Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein(RNP)electroporation

Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising cost.Furthermore,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in cattle.In this point of view,electroporation in embryos has been risen as an alternative.Results First,editing efficiency of our electroporation methods were tested for embryos.Presence of mutation on embryo was confirmed by T7E1 assay.With first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,respectively.In case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for BLG.Afterwards,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing.Thirteen recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double KO.Ten surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both genes.Conclusions These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied.Finally,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.

Robust Machine Learning Technique to Classify COVID-19 Using Fusion of Texture and Vesselness of X-Ray Images

Manual investigation of chest radiography(CXR)images by physicians is crucial for effective decision-making in COVID-19 diagnosis.However,the high demand during the pandemic necessitates auxiliary help through image analysis and machine learning techniques.This study presents a multi-threshold-based segmentation technique to probe high pixel intensity regions in CXR images of various pathologies,including normal cases.Texture information is extracted using gray co-occurrence matrix(GLCM)-based features,while vessel-like features are obtained using Frangi,Sato,and Meijering filters.Machine learning models employing Decision Tree(DT)and RandomForest(RF)approaches are designed to categorize CXR images into common lung infections,lung opacity(LO),COVID-19,and viral pneumonia(VP).The results demonstrate that the fusion of texture and vesselbased features provides an effective ML model for aiding diagnosis.The ML model validation using performance measures,including an accuracy of approximately 91.8%with an RF-based classifier,supports the usefulness of the feature set and classifier model in categorizing the four different pathologies.Furthermore,the study investigates the importance of the devised features in identifying the underlying pathology and incorporates histogrambased analysis.This analysis reveals varying natural pixel distributions in CXR images belonging to the normal,COVID-19,LO,and VP groups,motivating the incorporation of additional features such as mean,standard deviation,skewness,and percentile based on the filtered images.Notably,the study achieves a considerable improvement in categorizing COVID-19 from LO,with a true positive rate of 97%,further substantiating the effectiveness of the methodology implemented.

Clinicopathological Description of a Chronic Lymphocytic Leukemia in a Dog

Hematopoietic tumors are associated with diagnostic and therapeutic challenges and a poor prognosis. Furthermore, their pathophysiology in dogs is not well understood. Chronic lymphocytic leukemia has been rarely reported in golden retrievers, perhaps due to difficulties in diagnosis and care. An 8-year-old spayed female golden retriever was referred to our hospital with a 2-month history of fatigue, weakness, and selective appetite. The dog was diagnosed with chronic lymphocytic leukemia based on blood smear evaluation. Despite treatment, the dog deteriorated and the owners opted for euthanasia. Histopathological examination revealed atypical lymphocytes in the spleen, extramedullary hematopoiesis in the spleen and liver, bone marrow depletion, moderate neutrophilic myocarditis, and acute renal tubular necrosis. In an immunohistochemical panel, cells in the spleen and bone marrow were positive for anti-CD20, anti-CD45, anti-CD3, anti-IBA-1, anti-Ki67, anti-MUM-1, and anti-PAX-5. In conclusion, the leukemia diagnosis was confirmed through reactive histiocytosis, marked extramedullary hematopoiesis, and other clinical and laboratory findings. Herein, we report a case of chronic lymphocytic leukemia in a female dog with clinical, laboratory, and necroscopy findings.

Zinc oxide nanoparticles accelerate the healing of methicillin-resistant Staphylococcus aureus(MRSA)-infected wounds in rabbits

Objective:To synthesize zinc oxide nanoparticles(ZnONPs)and evaluate their antibacterial and wound healing effects against wounds infected with methicillin-resistant Staphylococcus aureus(MRSA).Methods:ZnONPs were prepared by sol-gel method and characterized by X-ray diffraction(XRD)analysis and scanning electron microscopy(SEM).A total of 18 rabbits were divided into three groups:the ZnONPs group,the gentamicin group and the control group.A wound of 3 cm^(2) was inflicted on each rabbit and contaminated with MRSA inoculum.Treatment was started from the fourth day post-surgery.Wound healing,microbiological analysis,and histopathological analysis were performed to assess the efficacy of ZnONPs ointment.Results:XRD analysis confirmed the hexagonal wurtzite structure of the ZnONPs with an average crystallite size of 29.23 nm.SEM revealed discoid-shaped ZnONPs with a rough surface and an average size of 48.36 nm.Energy-dispersive X-ray analysis confirmed the purity of ZnONPs.Moreover,the particle size ranged from 100-700 nm with a high agglomeration trend.Treatment with ZnONPs promoted MRSA-infected wound healing.In addition,ZnONPs showed a good antibacterial effect as evidenced by a dose-dependent increase in the zone of inhibition.Conclusions:ZnONPs accelerate the healing of MRSA-infected wounds.Therefore,it can be explored for the treatment of MRSA infection.

