Issue published June 15, 1999

R hoA is a low-molecular-weight GTPase that has been implicated in the regulation of hypertrophic cardiac muscle cell growth. To study the role of RhoA in control of cardiac function in vivo, transgenic mice expressing wild-type and constitutively activated forms of RhoA under the control of the cardiac-specific α-myosin heavy chain promoter were generated. Transgene-positive mice expressing high levels of either wild-type or activated RhoA showed pronounced atrial enlargement and manifested a lethal phenotype, often preceded by generalized edema, with most animals dying over the course of a few weeks. Echocardiographic analysis of visibly healthy wild-type RhoA transgenic mice revealed no significant change in left ventricular function. As their condition deteriorated, significant dilation of the left ventricular chamber and associated decreases in left ventricular contractility were detected. Heart rate was grossly depressed in both wild-type and activated RhoA-expressing mice, even prior to the onset of ventricular failure. Electrocardiography showed evidence of atrial fibrillation and atrioventricular block. Interestingly, muscarinic receptor blockade with atropine did not elicit a positive chronotropic response in the transgenic mice. We suggest that RhoA regulates cardiac sinus and atrioventricular nodal function and that its overexpression results in bradycardia and development of ventricular failure.J. Clin. Invest. 103:1627–1634 (1999)

T he emergence of resistant hepatitis B virus (HBV), with mutations in the YMDD motif of the polymerase gene after treatment with lamivudine, is becoming an important clinical problem. In this study, susceptibility of wild-type and lamivudine-resistant HBV M552I, M552V, and L528M/M552V mutants to other reverse transcriptase inhibitors was investigated by transient transfection of full-length HBV DNA into human hepatoma cells. HBV DNA replication was monitored by Southern blot hybridization, which showed the presence of a single-stranded band (representative of the HBV replicative intermediates) in the drug-free, wild-type HBV-transfected cells. This band was diminished in the samples of wild-type HBV DNA treated with either lamivudine, adefovir, or lobucavir. The band intensities from the lamivudine-resistant mutants were not decreased by treatment with lamivudine, but were decreased by the treatments with adefovir or lobucavir. In contrast, penciclovir and nevirapine did not diminish the intensity of the single-stranded band of wild-type HBV or the lamivudine-resistant mutants. These results demonstrate that lamivudine-resistant HBV is susceptible to adefovir and lobucavir. Lamivudine-resistant HBV should be treated with adefovir or lobucavir, and combination therapy with lamivudine and adefovir/lobucavir may prevent the emergence of lamivudine-resistant HBV.J. Clin. Invest. 103:1635–1640 (1999).

P reeclampsia, the major cause of maternal morbidity and mortality in developed countries, is associated with abnormalities of placenta function due to shallow invasion of the maternal decidua by trophoblasts. Data suggest that TGF-β may play a role in inhibiting trophoblast outgrowth or invasion, or both. We report that placental TGF-β₃ expression is high in early pregnancy but falls at around 9 weeks’ gestation. This pattern is inversely correlated with trophoblast outgrowth and fibronectin synthesis, markers of early trophoblast differentiation toward an invasive phenotype. We demonstrate that TGF-β₃ is overexpressed in preeclamptic placentae. In contrast to control placentae, explants from preeclamptic pregnancies fail to exhibit spontaneous invasion in vitro. Significantly, antisense-induced inhibition of TGF-β₃ expression, and inhibition of TGF-β₃ activity with antibodies, induces the formation of columns of trophoblast cells, which migrate out of the explant into the underlying Matrigel. To our knowledge, this is the first demonstration that the hypoinvasive placental phenotype characteristic of preeclampsia can be essentially normalized in vitro by biochemical manipulation. We speculate that a failure to downregulate expression of TGF-β₃ at around 9 weeks’ gestation results in shallow trophoblast invasion and predisposes the pregnancy to preeclampsia.

