Enhancing cancer immunotherapy with Anti-NKG2D/IL-15(N72D)/Sushi fusion protein: Targeting cytotoxic immune cells and boosting IL-15 efficacy.

BACKGROUND: There are a number of distinct challenges and complexities associated with administering IL-15 for cancer immunotherapy that must be taken into consideration. OBJECTIVE: The purpose of this study was to design a fusion protein for targeting cytotoxic immune cells and enhance IL-15 efficiency. METHODS: A fusokine that contains IL-15(N72D), a Sushi domain, and anti-NKG2D scFv was designed. The fusion protein was in-silico modeled using the Swiss model server, followed by docking and molecular dynamics simulations. The in-vitro purified fusokine was evaluated using dot blot and Western blot. Then, flow cytometry was employed to evaluate biological properties such as proliferation, cytotoxicity, and degranulation. RESULTS: Fusokine and IL-15(N72D)/Sushi, which had molecular weights of about 52 kDa and 26 kDa, respectively, were expressed in CHO-K1 cells. The fusokine binds 69.6 % of the CHO-NKG2D+ cells that express 83.1 % NKG2D. Both the fusokine and the IL-15(N72D)/Sushi significantly stimulate the proliferation of lymphocytes. After 14 days of growth, the vitality of untreated cells decreased to about 17.5 %, but 82.2 % and 56.6 % of cells were still alive when fusokine and IL-15(N72D)/Sushi were present. Furthermore, administration of fusokine was associated with the highest rates of target tumor cell cytotoxicity. Additionally, although it was not statistically significant, fusokine increased the expression of CD107a and granzyme B by 1.25 times and 2.4 times, respectively. CONCLUSION: The fusokine possesses the capability to stimulate the survival and multiplication of lymphocytes, as well as their ability to eliminate tumors. These characteristics have led to its consideration as a potential treatment for immunotherapy.

Identification of intrinsically disordered regions in hub genes of acute myeloid leukemia: A bioinformatics approach.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Over the past decades, there has been a great challenge in the treatment of AML. A combination of gene expression profiling with computational approaches can lead to the identification of hub genes in AML. However, it is important to study the structure of these hub genes considering their importance in the protein-protein interaction (PPI) network of specific cancer. In this study, we designed an integrated method to analyze the presence of intrinsically disordered regions (IDRs) in selected hub genes of AML. A gene expression profile of AML was obtained from Gene Expression Omnibus (GEO) database. Further analysis identified differentially expressed genes (DEGs) in AML. Additionally, the top 15 hub genes following construction and analysis of the PPI network of DEGs were selected. Validation of hub genes revealed that there is a reverse relationship between overexpression of FLT3, PPBP, and PF4 genes and the survival of AML patients. Based on IDRs investigation, FLT3 and PF4 are partially disordered, while PPBP is mostly disordered. Through clustering the network into structural modules, we identified two important modules in the PPI network of DEGs that showed the important position of PPBP in module 1. Based on further analysis of protein flexibility and its important role in biological processes, we suggest that PPBP can be considered as a potential drug target in AML.

Current Practices and Challenges in Method Validation.

Method validation is a cornerstone on which biomarker development and utilization rest. However, given the abundance of biomarker candidates that are being identified and characterized, validation of these entities for the use in nonclinical studies can be complex. The objective of this continuing education course was to review current practices and challenges encountered during the validation of methods for the analysis of novel biomarkers. Additionally, the importance of biological validation and correlation with pathology end points for biomarker candidates was discussed. This article is a summary of the materials presented at the 36th Annual Symposium of the Society of Toxicologic Pathology for a continuing education course titled "Current Practices and Challenges in Method Validation." The speakers were subject-matter experts in the validation of quantitative mass spectrometry, multiplex binding assays, biological biomarkers, and immunophenotyping and anatomic and clinical pathology considerations in biomarker qualification.

