Post-treatment Assay Research Articles

In Vitro Anti-rotaviral Activity of Achillea kellalensis

Achillea kellalensis, which is frequently used by Chaharmahal va Bakhtiarians residing in, Southwest of Iran, as a traditional herbal medicine for the treatment of acute diarrhea, has been selected to examine its antiviral activities against bovine rotavirus and cell toxicity activity in MA-104 cells. The aim of this study was to evaluate the in vitro cytotoxic and anti-rotavirus properties of crude extracts of A. kellalensis. The dried and powdered flowers of Achillea kellalensis were extracted with hot water and ethanol 50% (v/v). The cell viability and toxicity of the extracts were evaluated on MA-104 cells using four methods; trypan blue dye, NR, crystal violet and MTT assay. The in vitro anti-rotavirus properties were determined via four different assays, in order to evaluate the direct inhibition and/or the inhibition of viral replication. Cytotoxicity of two A. kellalensis extracts showed different concentrations. Hydro-alcoholic extract had low CC50 at 600 µg/mL by the NR assay while the aqueous extract had high CC50 at 1000µg/mL by the crystal violet method. In the simultaneous treatment assay and post treatment assay, the extracts were able to prevent viral replication and inhibit the viral CPE on MA-104 cells at 10 TCID50, but the extracts did not exhibit direct antiviral activity on rotavirus adsorption. The effective concentration (EC50) of both extracts was observed to be 100 µg/mL. These results indicate that A. kellalensis extracts exert potent anti-rotaviral activity only after viral adsorption. The two extracts from A. kellalensis showed a good selectivity index. Also these results suggest that extracts prepared from the flowers of A. kellalensis may be potential anti-rotaviral agents in vivo and be useful in veterinary medicine.

In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination

Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2–5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC50) with the ranging from 10.8±1.4 to 22.5±2.2μM against PEDV. Compounds 1–5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC50 values of 12.2±2.8 and 14.6±1.3μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24h) than in early stages (6 and 12h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection.

Antiviral activity of Alpinia katsumadai extracts against rotaviruses

In vitro anti-rotavirus activity of Alpinia katsumadai (AK) extracts were evaluated against bovine G8P[7] and porcine G5P[7] rotaviruses in two different assay strategies, a mixed treatment assay and a post treatment assay. In the mixed treatment assay, six AK extracts [AK-1 (EtOH extract), AK-3 (H2O layer), AK-5 (40% methanol fraction), and AK-9–11 (H2O extract, polysaccharide fraction, supernatant fraction)] exhibited inhibitory activities against G5P[7] rotavirus with the EC50 values ranging from 0.7±0.4 to 33.7±6.5μg/mL. Extracts AK-1, AK-3, and AK-5 inhibited rotavirus infection against G8P[7] rotavirus, the with EC50 values of 8.4±2.2μg/mL, 6.5±0.8μg/mL and 8.4±5.0μg/mL, respectively. By hemagglutination inhibition (HI) assay, six AK extracts completely inhibited viral adsorption onto human RBCs in both strains of rotaviruses at less than 11μg/mL. However, in the post treatment assay, there was no anti activity shown against both strains of rotaviruses. As a result, six AK extracts were attributed mainly to having a strong interaction with hemagglutinin protein on the outer surface of rotavirus, resulting to blockage of viral adsorption.

In Vitro inhibitory activity of Alpinia katsumadai extracts against influenza virus infection and hemagglutination

