Paul C. Stromberg

Pten in stromal fibroblasts suppresses mammary epithelial tumours

The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten–Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours.

Interleukin-10 Promotes Mycobacterium tuberculosis Disease Progression in CBA/J Mice

IL-10 is a potent immunomodulatory cytokine that affects innate and acquired immune responses. The immunological consequences of IL-10 production during pulmonary tuberculosis (TB) are currently unknown, although IL-10 has been implicated in reactivation TB in humans and with TB disease in mice. Using Mycobacterium tuberculosis-susceptible CBA/J mice, we show that blocking the action of IL-10 in vivo during chronic infection stabilized the pulmonary bacterial load and improved survival. Furthermore, this beneficial outcome was highly associated with the recruitment of T cells to the lungs and enhanced T cell IFN-γ production. Our results indicate that IL-10 promotes TB disease progression. These findings have important diagnostic and/or therapeutic implications for the prevention of reactivation TB in humans.

Mouse development with a single E2F activator

The E2F family is conserved from Caenorhabditis elegans to mammals, with some family members having transcription activation functions and others having repressor functions. Whereas C. elegans and Drosophila melanogaster have a single E2F activator protein and repressor protein, mammals have at least three activator and five repressor proteins. Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, we targeted the inactivation of the entire subset of activators, E2f1, E2f2, E2f3a and E2f3b, singly or in combination in mice. We demonstrate that E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus (E2f3a3bki or E2f3a1ki, respectively) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. We conclude that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development.

Studies of early hepatocellular proliferation and peroxisomal proliferation in Sprague-Dawley rats treated with tumorigenic doses of clofibrate

Clofibrate, a peroxisome proliferator, is hepatocarcinogenic in rats in a dose-dependent fashion. While there is a relationship between peroxisome proliferation and rodent liver carcinogenesis, recent evidence also suggests an association between the tumorigenicity of peroxisome proliferators and sustained cell proliferation. To investigate the role of early cell proliferation in clofibrate-induced carcinogenesis and the predictive potential of this endpoint, in a 3-month study, rats were fed clofibrate doses equivalent to those used in the chronic bioassay, and cell proliferation was determined after 1 week and 3 months, using a 1-week continuous bromodeoxyuridine (BrdU)-labeling technique. Adult Sprague-Dawley rats were fed clofibrate at 1500, 4500, or 9000 ppm. Six rats/sex/group were killed after 1 or 13 weeks of treatment. Osmotic minipumps containing BrdU were implanted into rats 7 days prior to necropsy to determine the cumulative 7-day hepatocyte labeling index immunohistochemically. A dose-related increase in hepatocyte labeling index was seen after 1 week of treatment. However, at 13 weeks, sustained increases in hepatocyte proliferation were not seen; but a dose-related decrease in the hepatocyte labeling index was observed. Liver stereology at 13 weeks demonstrated a dose-related increase in liver weight and volume, but a decrease in hepatocyte nuclei per unit volume, a minimal increase or no change in the total number of hepatocyte nuclei per liver, and an absolute decline in the total number of BrdU-labeled hepatocyte nuclei per liver. These data suggest that in rats, clofibrate may influence hepatocarcinogenicity by decreases in normal hepatocyte proliferation over time and this effect may influence the pathogenesis of tumors at time points beyond 13 weeks of treatment.

CCL5 participates in early protection against Mycobacterium tuberculosis

Control of M.tb, the causative agent of TB, requires immune cell recruitment to form lung granulomas. The chemokines and chemokine receptors that promote cell migration for granuloma formation, however, are not defined completely. As immunity to M.tb manifests slowly in the lungs, a better understanding of specific roles for chemokines, in particular those that promote M.tb‐protective TH1 responses, may identify targets that could accelerate granuloma formation. The chemokine CCL5 has been detected in patients with TB and implicated in control of M.tb infection. To define a role for CCL5 in vivo during M.tb infection, CCL5 KO mice were infected with a low dose of aerosolized M.tb. During early M.tb infection, CCL5 KO mice localized fewer APCs and chemokine receptor‐positive T cells to the lungs and had microscopic evidence of altered cell trafficking to M.tb granulomas. Early acquired immunity and granuloma function were transiently impaired when CCL5 was absent, evident by delayed IFN‐γ responses and poor control of M.tb growth. Lung cells from M.tb‐infected CCL5 KO mice eventually reached or exceeded the levels of WT mice, likely as a result of partial compensation by the CCL5‐related ligand, CCL4, and not because of CCL3. Finally, our results suggest that most T cells use CCR5 but not CCR1 to interact with these ligands. Overall, these results contribute to a model of M.tb granuloma formation dependent on temporal regulation of chemokines rather than on redundant or promiscuous interactions.

IL-10 Inhibits Mature Fibrotic Granuloma Formation during Mycobacterium tuberculosis Infection

Protective immunity and latent Mycobacterium tuberculosis infection in humans are associated with the formation of mature protective granulomas within the lung. Unfortunately, understanding the importance of such structures has been hindered by the lack of small-animal models that can develop mature granulomas. In this article, we describe for the first time, to our knowledge, the formation of mature, fibrotic M. tuberculosis–containing pulmonary granulomas in a mouse model of IL-10 deficiency (CBA/J IL-10−/−). Long-term control of M. tuberculosis infection in the absence of IL-10 was also associated with an early and enhanced capacity for Ag presentation and a significant increase in the generation of multifunctional T cells. Although IL-10 deficiency is known to enhance Th1 immune responses in general, we demonstrate in this study using transient anti–IL-10R treatment that it is the presence of IL-10 in vivo during the first month of M. tuberculosis infection that plays a definitive role in the inhibition of optimum protective immunity that can establish the environment for mature granuloma formation. Although the importance of IL-10 during M. tuberculosis infection has been debated, our data demonstrate that in CBA/J mice, IL-10 plays a significant early inhibitory role in preventing the development of protective immunity associated with containment of M. tuberculosis infection.