Quercetin Alleviates Lipopolysaccharide-Induced Cardiac Inflammation via Inhibiting Autophagy and Programmed Cell Death

Objective The aim of this study is to explore the potential modulatory role of quercetin against Endotoxin or lipopolysaccharide(LPS)induced septic cardiac dysfunction.Methods Specific pathogen-free chicken embryos(n=120)were allocated untreated control,phosphate buffer solution(PBS)vehicle,PBS with ethanol vehicle,LPS(500 ng/egg),LPS with quercetin treatment(10,20,or 40 nmol/egg,respectively),Quercetin groups(10,20,or 40 nmol/egg).Fifteenday-old embryonated eggs were inoculated with abovementioned solutions via the allantoic cavity.At embryonic day 19,the hearts of the embryos were collected for histopathological examination,RNA extraction,real-time polymerase chain reaction,immunohistochemical investigations,and Western blotting.Results They demonstrated that the heart presented inflammatory responses after LPS induction.The LPS-induced higher mRNA expressions of inflammation-related factors(TLR4,TNFα,MYD88,NF-κB1,IFNγ,IL-1β,IL-8,IL-6,IL-10,p38,MMP3,and MMP9)were blocked by quercetin with three dosages.Quercetin significantly decreased immunopositivity to TLR4 and MMP9 in the treatment group when compared with the LPS group.Quercetin significantly decreased protein expressions of TLR4,IFNγ,MMP3,and MMP9 when compared with the LPS group.Quercetin treatment prevented LPS-induced increase in the mRNA expression of Claudin 1 and ZO-1,and significantly decreased protein expression of claudin 1 when compared with the LPS group.Quercetin significantly downregulated autophagyrelated gene expressions(PPARα,SGLT1,APOA4,AMPKα1,AMPKα2,ATG5,ATG7,Beclin-1,and LC3B)and programmed cell death(Fas,Bcl-2,CASP1,CASP12,CASP3,and RIPK1)after LPS induction.Quercetin significantly decreased immunopositivity to APOA4,AMPKα2,and LC3-II/LC3-I in the treatment group when compared with the LPS group.Quercetin significantly decreased protein expressions of AMPKα1,LC3-I,and LC3-II.Quercetin significantly decreased the protein expression to CASP1 and CASP3 by immunohistochemical investigation or Western blotting in treatment group when compared with LPS group.Conclusion Quercetin alleviates cardiac inflammation induced by LPS through modulating autophagy,programmed cell death,and myocardiocytes permeability.

Development of genome engineering technologies in cattle: from random to specific

The production of transgenic farm animals(e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos(zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure.However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer(SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies(e.g.,ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies wil accelerate our understanding of genetic traits in bovine and wil be readily adapted for bio-medical applications in cattle.

Effect of Cadmium on Rat Leydig Cell Testosterone Production and DNA Integrity in vitro

Cadmium （Cd） is an elemental heavy metal with widely recognized toxicity. Its long-term use in industrial processes and daily activities has caused alarming levels of Cd contamination in the natural environment. According to the estimates by the Agency of Toxic Substances and Disease Registry in the US, 25 000 to 30 000 metric tons of Cd is annually released to the environment . Results of previous studies have demonstrated that several organs are targets of Cd, but the most important of these targeted organs may be the testes.