BackgroundAlpinia katsumadai (AK) extracts and fractions were tested for in vitro antiviral activities against influenza virus type A, specially human A/PR/8/34 (H1N1) and avian A/Chicken/Korea/MS96/96 (H9N2), by means of time-of-addition experiments; pre-treatment, simultaneous treatment, and post treatment.ResultsIn pre-treatment assay, the AK extracts and AK fractions did not show significant antiviral activity. During the simultaneous treatment assay, one AK extract and five AK fractions designated as AK-1 to AK-3, AK-5, AK-10, and AK-11 showed complete inhibition of virus infectivity against A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2). The 50% effective inhibitory concentrations (EC50) of these one AK extracts and five AK fractions with exception of the AK-9 were from 0.8 ± 1.4 to 16.4 ± 4.5 μg/mL against A/PR/8/34 (H1N1). The two AK extracts and three AK fractions had EC50 values ranging from <0.39 ± 0.4 to 2.3 ± 3.6 μg/mL against A/Chicken/Korea/MS96/96 (H9N2). By the hemagglutination inhibition (HI) assay, the two AK extracts and five AK fractions completely inhibited viral adsorption onto chicken RBCs at less than 100 μg/mL against both A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2). Interestingly, only AK-3 was found with inhibition for both viral attachment and viral replication after showing extended antiviral activity during the post treatment assay and quantitative real-time PCR.ConclusionsThese results suggest that AK extracts and fractions had strong anti-influenza virus activity that can inhibit viral attachment and/or viral replication, and may be used as viral prophylaxis.

Who would have thought it! Influence on outcome of radiotherapy, Ki67 and stroma. Introduction to Session 5

In breast cancer there are clearly a number of factors that counter-intuitively influence outcome or for which there is a change, after a considerable time, in longstanding evidence that outcome was not affected. The latter is well represented by the effect of postoperative radiotherapy in early breast cancer on overall survival. There was clear evidence that such treatment reduced local recurrence risk (by about two-thirds) and risk of death from breast cancer (by one-sixth [1]), but until 1997 evidence suggested treatment would not improve overall survival. The results in that year of two randomised trials in high-risk patients showed that the addition of postmastectomy radiotherapy to adjuvant chemotherapy improved overall survival [2,3]. Ragaz argues from the updated results of these pivotal Canadian and Danish trails, from survival trends observed in British Columbia, Canada and from the data presented consistently from 1990 to 2005 by the Early Breast Cancer Trialists' Collaborative Group that this effect on outcome is real [4]. The reality is explained by the record reduction over this period in post-radiotherapy acute and late morbidity due to improved techniques and dosimetry. Ragaz also indicates that, due to lack of radiotherapy trials, there is no clarity on what needs to be irradiated, while the question of who needs to be irradiated can no longer be answered simplistically on the basis of the number of nodes involved. The histopathological extent of nodal involvement judged by the percentage of involved nodes, by the spread within a node and by the presence of extracapsular spread - all indicators of risk of systemic relapse - is more critical than the indicators of increased risk of local failure. The need for more trials in radiotherapy is underlined by the fact that this paradigm shift in attitude and assessment of benefit occurred when the results of the pivotal randomised trials were published in 1997 showing that postoperative radiotherapy could improve long-term overall survival and not just cause long-term morbidity. That a single measurement of Ki67 does predict long-term outcome is discussed by Dowsett and colleagues [5], but with the caveat that such a single assay may not deliver all of the required information. For prognosis alone, a single pretreatment assay suffices - but for predictive information, a second post-treatment assay in the neoadjuvant case is needed. Further assays offer even more information for drug development studies or of drug resistance mechanisms. While breast stroma has been of passing interest to pathologists since at least the 1960s, when elastosis was considered a possible marker of prognosis, the hypothesis that malignant epithelial cells alone are prognostic and the supporting (stromal) tissues are of no prognostic influence has generally been the focus of research and clinical interest. This no longer stands up to scrutiny. Howell and colleagues describe well how stroma is recruited by tumours, is variable from tumour to tumour, and how tumour genes may be downregulated (for adhesion) or upregulated (facilitating cell movement) [6] - all potentially affecting outcome. Stroma does not, however, integrate with other prognostic factors. Stroma is already targeted in various ways in clinical situations. Bevacizumab and the bisphosphonates are already in regular and effective use. There is, therefore, opportunity for further improvement in outcomes as other stromal targets are explored. 'Who would have thought it!' is a statement and not a question. Many did think it, and that is why the critically important trials in postmastectomy radiotherapy were carried out even though some trialists expected the old paradigm to be confirmed. Newer technologies now enable pathologists and clinicians to revisit hypotheses on stroma mooted 40 years ago. Neoadjuvant treatments also facilitate assessment of the prognostic effects of molecular and genetic factors. Clearly, it is having the thought that counts.

