AID overexpression leads to aggressive murine CLL and nonimmunoglobulin mutations that mirror human neoplasms.

Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease that exemplifies this process and is a model for neoplasms in general, we created transgenic mice overexpressing the enzyme activation-induced deaminase (AID), which has a normal function of inducing DNA mutations in B lymphocytes. AID not only allows normal B lymphocytes to develop more effective immunoglobulin-mediated immunity, but is also able to mutate nonimmunoglobulin genes, predisposing to cancer. In CLL, AID expression correlates with poor prognosis, suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Eµ-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in nonimmunoglobulin genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically similar amino acid substitutions as in human CLL and lymphoma. Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

Human hydatid cyst fluid-induced therapeutic anti-cancer immune responses via NK1.1+ cell activation in mice.

Echinococcus granulosus is a cestode parasite which causes cystic echinococcosis disease. Previously we observed that vaccination with E. granulosus antigens from human hydatid cyst fluid (HCF) significantly inhibits colon cancer growth. In the present work, we evaluate the anti-tumor immune response induced by human HCF against LL/2 lung cancer in mice. HCF vaccination protected from tumor growth, both in prophylactic and therapeutic settings, and significantly increased mouse survival compared to control mice. Considering that tumor-associated carbohydrate antigens are expressed in E. granulosus, we oxidized terminal carbohydrates in HCF with sodium periodate. This treatment abrogates the anti-tumor activity induced by HCF vaccination. We found that HCF vaccination-induced IgG antibodies that recognize LL/2 tumor cells by flow cytometry. An antigen-specific immune response is induced with HCF vaccination in the tumor-draining lymph nodes and spleen characterized by the production of IL-5 and, in less extent, IFNÉ£. In the tumor microenvironment, we found that NK1.1 positive cells from HCF-treated mice showed higher expression of CD69 than control mice ones, indicating a higher level of activation. When we depleted these cells by administrating the NK-specific antibody NK1.1, a significantly decreased survival was observed in HCF-induced mice, suggesting that NK1.1+ cells mediate the anti-tumor protection induced by HCF. These results suggest that HCF can evoke an integrated anti-tumor immune response involving both, the innate and adaptive components, and provide novel insights into the understanding of the intricate relationship between HCF vaccination and tumor growth.

Establishment of an environmental microbiological monitoring program in a mice barrier facility.

Rodents used in biomedical research are maintained behind barriers to exclude microbial contaminants. Several check points have to be monitored to eliminate the potential of introducing adventitious agents into the facility. Microbiological monitoring of a mouse facility environment enables to evaluate the efficiency of sanitization and cleaning procedures, air quality, and technician good practices. At our SPF mouse facility, we implemented an environmental microbiological monitoring program based in sedimentation and swabbing, inexpensive and easy to use methods. The aim of this work was to evaluate the results and the efficiency of the monitoring program after seven years. The median for bacteria and fungi counts in the SPF sampled areas was ≤2 CFU/2 h for settle plates and <1 CFU per swabbing plate, satisfying the requirements for grade C of the EU-GMP, with some modifications. The environmental monitoring program was useful to detect early warning of problems and enabled us to define a safe range of microbiological counts. In addition, SPF status defined for our mice was maintained throughout this study, confirmed by our HM program. This work could encourage directors and technicians of other mouse facilities in Latin America and rest of the world to implement this kind of program.

Colony aging affects the reproductive performance of Swiss Webster females used as recipients for embryo transfer.

The objective was to evaluate the influence of colony aging in a Swiss Webster (SW) outbred stock used as recipients for embryo transfer. In the first study, a retrospective analysis was performed throughout several generations during a 38-month period in 2,398 embryos transferred to 108 SW recipients. A decrease in the percentage of live pups from transferred embryos was found at the end of the period. Impairment occurred due to the incidence of maternal cannibalism that increased from 0% to 67-100% (P<0.05), while pregnancy rate (pregnant/transferred recipients) and number of pups per delivered female were not affected throughout the period (P=NS). A following study was carried out to compare the reproductive performance of SW stock vs. B6D2F1 hybrid females in a 5-year interval. The study was conducted on a total of 893 embryos transferred to 40 females (20 SW and 20 B6D2F1) in Year #1, and 514 embryos transferred to 30 females (15 SW and 15 B6D2F1) in Year #5. No cases of maternal cannibalism were found on Year #1 in any of the strains (0/10 and 0/10). However, an incidence of 44,4% (4/9) was seen on Year #5 for SW, while for B6D2F1 the incidence was 0% (0/12) (P<0.05). Further examination of the uterus showed endometrial cysts and abnormal implantation sites in SW on Year #5 but not in B6D2F1 females. In conclusion, this study reports an impairment of the reproductive performance of an early aged SW outbred stock colony mainly due to the occurrence of maternal cannibalism. This finding has important implications for embryo transfer programs conducted in mouse facilities.

Long-Term Effect of Environmental Enrichment on Reproductive Performance of Swiss Webster Mice and Their Female Offspring.

The aim of this study was to evaluate the effect of an environmental enrichment (EE) plan on the reproductive performance of Swiss Webster mice and their female offspring used as recipients for embryo transfer. A total of 54 breeder mice and 60 F1 females, used as foster mothers, were allocated in two experimental groups to receive or not receive EE for physical well-being. Reproductive outcomes of the Swiss trios such as birth rate and pup number, litter size, pups' weight at weaning, interlitter interval and time to first litter were analyzed. Environmental enrichment significantly increased pups weight from breeding trios compared to the control group (14.4 ± 0.1 vs. 13.8g ± 0.1, EE vs. control, respectively; p < 0.01). Other parameters did not differ between both groups. Reproductive parameters of female offspring used as recipients for embryo transfer did not differ among groups subjected or not to EE. These data demonstrate that the EE protocol applied in Swiss Webster breeder mice positively enhanced pups weight, and did not interfere with other reproductive outcomes. In conclusion, this study supports the implementation of EE plans usually applied for animal welfare in mouse facilities with slight improvement in reproductive performance.