Issue published February 15, 1999

M yotonic dystrophy (DM) is the most common form of muscular dystrophy and is caused by expansion of a CTG trinucleotide repeat on human chromosome 19. Patients with DM develop atrioventricular conduction disturbances, the principal cardiac manifestation of this disease. The etiology of the pathophysiological changes observed in DM has yet to be resolved. Haploinsufficiency of myotonic dystrophy protein kinase (DMPK), DM locus-associated homeodomain protein (DMAHP) and/or titration of RNA-binding proteins by expanded CUG sequences have been hypothesized to underlie the multi-system defects observed in DM. Using an in vivo murine electrophysiology study, we show that cardiac conduction is exquisitely sensitive to DMPK gene dosage. DMPK^–/– mice develop cardiac conduction defects which include first-, second-, and third-degree atrioventricular (A–V) block. Our results demonstrate that the A–V node and the His-Purkinje regions of the conduction system are specifically compromised by DMPK loss. Importantly, DMPK^+/– mice develop first-degree heart block, a conduction defect strikingly similar to that observed in DM patients. These results demonstrate that DMPK dosage is a critical element modulating cardiac conduction integrity and conclusively link haploinsufficiency of DMPK with cardiac disease in myotonic dystrophy.

A key question in understanding the status of the immune system in HIV-1 infection is whether the adult thymus contributes to reconstitution of peripheral T lymphocytes. We analyzed the thymus in adult patients who died of HIV-1 infection. In addition, we studied the clinical course of HIV-1 infection in three patients thymectomized for myasthenia gravis and determined the effect of antiretroviral therapy on CD4⁺ T cells. We found that five of seven patients had thymus tissue at autopsy and that all thymuses identified had inflammatory infiltrates surrounding lymphodepleted thymic epithelium. Two of seven patients also had areas of thymopoiesis; one of these patients had peripheral blood CD4⁺ T-cell levels of <50/mm³ for 51 months prior to death. Of three thymectomized patients, one rapidly progressed to AIDS, one progressed to AIDS over seven years (normal progressor), whereas the third remains asymptomatic at least seven years after seroconversion. Both latter patients had rises in peripheral blood CD4⁺ T cells after antiretroviral therapy. Most patients who died of complications of HIV-1 infection did not have functional thymus tissue, and when present, thymopoiesis did not prevent prolonged lymphopenia. Thymectomy before HIV-1 infection did not preclude either peripheral CD4⁺ T-cell rises or clinical responses after antiretroviral therapy.

P atients with pemphigus foliaceus (PF) have blisters on skin, but not mucous membranes, whereas patients with pemphigus vulgaris (PV) develop blisters on mucous membranes and/or skin. PF and PV blisters are due to loss of keratinocyte cell–cell adhesion in the superficial and deep epidermis, respectively. PF autoantibodies are directed against desmoglein (Dsg) 1; PV autoantibodies bind Dsg3 or both Dsg3 and Dsg1. In this study, we test the hypothesis that coexpression of Dsg1 and Dsg3 in keratinocytes protects against pathology due to antibody-induced dysfunction of either one alone. Using passive transfer of pemphigus IgG to normal and DSG3^null neonatal mice, we show that in the areas of epidermis and mucous membrane that coexpress Dsg1 and Dsg3, antibodies against either desmoglein alone do not cause spontaneous blisters, but antibodies against both do. In areas (such as superficial epidermis of normal mice) where Dsg1 without Dsg3 is expressed, anti-Dsg1 antibodies alone can cause blisters. Thus, the anti-desmoglein antibody profiles in pemphigus sera and the normal tissue distributions of Dsg1 and Dsg3 determine the sites of blister formation. These studies suggest that pemphigus autoantibodies inhibit the adhesive function of desmoglein proteins, and demonstrate that either Dsg1 or Dsg3 alone is sufficient to maintain keratinocyte adhesion.

T ransplant-associated arteriosclerosis remains an obstacle to long-term graft survival. To determine the contribution to transplant arteriosclerosis of MHC and adhesion molecules from cells of the donor vasculature, we allografted carotid artery loops from six mutant mouse strains into immunocompetent CBA/CaJ recipients. The donor mice were deficient in either MHC I molecules or MHC II molecules, both MHC I and MHC II molecules, the adhesion molecule P-selectin, intercellular adhesion molecule (ICAM)-1, or both P-selectin and ICAM-1. Donor arteries in which ICAM-1, MHC II, or both MHC I and MHC II were absent showed reductions in neointima formation of 52%, 33%, and 38%, respectively, due primarily to a reduction in smooth muscle cell (SMC) accumulation. In P-selectin–deficient donor arteries, neointima formation did not differ from that in controls. In donor arteries lacking both P-selectin and ICAM-1, the size of the neointima was similar to that in those lacking ICAM-1 alone. In contrast, neointima formation increased by 52% in MHC I–deficient donor arteries. The number of CD4-positive T cells increased by 2.8-fold in MHC I–deficient arteries, and that of α-actin–positive SMCs by twofold. These observations indicate that ICAM-1 and MHC II molecules expressed in the donor vessel wall may promote transplant-associated arteriosclerosis. MHC I molecules expressed in the donor may have a protective effect.