The Effect of a<i>Lactobacillus</i>-Based Probiotic for the Control of Necrotic Enteritis in Broilers

Necrotic Enteritis (NE) caused by Clostridium perfringens (CP) in poultry is probably the most important bacterial disease in terms of economic implications. The disease is multi-factorial and is invariably associated with predisposing factors. The present study investigated the effect of a commercially available Lactobacillus-based probiotic (FM-B11) for the control of necrotic enteritis in broiler chickens. In experiment 1, one-day-of-hatch broiler chicks were randomly allocated to the following treatment groups: 1) Non-challenged (NC);2) Challenged (C);3) Challenged + probiotic (C+ FM-B11). Prior to placement, chicks in groups 2 and 3 received 0.25 mL of Salmonella typhimurium (ST) containing 105 cfu of viable cells by oral gavage. At 14, 15 and 16 days of age, all chicks in group 3 were treated with FM-B11 in the drinking water at a concentration of 106 cfu/ml. At 21d of age, all chicks in groups 2 and 3, were individually challenged with 5 × 104 sporulated oocysts of E. maxima by oral gavage. At 26d of age, all chicks in groups 2 and 3, were individually challenged with 108 cfu CP;body weight (BW) was recorded prior to challenge. The experiment was terminated at 29 days of age and the following parameters were evaluated: NE-associated mortality, CP lesion scores, CP concentrations in ileum, BW, and body weight gain (BWG). Chicks treated with FM-B11 had significantly (P < 0.05) higher body weight gain after challenge when compared to control challenge chickens. Total mortality was higher in the C group (48.8%) when compared to the C + FM-B11 (12.7%). Even though there was no significant (P > 0.05) difference in lesion score between C and C + FM-B11, group C + FM-B11 had significantly (P < 0.05) lower total number of cfu of CP recovered from the ileal mucosa and content samples when compared to group C. Experiment 2 was a unique and remarkable case report of a field outbreak of NE in a commercial broiler farm in Argentina. A reduction and control of the mortality associated with NE following 3 days of administration of FM-B11 was observed as compared with the control non treated house. These results imply that the commercially available Lactobacillus-based probiotic FM-B11 was able to reduce the severities of NE, as a secondary bacterial infection, in an experimental NE challenge model;as well as, in a commercial field outbreak of NE.

Evaluation of the Aurora Kinase Inhibitor, ZM447439, in Canine Malignant Lymphoid Cells <i>in Vitro</i>

Aurora kinases play an important role in the cell cycle. These enzymes help establish mitotic spindles by directing centrosome duplication and separation and by regulating the spindle assembly checkpoint thereby helping control cytokinesis. An over-expression of aurora kinases has been reported in a variety of human tumors. In this study, we identified the expression of aurora-A and aurora-B kinases in canine malignant lymphoid cells. We also evaluated the effects of the aurora kinase inhibitor (ZM447439), and found that this inhibitor decreases cell viability, increases DNA content change, and leads to apoptosis in canine B- and T-cell lymphoid cell lines. The lymphotoxicity induced by ZM447439 in these canine lymphoid cell lines suggests that further in vivo evaluation of aurora kinase inhibitors as a potential treatment for canine malignant lymphoid tumors is warranted.

The Influence of Culture Medium Type on Cellular Phenotype of Canine Adipose Derived Stem Cells

Canine adipose derived stem cells (ASCs) hold a great promise for the therapy of osteoarthritis in veterinary medicine. Current therapy is an autologous, stromal vascular fraction. Allogeneic ASCs provide many advantages, including efficient, cost-effective treatments while eliminating a surgical procedure in a diseased animal. Cultured ASCs can be expanded and characterized, allowing selection of desirable qualities. Use of allogeneic ASCs requires selection of a culture medium that provides consistent, desirable cellular products. The supplements within a medium can greatly influence cellular phenotypes. We hypothesized that medium type influenced cellular phenotype, allowing selection of a specified cellular product for clinical applications. We evaluated ASCs derived from adipose tissue of six dogs, assessing mRNA expression of proinflammatory: interleukin-1b, cyclooxygenase-2, and anti-inflammatory mediators: tissue inhibitor metalloproteinase-2 and interleukin -1 receptor antagonist, via quantitative RT-PCR prior to, and following culture in five cell culture media: basic cell growth medium (BGM), Keratinocyte N acetyl-L-cysteine supplemented (KNAC) medium, Multipotent Adult Progenitor Cell (MAPC) medium, serum free medium (SFM) and xeno-free medium. Major histocompatability complex I (MHCI), major histocompatability complex II (MHCII), CD44 and CD90 immunophenotypes were assessed via flow cytometry analysis. Tri-lineage differentiation (bone, adipose and cartilage tissue) was utilized to verify multipotency. SFM and xeno-free culture conditions did not produce cell expansion sufficient to assess phenotype. ASCs prior to culture had wide variability in all mediator levels, while culturing in the remaining conditions resulted in more predictable expression levels of inflammatory mediators, with a decrease in all levels. Cultured ASCs retained expression of cell surface markers MHCI, CD44 and CD90, while decreasing MHCII expression levels. KNAC and MAPC medium conditions consistently produced tri-lineage differentation;BGM, SFM and xeno-free medium did not. Culture condition will influence phenotype of ASCs, and should be selected according to the intended therapeutic effect.

