Avicenna Journal of Medical Biotechnology
Volume:12 Issue: 3, Jul-Sep 2020

Pasteurella multocida (P. multocida) is the highly contagious causative agent of a broad range of diseases in animals as well as an occasional human pathogen. Economically significant infections caused by P. multocida include avian fowl cholera, rabbit snuffles, and hemorrhagic septicemia in cattle, goats and pigs. Chemotherapy of pasteurellosis infections has some limitations, such as high cost of treatment, low efficacy, and the possibility of therapy failure due to antibiotic resistance. Prophylactic immunization offers a safe and effective preventive measure in case of zoonotic diseases. Bacterins, live attenuated and some old traditional vaccines against pasteurellosis remain in use today, beside their limitations. However, the past few years have seen significant progress in research to identify modern, effective vaccine candidates, but there is no new vaccine produced by new strategies. While scientists should struggle with a lot of aspects to design vaccine producing strategies, this review shows how pasteurellosis vaccine evolved and the limitations in its application which need to be overcome.

Circulating Tumor Cells (CTCs) detection in peripheral blood of epithelial cancer patients is an indicator of the presence of primary tumors and metastasis. The CTC phenotype detection uses epithelial markers in defining, detecting, and isolating CTCs. Circulating cell-separation technologies, with the epithelial origin, can be identified by epithelial biomarkers, with different techniques such as flow cytometry. The purpose of this study was to evaluate the expression of molecular Cytokeratins (CKs), CK7, CK8, CK18, CK19 (Pan-CK) and Epithelial Cell Adhesion Molecule (EpCAM) markers for CTC detection.

The Magnetic Activated Cell Sorting (MACS) was used to identify CTCs in the blood of patients. Specific antibodies to EpCAM and Pan-CK were used and analyzed by flow cytometry. In this study, 35 blood samples of patients with breast cancer were assessed before any treatment and 35 healthy blood samples as the control were evaluated.

Expression of CK markers in the peripheral blood of breast cancer patients was statistically significant with p≤0.05, specifically at stages II-IV, but it was not significant in patients at stage I and healthy controls. Biomarkers expression in the blood of patients and healthy controls was assessed along with the pathologic characteristics of patients.

CTC assessment by flow cytometry in patients with breast cancer could not only be used for detection but also can be considered as a source of specific and subjective evaluation for monitoring the therapy. Besides, the sensitivity and specificity of CTC detection were shown that could be enhanced by specific CK markers.

The fluoropyrimidine drug 5-Fluorouracil (5-FU) and the prodrug capecitabine have been extensively used for treatment of many types of cancer including colorectal, gastric, head and neck. Approximately, 10 to 25% of patients suffer from severe fluoropyrimidine-induced toxicity. This may lead to dose reduction and treatment discontinuation. Pharmacogenetics research could be useful for the identification of predictive markers in chemotherapy treatment. The aim of the study was to investigate the role of five genetic polymorphisms within two genes (DPYD, TYMS) in toxicity and efficacy of fluoropyrimidine-based chemotherapy.

Total genomic DNA was extracted from 83 cancer patients treated with fluoropyrimidine-based chemotherapy. In this study, three polymorphisms were genotyped in dihydropyrimidine dehydrogenase gene c.1905+1 G>A (DPYD*2A; rs3918290), c.1679 T>G (I560S; DPYD*13; rs55886062), and c.2846A>T (D949V; rs67376798) and two polymorphisms, besides the Variable Number of Tandem Repeat (VNTR) polymorphism and 6-bp insertion/deletion polymorphism in thymidylate synthase gene. The analysis of polymorphisms for rs3918290, rs55886062, rs67376798 and 6-bp insertion/ deletion in TYMS was done by Polymerase Chain Reaction-restriction Fragment Length Polymorphism (PCRRFLP) TYMS VNTR analysis. 5-FU-related toxicities such as anemia, febrile neutropenia, neurotoxicity, vomiting, nausea, and mucositis were evaluated according to NCI-CTC criteria version 4.0. T-test and chi-square were used and p-values less than 0.05 were considered statistically significant.

