Alternative to antibiotic growth promoters: beneficial effects of Saccharomyces cerevisiae and/or Lactobacillus acidophilus supplementation on the growth performance and sustainability of broilers' production.

Although antibiotics growth promoters (AGPs), including zinc-bacitracin (ZnB), can threaten human health due to developing antimicrobial resistance, as well as drug residue in animal and poultry products, ZnB is still widely used, particularly in developing countries, for the sustainability of poultry farming. The present investigation aims to assess the use of Saccharomyces cerevisiae and Lactobacillus acidophilus, with or without a prebiotic (mannooligosaccharide, MOS), as alternatives to ZnB. For this reason, 150 one-day-old chicks were grouped into six groups, designated negative control, LA, SC, ZnB, SA + MOS, and LA + MOS (5 replicates of 5 chicks for each group). Chicks kept in the control group were fed the basal diet. Chickens kept in LA and SC groups received L. acidophilus, S. cerevisiae at a 1 g/kg diet and 2 g/Kg, respectively. Chickens kept in ZnB received ZnB at 0.5 g/kg. Chicks kept in the SC + MOS and LA + MOS were fed a basal diet containing 2 g S. cerevisiae + 1 g MOS/kg or 1 g L. acidophilus + 1 g MOS /kg, respectively. The efficacy was assessed based on the growth performance, carcass traits, meat quality, nutrient digestibility, and blood biochemistry composition during the entire trial 1-36 days of age. Results showed that chicks kept in the SC group had greater BW than the control (p < 0.05). Chicks kept in the SC, LA, SC + MOS, and LA + MOS consumed less feed than the control and Zn-B groups (p < 0.05). Supplementation with S. cerevisiae resulted in a better (p < 0.05) feed conversion rate (FCR) than the control group. Supplementation with L. acidophilus + MOS significantly increased (p < 0.05) the relative liver weight compared to those supplemented with ZnB, S. cerevisiae, and L. acidophilus. In addition, supplementation with ZnB-induced spleen hypertrophy compared to S. cerevisiae and L. acidophilus-supplemented groups (p < 0.05). Plasma, meat, and liver cholesterol, as well as the cholesterol-to-lipid ratio of meat and liver, were significantly decreased (p < 0.05) in both SC and LA groups compared to the control group. Our research indicates that adding 2 g/kg of S. cerevisiae to broiler feed can effectively replace ZnB and enhance productive performance and economic profits, making it a viable and sustainable option for broiler farming.

Genomic insight into Campylobacter jejuni isolated from commercial turkey flocks in Germany using whole-genome sequencing analysis.

Campylobacter (C.) jejuni is a zoonotic bacterium of public health significance. The present investigation was designed to assess the epidemiology and genetic heterogeneity of C. jejuni recovered from commercial turkey farms in Germany using whole-genome sequencing. The Illumina MiSeq® technology was used to sequence 66 C. jejuni isolates obtained between 2010 and 2011 from commercial meat turkey flocks located in ten German federal states. Phenotypic antimicrobial resistance was determined. Phylogeny, resistome, plasmidome and virulome profiles were analyzed using whole-genome sequencing data. Genetic resistance markers were identified with bioinformatics tools (AMRFinder, ResFinder, NCBI and ABRicate) and compared with the phenotypic antimicrobial resistance. The isolates were assigned to 28 different sequence types and 11 clonal complexes. The average pairwise single nucleotide-polymorphisms distance of 14,585 SNPs (range: 0-26,540 SNPs) revealed a high genetic distinction between the isolates. Thirteen virulence-associated genes were identified in C. jejuni isolates. Most of the isolates harbored the genes flaA (83.3%) and flaB (78.8%). The wlaN gene associated with the Guillain-Barré syndrome was detected in nine (13.6%) isolates. The genes for resistance to ampicillin (bla OXA), tetracycline [tet(O)], neomycin [aph(3')-IIIa], streptomycin (aadE) and streptothricin (sat4) were detected in isolated C. jejuni using WGS. A gene cluster comprising the genes sat4, aph(3')-IIIa and aadE was present in six isolates. The single point mutation T86I in the housekeeping gene gyrA conferring resistance to quinolones was retrieved in 93.6% of phenotypically fluoroquinolone-resistant isolates. Five phenotypically erythromycin-susceptible isolates carried the mutation A103V in the gene for the ribosomal protein L22 inferring macrolide resistance. An assortment of 13 ß-lactam resistance genes (bla OXA variants) was detected in 58 C. jejuni isolates. Out of 66 sequenced isolates, 28 (42.4%) carried plasmid-borne contigs. Six isolates harbored a pTet-like plasmid-borne contig which carries the tet(O) gene. This study emphasized the potential of whole-genome sequencing to ameliorate the routine surveillance of C. jejuni. Whole-genome sequencing can predict antimicrobial resistance with a high degree of accuracy. However, resistance gene databases need curation and updates to revoke inaccuracy when using WGS-based analysis pipelines for AMR detection.