Molecular characteristics,antibiogram and prevalence of multi-drug resistant Staphylococcus aureus (MDRSA) isolated from milk obtained from culled dairy cows and from cows with acute clinical mastitis

Objectives: To study the molecular characteristics, antibiogram and prevalence of multidrug resistant Staphylococcus aureus(S. aureus)(MDRSA) isolated from milk obtained from culled dairy cows and from cows with acute clinical mastitis.Methods: Bacteria were cultured from 188 quarter milk samples obtained from cows before culling(n=139) and from cows affected with acute mastitis(n=49) belonging to 10 dairy farms. The bacteria were identified using colony moiphology, Gram staining and biochemical characteristics. S. aureus isolates were then subjected to molecular characterization using PCR targeting 16 S rRNA and mecA gene to identify Methicillin resistant S. aureus(MRSA). The antibiogram of all isolates was performed using the Kirby-Bauer disk diffusion method against 10 commonly used antibiotics in dairy farms.Results: S. aureus was isolated from 19(13.7%) samples obtained from culled cows and 11(22.4%) samples obtained from cows with acute mastitis. In both culled cows and cows with acute mastitis, in vitro antibiogram revealed that 100% of S. aureus isolates were resistant to erythromycin, penicillin G, streptomycin, doxycyclin, and trimethoprim/sulpha. The prevalence of MRSA in milk of culled cows and cows with acute mastitis was 26.3% and 18.2%, respectively, with an overall prevalence of 3.7% among all samples. All MRSA isolates were completely resistant to all tested antibiotics. All MRSA isolates were positive for the presence of the mecA gene.Conclusions: MRSA carrying the mecA gene were isolated from mastitic milk from dairy cows in Jordan for the first time. MRSA may pose a potential health risk to the public, farm workers and veterinarians.

IN VIVO TRANS-RECTAL ULTRASOUND-COUPLED NEAR-INFRARED OPTICAL TOMOGRAPHY OF INTACT NORMAL CANINE PROSTATE

This is the first tomography-presentation of the optical properties of a normal canine prostate,in vivo,in its native intact environment in the pelvic canal.The imaging was performed by trans-rectal near-infrared(NIR)optical tomography in steady-state measurement at 840 nm on three sagittal planes across the right lobe,middle-line,and left lobe,respectively,of the prostate gland.The NIR imaging planes were position-correlated with concurrently applied trans-rectal ultrasound,albeit there was no spatial prior employed in the NIR tomography reconstruction.The reconstructed peak absorption coefficients of the prostate on the three planes were 0.014,0.012,and 0.014mm^(−1).The peak reduced scattering coefficients were 5.28,5.56,and 6.53 mm^(−1).The peak effective attenuation coefficients were 0.45,0.43,and 0.50 mm^(−1).The absorption and effective attenuation coefficients were within the ranges predictable at 840 nm by literature values which clustered sparsely from 355 nm to 1064 nm,none of which were performed on a canine prostate with similar conditions.The effective attenuation coefficients of the gland were shown to be generally higher in the internal aspects than in the peripheral aspects,which is consistent with the previous findings that the urethral regions were statistically more attenuating than the capsular regions.

