC) was calculated for CSF samples. No neurological deficits were caused by MTX infusions. One piglet died from complications of anesthesia induction for MRI scanning. MRI scans showed accurate catheter placement without signal changes in the brainstem or cerebellum. One piglet had asymptomatic ventriculomegaly. Pathological analysis demonstrated meningitis and choroid plexitis consisting predominantly of CD-3 positive T-lymphocytes in all piglets and a small focal area of subependymal necrosis in one. In all piglets, mean peak MTX level in fourth ventricular CSF exceeded that in lumbar CSF by greater than five-fold. Serum MTX levels were undetectable or negligible. Statistically significant differences between fourth ventricle and lumbar AUC were detected at peaks (P = 0.01) and at all collection time points (P = 0.01) but not at troughs (P = 0.36). MTX can be infused into the fourth ventricle without clinical or radiographic evidence of damage. An inflammatory response without clinical correlate is observed. Significantly higher peak MTX levels are observed in the fourth ventricle than in the lumbar cistern.

Role of cytochrome C in apoptosis: increased sensitivity to tumor necrosis factor alpha is associated with respiratory defects but not with lack of cytochrome C release.

Although the role of cytochrome c in apoptosis is well established, details of its participation in signaling pathways in vivo are not completely understood. The knockout for the somatic isoform of cytochrome c caused embryonic lethality in mice, but derived embryonic fibroblasts were shown to be resistant to apoptosis induced by agents known to trigger the intrinsic apoptotic pathway. In contrast, these cells were reported to be hypersensitive to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis, which signals through the extrinsic pathway. Surprisingly, we found that this cell line (CRL 2613) respired at close to normal levels because of an aberrant activation of a testis isoform of cytochrome c, which, albeit expressed at low levels, was able to replace the somatic isoform for respiration and apoptosis. To produce a bona fide cytochrome c knockout, we developed a mouse knockout for both the testis and somatic isoforms of cytochrome c. The mouse was made viable by the introduction of a ubiquitously expressed cytochrome c transgene flanked by loxP sites. Lung fibroblasts in which the transgene was deleted showed no cytochrome c expression, no respiration, and resistance to agents that activate the intrinsic and to a lesser but significant extent also the extrinsic pathways. Comparison of these cells with lines with a defective oxidative phosphorylation system showed that cells with defective respiration have increased sensitivity to TNF-alpha-induced apoptosis, but this process was still amplified by cytochrome c. These studies underscore the importance of oxidative phosphorylation and apoptosome function to both the intrinsic and extrinsic apoptotic pathways.

Azidothymidine inhibits NF-kappaB and induces Epstein-Barr virus gene expression in Burkitt lymphoma.

The antiviral compound azidothymidine (AZT), alone or in combination with other agents, induces apoptosis in early-passage, Epstein-Barr virus-positive Burkitt lymphoma (EBV+ BL) lines and has clinical activity in EBV+ BL. We report here a mechanism of AZT's antitumor activity. The nuclei of these cells contain activated nuclear factor-kappaB (NF-kappaB) subunits p50, c-Rel, RelB, and p52, but not p65. Treatment of primary EBV+ BL lines with AZT inhibited NF-kappaB within 1 to 2 hours. This was followed by up-regulation of EBV gene expression including viral thymidine kinase (vTK) and apoptosis. Subclones of EBV+ BL cells that demonstrated activated p65 were resistant to AZT. In EBV+ BLs, AZT but not ganciclovir (GCV) was highly phosphorylated to its monophosphate form (AZT-MP). Phosphorylation, as well as apoptosis, was markedly enhanced in the presence of hydroxyurea. AZT inhibits NF-kappaB and up-regulates EBV gene expression in primary EBV+ BLs. AZT with hydroxyurea may represent an inexpensive, targeted regimen for endemic BL.

The oncolytic effect of recombinant vesicular stomatitis virus is enhanced by expression of the fusion cytosine deaminase/uracil phosphoribosyltransferase suicide gene.

Vesicular stomatitis virus (VSV) has recently been demonstrated to exhibit significant oncolytic capabilities against a wide variety of tumor models in vitro and in vivo. To potentially enhance the oncolytic effect, we generated a novel recombinant VSV (rVSV) that expressed the fusion suicide gene Escherichia coli cytosine deaminase (CD)/uracil phosphoribosyltransferase (UPRT). rVSV encoding the CD/UPRT fusion gene (VSV-C:U) exhibited normal growth properties and generated high levels of biologically active CD/UPRT that could catalyze the modification of 5-fluorocytosine into chemotherapeutic 5-fluorouracil (5-FU), which exhibited considerable bystander effect. Intratumoral inoculation of VSV-C:U in the presence of the systemically administered prodrug 5-fluorocytosine produced statistically significant reductions in the malignant growth of syngeneic lymphoma (A20) or mammary carcinoma (TSA) in BALB/c mice compared with rVSV treatments or with control 5-FU alone. Aside from detecting prolonged therapeutic levels of 5-FU in VSV-C:U-treated animals harboring TSA tumors and enhancing bystander killing of tumor cells, we demonstrated marked activation of IFN-gamma-secreting cytotoxic T cells by enzyme-linked immunospot analysis that may have also facilitated tumor killing. In conclusion, the insertion of the fusion CD/UPRT suicide gene potentiates the oncolytic efficiency of VSV by generating a strong bystander effect and by contributing to the activation of the immune system against the tumor without detrimentally altering the kinetics of virus-mediated oncolysis and may be useful in the treatment of malignant disease.

Potentiation of TRAIL-induced apoptosis in primary effusion lymphoma through azidothymidine-mediated inhibition of NF-kappa B.

The survival of viral mediated lymphomas depends upon constitutive nuclear factor kappa B (NF-kappaB) activity. AIDS-related human herpesvirus type 8-associated primary effusion lymphoma (PEL) responds poorly to chemotherapy and is almost invariably fatal. We have previously demonstrated that the antiviral combination of interferon alpha (IFN-alpha) and azidothymidine (AZT) induces apoptosis in PEL cell lines. We therefore used these agents as therapy for an AIDS patient with PEL. The patient had a dramatic response, with complete resolution of his malignant effusion in 5 days. In PEL cells, the death receptor ligand known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is markedly up-regulated by IFN-alpha; however, signals transduced by death receptors may also activate an antiapoptotic response mediated by NF-kappaB. In both the primary tumor cells from our patient and PEL cell lines, AZT selectively blocked nuclear entry of the NF-kappaB heterodimer p50 and p65, an effect not seen with other nonthymidine antiviral nucleosides. AZT monophosphate, the principal intracellular metabolite, inhibited phosphorylation and degradation of IkappaB by the IkappaB kinase complex. AZT- and IFN-alpha-mediated apoptosis was blocked by expression and nuclear localization of an IkappaB-resistant form of NF-kappaB (the p50 subunit linked to the transactivation domain of herpes simplex virus VP16). The proapoptotic effect of AZT and IFN-alpha in PEL occurs through the concomitant activation of TRAIL and blockade of NF-kappaB and represents a novel antiviral therapy for a virally mediated tumor.