D isorders of carbohydrate and lipid metabolism have been reported to cluster in patients with essential hypertension and in spontaneously hypertensive rats (SHRs). A deletion in the Cd36 gene on chromosome 4 has recently been implicated in defective carbohydrate and lipid metabolism in isolated adipocytes from SHRs. However, the role of Cd36 and chromosome 4 in the control of blood pressure and systemic cardiovascular risk factors in SHRs is unknown. In the SHR.BN-Il6/Npy congenic strain, we have found that transfer of a segment of chromosome 4 (including Cd36) from the Brown Norway (BN) rat onto the SHR background induces reductions in blood pressure and ameliorates dietary-induced glucose intolerance, hyperinsulinemia, and hypertriglyceridemia. These results demonstrate that a single chromosome region can influence a broad spectrum of cardiovascular risk factors involved in the hypertension metabolic syndrome. However, analysis of Cd36 genotypes in the SHR and stroke-prone SHR strains indicates that the deletion variant of Cd36 was not critical to the initial selection for hypertension in the SHR model. Thus, the ability of chromosome 4 to influence multiple cardiovascular risk factors, including hypertension, may depend on linkage of Cd36 to other genes trapped within the differential segment of the SHR.BN-Il6/Npy strain.J. Clin. Invest. 103:1651–1657 (1999).

C 57BL/6 mice injected with the 145-2C11 anti-CD3 mAb and grafted with MHC class II disparate bm12 skin develop a chronic rejection characterized by interstitial dermal fibrosis, a marked eosinophil infiltrate, and an obliterative intimal vasculopathy. Because these changes occur in the absence of alloreactive antibodies, we examined the contribution of cytokines in their pathogenesis. Chronically rejected grafts showed a marked accumulation of both IL-4 and IL-5 mRNA. Mixed lymphocyte reaction experiments established that mice undergoing chronic rejection were primed for IL-4, IL-5, and IL-10 secretion. In vivo administration of anti–IL-4 mAb completely prevented allograft vasculopathy as well as graft eosinophil infiltration and dermal fibrosis. Injection of anti–IL-5 mAb or the use of IL-5–deficient mice as recipients also resulted in the lack of eosinophil infiltration or dermal fibrosis, but these mice did develop allograft vasculopathy. Administration of anti–IL-10 mAb did not influence any histologic parameter of chronic rejection. Thus, in this model, IL-4– and IL-5–mediated tissue allograft eosinophil infiltration is associated with interstitial fibrosis. IL-4, but not eosinophils, is also required for the development of obliterative graft arteriolopathy.J. Clin. Invest. 103:1659–1667 (1999).

C hromosome translocations involving antigen receptor loci are a genetic hallmark of non-Hodgkin’s lymphomas in humans. Most commonly, these translocations result in juxtaposition of the immunoglobulin heavy-chain (IgH) locus with one of several cellular proto-oncogenes, leading to deregulated oncogene expression. The V(D)J recombinase, which mediates physiologic rearrangements of antigen receptor genes, may play a mechanistic role in some lymphoma translocations, although evidence is indirect. A high incidence of B-lineage lymphomas has been observed in mice with severe combined immunodeficiency (SCID) and p53-null mutations. We show that these tumors are characteristic of the pro–B-cell stage of development and that they harbor recurrent translocations involving chromosomes 12 and 15. Fluorescence in situ hybridization (FISH) shows retention of IgH sequences on the derivative chromosome 12, implying that breakpoints involve the IgH locus. Pro–B-cell lymphomas were suppressed in SCID p53^–/– mice by a Rag-2–null mutation, demonstrating that DNA breaks generated during V(D)J recombination are required for oncogenic transformation, and suggesting that t(12;15) arise during attempted IgH rearrangement in pro-B cells. These studies indicate that the oncogenic potential inherent in antigen receptor diversification is controlled in vivo by efficient rejoining of DNA ends generated during V(D)J recombination and an intact cellular response to DNA damage.J. Clin. Invest. 103:1669–1675 (1999).