Biological analysis of an innovative biodegradable antibiotic eluting bioactive glass/gypsum composite bone cement for treating experimental chronic MRSA osteomyelitis

A multi-barrier antibiotics loaded biodegradable composite bone cement for resolving chronic osteomyelitis has been studied to understand the physico-mechanical properties,drug loading/eluting efficiency,and different merits and demerits prior to clinical application.After successful induction of bone infection in 28 rabbits using methicillin-resistant Staphylococcus aureus(MRSA)strains,calcium sulfate/bioactive glass based composite cement was implanted in 12 defects to assess its performance over parenteral therapy with microscopic and radiological examination for 90 days.The composite cement revealed acceptable physico-mechanical properties and controlled drug elution kinetics.Furthermore,the antibiotics concentrations in bone up to 42 days were sufficient to kill MRSA without eliciting adverse drug reactions.The striking feature of platelets aggregation by composite cement could assist bone healing.The controlled degradation with simultaneous entrapment of composite cement within the osteoid tissues and complete repair of infected cortical defects(holes)in rabbit tibia at 6 weeks indicated the excellent anti-infective and osteoconductive properties of composite cement.Thus,the animal study demonstrated the superiority of composite over injectable antibiotic therapy based on infection resolution and bone regeneration.We thereby conclude that the composite cement can be effectively applied in the treatment of resistant cases of chronic osteomyelitis.

In vivo percutaneous reflectance spectroscopy of fatty liver development in rats suggests that the elevation of the scattering power is an early indicator of hepatic steatosis

This study assessed whether there was a scat tering spectral mar ker quantifiable by reflectance measurements that could indicate early development of hepatic steatosis in rats for potential applications to pre procurement organ evaluation.Sixteen rats were fed a methionine choline-deficient(MCD)diet and eight rats were fed a normal diet.Direct assessment of the liver parenchyma of rats in vivo was performed by percut aneous reflect ance spectroscopy using a single fiber probe at the beginning of diet-intake and arbitrary post-diet-intake times up to 11 weeks to render longitudinal comparison.Histological sampling of the liver over the duration of diet adm inistration was performed on two MCD diet treated rats and one control rat eutha-nized after reflectance spectroscopy measurement.The images of hematoxylin/eosin-stained liver specimens were analyzed morphometrically to evahuate the lipid size changes associated with the level of steatosis.The MCD-diet-treated group(n=16)had mild steatosis in seven rats,moderate in three rats,severe in six rats,and no other significant pathology.No control rats(n=8)developed hepatic steatosis.Among the parameters retrieved from per-SfS,only the scat tering power(can be either positive or negative)appeared to be statist ically diferent between MCD-treated and control livers.The scattering power for the 16 MCD-diet-treated livers at the time of euthanasia and presenting various levels of steatosis was 033±0.21,in comparison to 0.036±0.25 of the eight control livers(p=0.0189).When evaluated at days 12 and 13 combined,the scattering power of the 16 MCD-diet-treated livers was 032±0.17,in comparison to 0.10±0.11 of the eight control livers(p=0.0017).All of four MCD-treated livers harvested at days 12 and 13 presented mild steatosis with sub-micron size lipid droplets,even though none of the MCD-treated livers were sonogr aphically remarkable for fatty changes.The elevation of the scattering power may be a valuable marker indicating early hepatic steatosis before the steatosis is sonographically detectable.

Development of <i>in vitro</i>culture of rat Leydig cells after purification with Nycodenz gradient

The study examined the effect of human Chorionic Gonadotrophin (hCG) and/or Insulin Transferring Sodium Selenite (ITS) on the proliferation and development of rat Leydig cells purified by Nycodenz gradient. Leydig cells purity, viability and proliferation after purification and 3 days of cultured were evaluated. Leydig cells 1 × 106 cells/ml were cultured in DMEM containing 10% Newborn Calf Serum (NBCS) and divided into four kinds of treatments 1) as a control, 2) control + 2.5 IU/ml hCG, 3) control + ITS (5 μg/ml insulin, 10 μg/ml transferrin, 5 μg/ml Se), and 4) control + hCG + ITS. Leydig cells purification results showed 91.40% of purity, viability was 98.17% and concentration 7.03 × 106 cells/ml. The addition of ITS and hCG + ITS in DMEM produced Leydig cell proliferation by 88.35% and 90.64% higher than in controls (86.82%) (p p

Review of immunological responses to porcine coronaviruses and implications on population based control strategies in epidemic and endemic infections