Assessment of three alternative methods for bacterial disinfection of hatching eggs in comparison with conventional approach in commercial broiler hatcheries.

The disinfection of commercial hatching eggs before incubation is a common strategy to reduce potential vertical transmission of bacterial and fungal infections from the eggshell to one-day-old chicks that may prevail in poultry products and eventually reach the end consumer. The present investigation focuses on the parallel testing and application of four different disinfection methods (conventional and alternative) under commercial hatchery conditions against natural eggshell bacterial contamination. Hatching eggs from two ROSS 308 broiler breeder flocks were selected and divided into six different groups: two groups were not disinfected and served as negative controls, and four were independently disinfected following product specifications and protocols. From each group, a sample of 100 hatching eggs was selected for bacterial re-isolation, utilizing a modified shell rinse method. Colony-forming units (cfu) from the shell rinse suspensions were determined and analyzed to establish cfu values for each tested egg. These values were analyzed to determine the bacterial disinfection capacity of the four disinfection methods under commercial hatchery conditions. The tested methods were hydrogen peroxide + alcohol, peracetic acid, low energy electron beam, and the gold standard in practice: formaldehyde. Among these methods, formaldehyde, peracetic acid, and low energy electron beam showed a significant difference when compared to the non-disinfected groups whereas hydrogen peroxide + alcohol did not. The bacterial disinfection capacity of the tested methods was compared as well to the gold standard method formaldehyde fumigation and only low energy electron beam achieved similar disinfection levels as formaldehyde. According to our data, three methods significantly reduce the bacterial load on the eggshell of hatching eggs under commercial hatching conditions, including potential alternative methods such as low energy electron beam that perform similar to the gold standard in practice.

Cryptosporidiosis: From Prevention to Treatment, a Narrative Review.

Cryptosporidiosis is a water- and food-borne zoonotic disease caused by the protozoon parasite of the genus Cryptosporidium. C. hominis and C. parvum are the main two species causing infections in humans and animals. The disease can be transmitted by the fecal-oral route as well as the respiratory route. The infective stage (sporulated oocysts) is resistant to different disinfectants including chlorine. Currently, no effective therapeutic drugs or vaccines are available to treat and control Cryptosporidium infection. To prevent cryptosporidiosis in humans and animals, we need to understand better how the disease is spread and transmitted, and how to interrupt its transmission cycle. This review focuses on understanding cryptosporidiosis, including its infective stage, pathogenesis, life cycle, genomics, epidemiology, previous outbreaks, source of the infection, transmission dynamics, host spectrum, risk factors and high-risk groups, the disease in animals and humans, diagnosis, treatment and control, and the prospect of an effective anti-Cryptosporidium vaccine. It also focuses on the role of the One Health approach in managing cryptosporidiosis at the animal-human-environmental interface. The summarized data in this review will help to tackle future Cryptosporidium infections in humans and animals and reduce the disease occurrence.

Influence of COVID-19 on the sustainability of livestock performance and welfare on a global scale.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently spreading worldwide. The pandemic has already had significant adverse effects on human civilization, the environment, and the ecosystem at national and global levels. Moreover, the various sectors of the food production chain, particularly agriculture and livestock, have also been significantly affected in terms of production sustainability and economic losses. The global pandemic has already resulted in a sharp drop in meat, milk, and egg production. Restrictions of movement at national and international levels, implemented as a part of control strategies by public health sectors, have negatively impacted business related to the supply of raw materials for livestock farmers and farm outputs, veterinary services, farmworkers, and animal welfare. This review highlights the significant impacts of COVID-19 on the sustainability of livestock performance, welfare on a global scale, and strategies for mitigating these adverse effects.