T he cystic fibrosis (CF) gene encodes a cAMP-gated Cl^– channel (cystic fibrosis transmembrane conductance regulator [CFTR]) that mediates fluid transport across the luminal surfaces of a variety of epithelial cells. We have previously shown that gap junctional communication and Cl^– secretion were concurrently regulated by cAMP in cells expressing CFTR. To determine whether intercellular communication and CFTR-dependent secretion are related, we have compared gap junctional coupling in a human pancreatic cell line harboring the ΔF508 mutation in CFTR and in the same cell line in which the defect was corrected by transfection with wild-type CFTR. Both cell lines expressed connexin45 (Cx45), as evidenced by RT-PCR, immunocytochemistry, and dual patch-clamp recording. Exposure to agents that elevate intracellular cAMP or specifically activate protein kinase A evoked Cl^– currents and markedly increased junctional conductance of CFTR-expressing pairs, but not in the parental cells. The latter effect, which was caused by an increase in single-channel activity but not in unitary conductance of Cx45 channels, was not prevented by exposing CFTR-expressing cells to a Cl^– channel blocker. We conclude that expression of functional CFTR restored the cAMP-dependent regulation of junctional conductance in CF cells. Direct intercellular communication coordinates multicellular activity in tissues that are major targets of CF manifestations. Consequently, defective regulation of gap junction channels may contribute to the altered functions of tissues affected in CF.J. Clin. Invest. 103:1677–1684 (1999).

< i>Sle1 and Sle3 are 2 loci that confer susceptibility to lupus nephritis in the NZM2410 strain of mice. Our previous work has shown that B6.NZMc1 mice, congenic for Sle1, exhibit loss of tolerance to chromatin but do not develop any pathogenic autoantibodies or disease. B6.NZMc7 mice, congenic for Sle3, exhibit low-grade polyclonal B- and T-cell activation, elevated CD4/CD8 ratios, and mildly penetrant glomerulonephritis. In contrast to these monocongenics, the present study reveals that B6.NZMc1|c7 mice, bicongenic for Sle1 and Sle3, exhibit splenomegaly, significantly expanded populations of activated B and CD4⁺ T cells, and a robust, variegated IgG autoantibody response targeting multiple components of chromatin (including double-stranded DNA), intact glomeruli, and basement membrane matrix antigens. As one might predict, these mice, particularly the females, exhibit highly penetrant glomerulonephritis.These findings lend strong support to a two-step epistatic model for the formation of pathogenic, nephrophilic autoantibodies in lupus. Whereas loci such as Sle1 may serve to breach tolerance to chromatin, full-blown pathogenic maturation of the autoantibody response appears to require additional input from other loci (such as Sle3) and gender-based factors.J. Clin. Invest. 103:1685–1695 (1999).

E xpression of lipoprotein lipase (LPL) by the macrophage has been proposed to promote foam cell formation and atherosclerosis, primarily on the basis of in vitro studies. LPL-deficient mice might provide a model for testing the role of LPL secretion by the macrophage in an in vivo system. Unfortunately, homozygous deficiency of LPL in the mouse is lethal shortly after birth. Because the fetal liver is the major site of hematopoiesis in the developing fetus, transplantation of C57BL/6 mice with LPL^–/– fetal liver cells (FLCs) was used to investigate the physiologic role of macrophage LPL expression in vivo. Thirty-four female C57BL/6 mice were lethally irradiated and reconstituted with FLCs from day 14 LPL^+/+, LPL^+/–, and LPL^–/– donors. No significant differences were detected in plasma levels of post-heparin LPL activity or in serum cholesterol or triglyceride levels between the 3 groups on either a chow diet or an atherogenic diet. After 19 weeks on the atherogenic diet, aortae were collected for quantitative analysis of the extent of aortic atherosclerosis. LPL expression was detected by immunocytochemistry and in situ hybridization in macrophages of aortic atherosclerotic lesions of LPL^+/+→C57BL/6 and LPL^+/–→C57BL/6 mice, but not in LPL^–/–→C57BL/6 mice, whereas myocardial cells expressed LPL in all groups. The mean aortic lesion area was reduced by 55% in LPL^–/–→C57BL/6 mice compared with LPL^+/+→C57BL/6 mice and by 45% compared with LPL^+/–→C57BL/6 mice, respectively. These data demonstrate in vivo that LPL expression by macrophages in the artery wall promotes foam cell formation and atherosclerosis.J. Clin. Invest. 103:1697–1705 (1999).

T he human leukocyte antigen (HLA) restriction of the IgE response to different allergens in humans has been a subject of numerous published studies. However, the role and contribution of specific HLA class II molecules in the pathogenesis of allergic airway inflammation are unknown and difficult to assess. HLA-DQ6 and HLA-DQ8 transgenic mice lacking endogenous mouse class II gene expression were actively immunized and later challenged intranasally with short ragweed (SRW) allergenic extract. The HLA-DQ transgenic mice developed pulmonary eosinophilia and lung tissue damage. We also found an increase in total protein (TP) level and IL-5 production in bronchoalveolar lavage (BAL) fluid and an increase in SRW-specific Th2-type immunoglobulins (IgG1, IgG2b) and total serum IgE levels. Under similar treatment, DQ-negative full-sib control mice were normal. The allergic response could be significantly inhibited or abrogated in HLA-DQ mice by systemic treatment with anti-DQ mAb. The in vivo responses of HLA-DQ6 and HLA-DQ8 mice showed differences in terms of levels of eosinophilia, BAL protein, IL-5 concentration, and lung hyperreactivity to inhaled methacholine. These findings demonstrate the crucial role for specific HLA-DQ molecules in SRW-specific CD4⁺ T-cell activation and resulting recruitment of eosinophils into the airways.J. Clin. Invest. 103:1707–1717 (1999).

T he histological identification of increased eosinophils in the gastrointestinal tract occurs in numerous clinical disorders; however, there is a limited understanding of the mechanisms regulating eosinophil trafficking into this mucosal surface. The results presented in this study characterize the processes regulating eosinophil homing into the gastrointestinal tract at baseline. Eosinophils were found to be present in the lamina propria of 19-day-old embryos and germ-free adult mice at concentrations comparable to those present in non–germ-free adult mice. Furthermore, eosinophil gastrointestinal levels were not altered by increasing circulating eosinophils after pulmonary allergen challenge. Gastrointestinal eosinophil levels were partially reduced in mice deficient in recombinase activating gene-1 (RAG-1), IL-5, or the β common chain (βc), but these reductions paralleled reductions in circulating eosinophils. In contrast, mice deficient in eotaxin had a marked reduction in gastrointestinal eosinophils but normal levels of eosinophils in the hematopoietic compartments. Furthermore, eotaxin was important for regulating eosinophil levels, even in the presence of high levels of IL-5. These investigations demonstrate eosinophil homing into the gastrointestinal tract during embryonic development occurring independently of viable intestinal flora. Furthermore, eotaxin is identified as the primary regulator of eosinophil gastrointestinal homing under homeostatic states, and may therefore have a fundamental role in innate immune responses.J. Clin. Invest. 103:1719–1727 (1999).

H uman retinoid X receptor α (hRXRα) is a member of the nuclear receptor family of transcriptional regulators. It regulates transcription through its association with several heterodimeric partners, including the vitamin D₃ receptor (VDR). Signaling through the VDR is essential for normal calcium homeostasis and has been shown to inhibit the proliferation of cancer cells derived from a number of tissues. Here we show that phosphorylation of hRXRα in ras-transformed human keratinocytes through the activated Ras–Raf–mitogen-activated protein kinase (Ras-Raf-MAP kinase) pathway results in attenuated transactivation by the VDR and resistance to the growth inhibitory action of 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] and RXR-specific agonist LG1069 (4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphtalenyl) ethenyl]-benzoic acid). Phosphorylation of hRXRα occurs at serine 260, a consensus MAP kinase site. Inhibition of MAP kinase activity or point mutagenesis of serine 260 of hRXRα reverses the observed resistance to 1,25(OH)₂D₃ and LG1069. Thus, hRXRα is a downstream target of MAP kinase, and its phosphorylation may play an important role in malignant transformation.J. Clin. Invest. 103:1729–1735 (1999).