Five major porcine coronaviruses(COVs)have been identified which cause severe gastrointestinal(GI)and respiratory disease in pigs.They include transmissible gastroenteritis(TGEV),porcine epidemic diarrhea virus(PEDV),porcine deltacoronavirus,porcine respiratory coronavirus,and porcine hemagglutinating encephalomyelitis.These diseases,especially TGEV and PEDV,have caused epidemics in Europe,Asia,and the Americas over the past 50 years,causing significant economic losses to swine producers.As pigs are a major protein source worldwide there is great interest in understanding,controlling,and preventing these diseases.These diseases have no cure,and current vaccines are not fully protective.On-farm prevention and biosecurity are difficult to enforce and have not stopped the spread of these diseases between herds.Recent advances in the immunology of porcine COVs has revealed that the immune response to porcine COVs shares many similarities with the response to human COVs,leading to increased interest in pigs as models for human disease.Highlights of these advances include the key role of local antigen presenting cells in the gastrointestinal tract in stimulating a protective immune response.This understanding has lead to new proposed vaccines.Advances in the understanding of the ways the viruses evade and degrade the host immune system have also lead to novel proposed therapies.Many of these therapies are in the early development stages,as researchers attempt to create efficacious,cost-effective,and practical therapies for these diseases.

Spontaneous Mammary Carcinomas in Female Dogs: Association between Immunohistochemical Degrees of Neoplasia Aggressiveness and Residual Pyrethroids

Of the tumors diagnosed in the female dogs have the highest mammary neoplasias incidence. These neoplasias can be influenced by environmental contaminants. Despite evidence of pyrethroid toxicity, carcinogenic potential has not yet been sufficiently elucidated, there is a need to investigate their involvement in mammary tumor. In previous studies, pyrethroid residues were detected in female dogs with mammary neoplasia, however was not investigate the influence of this insecticide in the genesis and aggressiveness of mammary cancer. This study aimed to investigate possible relations between pyrethroid residues and aggressiveness of mammary carcinoma in female dogs. Fifty selected female dogs were divided into five groups of 10 animals each: the Control group, female dogs without mammary neoplasia;the groups Luminal A, Luminal B, HER-2 Superexpression and Basal were constituted by female dogs that presented inguinal mammary carcinoma classified immunohistochemically. The aggressiveness of carcinomas was evaluated by immunohistochemistry (HER-2, p63, estrogen receptor). Residual concentrations of the pyrethroids from the mammary gland and fat tissue adjacent to it were determined by HPLC. Data were analyzed by Chi-Square test. Of the all animals, six presented residues of pyrethroids in mammary samples and 10 presented it in fat tissue samples. There was no statistical evidence that pyrethroids are involved in mammary carcinoma aggressiveness in female dogs.

Antitumor Effects and Acute Oral Toxicity Studies of a Plant Extract Mixture Containing <i>Rhus verniciflua</i>and Some Other Herbs

A novel antitumor agent was developed from six kinds of herbs containing Rhus verniciflua (Rv-PEM01). The components were traditionally established for each formula for traditional medicine. The formula was designed to affect antitumor effect as well as maintain host immune functions. First, we investigated the antiproliferative activities of Rv-PEM01 on human and canine tumor cell lines in vitro, and on antitumor effects using BALB/cAJcl-nu/nu mice in vivo. Acute oral toxicity of Rv-PEM01 was also investigated in vivo in ddY mice. Rv-PEM01 exhibited antiproliferative activities against PC-3 (IC50: 0.328 ± 0.081 mg/ml), A549 (IC50: 0.520 ± 0.070 mg/ml), D-17 (IC50: 0.124 ± 0.037 mg/ml) and MRC-5 (IC50: 0.505 ± 0.058 mg/ml) cells. Luteolin 7-β-D-glucopyranoside and apigenin 7-β-D-glucopyranoside were identified as the main active compounds in Rv-PEM01 by HPLC analysis. The single dose toxicity study of Rv-PEM01 did not result in any deaths or abnor-malities in daily behavior, body weight gain, or anatomical observations at necropsy. Thus, so we could not calculate the 50% lethal dose (LD50) in mice, but it would be higher than 5.0 g/kg. Treat- ment with Rv-PEM01 at a dose of 2.5 g/kg tended to show antitumor activities on mice bearing Colon26 tumors compared with the control group. It was concluded that the formula was a safe antitumor agent with no side effects on mouse physiological function as judged by survival and organ weight.