Cynthia Besch-Williford

Inflammatory central nervous system disease in lupus-prone MRL/lpr mice: comparative histologic and immunohistochemical findings

The brains of pathogen-free autoimmune MRL/lpr, NZBWF1 and NZB mice were examined for central nervous system (CNS) inflammation in premoribund 8-week-old animals and at ages when active systemic lupus erythematosus (SLE) was present. CNS inflammation was observed only in MRL/lpr mice. Immunohistochemical studies of brains from young MRL/lpr mice found that infiltrates were composed primarily of CD4+ cells. Older MRL/lpr mice (22 and 26 weeks of age) had CD4+ cells predominantly, but CD8+ and B220+ cells were also present. Perivascular leakage of IgG was a prominent and unexpected finding in the MRL/lpr model. Congenic MRL/+ mice with late-onset autoimmunity had no inflammatory cells in brain tissue, and there was no perivascular staining with IgG or albumin. Our findings suggest that MRL/lpr mice are a useful model for studies of lupus-associated CNS inflammatory disease, and perivascular leakage may be a primary mechanism for entry of IgG into the brain.

Progestin-Dependent Progression of Human Breast Tumor Xenografts: A Novel Model for Evaluating Antitumor Therapeutics

Recent clinical trials indicate that synthetic progestins may stimulate progression of breast cancer in postmenopausal women, a result that is consistent with studies in chemically-induced breast cancer models in rodents. However, progestin-dependent progression of breast cancer tumor xenografts has not been shown. This study shows that xenografts obtained from BT-474 and T47-D human breast cancer cells without Matrigel in estrogen-supplemented nude mice begin to regress within days after tumor cell inoculation. However, their growth is resumed if animals are supplemented with progesterone. The antiprogestin RU-486 blocks progestin stimulation of growth, indicating involvement of progesterone receptors. Exposure of xenografts to medroxyprogesterone acetate, a synthetic progestin used in postmenopausal hormone replacement therapy and oral contraception, also stimulates growth of regressing xenograft tumors. Tumor progression is dependent on expression of vascular endothelial growth factor (VEGF); growth of progestin-dependent tumors is blocked by inhibiting synthesis of VEGF or VEGF activity using a monoclonal anti-VEGF antibody (2C3) or by treatment with PRIMA-1, a small-molecule compound that reactivates mutant p53 into a functional protein and blocks VEGF production. These results suggest a possible model system for screening potential therapeutic agents for their ability to prevent or inhibit progestin-dependent human breast tumors. Such a model could potentially be used to screen for safer antiprogestins, antiangiogenic agents, or for compounds that reactivate mutant p53 and prevent progestin-dependent progression of breast disease.

Molecular characterization of newly recognized rodent parvoviruses

Several autonomous rodent parvoviruses distinct from the prototypic rodent parvoviruses have been isolated. These include variants of a mouse parvovirus (MPV), a hamster isolate designated hamster parvovirus (HaPV), and a variant strain of minute virus of mice (MVM) designated MVM-Cutter or MVM(c). In this study, the DNA sequence of the coding regions of the viral genome and the predicted protein sequences for each of these new isolates were determined and compared to the immunosuppressive and prototypic strains of MVM [MVM(i) and MVM(p)], the rodent parvovirus H-1, and LuIII, an autonomous parvovirus of uncertain host origin. Sequence comparisons showed that the MPV isolates were almost identical, HaPV was very similar to MPV, and MVM(c) was most similar to MVM(i) and MVM(p). Haemagglutination inhibition assays revealed that MPV and HaPV represent two serotypes distinct from previously characterized rodent parvovirus serotypes while MVM(c) belongs to the MVM serotype. Each of the newly isolated rodent parvoviruses was shown to encapsidate a predominantly negative-sense 5 kb DNA genome and to encode two nonstructural proteins (NS1 and NS2) and two structural viral proteins (VP1 and VP2). These studies indicate that MPV and HaPV are autonomous parvoviruses distinct from previously characterized parvoviruses and MVM(c) is a variant strain of MVM distinct from MVM(i) and MVM(p).

Centrosome-centriole abnormalities are markers for abnormal cell divisions and cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model.

We utilized the transgenic adenocarcinoma mouse prostate (TRAMP) model to study the formation of abnormal mitosis in malignant tumors of the prostate. The results presented here are focused on centrosome and centriole abnormalities and the implications for abnormal cell divisions, genomic instability, and apoptosis. Centrosomes are microtubule organizing organelles which assemble bipolar spindles in normal cells but can organize mono‐, tri‐, and multipolar mitoses in tumor cells, as shown here with histology and electron microscopy in TRAMP neoplastic tissue. These abnormalities will cause unequal distribution of chromosomes and can initiate imbalanced cell cycles in which checkpoints for cell cycle control are lost. Neoplastic tissue of the TRAMP model is also characterized by numerous apoptotic cells. This may be the result of multipolar mitoses related to aberrant centrosome formations. Our results also reveal that centrosomes at the poles in mitotic cancer cells contain more than the regular perpendicular pair of centrioles which indicates abnormal distribution of centrioles during separation to the mitotic poles. Abnormalities in the centriole‐centrosome complex are also seen during interphase where the complex is either closely associated with the nucleus or loosely dispersed in the cytoplasm. An increase in centriole numbers is observed during interphase, which may be the result of increased centriole duplication. Alternatively, these centrioles may be derived from basal bodies that have accumulated in the cells cytoplasm, after the loss of cell borders. The supernumerary centrioles may participate in the formation of abnormal mitoses during cell division. These results demonstrate multiple abnormalities in the centrosome‐centriole complex during prostate cancer that result in abnormal mitoses and may lead to increases in genomic instability and/or apoptosis.

Serodiagnosis of Mice Minute Virus and Mouse Parvovirus Infections in Mice by Enzyme-Linked Immunosorbent Assay with Baculovirus-Expressed Recombinant VP2 Proteins

ABSTRACT Mice minute virus (MMV) and mouse parvovirus (MPV) type 1 are the two parvoviruses known to naturally infect laboratory mice and are among the most prevalent infectious agents found in contemporary laboratory mouse colonies. Serologic assays are commonly used to diagnose MMV and MPV infections in laboratory mice; however, highly accurate, high-throughput serologic assays for the detection of MMV- and MPV-infected mice are needed. To this end, the major capsid viral protein (VP2) genes of MMV and MPV were cloned and MMV recombinant VP2 (rVP2) and MPV rVP2 proteins were expressed by using a baculovirus system. MMV rVP2 and MPV rVP2 spontaneously formed virus-like particles that were morphologically similar to empty parvovirus capsids. These proteins were used as antigens in enzyme-linked immunosorbent assays (ELISAs) to detect anti-MMV or anti-MPV antibodies in the sera of infected mice. Sera from mice experimentally infected with MMV (n = 43) or MPV (n = 35) and sera from uninfected mice (n = 30) were used to evaluate the ELISAs. The MMV ELISA was 100% sensitive and 100% specific in detecting MMV-infected mice, and the MPV ELISA was 100% sensitive and 98.6% specific in detecting MPV-infected mice. Both assays outperformed a parvovirus ELISA that uses a recombinant nonstructural protein (NS1) of MMV as antigen. The MMV rVP2 and MPV rVP2 proteins provide a ready source of easily produced antigen, and the ELISAs developed provide highly accurate, high-throughput assays for the serodiagnosis of MMV and MPV infections in laboratory mice.

Synthetic progestins induce growth and metastasis of BT-474 human breast cancer xenografts in nude mice

Objective: Previous studies have shown that sequential exposure to estrogen and progesterone or medroxyprogesterone acetate (MPA) stimulates vascularization and promotes the progression of BT-474 and T47-D human breast cancer cell xenografts in nude mice (Liang et al, Cancer Res 2007, 67:9929). In this follow-up study, the effects of progesterone, MPA, norgestrel (N-EL), and norethindrone (N-ONE) on BT-474 xenograft tumors were compared in the context of several different hormonal environments. N-EL and N-ONE were included in the study because synthetic progestins vary considerably in their biological effects and the effects of these two progestins on the growth of human tumor xenografts are not known. Methods: Estradiol-supplemented intact and ovariectomized immunodeficient mice were implanted with BT-474 cells. Progestin pellets were implanted simultaneously with estradiol pellets either 2 days before tumor cell injection (ie, combined) or 5 days after tumor cell injections (ie, sequentially). Results: Progestins stimulated the growth of BT-474 xenograft tumors independent of exposure timing and protocol, MPA stimulated the growth of BT-474 xenograft tumors in ovariectomized mice, and progestins stimulated vascular endothelial growth factor elaboration and increased tumor vascularity. Progestins also increased lymph node metastasis of BT-474 cells. Therefore, progestins, including N-EL and N-ONE, induce the progression of breast cancer xenografts in nude mice and promote tumor metastasis. Conclusions These observations suggest that women who ingest progestins for hormone therapy or oral contraception could be more at risk for developing breast cancer because of proliferation of existing latent tumor cells. Such risks should be considered in the clinical setting.

Aggressive Prostate Cancer Is Prevented in ERαKO Mice and Stimulated in ERβKO TRAMP Mice

Previous evidence suggests soy genistein may be protective against prostate cancer, but whether this protection involves an estrogen receptor (ER)-dependent mechanism is unknown. To test the hypothesis that phytoestrogens may act through ERα or ERβ to play a protective role against prostate cancer, we bred transgenic mice lacking functional ERα or ERβ with transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Dietary genistein reduced the incidence of cancer in ER wild-type (WT)/transgenic adenocarcinoma of mouse prostate mice but not in ERα knockout (KO) or ERβKO mice. Cancer incidence was 70% in ERWT mice fed the control diet compared with 47% in ERWT mice fed low-dose genistein (300 mg/kg) and 32% on the high-dose genistein (750 mg/kg). Surprisingly, genistein only affected the well differentiated carcinoma (WDC) incidence but had no effect on poorly differentiated carcinoma (PDC). No dietary effects have been observed in either of the ERKO animals. We observed a very strong genotypic influence on PDC incidence, a protective effect in ERαKO (only 5% developed PDC), compared with 19% in the ERWT, and an increase in the incidence of PDC in ERβKO mice to 41%. Interestingly, immunohistochemical analysis showed ERα expression changing from nonnuclear in WDC to nuclear in PDC, with little change in ERβ location or expression. In conclusion, genistein is able to inhibit WDC in the presence of both ERs, but the effect of estrogen signaling on PDC is dominant over any dietary treatment, suggesting that improved differential targeting of ERα vs. ERβ would result in prevention of advanced prostate cancer.

Natural and Synthetic Progestins Accelerate 7,12-Dimethylbenz[a]Anthracene-Initiated Mammary Tumors and Increase Angiogenesis in Sprague-Dawley Rats

Purpose: Synthetic progestins are widely used therapeutically; however, there is controversy regarding their proliferative effects. We used a rat 7,12-dimethylbenz[a]anthracene (DMBA)–induced mammary tumor model to test the hypothesis that progestins increase angiogenesis and as a result decrease the latency period and increase the multiplicity of mammary tumors. Experimental Design: Medroxyprogesterone acetate (MPA) pellets were implanted 2, 4, or 6 weeks after DMBA exposure; RU-486 was given 3 days before MPA. Experiments were concluded 70 days after DMBA administration. Results: MPA exposure 4 or 6 weeks after DMBA reduced the latency period for appearance of tumors in a dose-dependent manner and increased tumor incidence. Administration of MPA 2 weeks after DMBA administration reduced tumor incidence and was protective. Progesterone did not reduce the latency period but significantly increased tumor incidence. RU-486 delayed the latency period and decreased tumor incidence in animals exposed to MPA at 4 weeks after DMBA treatment, indicating that the progesterone receptor may be partially responsible for transmission of proliferative signals. RU-486 also delayed the latency period but failed to reduce overall tumor incidence when animals were exposed to MPA at 6 weeks after DMBA treatment, indicating that other factors may also control MPA-induced acceleration. Whereas MPA-accelerated tumors were both intraductal and tubular, progesterone-accelerated and/or DMBA-induced tumors were tubular. Progestin treatment increased vascular endothelial growth factor expression within tumors in a ligand- and cell type–dependent manner and increased angiogenesis in correlation with vascular endothelial growth factor expression. No mammary tumors or progesterone receptor were detected in DMBA-treated ovariectomized rats regardless of progestin administration. Conclusions: We propose that progestins can accelerate the development of mammary tumors and that antiangiogenic agents and/or the use of antiprogestins that can reduce tumor incidence might be a viable therapeutic option for treatment of progestin-accelerated tumors. The model described here is a potentially useful preclinical model for rapidly screening such compounds.

Biology and medicine of the ferret.

There is still much more to be learned about ferret behavior, nutrition, and physiologic responses associated with aging, disease, and environmental stimuli. The many similarities in the clinical disorders of ferrets and other small companion animals should emphasize the importance of using the same diagnostic methods developed for common companion animals in examinations of ferrets. There is still very little known of the efficacy of a wide spectrum of drugs in ferrets, and judicious use of pharmacodynamic agents that are safe for cats would be a reasonable approach to drug therapy.

Chemokine Receptor CXCR2 Mediates Bacterial Clearance Rather Than Neutrophil Recruitment in a Murine Model of Pneumonic Plague

Pulmonary infection by Yersinia pestis causes pneumonic plague, a necrotic bronchopneumonia that is rapidly lethal and highly contagious. Acute pneumonic plague accompanies the up-regulation of pro-inflammatory cytokines and chemokines, suggesting that the host innate immune response may contribute to the development of disease. To address this possibility, we sought to understand the consequences of neutrophil recruitment during pneumonic plague, and we studied the susceptibility of C3H-HeN mice lacking the CXC chemokine KC or its receptor CXC receptor 2 (CXCR2) to pulmonary Y. pestis infection. We found that without Kc or Cxcr2, disease progression was accelerated both in bacterial growth and development of primary bronchopneumonia. When examined in an antibody clearance model, Cxcr2(-/-) mice were not protected by neutralizing Y. pestis antibodies, yet bacterial growth in the lungs was delayed in a manner associated with a neutrophil-mediated inflammatory response. After this initial delay, however, robust neutrophil recruitment in Cxcr2(-/-) mice correlated with bacterial growth and the development of fulminant pneumonic and septicemic plague. In contrast, attenuated Y. pestis lacking the conserved pigmentation locus could be cleared from the lungs in the absence of Cxcr2 indicating virulence factors within this locus may inhibit CXCR2-independent pathways of bacterial killing. Together, the data suggest CXCR2 uniquely induces host defense mechanisms that are effective against virulent Y. pestis, raising new insight into the activation of neutrophils during infection.