Gregory P. Boivin

The Na,K-ATPase α2 Isoform Is Expressed in Neurons, and Its Absence Disrupts Neuronal Activity in Newborn Mice

Na,K-ATPase is an ion transporter that impacts neural and glial physiology by direct electrogenic activity and the modulation of ion gradients. Its three isoforms in brain have cell-type and development-specific expression patterns. Interestingly, our studies demonstrate that in late gestation, the α2 isoform is widely expressed in neurons, unlike in the adult brain, in which α2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of α2 isoform expression in neurons is interesting in light of our examination of mice lacking the α2 isoform which fail to survive after birth. These animals showed no movement; however, defects in gross brain development, muscle contractility, neuromuscular transmission, and lung development were ruled out. Akinesia suggests a primary neuronal defect and electrophysiological recordings in the pre-Bötzinger complex, the brainstem breathing center, showed reduction of respiratory rhythm activity, with less regular and smaller population bursts. These data demonstrate that the Na,K-ATPase α2 isoform could be important in the modulation of neuronal activity in the neonate.

Modulation of Immune Responses in Murine Pulmonary Histoplasmosis

The influence of endogenous interleukin (IL)-12 on the course of pulmonary histoplasmosis was examined in naive and immune mice. All naive animals pretreated with anti-IL-12 monoclonal antibody (MAb) died by day 14. All mice died when anti-IL-12 MAb was initiated as late as postinfection day 3. Unlike those of controls, lungs of naive mice given anti-IL-12 MAb had depressed levels of interferon (IFN)-gamma and increased tumor necrosis factor (TNF)-alpha. The 2 groups had similar IL-4 levels. Administration of anti-IL-4 MAb rescued mice from the inimical effects of anti-IL-12 MAb. Survival of mice given both anti-IL-12 and anti-IL-4 MAb was associated with a blunted TNF-alpha response. In reinfection histoplasmosis, treatment with anti-IL-12 MAb did not alter survival. Fungus burden in lungs, livers, and spleens differed at week 2, but not at week 1, of infection. Thus, endogenous IL-12 is critical for optimal generation of a protective immune response in pulmonary histoplasmosis.

TGF-β1 Regulates Lymphocyte Homeostasis by Preventing Activation and Subsequent Apoptosis of Peripheral Lymphocytes

TGF-β1 plays an important role in the maintenance of immune homeostasis and self-tolerance. To determine the mechanism by which TGF-β1 prevents autoimmunity we have analyzed T cell activation in splenic lymphocytes from TGF-β1-deficient mice. Here we demonstrate that unlike wild-type splenic lymphocytes, those from Tgfb1−/− mice are hyporesponsive to receptor-mediated mitogenic stimulation, as evidenced by diminished proliferation and reduced IL-2 production. However, they have elevated levels of IFN-γ and eventually undergo apoptosis. Receptor-independent stimulation of Tgfb1−/− T cells by PMA plus ionomycin induces IL-2 production and mitogenic response, and it rescues them from anergy. Tgfb1−/− T cells display decreased CD3 expression; increased expression of the activation markers LFA-1, CD69, and CD122; and increased cell size, all of which indicate prior activation. Consistently, mutant CD4+ T cells have elevated intracellular Ca2+ levels. However, upon subsequent stimulation in vitro, increases in Ca2+ levels are less than those in wild-type cells. This is also consistent with the anergic phenotype. Together, these results demonstrate that the ex vivo proliferative hyporesponsiveness of Tgfb1−/− splenic lymphocytes is due to prior in vivo activation of T cells resulting from deregulated intracellular Ca2+ levels.

Haploinsufficiency of Atp2a2, Encoding the Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2 Ca2+ Pump, Predisposes Mice to Squamous Cell Tumors via a Novel Mode of Cancer Susceptibility

A null mutation in one copy of the Atp2a2 or ATP2A2 gene, encoding sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2), leads to squamous cell tumors in mice and to Darier disease in humans, a skin disorder that also involves keratinocytes. Here, we examined the time course and genetic mechanisms of tumor development in the mutant animals. Atp2a2+/- mice overexpressed keratins associated with keratinocyte hyperactivation in normal forestomachs as early as 2 months of age. By the age of 5 to 7 months, 22% of mutants had developed papillomas of the forestomach, and 89% of mutants older than 14 months had developed squamous cell papillomas and/or carcinomas, with a preponderance of the latter. Tumors occurred in regions that had keratinized epithelium and were subjected to repeated mechanical irritation. The genetic mechanism of tumorigenesis did not involve loss of heterozygosity, as tumor cells analyzed by laser capture microdissection contained the wild-type Atp2a2 allele. Furthermore, immunoblot and immunohistochemical analysis showed that tumor keratinocytes expressed the SERCA2 protein. Mutations were not observed in the ras proto-oncogenes; however, expression of wild-type ras was up-regulated, with particularly high levels of K-ras. Loss of the p53 tumor suppressor gene occurred in a single massive tumor, whereas other tumors had increased levels of p53 protein but no mutations in the p53 gene. These findings show that SERCA2 haploinsufficiency predisposes mice to tumor development via a novel mode of cancer susceptibility involving a global change in the tumorigenic potential of keratinized epithelium in Atp2a2+/- mice.