Scientific and Regulatory Policy Committee Points to Consider Review: Inclusion of Reproductive and Pathology End Points for Assessment of Reproductive and Developmental Toxicity in Pharmaceutical Drug Development.

Standard components of nonclinical toxicity testing for novel pharmaceuticals include clinical and anatomic pathology, as well as separate evaluation of effects on reproduction and development to inform clinical development and labeling. General study designs in regulatory guidances do not specifically mandate use of pathology or reproductive end points across all study types; thus, inclusion and use of these end points are variable. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) formed a Working Group to assess the current guidelines and practices on the use of reproductive, anatomic pathology, and clinical pathology end points in general, reproductive, and developmental toxicology studies. The Working Group constructed a survey sent to pathologists and reproductive toxicologists, and responses from participating organizations were collected through the STP for evaluation by the Working Group. The regulatory context, relevant survey results, and collective experience of the Working Group are discussed and provide the basis of each assessment by study type. Overall, the current practice of including specific end points on a case-by-case basis is considered appropriate. Points to consider are summarized for inclusion of reproductive end points in general toxicity studies and for the informed use of pathology end points in reproductive and developmental toxicity studies.

Clinical, hematological and molecular evaluation of piroplasma and Anaplasma infections in small ruminants and tick vectors from Kurdistan province, western Iran.

Tick-borne haemoparasite infections are a major challenge in small ruminant (SR) production across tropical areas. The present study evaluated the prevalence of Theileria, Babesia and Anaplasma in SRs and their tick vectors and estimated the association between pathogen prevalence with clinical hematological findings among SR populations in Kurdistan province, western Iran. In total, 250 blood samples and 250 tick species (one per animal) were collected from SR populations, along with clinical and hematological examinations. Microscopy of blood smears and molecular analysis were performed to detect potential infection with Theileria, Babesia and Anaplasma. Moreover, haemoparasites were explored in the isolated ticks using semi-nested PCR. Based on microscopy, the prevalence of Theileria, Anaplasma and Babesia infections was 91.2%, 23.2% and 2.4%, respectively. Semi-nested PCR analysis of blood samples demonstrated 86.8%, 78.8% and 14% prevalence for T. ovis, A. ovis and B. ovis, respectively. Dermacentor marginatus and Rhipicephalus turanicus were predominant isolated tick vectors from SR, while D. marginatus was the most contaminated tick in all investigated counties. There were, also, a statistically significant association between the estimated molecular prevalence rates with semi-yellow conjunctiva (A. ovis), body temperature (T. ovis and A. ovis), heart rate (T. ovis and B. ovis), mean white blood cell count (T. ovis and A. ovis), mean red blood cell count (T. ovis and B. ovis), as well as mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration in all haemoparasite infections. Future studies are recommended to reveal the epidemiology of such infections in SRs in Iran.

A survey of aflatoxin M1 contamination in bulk milk samples from dairy bovine, ovine, and caprine herds in Iran.

A total of 150 bovine (60), ovine (42), and caprine (48) bulk milk samples were analyzed using a commercially available competitive ELISA kit. Overall, AFM1 was found in 46.7 % of the analyzed samples by an average concentration of 40.3 ± 22.2 ng/L. The incidence rates of AFM1 contamination in bovine, ovine, and caprine bulk milk samples were 66.7, 31.0, and 35.4 %, respectively. The concentration of AFM1 in 37.5 % of AFM1-positive bovine milk samples and 5.9 % of AFM1-positive caprine milk samples were higher than 50 ng/L.

Prevalence of Coxiella burnetii in bulk milk samples from dairy bovine, ovine, caprine, and camel herds in Iran as determined by polymerase chain reaction.