Effect of cell cycle specificity of chemotherapeutic agents on the gonadal functions of a pediatric cancer population: role of anti-mullerian hormone (AMH)

OBJECTIVE: The objectives of the study are to 1) determine whether AMH levels could be used to quantify gonadal damage inflicted by gonadotoxic chemotherapy and 2) to examine the effect of cell cycle specificity of the chemotherapeutic agents on the gonadal reserve as evidenced by the post-treatment AMH assay.DESIGN: Cohort study.MATERIALS AND METHODS: Baseline blood samples were obtained from 34 cancer patients (group I: 27 males and 7 females) and 10 normal controls (group II: 7 males and 3 females). Group I was composed of patients treated with chemotherapy for hematological, musculoskeletal and soft tissue cancers in 18,6 and 10 patients, respectively. Patients were followed until the completion of chemotherapy (range1-3 years). A second blood sample was collected at the completion of chemotherapy. AMH assay was completed using a commercially available kit.RESULTS: Using Spearman correlation, baseline AMH levels were significantly correlated with age of the study population (R=0.41, P=0.007). Patients and controls were comparable regarding their age at the initiation of chemotherapy. Baseline AMH levels were comparable between patients and controls irrespective of gender, age, pubertal status and type of malignancy (Boys7.83±4.75ng/ml vs 17.24±7.77 ng/ml; P=0.056) and (Girls 1.53±1.39 ng/ml vs 1.86±0.55 ng/ml; P=0.12)}. Post-chemotherapy AMH levels were not significantly different from baseline in boys (9.86±7.30 ng/ml vs 7.83±4.75 ng/ml, P=0.28). On the other side, post-chemotherapy AMH levels were significantly lower from baseline in girls (0.81±1.11 ng/ml vs 1.53±1.39 ng/ml, P=0.02). In boys and girls there was no significant reduction of post-chemotherapy AMH levels in subjects who received cell cycle specific chemotherapy. Similarly, there was no significant reduction of post-chemotherapy AMH levels in male subjects who received cell cycle non specific chemotherapy, P=0.06. On the contrary, girls who received cell cycle non specific chemotherapy showed significantly lower AMH levels compared to baseline (0.22 0.35 ng/ml vs 1.28 ±1.43 ng/ml; P=0.01).CONCLUSIONS: AMH assay is a sensitive marker for ovarian functions before and after puberty in patients at risk of gonadal damage secondary to chemotherapy. Ovarian reserve appears to be more sensitive to the insult of cell cycle non specific chemotherapeutic agents than the testicular reserve. AMH assay could be added to the initial work up of all patients undergoing chemotherapy to assess the damage to their reproductive potential. OBJECTIVE: The objectives of the study are to 1) determine whether AMH levels could be used to quantify gonadal damage inflicted by gonadotoxic chemotherapy and 2) to examine the effect of cell cycle specificity of the chemotherapeutic agents on the gonadal reserve as evidenced by the post-treatment AMH assay. DESIGN: Cohort study. MATERIALS AND METHODS: Baseline blood samples were obtained from 34 cancer patients (group I: 27 males and 7 females) and 10 normal controls (group II: 7 males and 3 females). Group I was composed of patients treated with chemotherapy for hematological, musculoskeletal and soft tissue cancers in 18,6 and 10 patients, respectively. Patients were followed until the completion of chemotherapy (range1-3 years). A second blood sample was collected at the completion of chemotherapy. AMH assay was completed using a commercially available kit. RESULTS: Using Spearman correlation, baseline AMH levels were significantly correlated with age of the study population (R=0.41, P=0.007). Patients and controls were comparable regarding their age at the initiation of chemotherapy. Baseline AMH levels were comparable between patients and controls irrespective of gender, age, pubertal status and type of malignancy (Boys7.83±4.75ng/ml vs 17.24±7.77 ng/ml; P=0.056) and (Girls 1.53±1.39 ng/ml vs 1.86±0.55 ng/ml; P=0.12)}. Post-chemotherapy AMH levels were not significantly different from baseline in boys (9.86±7.30 ng/ml vs 7.83±4.75 ng/ml, P=0.28). On the other side, post-chemotherapy AMH levels were significantly lower from baseline in girls (0.81±1.11 ng/ml vs 1.53±1.39 ng/ml, P=0.02). In boys and girls there was no significant reduction of post-chemotherapy AMH levels in subjects who received cell cycle specific chemotherapy. Similarly, there was no significant reduction of post-chemotherapy AMH levels in male subjects who received cell cycle non specific chemotherapy, P=0.06. On the contrary, girls who received cell cycle non specific chemotherapy showed significantly lower AMH levels compared to baseline (0.22 0.35 ng/ml vs 1.28 ±1.43 ng/ml; P=0.01). CONCLUSIONS: AMH assay is a sensitive marker for ovarian functions before and after puberty in patients at risk of gonadal damage secondary to chemotherapy. Ovarian reserve appears to be more sensitive to the insult of cell cycle non specific chemotherapeutic agents than the testicular reserve. AMH assay could be added to the initial work up of all patients undergoing chemotherapy to assess the damage to their reproductive potential.