DPYD gene polymorphisms were not observed in this study. The frequency of the TYMS +6 bp allele was 40.35% and the -6 bp allele was 59.65% in this study. The frequency of VNTR 2R allele was 48.75% and 3R allele was 51.15%. Toxicity grade II diarrhea, mucositis, nausea, vomiting, and neurotoxicity was 2.2, 24.1, 15.7, 6, and 51.8%, respectively. Thymidylate synthase ins/del polymorphisms were associated with increased grade III neurotoxicity (p=0.02). Furthermore, anemia grade III was significantly associated with 2R/2R genotype (0.009).

Thymidylate synthase gene polymorphisms may play a key role in fluoropyrimidne -based chemotherapy. Although rare DPYD polymorphisms were not observed in our study, according to large population studies, DPYD gene polymorphisms could be used as a predictive biomarker for patient treatments.

Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is one of the promising cell surface antigens for targeting cancer cells. The aim of this study was to evaluate ROR1 cell surface expression in bladder cancer cells using a murine anti-ROR1 monoclonal antibody (mAb) called 5F1-B10 as well as investigate its potential in apoptosis induction.

Expression of ROR1 in two human bladder cell lines, 5637 and EJ138, as well as a non-cancerous human cell line, Human Fetal Foreskin Fibroblast (HFFF), was examined by flow cytometry and immunocytochemistry. Immunohistochemical staining of cancer and normal bladder tissues was also performed.

The flow cytometry results showed that 5F1-B10 mAb could recognize ROR1 molecules in 86.1% and 45.6% of 5637 and EJ138 cells, respectively. The expression level of ROR1 was 5.49% in HFFF cells. The immunocytochemistry and immunohistochemistry staining results also confirmed the presence of ROR1 on the surface of both bladder cancer cells and tissues, respectively. The obtained data from apoptosis assay demonstrated that 5F1-B10 mAb could induce apoptosis in both 5637 and EJ138 cell lines.

Taken together, our finding indicates the role of ROR1 in bladder cancer cell survival and suggests this receptor might be a promising target for developing novel therapeutic agents against bladder carcinoma.

Osteoarthritis (OA) is a chronic disease that attacks joints and bones
which can be caused by trauma or other joint diseases. Stem cell and Conditioned
Medium (CM) of stem cells are developed for OA therapy, which is minimally invasive. It can decrease inflammation and be a replacement for knee surgery. This study
aimed to utilize human Wharton’s Jelly-Mesenchymal Stem Cells (hWJMSCs) as an alternative OA therapy.

CM from hWJMSCs induced by IGF1 was collected. The OA cells model (IL1βCHON002) culture was treated as follows: 1) with hWJMSCs-CM 15% (v/v); 2) with
hWJMSCs-CM 30% (v/v); 3) with IGF1-hWJMSCs (IGF1-hWJMSCs-CM) 15% (v/v); 4)
with IGF1-hWJMSCs-CM 30% (v/v). Parameters including inflammatory cytokines
(IL10 and TNFα), extracellular matrix degradation (MMP3 expression), and chondrogenic marker (COL2 expression) were determined.

The most significant increase in COL2 chondrogenic markers was found in
IL1β-CHON002 treatment using 15% CM of hWJMSCs induced with IGF1. CM of hWJMSCs can reduce inflammatory cytokines (TNFα and IL10) and matrix degradation
mediator MMP3. Better result was gained from IGF1-induced hWJMSCs-CM.

CM of IGF1-hWJMSCs reduce inflammation while repairing injured joint in
the human chondrocyte OA model.

Cone snails are a natural source of complex peptides with analgesic properties called conotoxins. These peptides are secreted in a complex venomic mixture and are predominantly smaller than 5 kDa. The present study aimed to document the analgesic activity of two species of Conus coronatus (C. coronatus) and Conus frigidus (C. frigidus) venom collected off the Iranian coast in a mouse behavioral test.