Occurrence, Phenotypic and Molecular Characteristics of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Healthy Turkeys in Northern Egypt.

Poultry is one of the most important reservoirs for zoonotic multidrug-resistant pathogens. The indiscriminate use of antimicrobials in poultry production is a leading factor for development and dissemination of antimicrobial resistance. This study aimed to describe the prevalence and antimicrobial resistance of E. coli isolated from healthy turkey flocks of different ages in Nile delta region, Egypt. In the current investigation, 250 cloacal swabs were collected from 12 turkey farms in five governorates in the northern Egypt. Collected samples were cultivated on BrillianceTM ESBL agar media supplemented with cefotaxime (100 mg/L). The E. coli isolates were identified using MALDI-TOF-MS and confirmed by a conventional PCR assay targeting 16S rRNA-DNA. The phenotypic antibiogram against 14 antimicrobial agents was determined using the broth micro-dilution method. DNA-microarray-based assay was applied for genotyping and determination of both, virulence and resistance-associated gene markers. Multiplex real-time PCR was additionally applied for all isolates for detection of the actual most relevant Carbapenemase genes. The phenotypic identification of colistin resistance was carried out using E-test. A total of 26 E. coli isolates were recovered from the cloacal samples. All isolates were defined as multidrug-resistant. Interestingly, two different E. coli strains were isolated from one sample. Both strains had different phenotypic and genotypic profiles. All isolates were phenotypically susceptible to imipenem, while resistant to penicillin, rifampicin, streptomycin, and erythromycin. None of the examined carbapenem resistance genes was detected among isolates. At least one beta-lactamase gene was identified in most of isolates, where blaTEM was the most commonly identified determinant (80.8%), in addition to blaCTX-M9 (23.1%), blaSHV (19.2%) and blaOXA-10 (15.4%). Genes associated with chloramphenicol resistance were floR (65.4%) and cmlA1 (46.2%). Tetracycline- and quinolone-resistance-associated genes tetA and qnrS were detected in (57.7%) and (50.0%) of isolates, respectively. The aminoglycoside resistance associated genes aadA1 (65.4%), aadA2 (53.8%), aphA (50.0%), strA (69.2%), and strB (65.4%), were detected among isolates. Macrolide resistance associated genes mph and mrx were also detected in (53.8%) and (34.6%). Moreover, colistin resistance associated gene mcr-9 was identified in one isolate (3.8%). The class 1 integron integrase intI1 (84.6%), transposase for the transposon tnpISEcp1 (34.6%) and OqxB -integral membrane and component of RND-type multidrug efflux pump oqxB (7.7%) were identified among the isolates. The existing high incidence of ESBL/colistin-producing E. coli identified in healthy turkeys is a major concern that demands prompt control; otherwise, such strains and their resistance determinants could be transmitted to other bacteria and, eventually, to people via the food chain.

Poultry Production and Sustainability in Developing Countries under the COVID-19 Crisis: Lessons Learned.

Poultry farming is a significant source of revenue generation for small farmers in developing countries. It plays a vital role in fulfilling the daily protein requirements of humans through meat and eggs consumption. The recently emerged pandemic Coronavirus Disease-19 (COVID-19) impacts the poultry production sector. Although the whole world is affected, these impacts may be more severe in developing countries due to their dependency on exporting necessary supplies such as feed, vaccines, drugs, and utensils. In this review, we have discussed poultry production in developing countries under the COVID-19 crisis and measures to regain the loss in the poultry industries. Generally, due to the lockdown, trade limitations have negatively impacted poultry industries, which might exacerbate global poverty. Coordinated activities have to be taken at the private and government levels to arrange soft loans so that these farms can restore their production and marketing to normal levels. In addition, here, we have focused on the supply of farm input, feed, other raw materials, management system, improved breeding efficiency, veterinary services, and marketing of egg and meat, which have to be ensured to secure a sustainable poultry production chain.

Improved Subtyping of Avian Influenza Viruses Using an RT-qPCR-Based Low Density Array: 'Riems Influenza a Typing Array', Version 2 (RITA-2).

Avian influenza virus (AIV) variants emerge frequently, which challenges rapid diagnosis. Appropriate diagnosis reaching the sub- and pathotype level is the basis of combatting notifiable AIV infections. Real-time RT-PCR (RT-qPCR) has become a standard diagnostic tool. Here, a total of 24 arrayed RT-qPCRs is introduced for full subtyping of 16 hemagglutinin and nine neuraminidase subtypes of AIV. This array, designated Riems Influenza A Typing Array version 2 (RITA-2), represents an updated and economized version of the RITA-1 array previously published by Hoffmann et al. RITA-2 provides improved integration of assays (24 instead of 32 parallel reactions) and reduced assay volume (12.5 µL). The technique also adds RT-qPCRs to detect Newcastle Disease (NDV) and Infectious Bronchitis viruses (IBV). In addition, it maximizes inclusivity (all sequences within one subtype) and exclusivity (no intersubtypic cross-reactions) as shown in validation runs using a panel of 428 AIV reference isolates, 15 reference samples each of NDV and IBV, and 122 clinical samples. The open format of RITA-2 is particularly tailored to subtyping influenza A virus of avian hosts and Eurasian geographic origin. Decoupling and re-arranging selected RT-qPCRs to detect specific AIV variants causing epizootic outbreaks with a temporal and/or geographic restriction is possible.

Probiotics, Prebiotics, and Phytogenic Substances for Optimizing Gut Health in Poultry.

The gut microbiota has been designated as a hidden metabolic 'organ' because of its enormous impact on host metabolism, physiology, nutrition, and immune function. The connection between the intestinal microbiota and their respective host animals is dynamic and, in general, mutually beneficial. This complicated interaction is seen as a determinant of health and disease; thus, intestinal dysbiosis is linked with several metabolic diseases. Therefore, tractable strategies targeting the regulation of intestinal microbiota can control several diseases that are closely related to inflammatory and metabolic disorders. As a result, animal health and performance are improved. One of these strategies is related to dietary supplementation with prebiotics, probiotics, and phytogenic substances. These supplements exert their effects indirectly through manipulation of gut microbiota quality and improvement in intestinal epithelial barrier. Several phytogenic substances, such as berberine, resveratrol, curcumin, carvacrol, thymol, isoflavones and hydrolyzed fibers, have been identified as potential supplements that may also act as welcome means to reduce the usage of antibiotics in feedstock, including poultry farming, through manipulation of the gut microbiome. In addition, these compounds may improve the integrity of tight junctions by controlling tight junction-related proteins and inflammatory signaling pathways in the host animals. In this review, we discuss the role of probiotics, prebiotics, and phytogenic substances in optimizing gut function in poultry.

Diversity of Coronaviruses with Particular Attention to the Interspecies Transmission of SARS-CoV-2.

In December 2019, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China with serious impacts on global health and economy that is still ongoing. Although interspecies transmission of coronaviruses is common and well documented, each coronavirus has a narrowly restricted host range. Coronaviruses utilize different receptors to mediate membrane fusion and replication in the cell cytoplasm. The interplay between the receptor-binding domain (RBD) of coronaviruses and their coevolution are determinants for host susceptibility. The recently emerged SARS-CoV-2 caused the coronavirus disease 2019 (COVID-19) pandemic and has also been reported in domestic and wild animals, raising the question about the responsibility of animals in virus evolution. Additionally, the COVID-19 pandemic might also substantially have an impact on animal production for a long time. In the present review, we discussed the diversity of coronaviruses in animals and thus the diversity of their receptors. Moreover, the determinants of the susceptibility of SARS-CoV-2 in several animals, with special reference to the current evidence of SARS-CoV-2 in animals, were highlighted. Finally, we shed light on the urgent demand for the implementation of the One Health concept as a collaborative global approach to mitigate the threat for both humans and animals.

Impact of Nano-Bromocriptine on Egg Production Performance and Prolactin Expression in Layers.

The current study aimed to investigate the potential use of nano-bromocriptine in improving the laying performance of late laying hens by modulating the prolactin gene expression. A total of 150 NOVOgen brown laying hens aged 70 weeks were randomly allocated into three groups of 50 birds each. The first group was kept as a control, while the second and the third groups were treated with bromocriptine and nano-bromocriptine, respectively, at a dose of 100 µg/kg body weight per week. The pause days, egg production, feed per dozen egg, and Haugh unit were determined on a monthly basis. Also, the relative prolactin gene expression in the pituitary gland was quantified using qPCR and the number of the ovarian follicles was determined after slaughtering at the 84th week of age. It was found that nano-bromocriptine and bromocriptine improved egg laying performance with minimal pause days, reduced feed per dozen egg, and depressed the relative prolactin gene expression; however, nano-bromocriptine treatment was significantly effective compared to bromocriptine. In conclusion, nano-bromocriptine might be beneficial for elongating sequences and reducing pauses.

Correction: Efficacy of six disinfection methods against extended-spectrum beta-lactamase (ESBL) producing E. coli on eggshells in vitro.

[This corrects the article DOI: 10.1371/journal.pone.0238860.].

Seroprevalences of specific antibodies against avian pathogens in free-ranging ring-necked pheasants (Phasianus colchicus) in Northwestern Germany.

Infectious diseases in captive pheasants (Phasianus colchicus) are well known, but there is a lack of knowledge about occurrence and distribution of pathogens in free-ranging pheasants in Germany. We investigated 604 sera from hunted pheasants and 152 sera from wild caught pheasants between 2011 to 2015, with the aim to determine the prevalence of specific antibodies against different viruses: Avian influenza virus (AIV) of subtypes H5, H7, H9, paramyxovirus type 1 (PMV-1), avian encephalomyelitis virus (AEV), infectious bursitis disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (aMPV) and Salmonella sp., Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG). In addition, 178 caeca were investigated for Histomonas meleagridis. The study reveals an ongoing circulation of IBV in the wild pheasant population during the study. Also high seroprevalences of specific antibodies against aMPV depending on the area and a strong increase in prevalence of IBDV antibodies in sera of pheasants in Lower Saxony were detected. ILTV antibody prevalences differed between areas and AEV antibody detection differed between years significantly, whereas specific antibodies against PMV-1 could not be detected and antibodies against AIV-H5, -H7 and -H9 and Mycoplasma spp. were detected in very few cases.

Poult Enteritis and Mortality Syndrome in Turkey Poults: Causes, Diagnosis and Preventive Measures.

Poult enteritis and mortality syndrome (PEMS) is one of the most significant problem affecting turkeys and continues to cause severe economic losses worldwide. Although the specific causes of PEMS remains unknown, this syndrome might involve an interaction between several causative agents such as enteropathogenic viruses (coronaviruses, rotavirus, astroviruses and adenoviruses) and bacteria and protozoa. Non-infectious causes such as feed and management are also interconnected factors. However, it is difficult to determine the specific cause of enteric disorders under field conditions. Additionally, similarities of clinical signs and lesions hamper the accurate diagnosis. The purpose of the present review is to discuss in detail the main viral possible causative agents of PEMS and challenges in diagnosis and control.

Evolutionary insights into the furin cleavage sites of SARS-CoV-2 variants from humans and animals.

The SARS-CoV-2 spike protein Q677P/H mutation and furin cleavage site (FCS) have been shown to affect cell tropism and virus transmissibility. Here, we analyzed the frequency of Q677P/H and FCS point mutations in 1,144,793 human and 1042 animal spike protein sequences and from those of the emergent variants B.1.1.7, B.1.351, P.1, B.1.429 + B.1.427, and B.1.525, which were deposited in the database of the GISAID Initiative. Different genetic polymorphisms, particularly P681H and A688V, were detected in the FCS, mainly in human isolates, and otherwise, only pangolin and bat sequences had these mutations. Multiple FCS amino acid deletions such as Δ680SPRRA684 and Δ685RSVA688 were only detected in eight and four human isolates, respectively. Surprisingly, deletion of the entire FCS motif as Δ680SPRRARSVA688 and Δ680SPRRARSVAS689 was detected only in three human isolates. On the other hand, analysis of FCS from emergent variants showed no deletions in the FCS except for spike P681del, which was detected in seven B.1.1.7 isolates from the USA. Spike Q677P was detected only once in variant, B.1.1.7, whereas Q677H was detected in all variants, i.e., B.1.1.7 (n = 1938), B.1.351 (n = 28), P.1 (n = 9), B.1.429 + B.1.427 (n = 132), and B.1.525 (n = 1584). Structural modeling predicted that mutations or deletions at or near the FCS significantly alter the cleavage loop structure and would presumably affect furin binding. Taken together, our results show that Q677H and FCS point mutations are prevalent and may have various biological effects on the circulating variants. Therefore, we recommend urgent monitoring and surveillance of the investigated mutations, as well as laboratory assessment of their pathogenicity and transmissibility.

Influence of COVID-19 on the poultry production and environment.

Although chickens are not susceptible to SARS-CoV-2, several coronavirus disease outbreaks have been described concerning poultry processing facilities in different countries. The COVID-19 pandemic and the developed strain caused 2nd, 3rd, and recent Indian strain waves of epidemics that have led to unexpected consequences, such as forced reductions in demands for some industries, transportation systems, employment, and businesses due to public confinement. Besides, poultry processing plants' conditions exacerbate the risks due to the proximity on the line, cold, and humidity. Most workers do not have access to paid sick time or adequate health care, and because of the low wages, they have limited reserves to enable them to leave steady employment. In addition, workers in meat and poultry slaughterhouses may be infected through respiratory droplets in the air and/or from touching dirty surfaces or objects such as workstations, break room tables, or tools. Egg prices have increased dramatically during the lockdown as consumers have started to change their behaviors and habits. The COVID pandemic might also substantially impact the international poultry trade over the next several months. This review will focus on the effect of COVID-19 on poultry production, environmental sustainability, and earth systems from different process points of view.

Monoclonal antibodies specific for the hemagglutinin-neuraminidase protein define neutralizing epitopes specific for Newcastle disease virus genotype 2.VII from Egypt.

BACKGROUND: Newcastle disease is a devastating disease in poultry caused by virulent Newcastle disease virus (NDV), a paramyxovirus endemic in many regions of the world despite intensive vaccination. Phylogenetic analyses reveal ongoing evolution of the predominant circulating genotype 2.VII, and the relevance of potential antigenic drift is under discussion. To investigate variation within neutralization-sensitive epitopes within the protein responsible for receptor binding, i.e. the Hemagglutinin-Neuraminidase (HN) spike protein, we were interested in establishing genotype-specific monoclonal antibodies (MAbs). METHODS: An HN-enriched fraction of a gradient-purified NDV genotype 2.VII was prepared and successfully employed to induce antibodies in BalbC mice that recognize conformationally intact sites reactive by haemagglutination inhibition (HI). For subsequent screening of mouse hybridoma cultures, an NDV-ELISA was established that utilizes Concanavalin A (ConA-ELISA) coupled glycoproteins proven to present conformation-dependent epitopes. RESULTS: Six out of nine selected MAbs were able to block receptor binding as demonstrated by HI activity. One MAb recognized an epitope only present in the homologue virus, while four other MAbs showed weak reactivity to selected other genotypes. On the other hand, one broadly cross-reacting MAb reacted with all genotypes tested and resembled the reactivity profile of genotype-specific polyclonal antibody preparations that point to minor antigenic differences between tested NDV genotpyes. CONCLUSIONS: These results point to the concurrent presence of variable and conserved epitopes within the HN molecule of NDV. The described protocol should help to generate MAbs against a variety of NDV strains and to enable in depth analysis of the antigenic profiles of different genotypes.

Fluoropyrimdine therapy induced alterations in interleukins expression in colorectal cancer patients.

This study monitored the changes in the expression of inflammatory IL-6 and IL-1ß during the treatment period of Fluoropyrimidine (FP) based therapy. RNA was extracted from the peripheral blood of 102 CRC patients before treatment with FP therapy, and from 48 and 32 patients after 3 and 6 months of treatment, respectively. The genetic transcription of IL-6 and IL-1ß was determined by real time PCR. Patients were stratified according to their levels of IL-6 and IL-1ß genes expression for subgroup and survival analyses. Baseline CRC patients showed overexpression of IL-6 and IL-1ß compared to healthy control. FP therapy significantly induced IL-6 and IL-1ß expression. Subgroup analysis showed that patients with right colon tumors had significant elevation in both IL-6 and IL-1ß with FP therapy. FP therapy significantly induced IL-1ß expression in patients ⩽45 years, smokers, with high baseline level of CA19.9, right colon tumors, low grade pathology, T3 tumors and positive lymph nodes. Survival analysis showed that baseline levels of interleukins expression had insignificant effect on overall survival and event free survival. FP therapy has an impact on the level of interleukins expression declared in certain clinicopathological subgroups of CRC patients, but without a prognostic significance on patients' survival.