Q fever is a widespread zoonosis caused by the obligate intracellular micro-organism Coxiella burnetii. The objective of this study was to determine the prevalence rate of C. burnetii in bulk milk samples from dairy bovine, ovine, caprine, and camel herds in Isfahan province, Iran. In the present study, 567 bulk milk samples from 186 dairy bovine, ovine, caprine, and camel herds were tested for C. burnetii using a nested polymerase chain reaction assay. The animals whose milk samples collected for this study were clinically healthy. In total, 8 of 247 (3.2%) bovine milk samples were positive; the positive samples originated from 6 of 90 (6.7%) dairy herds. Eight of 140 (5.7%) ovine bulk milk samples from 42 sheep breeding farms and 5 of 110 (4.5%) caprine bulk milk samples from 32 goat breeding farms were positive for C. burnetii. One of 70 (1.4%) camel bulk milk samples from 22 camel breeding farms was also positive for C. burnetii. Although no extensive prevalence study was undertaken, the results of this study indicate that clinically healthy dairy animals are important sources of C. burnetii infection in Iran. To the authors' knowledge, this study is the first report of direct identification of C. burnetii using polymerase chain reaction in bulk milk samples from dairy ovine herds in Iran and the first report of direct identification of C. burnetii in bulk milk samples from dairy camel herds. Further intensive prevalence studies on Coxiella infection and on possible risks of dairy products will be needed to elucidate the epidemiology of Q fever in Iran.

An Overview of Aptamer: The Prominent Applications and Different Computational Tools for its Design.

BACKGROUND: Aptamers are short single-stranded oligonucleotides; due to their 3D structure, they can specifically bind to various targets. They can target several molecules ranging from metal ions, organic components to proteins, and large cells. METHODS: According to the high affinity of aptamers, they can be used for diagnosis, therapeutic, vaccine development, and gene silencing applications. The conventional method for aptamer selection is known as the Systematic Evolution of Ligands by Exponential (SELEX). However, despite the efficiency of SELEX as a screening procedure, it is beneficial to develop more rational procedures for aptamer selection. RESULTS & DISCUSSION: Herein, in silico approaches can play an effective role given their potential in representing an efficient, cost-effective, parallelizable, and rapid strategy. In recent years, several attempts have been applied to develop algorithms and software for the rational selection of aptamers. However, there is still a need for more efforts to achieve the most efficient techniques in this area. CONCLUSION: In this review, we aim to overview different computational approaches that are used for aptamer selection.

Prevalence and antimicrobial resistance of Campylobacter species isolated from raw camel, beef, lamb, and goat meat in Iran.

Campylobacter spp. are one of the most common causes of acute bacterial gastroenteritis in human beings which are transmitted mostly via food originating from animals. This study was conducted to determine the prevalence and antimicrobial resistance of Campylobacter spp. isolated from retail raw meats in Iran. From June 2008 to June 2009, a total of 722 raw meat samples from camel (n = 107), beef (n = 190), lamb (n = 225), and goat (n = 180) were purchased from randomly selected retail outlets in Isfahan and Yazd, Iran, and were evaluated for the presence of Campylobacter spp. In this study, 50 of the 722 meat samples (6.9%) were contaminated with Campylobacter spp. The highest prevalence of Campylobacter spp. was found in lamb meat (12.0%), followed by goat meat (9.4%), beef meat (2.4%), and camel meat (0.9%). The most prevalent Campylobacter spp. isolated from the meat samples was Campylobacter jejuni (84.0%); the remaining isolates were Campylobacter coli (16.0%). Susceptibilities of 50 Campylobacter isolates were determined for 10 antimicrobial drugs using the disk-diffusion assay. Resistance to tetracycline was the most common finding (68.0%), followed by resistance to ciprofloxacin (46.0%) and nalidixic acid (40.0%). All of the isolates were susceptible to erythromycin, gentamicin, and chloramphenicol. Significantly higher prevalence rates of Campylobacter spp. (p < 0.05) were found in the lamb meat samples taken in spring (20.0%) and summer (18.9%). To our knowledge, this study is the first report of the isolation of Campylobacter spp. from raw camel, lamb, and goat meat in Iran.

Occurrence of toxigenic Vibrio parahaemolyticus strains in shrimp in Iran.