Allele-specific tumor spectrum in Pten knockin mice

Germline mutations in the tumor suppressor gene PTEN (phosphatase and tensin homology deleted on chromosome 10) cause Cowden and Bannayan–Riley–Ruvalcaba (BRR) syndromes, two dominantly inherited disorders characterized by mental retardation, multiple hamartomas, and variable cancer risk. Here, we modeled three sentinel mutant alleles of PTEN identified in patients with Cowden syndrome and show that the nonsense Pten∆4–5 and missense PtenC124R and PtenG129E alleles lacking lipid phosphatase activity cause similar developmental abnormalities but distinct tumor spectra with varying severity and age of onset. Allele-specific differences may be accounted for by loss of function for Pten∆4–5, hypomorphic function for PtenC124R, and gain of function for PtenG129E. These data demonstrate that the variable tumor phenotypes observed in patients with Cowden and BRR syndromes can be attributed to specific mutations in PTEN that alter protein function through distinct mechanisms.

Exposure to Mycobacterium avium can modulate established immunity against Mycobacterium tuberculosis infection generated by Mycobacterium bovis BCG vaccination

Mycobacterium bovis bacille Calmette Guerin (BCG), the current vaccine against infection with Mycobacterium tuberculosis, offers a variable, protective efficacy in man. It has been suggested that exposure to environmental mycobacteria can interfere with the generation of BCG‐specific immunity. We hypothesized that exposure to environmental mycobacteria following BCG vaccination would interfere with established BCG immunity and reduce protective efficacy, thus modeling the guidelines for BCG vaccination within the first year of life. Mice were vaccinated with BCG and subsequently given repeated oral doses of live Mycobacterium avium to model exposure to environmental mycobacteria. The protective efficacy of BCG with and without subsequent exposure to M. avium was determined following an aerogenic challenge with M. tuberculosis. Exposure of BCG‐vaccinated mice to M. avium led to a persistent increase in the number of activated T cells within the brachial lymph nodes but similar T cell activation profiles in the lungs following infection with M. tuberculosis. The capacity of BCG‐vaccinated mice to reduce the bacterial load following infection with M. tuberculosis was impaired in mice that had been exposed to M. avium. Our data suggest that exposure to environmental mycobacteria can negatively impact the protection afforded by BCG. These findings are relevant for the development of a vaccine administered in regions with elevated levels of environmental mycobacteria.

Population biology of Camallanus oxycephalus Ward and Magath, 1916 (Nematoda: Camallanidae) in white bass in western Lake Erie.

The distribution and abundance of the nematode Camallanus oxycephalus infecting white bass, Morone chrysops, in western Lake Erie was studied for over 2 years. Infection was generally more frequent and of higher intensity in large fish. The frequency distributions of nematode abundance in all segments of the fish population followed the negative binomial distribution. The data show seasonal cycles in population structure, site selection, intensity of infection, maturation, and reproduction. Infection occurs during July and August with a resulting peak in population density; during late summer and autumn, mortality, probably density-dependent, reduces the population by 30 to 60%; surviving worms are eliminated at 1 year of age. Growth and development of female worms is arrested from November to April, then proceeds at a rapid rate until the worms release their larvae and die. This growth pattern is probably related to temperature but may also involve host hormone cycles. The dispersal period of the nematode coincides with the annual maximum density of the intermediate host, a cyclopoid copepod,and is interpreted as an adaptation which increases the probability of successful transmission. Because the number of larvae produced by each female worm is a function of body volume, natural selection has favored rapid spring growth and attainment of large body size relative to the male worm. Both seasonal timing in the life cycle and the number of larvae produced are important factors in determining the abundance of this and perhaps other parasites. Evidence is presented suggesting that fluctuations of environmental parameters may disrupt the timing of transmission and alter the distribution and abundance of the parasite. It is hypothesized that the magnitude of such changes in parasite abundance may be related to the complexity of the host-parasite system.

Cutaneous neosporosis in two adult dogs on chronic immunosuppressive therapy

Antemortem diagnosis of generalized ulcerative and pyogranulomatous dermatitis with numerous intralesional tachyzoites was made from skin biopsy specimens from 2 adult dogs on chronic immunosuppressive therapy. A 9-year-old Italian Greyhound was on long-term corticosteroid therapy for the treatment of a lupus-like systemic autoimmune disorder, and a 7-year-old Labrador Retriever had received several months of chemotherapy for lymphosarcoma. The tachyzoites were identified as Neospora caninum by immunoperoxidase immunohistochemistry. Both dogs were treated with clindamycin. Lesions in the Greyhound resolved; however, the Labrador Retriever was euthanized because of evidence of neuromuscular disease, despite improvement of the skin lesions. These 2 cases indicate that cutaneous neosporosis can occur in adult dogs on chronic immu-nosuppressive therapy. The disease may result from reactivation of a congenital infection and/or a recently acquired primary infection.