Expression of angiogenic factors in juvenile rheumatoid arthritis: Correlation with revascularization of human synovium engrafted into SCID mice

OBJECTIVEnAlthough increased vascularity was noted in early histopathologic studies of juvenile rheumatoid arthritis (JRA) synovium, the available data on angiogenesis in JRA are very limited. The main purposes of this study were to assess expression of the key angiogenic factors in JRA synovium, and to evaluate a SCID mouse model of JRA as an approach to study in vivo regulation of the expression of these factors in JRA.nnnMETHODSnRNase protection assay was used to assess the expression of the key angiogenic factors in fresh JRA synovium and in JRA synovial tissue fragments that had been minced and then implanted into SCID mice. Vascularity of the samples was assessed by immunohistochemical staining for von Willebrand factor. Synovial specimens obtained from patients with osteoarthritis (OA) or other noninflammatory arthropathies were used as controls.nnnRESULTSnDetectable levels of messenger RNA for vascular endothelial growth factor and angiopoietin 1 and their respective receptors, as well as endoglin and thrombin receptors, were present in all JRA tissue specimens studied. The levels of expression of these factors in JRA tissues were significantly higher than those in tissues obtained from patients with OA or other noninflammatory arthropathies. Furthermore, increased expression of the key angiogenic factors in the fresh JRA tissues correlated with the exuberant revascularization of JRA minced tissue fragments implanted into SCID mice. This was in sharp contrast to the poor revascularization of implanted OA tissues.nnnCONCLUSIONnJRA synovium is characterized by high angiogenic activity. SCID mouse-human JRA synovium chimeras may provide a good approach to study the in vivo regulation of angiogenesis in JRA.

TGFβ1 Inhibits Ca2+-Calcineurin-Mediated Activation in Thymocytes

TGFβ1 is a polypeptide growth modulatory and differentiation factor involved in many biological processes including immune homeostasis and self-tolerance. Tgfb1 knockout mice die around weaning age due to severe inflammation in most major organ systems, but the mechanism underlying this disease is not understood. In this study we demonstrate that Tgfb1−/− CD4+CD8+ and CD4+CD8− thymocytes are hyperresponsive to receptor-mediated and receptor-independent mitogenic stimulation. A suboptimal concentration of ionomycin in the presence of PMA fully activates Tgfb1−/− thymocytes, whereas the inhibitors of Ca2+ influx and calcineurin, EGTA and FK506, eliminate the hyperresponsiveness. Hence, the hypersensitivity of Tgfb1−/− thymocytes is due to a lowered threshold for Ca2+-dependent activation. Further, we demonstrate that the hypersensitivity of thymocytes results from the absence of TGFβ1 and not from the inflammatory environment because the thymocytes are hyperresponsive in preinflammatory-stage Tgfb1−/− mice. Our results suggest for the first time that TGFβ1 functions to inhibit aberrant T cell expansion by maintaining intracellular calcium concentration levels low enough to prevent a mitogenic response by Ca2+-independent stimulatory pathways alone. Consequently, TGFβ1 prevents autoimmune disease through a Ca2+ regulatory pathway that maintains the activation threshold above that inducible by self-MHC-TCR interactions.

Germ-free and barrier-raised TGFβ1-deficient mice have similar inflammatory lesions

Barrier-raised transforming growth factor β1 (TGFβ1)-deficient mice consistently die before 35 days of age of a severe multiorgan inflammatory disease that can affect the skeletal muscle, heart, liver, pancreas, salivary gland, lung, oesophagus and stomach. The underlying cause of this disease is not known. To determine whether abnormal responsiveness of the immune system to the presence of enteric flora plays a causative role, a colony of TGFβ1-deficient and wild-type mice were raised in a sterile environment. Seven germ-free TGFβ1-deficient and 5 germ-free TGFβ1 wild-type mice were examined. Lesion development was analysed and compared with historical data on 50 barrier-raised TGFβ1 mutant mice and 32 barrier-raised wild-type mice. All germ-free TGFβ1-deficient mice died shortly after weaning, as do their barrier-raised counterparts. There was a significant delay in death in germ-free TGFβ1-deficient mice compared with barrier-raised mutant mice. However, there was no difference in the type, severity or incidence of lesions between TGFβ1 mutant mice raised under germ-free or barrier conditions. Germ- free wild-type mice had no lesions. It is concluded that microorganisms play a minimal role in disease induction in TGFβ1-deficient mice

Influence of the α-Tropomyosin 180 mutation on in vivo left ventricular systolic and diastolic function

Familial hypertrophic cardiomyopathy (FHC) is a genotypically heterogenous disease produced by mutations in sarcomeric proteins. A missense mutation in the o~-tropomyosin gene at amino acid residue 180 (TMGIul80GIy) is a recently reported cause of FftC. Transgenic mice (TG) with mutant TM driven by the MHC promotor (4 lines, 10-40 copy number), die at 3-4 months of age, and at necropsy exhibit 3-fold cardiac enlargement, ventricular hypertrophy, and atrial fibrosis. In order to evaluate in vivo effects of TMGIuI80Gly on LV function and geometry, 2D-guided M-mode and color flow-guided Doppler were performed in 6 TG and 6 littermate controls (WT) aged 3 months. LV mass normalized to body weight (LV/BW) and the ratio of LV wall thickness to cavity (h/r) were significantly greater in TG than WT: