The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.Infection with human papillomavirus type 16 (HPV-16) is the main factor associated with development of cervical cancer (42). The HPV-16 E6 and E7 proteins are constitutively produced in cervical carcinomas, and E7 was shown to interact with several cell compounds, causing deregulation of the cell cycle and cell transformation (43). E7 is a 98-amino-acid nuclear phosphoprotein that is devoid of any known enzymatic activity (36). In eukaryotic cells, E7's half-life is short (30 to 40 min); its degradation is mediated by the ubiquitin-proteasome pathway (31). E7 protein is widely studied because of its implication in carcinoma onset. It is also considered to be a good antigen candidate for the development of new vaccines against cervical cancer.E7 production systems have been developed in both eukaryotes (1, 39) and prokaryotes (27,34). Since the 1990s, several workers have investigated the use of bacteria as E7 antigen delivery vehicles to elicit an immune response against 22). The gram-positive and generally regarded as safe (GRAS) commensal bacterium Streptococcus gordonii was used for this purpose to display E7 protein at the cell surface in fusion with export signals (30). These recombinant S. gordonii strains could elicit an immune response in mice and monkeys (23,26). Although encouraging, these results rely on a commensal, GRAS but non-food-grade bacterium. One risk of commensal, and hence persistent, microorganisms is the induction of immunotolerance. Thus, a transient presentation of the antigen to the immune system by a noncommensal bacterium may be needed to avoid this risk.None of the systems mentioned above seems to provide the combination of safety, sufficient yields, and simplified methods that would allow both purification and eventual oral immunization using E7. We therefore considered an alternative system for native E7 production based on a food-grade lactic acid bacterium. The best-known lactic acid bacterium, Lactococcus lactis, has been extensively engineered for the production of heterologous proteins (5,6,10,18,20,21,28,29,35,37). Protein production in L. lactis offers advantages: L. lactis is a food-grade gram-positive bacterium that produces very low amounts of native exoproteins. It is therefore a good candidate for heterologous protein secretion in different applications ranging from industrial production of high-added-value proteins to in vivo use as a live vaccine. As L. lactis is a noncommensal and transient bacterium in the digestive tract (12), t...

Introduction: Canine transmissible venereal tumour (CTVT) is a sexually transmitted tumour affecting dogs worldwide, imposing a financial burden on dog owners. A stable culture cell line in continuous passages for >18 months has only been achieved once. The present study investigated a stable CTVT cell line isolated from a bitch and its potential as a vaccine. Material and Methods: A biopsy from a 2-year-old mongrel bitch with CTVT was obtained for histopathological confirmation and isolation of tumour cells. The isolated cells were cultured to passage 55 and characterised by flow cytometry, with karyotyping by GTG-banding and by PCR detection of myc S-2 and LINE AS1. The isolated CTVT cell line was also used as a preventive vaccine in a canine model. Results: Histopathological analysis of the isolated tumour cells revealed typical CTVT characteristics. Constant proliferation and stable morphological characteristics were observed during culture. Phenotypic analysis determined the expression of HLA-DR+, CD5.1+, CD14+, CD45+, CD83+, CD163+, and Ly-6G-Ly-6C+. GTG-banding revealed a mean of 57 chromosomes in the karyotype with several complex chromosomal rearrangements. LINE-c-myc insertion in the isolated CTVT cell line at 550 bp was not detected. However, a 340-bp band was amplified. Isolated CTVT cell line inoculation at a concentration of 1×108 did not induce tumour growth in bitches, nor did a challenge with primary CTVT cells. Conclusion: The present study successfully identified and isolated a stable CTVT cell line that may be useful in CTVT prevention.

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Measurement of cytokine levels has yielded useful information on the pathological process of different diseases such as AIDS, endotoxic shock, sepsis, asthma, and cancer. It may also be of use in the monitoring of disease progression and/or inflammation. To determine spontaneous cytokine gene expression in whole blood and PBMCs, whole blood was obtained from healthy volunteers and total mRNA was isolated from PBMCs. The kinetics of response were determined by sequential testing of cytokine gene expression by RT-PCR analysis. Our results demonstrated that isolated and incubated PBMCs expressed TNF-alpha and high levels of IL-1beta, IL-6, IL-8, and IL-10. In contrast, WB only expressed the mRNA cytokines of TNF-alpha and IL-8 (p < 0.05). These results suggest that spontaneous myriad mRNA cytokine expression can be avoided with the use of WB incubation and the rapid collection of PBMCs. Furthermore, this method should be employed in all cases where the levels of cytokine gene expression can be evaluated.

Breast cancer is the most frequent malignant tumor among women and is the leading cause of death from cancer worldwide. In 2020, 90,222 deaths from breast cancer were reported in México. However, cancer cells resistant to antineoplastic drugs are frequent, which compromises the patient’s survival. For this reason, in oncology, it is essential to search for new sources of anticancer compounds. Endophytic fungi are currently considered potential reservoirs for compounds with antitumor activity. More than 100 different classes of secondary metabolites have been reported with activity against different types of cancer, including breast cancer. Therefore, the objective of this study was to evaluate the activity of methanol extracts of endophytic fungi isolated from Lophocereus marginatus against the MCF-7 breast cancer cell line, using as controls the MA-104 cell line human peripheral blood mononuclear cells. Fungal strains were isolated from stems of L. marginatus and molecularly identified from ribosomal DNA internal transcript spacer region sequencing. Extraction of the secondary metabolites was performed from the maceration of mycelium fungus in methanol. The solvent was removed with a rotary evaporator and the extract was reconstituted with dimethyl sulfoxide (DMSO). Next, cells were incubated with the extract at concentrations ranging from 31.25 µg/mL to 250 µg/mL for 48 h at 37 °C and 5% CO2. Growth inhibition was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay, using 1% DMSO was used as a negative control. IC50 values and selectivity indexes (SI) were then calculated. It was found that tumor cells growth inhibition by the extracts increased in a concentration-dependent manner. A. versicolor PME-H005 strain extract showed the highest antitumor activity, with up to 58.9% growth inhibition at 250 µg/mL and IC50 value of 95.21 ± 1 µg/mL against MCF-7 cells. The highest SI was obtained with MCF-7 cell s with 2.77, as compared with normal PBMC with an IC50 of 264 ± 1.5 µg/mL. In addition, the M. anisopliae PME-H007 strain presented a high SI value of 2.1 and an IC50 of 245.9 ± 1.9 µg/mL, using the MCF-7 cell line. This study shows the potential of the endophytic fungus A. versicolor PME-H005 isolated from L. marginatus for production of secondary metabolites with antitumor activity against MCF-7 cells. Citation Format: Jesica M. Ramírez Villalobos, Cesar I. Romo Sáenz, Karla S. Morán Santibañez, Patricia Tamez Guerra, Orquídea Pérez González, Reyes Tamez Guerra, Cristina Rodríguez-Padilla, Ricardo A. Gomez Flores. In vitro antitumoral activity of endophytic fungi isolated from Lophocereus marginatus against the human breast cancer cell line MCF-7 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-27.

ARTICLE INFOIMMUNEPOTENT CRP is a biological response modifier with intrinsic antioxidant, antiinflammatory, and antitumor properties. In the following review, we have summarized almost 20 years of research with this bio-compound regarding its obtention method, action mechanism, molecular target and in vitro, preclinical and clinical research.