Vibrio parahaemolyticus, a common cause of foodborne gastroenteritis in people, is frequently isolated from a variety of seafood, including shrimp. The virulence of clinical V. parahaemolyticus strains is commonly associated with expression of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), which are encoded by the tdh and trh genes. This study was conducted to determine the prevalence rate of total and toxigenic V. parahaemolyticus in shrimp caught off the south coast of Iran. Three hundred freshly caught shrimp from four different species, Penaeus monodon, Penaeus semisulcatus, Penaeus indicus, and Penaeus merguiensis, were collected in three provinces along Persian Gulf in the south coast of Iran. Shrimp were collected at the end of each month from July 2008 to July 2009. The samples were analyzed for the presence of V. parahaemolyticus and the occurrence of toxigenic strains. Using conventional bacteriological techniques, 29 V. parahaemolyticus isolates were identified in which 28 strains were confirmed by a polymerase chain reaction assay targeting the tlh gene of V. parahaemolyticus. Using polymerase chain reaction assays targeting the tdh and trh genes, five (1.7%) and two (0.7%) isolates were tdh positive and trh positive, respectively. The tdh-positive isolates were commonly detected in summer, whereas no toxigenic strain was isolated in winter. To the best of our knowledge, the present study is the first report of the presence of toxigenic tdh- and trh-positive V. parahaemolyticus strains in the seafood in Iran.

Biosensors for detection of Tau protein as an Alzheimer's disease marker.

Known as a main neural MAP (microtubule associated protein), tau protein contributes to stabilizing microtubules involved in cellular transmission. Tau dysfunction is mainly associated with neurodegenerative diseases, particularly Alzheimer's disease (AD). In these patients, all the six tau isoforms, which are in hyperphosphorylated form, are first aggregated and then polymerized into neurofibrillary tangles inside the brain. Tau protein detected in cerebrospinal fluid (CSF) is significantly correlated with AD and is well recognized as a hallmark of the disease. Served for detection of analytes of interest, biosensor device comprises a physical transducer and a keen biological recognition component. Qualitative and quantitative evaluations may be performed through analyzation of the data, which is gathered by measurable signals converted from biological reaction. Antibodies, receptors, microorganisms, nucleic acids, enzymes, cells and tissues, as well as some biomimetic structures, normally constitute the biosensor biological recognition part. Production of nanobiosensor, which was made possible through several accomplishments in nano- and fabrication technology, opens up new biotechnological horizons in diagnosis of multiple diseases. In recent years, many researches have been focused on developing novel and effective tau protein biosensors for rapid and accurate detection of AD. In this review, tau protein function and correlation with AD as well as the eminent research on developing nanobiosensor based on optical, electrochemical and piezoelectric approaches will be highlighted.

Comparison of two commercial radial immunodiffusion assays for detection of bovine immunoglobulin G in newborn calves.

Bovine failure of passive transfer (FPT), defined as inadequate transfer of colostral immunoglobulins from the dam to the calf, has been associated with increased risk in neonatal mortality. Currently, radial immunodiffusion (RID) assay is considered to be the gold standard in determining FPT in serum samples from calves. There are 2 commercial RID assays routinely used for serodiagnosis of FPT in calves: VET-RID and SRID. Discrepancies between results of these RID assays were observed in the authors' laboratory. The objective of this study was to compare 2 commercial RID assays by testing a paired panel of 30 blood samples collected from newborn Holsteins at birth before, and 24 hr after, ingestion of colostrum, a commercial bovine reference serum, and a panel of different concentrations of 2 purified bovine immunoglobulin G (IgG) products. Overall, the results of this study showed a high level of discrepancy and poor agreement between the 2 RID kits. The interassay precision study revealed lower between-run coefficients of variation for the VET-RID kit compared with the SRID kit. The spiking and recovery study using purified bovine IgG products demonstrated that the VET-RID kit more closely approximates the expected concentrations of the purified bovine IgG products, whereas the SRID kit consistently overestimates the concentration of purified bovine IgG products. It was concluded that this may be due to inaccuracies in the internal standards of the SRID kit.

Western blot immunoassay as a confirmatory test for the presence of anti-Mycoplasma hyopneumoniae antibodies in swine serum.

A Western blot immunoassay (WBI) was developed as a confirmatory test for 2 commercial Mycoplasma hyopneumoniae (Mhyo) ELISAs. The WBI detected at least 5 antigen bands (150, 130, 74, 70, and 30 kDa) from Mhyo whole membrane proteins that were not present in the antigens prepared from M. hyorhinis and M. hyosynoviae. Among discrepant sera from vaccinated pigs (n = 17) and field samples (n = 91) assayed by WBI: 1) 2 of the ELISA-positive samples reacted with all 5 antigens bands; 2) all blocking ELISA-positive samples (n = 53) bound 150-, 130-, 74-, and 70-kD antigen bands; and 3) all indirect ELISA-positive samples (n = 55) bound 150-, 130-, and 30-kD antigens. We conclude that the WBI targeting the top 4 antigen bands is a useful confirmatory test for samples initially screened using the commercial Mhyo ELISAs.

AZDAST the new horizon in antimicrobial synergism detection.

The attempts via introducing many methods have been conducted to select the best antibiotic combination in the treatment of seriously ill patients. Operational or interpretational complexity or time-consuming along with sufficient accuracy led to postpone routine clinical use of these tests until today, despite the urgent need for them. By this study and proposed method, selection of the best double antibiotic synergistic combination against resistant pathogen is simply same as Kirby-Bauer antibiotic susceptibility test. It seems, precise and reliable results (very low coefficient of variation) will be introduced it as a routine accurate diagnostic doubled antimicrobial synergism test.•The objective of this study was to introduce a novel method in antibiotic interaction detection.•It demonstrates high sensitivity and accuracy.•Easy implementation by routine microbiology labs materials and equipment and so easy stand-alone interpretation seems to make it friendly test be able to replacing the previous methods.

Best practices for veterinary toxicologic clinical pathology, with emphasis on the pharmaceutical and biotechnology industries.

The purpose of this paper by the Regulatory Affairs Committee (RAC) of the American Society for Veterinary Clinical Pathology (ASVCP) is to review the current regulatory guidances (eg, guidelines) and published recommendations for best practices in veterinary toxicologic clinical pathology, particularly in the pharmaceutical and biotechnology industries, and to utilize the combined experience of ASVCP RAC to provide updated recommendations. Discussion points include (1) instrumentation, validation, and sample collection, (2) routine laboratory variables, (3) cytologic laboratory variables, (4) data interpretation and reporting (including peer review, reference intervals and statistics), and (5) roles and responsibilities of clinical pathologists and laboratory personnel. Revision and improvement of current practices should be in alignment with evolving regulatory guidance documents, new technology, and expanding understanding and utility of clinical pathology. These recommendations provide a contemporary guide for the refinement of veterinary toxicologic clinical pathology best practices.

Comparison of the vanadate oxidase method with the diazo method for serum bilirubin determination in dog, monkey, and rat.

The most widely used method for bilirubin concentration determination is the diazo method, which measures the color of azobilirubin. The vanadate oxidase method is based on oxidation of bilirubin to biliverdin by vanadate. The objective of this study was to compare total and direct bilirubin concentration ([Bt] and [Bd], respectively) determined by the diazo and vanadate oxidase methods in pooled serum samples from dogs, monkeys, and rats spiked with panels of different concentrations of bilirubin standards. Pooled serum samples from 40 dogs, 40 monkeys, and 60 rats were spiked with either ditaurine conjugates of bilirubin or a standard reference material. The results obtained from both assays were compared using Deming regression analysis. The intra- and interassay precision, expressed as a percentage of the coefficient of variation (%CV), was determined for [Bt] and [Bd], and the mean percentage of recovery was calculated. The vanadate oxidase method displayed an excellent correlation (r  =  0.99-1.00) with the diazo method. Using Deming regression, there were minimal negative or positive constant and proportional biases for [Bt] and [Bd]. The precision studies revealed that the vanadate oxidase method has comparable between-run and within-run CVs to those of the diazo method. The recovery study demonstrated that the diazo method more closely approximates the expected values of [Bt]. In conclusion, the vanadate oxidase method is a simple and rapid method that can be employed as an alternative to the diazo method when interfering substances are present in the serum samples of dog, monkey, and rat.

