MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines

Analyses of MUC1-specific cytotoxic T cell precursor (CTLp) frequencies were performed in mice immunized with three different MUC1 vaccine immunotherapeutic agents. Mice were immunized with either a fusion protein comprising MUC1 and glutathione S-transferase (MUC1-GST), MUC1-GST fusion protein coupled to mannan (MFP) or with a recombinant vaccinia virus expressing both MUC1 and interleukin-2. Mouse strain variations in immune responsiveness have been observed with these vaccines. We have constructed mice transgenic for the human MUC1 gene to study MUC1-specific immune responses and the risk of auto-immunity following MUC1 immunization. Transgenic mice immunized with MUC1 were observed to be partially tolerant in that the MUC1-specific antibody response is lower than that observed in syngeneic but non-transgenic mice. However, a significant MUC1-specific CTLp response to all three vaccines was observed, indicating the ability to overcome T cell, but to a lesser extent B cell, tolerance to MUC1 in these mice. Histological analysis indicates no evidence of auto-immunity to the cells expressing the human MUC1 molecule. These results suggest that it is possible to generate an immune response to a cancer-related antigen without damage to normal tissues expressing the antigen. Received: 7 July 1999 / Accepted: 26 August 1999     

Oxysterol nuclear receptor LXRbeta regulates cholesterol homeostasis and contractile function in mouse uterus

The uterus is an organ where lipid distribution plays a critical role for its function. Here we show that nuclear receptor for oxysterols LXRbeta prevents accumulation of cholesteryl esters in mouse myometrium by controlling expression of genes involved in cholesterol efflux and storage (abca1 and abcg1). Upon treatment with an LXR agonist that mimics activation by oxysterols, expression of these target genes was increased in wild-type mice, whereas under basal conditions, lxralpha;beta(-/-) mice exhibited a marked decrease in abcg1 accumulation. This change resulted in a phenotype of cholesteryl ester accumulation. Besides, a defect of contractile activity induced by oxytocin or PGF2alpha was observed in mice lacking LXRbeta. These results imply that LXRbeta provides a safety valve to limit cholesteryl ester levels as a basal protective mechanism in the uterus against cholesterol accumulation and is necessary for a correct induction of contractions.     

Myopathy-associated alphaB-crystallin mutants: abnormal phosphorylation, intracellular location, and interactions with other small heat shock proteins

Three mutations (R120G, Q151X, and 464delCT) in the small heat shock protein alphaB-crystallin cause inherited myofibrillar myopathy. In an effort to elucidate the molecular basis for the associated myopathy, we have determined the following for these mutant alphaB-crystallin proteins: (i) the formation of aggregates in transfected cells; (ii) the partition into different subcellular fractions; (iii) the phosphorylation status; and (iv) the ability to interact with themselves, with wild-typealphaB-crystallin, and with other small heat shock proteins that are abundant in muscles. We found that all three alphaB-crystallin mutants have an increased tendency to form cytoplasmic aggregates in transfected cells and significantly increased levels of phosphorylation when compared with the wild-type protein. Although wild-type alphaB-crystallin partitioned essentially into the cytosol and membranes/organelles fractions, mutant alphaB-crystallin proteins partitioned additionally into the nuclear and cytoskeletal fractions. By using various protein interaction assays, including quantitative fluorescence resonance energy transfer measurements in live cells, we found abnormal interactions of the various alphaB-crystallin mutants with wild-type alphaB-crystallin, with themselves, and with the other small heat shock proteins Hsp20, Hsp22, and possibly with Hsp27. The collected data suggest that eachalphaB-crystallin mutant has a unique pattern of abnormal interaction properties. These distinct properties of the alphaB-crystallin mutants identified are likely to contribute to a better understanding of the gradual manifestation and clinical heterogeneity of the associated myopathy in patients.     

Biochemical characterization of USP7 reveals post-translational modification sites and structural requirements for substrate processing and subcellular localization

Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinating enzymes. Among other functions, USP7 is involved in the regulation of stress response pathways, epigenetic silencing and the progress of infections by DNA viruses. USP7 is a 130-kDa protein with a cysteine peptidase core, N- and C-terminal domains required for protein-protein interactions. In the present study, recombinant USP7 full length, along with several variants corresponding to domain deletions, were expressed in different hosts in order to analyze post-translational modifications, oligomerization state, enzymatic properties and subcellular localization patterns of the enzyme. USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in mammalian cells. In in vitro activity assays, N- and C-terminal truncations affected the catalytic efficiency of the enzyme different. Both the protease core alone and in combination with the N-terminal domain are over 100-fold less active than the full length enzyme, whereas a construct including the C-terminal region displays a rather small decrease in catalytic efficiency. Limited proteolysis experiments revealed that USP7 variants containing the C-terminal domain interact more tightly with ubiquitin. Besides playing an important role in substrate recognition and processing, this region might be involved in enzyme dimerization. USP7 constructs lacking the N-terminal domain failed to localize in the cell nucleus, but no nuclear localization signal could be mapped within the enzyme's first 70 amino acids. Instead, the tumor necrosis factor receptor associated factor-like region (amino acids 70-205) was sufficient to achieve the nuclear localization of the enzyme, suggesting that interaction partners might be required for USP7 nuclear import.     

Heat shock response in CHO mammalian cells is controlled by a nonlinear stochastic process

In many biological systems, the interactions that describe the coupling between different units in a genetic network are nonlinear and stochastic. We study the interplay between stochasticity and nonlinearity using the responses of Chinese hamster ovary (CHO) mammalian cells to different temperature shocks. The experimental data show that the mean value response of a cell population can be described by a mathematical expression (empirical law) which is valid for a large range of heat shock conditions. A nonlinear stochastic theoretical model was developed that explains the empirical law for the mean response. Moreover, the theoretical model predicts a specific biological probability distribution of responses for a cell population. The prediction was experimentally confirmed by measurements at the single-cell level. The computational approach can be used to study other nonlinear stochastic biological phenomena.     

Sterile protection against malaria is independent of immune responses to the circumsporozoite protein

Background

Research aimed at developing vaccines against infectious diseases generally seeks to induce robust immune responses to immunodominant antigens. This approach has led to a number of efficient bacterial and viral vaccines, but it has yet to do so for parasitic pathogens. For malaria, a disease of global importance due to infection by Plasmodium protozoa, immunization with radiation-attenuated sporozoites uniquely leads to long lasting sterile immunity against infection. The circumsporozoite protein (CSP), an important component of the sporozoite''s surface, remains the leading candidate antigen for vaccines targeting the parasite''s pre-erythrocytic stages. Difficulties in developing CSP-based vaccines that reproduce the levels of protection afforded by radiation-attenuated sporozoites have led us to question the role of CSP in the acquisition of sterile immunity. We have used a parasite transgenic for the CSP because it allowed us to test whether a major immunodominant Plasmodium antigen is indeed needed for the induction of sterile protective immunity against infection.

Methodology/Main Findings

We employed a P. berghei parasite line that expresses a heterologous CSP from P. falciparum in order to assess the role of the CSP in the protection conferred by vaccination with radiation-attenuated P. berghei parasites. Our data demonstrated that sterile immunity could be obtained despite the absence of immune responses specific to the CSP expressed by the parasite used for challenge.

Conclusions

We conclude that other pre-erythrocytic parasite antigens, possibly hitherto uncharacterised, can be targeted to induce sterile immunity against malaria. From a broader perspective, our results raise the question as to whether immunodominant parasite antigens should be the favoured targets for vaccine development.