The Clinical Significance of Promoter Methylation of Fluoropyrimidine Metabolizing and Cyclooxygenase Genes in Colorectal Cancer.

AIMS: This study investigated the impact of promoter methylation of flouropyrimidine (FP) metabolizing and cyclooxygenase 2 (COX2) genes on their mRNA expression and on the clinical outcome of colorectal cancer (CRC) patients. METHODS: Methylation specific-PCR and real time-PCR of thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and COX2 were performed at baseline and after 3 and 6 months of FP therapy. Pairwise comparisons were conducted between the subgroups of CRC patients. The event free survival (EFS) and the hazard of progression were estimated by univariate and multivariate analyses. RESULTS: At baseline CRC patients, both TS and TP were overexpressed, in spite of the unmethylation of TS and the full methylation of TP genes. Significant downexpression of DPD and COX2 were associated their promoter's methylation. At the end of FP therapy, TS, DPD and COX2 were overexpressed by 7.52, 2.88 and 3.45 folds, respectively, while TP was downexpressed by 0.54 fold. However, no change was observed in the methylation status of genes with FP therapy. Pairwise comparisons revealed significant difference in the expression and the methylation status of genes according to the clinicopathological characters of CRC patients either at baseline or after FP therapy. The overexpression of DPD and COX2 genes were indicators for a poor EFS of CRC patients. Also, the high level of COX2 expression was found to be significantly correlated with the hazard of progression (HR = 1.73, 95% CI = 1.02-3.03). CONCLUSION: The promoter methylation of FP metabolizing and COX2 genes has significant impact on the expression and the treatment outcome of CRC patients.

Epigenetic immunomodulatory effect of eugenol and astaxanthin on doxorubicin cytotoxicity in hormonal positive breast Cancer cells.

BACKGROUND: Hormonal receptor positive (HR+) breast cancer is the most commonly diagnosed molecular subtype of breast cancer; which showed good response to doxorubicin (DOX)-based chemotherapy. Eugenol (EUG) and astaxanthin (AST) are natural compounds with proved epigenetic and immunomodulatory effects in several cancer cell lines. This study has been initiated to investigate the molecular mechanism (s) whereby EUG and AST could enhance DOX cytotoxicity in MCF7 cells. METHODS: Cytotoxic activity of DOX alone and combined with either 1 mM EUG or 40 µM AST was performed using sulphorhodamine-B assay in MCF7 cells. Global histones acetylation and some immunological markers were investigated using ELISA, western blotting and quantitative RT-PCR techniques. Functional assay of multidrug resistance was performed using rhodamine 123 and Hoechst 3342 dyes. Flow cytometry with annexin V and propidium iodide were used to assess the change in cell cycle and apoptosis along with the expression of some differentiation, apoptosis and autophagy proteins. RESULTS: DOX alone resulted in concentration-dependent cytotoxicity with IC50 of 0.5 µM. Both EUG and AST significantly increased DOX cytotoxicity which is manifested as a significant decrease in DOX IC50 from 0.5 µM to 0.088 µM with EUG and to 0.06 µM with AST. Combinations of DOX with 1 mM EUG or 40 µM AST significantly increased the level of histones acetylation and histone acetyl transferase expression, while reduced the expression of aromatase and epidermal growth factor receptor (EGFR) when compared with 0.25 µM DOX alone. Also both combinations showed higher uptake of rhodamine but lower of Hoechst stains, along with increased the percentage of caspase 3, and decreased the expression of CK7 and LC3BI/II ratio. EUG combination induced IFγ but reduced TNFα causing shifting of cells from G2/M to S and G0/ G1 phases. Combination of DOX with EUG induced apoptosis through the higher BAX/ BCl2 ratio, while with AST was through the increase in caspase 8 expressions. CONCLUSION: EUG and AST potentiated the anticancer activity of DOX through epigenetic histones acetylation along with the immunonomodulation of different apoptotic approaches in MCF7 cells.