Stability of hematologic analytes in monkey, rabbit, rat, and mouse blood stored at 4°C in EDTA using the ADVIA 120 hematology analyzer.

BACKGROUND: The time from sampling to analysis can be delayed when blood samples are shipped to distant reference laboratories or when analysis cannot be readily performed. OBJECTIVE: The objective of this study was to evaluate the stability of hematologic analytes in blood samples from monkeys, rabbits, rats, and mice when samples were stored for up to 72 hours at 4°C. METHODS: Blood samples from 30 monkeys, 15 rabbits, 20 rats, and 30 mice were collected into EDTA-containing tubes and were initially analyzed within 1 hour of collection using the ADVIA 120 analyzer. The samples were then stored at 4°C and reanalyzed at 24, 48, and 72 hours after collection. RESULTS: Significant (P<.0003) changes in hematologic analytes and calculations included increased HCT and MCV and decreased MCHC and cell hemoglobin concentration mean (CHCM) at 72 hours and increased MPV at 24 hours in monkeys; increased MCV at 72 hours and MPV at 48 hours and decreased monocyte count at 24 hours in rabbits; increased MCV and decreased MCHC, CHCM, and monocyte count at 24 hours in rats; increased MCV, red cell distribution width, and MPV and decreased MCHC, CHCM, and monocyte count at 24 hours in mice. CONCLUSIONS: Although most of the changes in the hematologic analytes in blood from monkeys, rabbits, rats, and mice when samples were stored at 4°C were analytically acceptable and clinically negligible, the best practice in measuring hematologic analytes in these animals is timely processing of blood samples, preferably within 1 hour after collection.

Determination of plasma fibrinogen concentrations in beagle dogs, cynomolgus monkeys, New Zealand white rabbits, and Sprague-Dawley rats by using Clauss and prothrombin-time-derived assays.

The most widely used technique for determination of fibrinogen concentration is the Clauss fibrinogen (FIB(Clauss)) assay, which measures the clotting time of plasma after addition of excess thrombin. More recently, the PT-derived fibrinogen (FIB(PT)) assay has been developed, based on the relationship between fibrinogen concentration and the kinetics of clot formation during the prothrombin time. The objective of this study was to compare the fibrinogen concentration determined by the FIB(Clauss) and FIB(PT) assays in citrated plasma samples from healthy dogs (n = 40), monkeys (n = 40), rabbits (n = 26), and rats (n = 58) by using an automated coagulation analyzer. Results of a t test analysis indicated that the mean plasma fibrinogen concentrations measured by the 2 assays for all 4 species were significantly different. According to Pearson correlation coefficients, the FIB(PT) assay displayed a high correlation (0.93 to 0.98) with the FIB(Clauss) assay for all species. When the FIB(PT) and FIB(Clauss) assays were compared by using Deming regression, positive or negative constant and proportional biases emerged for all species. Intra- and interassay coefficients of variation for the FIB(PT) and FI(BClauss) assays were 0.8% to 2.3% and 1.8% to 7.4%, respectively. In conclusion, the FIB(PT) assay is a rapid and economical method for estimating fibrinogen concentration in plasma samples from dogs, monkeys, rabbits, and rats. However, it should not be used without restriction. Further studies are required to investigate the performance of this assay in animals with various pathologic states, including coagulopathy, dysfibrinogenemia, and hypo- or hyperfibrinogenemia.