Conotoxin containing fractions was extracted from the venom ducts and initially purified by column chromatography. The analgesic effect of the fractions was determined on formalin pain model and hot-plate test.

The results led to the identification of four fractions with analgesic activity in C. coronatus and two in C. frigidus. Only one fraction was able to reduce the flinching and licking in both acute pain and chronic pain phases of the formalin test. Moreover, the activity of this fraction remained 30 minutes on the hot-plate test. Purification of the fractions was carried out by RP-HPLC. LC-ESI-MS analysis of the fractions showed that the conotoxins of the analgesic fraction had molecular weights not previously reported.

The findings give insight into the venom of two previously under-investigated Conus species and reveal the therapeutic potential of the containing conopeptides.

Recently, using antibacterial peptides has been considered as a strategy to manage the worldwide antibiotic-resistance crisis. Screening of Dasht-Desert Bacterial Culture Collection (DDBCC) for bacteriocin or bacteriocin-like producer was aimed in this study to introduce native antibacterial agent(s).

In this study, 170 isolates were examined by the cross-streak method against G+ and G- indicators. Isolates with antimicrobial activity were compared using turbidity and well diffusion tests. The candidate isolate, DDBCC70, was molecularly and biochemically characterized. Then, the production of an antibacterial agent was physicochemically optimized. The supernatant was saturated ammonium sulfate. SDSPAGE and Thin-Layer Chromatography (TLC) analyses, cytotoxicity, and hemagglutination tests were performed.

First, 23 isolates were detected with antimicrobial activity against at least three of the indicator strains. DDBCC70 was distinguished with the broad-spectrum of antibacterial effects of the Cell-Free Supernatants (CFSs). The black pigments on BHI and a 98% similarity in 16S rDNA and similarity in biochemical tests confirmed the strain of DDBCC70 as Bacillus atrophaeus (B. atrophaeus). The highest amount of the antibacterial agent, Bac70, was obtained from the modified brain heart infusion medium. It was revealed that 70% ammonium sulfate-saturated Bac70 was 3.8 and 1.6 times more effective on Pseudomonas aeuroginosa (P. aeuroginosa) and Klebsiella pneumoniae (K. pneumoniae). Bac70, a >25 kDa protein and a safe compound for blood cells, neither agglutinated human erythrocyte nor lysed sheep blood. The purified bacteriocin-like molecule destroyed biofilms from P. aeruginosa and Staphylococcus aureus (S. aureus). Moreover, the fraction of Bac70 from the TLC plate showed higher inhibitory effects against K. pneumoniae.

Based on the above-mentioned features, Bac70 is a potential alternative therapeutic agent in pharmaceutical, food preservative and biotech-related industries.

Enterohemorrhagic Escherichia coli (E. coli) (EHEC) O157:H7 is a major foodborne pathogen causing severe disease in humans worldwide. Cattle are important reservoirs of E. coli O157:H7 and developing a specific immunity in animals would be invaluable. The administration of Whole Cell Vaccines (WCV) is a wellestablished method of vaccination against bacterial infections. Route of administration, inactivation and using suitable adjuvant have significant effects on the characteristics and efficacy of WCV.

In the present study, an attempt was made to evaluate the immunogenic potency of heat and formalin inactivated cells administered orally and subcutaneously in mouse model by ELISA. Mice pretreated with streptomycin were used as a model to evaluate the efficacy of subcutaneous versus oral administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding.

Both forms of inactivated cells induced immune response and hence protection against infectious diseases caused by E. coli O157:H7. However, formalin inactivated cells of E. coli O157:H7 showed superior antigenicity compared to heat inactivated cells. Subcutaneous immunization of mice with both heat and formalin inactivated E. coli O157:H7 induced significant specific levels of IgG antibodies but did not lead to significant antigen-specific IgA rise in feces, whereas oral immunization elicited significant levels of IgG antibodies with some animals developing antigen-specific IgA in feces.

Inactivated E. coli O157:H7 is highly immunogenic and can induce protective immune responses via oral immunization.