In Vitro Antiviral Activity of Marine Sponges Collected Off Brazilian Coast

This paper describes the in vitro antiviral evaluation of 27 different marine sponges (Porifera) collected off Brazilian coastline in the search for novel drug leads. With these sponges aqueous and organic extracts were prepared and tested for anti-herpetic (HSV-1, KOS strain), anti-adenovirus (human AdV serotype 5) and anti-rotavirus (simian RV SA11) activities. The evaluation of the cytotoxicity and potential antiviral activity of these extracts were performed by using MTT assay. Results were expressed as 50% cytotoxicity (CC50) and 50% effective (EC50) concentrations, respectively, in order to calculate the selectivity indices (SI=CC50/EC50) of each extract. From the 40 sponge extracts tested, 17 extracts showed antiviral action in different degrees. The results concerning the antiviral activity were obtained by using three different strategies: (1) simultaneous assay, when sponge extracts were added to the cells at the same time of the viruses; (2) pre treatment assay, when sponge extracts were added to the cells 15 h prior to the viruses infection; and (3) post treatment assay, when the viruses were added to the cells and remained during 2 h prior to the addition of sponge extracts. The antiviral assays with HSV-1/KOS and AdV-5 showed more promising results when the pre treatment test was employed. In relation to the RV-SA11 virus, only the simultaneous assay showed antiviral activity. The extracts presenting the most promising results were the aqueous extracts of Cliona sp., Agelas sp.2, Tethya sp., Axinella aff corrugata, Polymastia janeirensis and Protosuberites sp., and these extracts deserve special attention in further studies.

Different Results of the Salmonella umu Test Between Three Isomers of Phenylenediamine (PDA) Derivatives

A post-treatment assay of the umu test was performed to detect genotoxicity of 10 phenylenediamine (PDA) derivatives using Salmonella typhimurium TA1535/pSK1002 with/without S9 mix. Seven chemicals (o-PDA, 4-chloro-o-PDA, 4-nitro-o-PDA, p-PDA, 2-chloro-p-PDA, 2-nitro-p-PDA, and 2,5-diaminotoluene) showed positive results with S9 mix, but three chemicals (m-PDA, 4-chloro-m-PDA, and 2,4-diaminotoluene) were negative with and without S9 mix. Four of 7 chemicals (o-PDA, 4-chloro-o-PDA, 4-nitro-o-PDA, and 2-nitro-p-PDA) that gave positive results with S9 mix were also positive without S9 mix. These results indicate that the genotoxicity of PDA derivative possessing m-position amino substituents was not detected in the umu post-treatment assay using TA1535/pSK1002.

ANTIVIRAL EFFECT OF SELECTED MEDICINAL PLANTS 1: EFFECT OF DIOSPYROS BATERI, DIOSPYROS MONBUTENSIS AND SPHENOCENTRUM JOLLYANUM ON POLIO VIRUSES

Methanolic extracts of the different morphological parts of three medicinal plants, Diospyros bateri, Diospyros monbutensis and Sphenocentrum jollyanum were evaluated for their antiviral activities on polio virus Types 1, 2, and 3. The leaf and root extracts of S. jollyanum, the seed extracts of D. monbutensis as well as the leaf extract of D. bateri were active against polio virus Type 2 in both the ‘post-treatment assay' and ‘pre-treatment assay' protocols. The seed extract of D. bateri were active against polio virus Type 2 in ‘post-treatment assay only'. Toxicity level of each plant extract or indicator cell lines were also determined and found to range from Minimum Cytotoxic Dose (MCD50) of 3.9x10-3 mg ml-1 for crude methanol leaf extract of S. jollyanum on Hep-2 (Human Epithelia cell line) to MCD50 of 1.2x10-7 mg ml-1 for leaf methanol extract of D. bateri on Vero cell line (African Green Monkey Fibroblast). Phytochemical screening of the plants revealed that tannins and saponins were present in the three plants while only D. monbutensis contain both combined and free anthraquinones. KEY WORDS: Diospyrus bateri, Diospyros monbutensis, Sphenocentrum jollyanum, polio virus [Nig J Natural Products & Medicine Vol.6 2002: 4-6]

Evaluation of the SOS/ umu-test post-treatment assay for the detection of genotoxic activities of pure compounds and complex environmental mixtures

This study presents an evaluation of the SOS/ umu-test after introducing an additional dilution and incubation in the post-treatment assay. This treatment reduces the influence of coloured test compounds that otherwise affect the colorimetric determination of the β-galactosidase activity and the bacterial growth measurement during the testing of complex environmental samples. The post-treatment assay significantly increased the β-galactosidase activity and consequently the enzyme induction ratios at higher doses of model genotoxins 4-nitroquinoline- N-oxide, N-methyl- N′-nitro- N-nitrosoguanidine, 2-aminoanthracene, benzo( a)pyrene with low or no effect on the sensitivity of the test itself. On the other hand tests of environmental extracts indicated significant increases in sensitivity after additional incubation. 4-Nitroquinoline- N-oxide treatments of bacteria in the test affected cell division and caused filamentous growth. The size of filamentous bacteria and incidence rate of the length categories was positively correlated with the concentrations of genotoxins. Presence of filamentous tester bacteria proved induction of SOS response and genotoxic activity of environment samples in SOS/ umu-test.

Toxicities of Three Synthetic Pyrethroids and Selected Orchard Insecticides to the Tufted Apple Budmoth, Platynota ideausalis (Lepidoptera: Tortricidae)1

Laboratory tests using residue exposure methods showed that the synthetic pyrethroids permethrin, fenvalerate, and flucythrinate were highly toxic to tufted apple budmoth, Platynota idaeusalis (Walker), larvae. Piperonyl butoxide used in conjunction with the pyrethroids showed neither synergistic nor antagonistic action for larvae of P. ideausalis or redbanded leafroller, Argyrotaenia velutinana (Walker). Field-laboratory tests with residues of permethrin and 5 nonpyrethroid insecticides showed that chlorpyrifos was the most toxic to neonate and 7-day-old P. idaeusalis larvae. All treatments gave poor larval mortality (≤65%) at 15-day posttreatment assay, and none of the insecticide residues was toxic to the egg. Laboratory tests using residue methods for eight nonpyrethroid insecticides showed chlorpyrifos, azinphosmethyl, and methomyl to be most toxic to 9-day-old P. idaeusalis larvae. Laboratory tests for ovicidal activity of azinphosmethyl, chlorpyrifos, methomyl, and permethrin showed that none of the treatment residues was toxic to the egg and only methomyl was toxic by contact.