M ice doubly heterozygous for a modified tissue factor pathway inhibitor (TFPI) allele (tfpi^δ) lacking its Kunitz-type domain-1 (TFPI^+/δ) and for a deficiency of the factor VII gene (FVII^+/–) were mated to generate 309 postnatal and 205 embryonic day 17.5 (E17.5) offspring having all the predicted genotypic combinations. Progeny singly homozygous for the tfpi^δ modification but with the wild-type fVII allele (FVII^+/+/TFPI^δ/δ), and mice singly homozygous for the fVII deficiency and possessing the wild-type tfpi allele (FVII^–/–/TFPI^+/+), displayed previously detailed phenotypes (i.e., a high percentage of early embryonic lethality at E9.5 or normal development with severe perinatal bleeding, respectively). Surprisingly, mice of the combined FVII^–/–/TFPI^δ/δ genotype were born at the expected mendelian frequency but suffered the fatal perinatal bleeding associated with the FVII^–/– genotype. Mice carrying the FVII^+/–/TFPI^δ/δ genotype were also rescued from the lethality associated with the FVII^+/+/TFPI^δ/δ genotype but succumbed to perinatal consumptive coagulopathy. Thus, the rescue of TFPI^δ/δ embryos, either by an accompanying homozygous or heterozygous FVII deficiency, suggests that diminishment of FVII activity precludes the need for TFPI-mediated inhibition of the FVIIa/tissue factor coagulation pathway during embryogenesis. Furthermore, the phenotypes of these combined deficiency states suggest that embryonic FVII is produced in mice as early as E9.5 and that any level of maternal FVII in early-stage embryos is insufficient to cause a coagulopathy in TFPI^δ/δ mice.

< i>Mycobacterium tuberculosis attaches to, enters, and replicates within alveolar macrophages (AMs). Our previous studies suggest that surfactant protein A (SP-A) can act as a ligand in the attachment of M. tuberculosis to AMs. Reactive nitrogen intermediates (RNIs) play a significant role in the killing of mycobacteria. We have demonstrated that RNI levels generated by AMs were significantly increased when interferon-γ–primed AMs were incubated with M. tuberculosis. However, the RNI levels were significantly suppressed in the presence of SP-A (10 μg/ml). The specificity of SP-A's effect was demonstrated by the use of F(ab′)₂ fragments of anti–SP-A monoclonal antibodies and by the use of mannosyl-BSA, which blocked the suppression of RNI levels by SP-A. Furthermore, incubation of deglycosylated SP-A with M. tuberculosis failed to suppress RNI by AMs, suggesting that the oligosaccharide component of SP-A, which binds to M. tuberculosis, is necessary for this effect. These results show that SP-A–mediated binding of M. tuberculosis to AMs significantly decreased RNI levels, suggesting that this may be one mechanism by which M. tuberculosis diminishes the cytotoxic response of activated AMs.

I t has been controversial whether high water permeability in the thin descending limb of Henle (TDLH) is required for formation of a concentrated urine by the kidney. Freeze-fracture electron microscopy (FFEM) of rat TDLH has shown an exceptionally high density of intramembrane particles (IMPs), which were proposed to consist of tetramers of aquaporin-1 (AQP1) water channels. In this study, transepithelial osmotic water permeability (P_f) was measured in isolated perfused segments (0.5–1 mm) of TDLH in wild-type (+/+), AQP1 heterozygous (+/–), and AQP1 null (–/–) mice. P_f was measured at 37°C using a 100 mM bath-to-lumen osmotic gradient of raffinose, and fluorescein isothiocyanate (FITC)–dextran as the luminal volume marker. P_f was (in cm/s): 0.26 ± 0.02 ([+/+]; SE, n = 9 tubules), 0.21 ± 0.01 ([+/–]; n = 12), and 0.031 ± 0.007 ([–/–]; n = 6) (P < 0.02, [+/+] vs. [+/–]; P < 0.0001, [+/+] vs. [–/–]). FFEM of kidney medulla showed remarkably fewer IMPs in TDLH from (–/–) vs. (+/+) and (+/–) mice. IMP densities were (in μm^–2, SD, 5–12 micrographs): 5,880 ± 238 (+/+); 5,780 ± 450 (+/–); and 877 ± 420 (–/–). IMP size distribution analysis revealed mean IMP diameters of 8.4 nm ([+/+] and [+/–]) and 5.2 nm ([–/–]). These results demonstrate that AQP1 is the principal water channel in TDLH and support the view that osmotic equilibration along TDLH by water transport plays a key role in the renal countercurrent concentrating mechanism. The similar P_f and AQP1 expression in TDLH of (+/+) and (+/–) mice was an unexpected finding that probably accounts for the unimpaired urinary concentrating ability in (+/–) mice.

I nherited defects in the degradation of glycosphingolipids (GSLs) cause a group of severe diseases known as GSL storage disorders. There are currently no effective treatments for the majority of these disorders. We have explored a new treatment paradigm, substrate deprivation therapy, by constructing a genetic model in mice. Sandhoff's disease mice, which abnormally accumulate GSLs, were bred with mice that were blocked in their synthesis of GSLs. The mice with simultaneous defects in GSL synthesis and degradation no longer accumulated GSLs, had improved neurologic function, and had a much longer life span. However, these mice eventually developed a late-onset neurologic disease because of accumulation of another class of substrate, oligosaccharides. The results support the validity of the substrate deprivation therapy and also highlight some limitations.

W e examined the role of the interleukin-8 (IL-8) receptor in a murine model of allergen-induced pulmonary inflammation using mice with a targeted deletion of the murine IL-8 receptor homologue (IL-8r^–/–). Wild-type (Wt) and IL-8r^–/– mice were systemically immunized to ovalbumin (OVA) and were exposed with either single or multiple challenge of aerosolized phosphate-buffered saline (OVA/PBS) or OVA (OVA/OVA). Analysis of cells recovered from bronchoalveolar lavage (BAL) revealed a diminished recruitment of neutrophils to the airway lumen after single challenge in IL-8r^–/– mice compared with Wt mice, whereas multiply challenged IL-8r^–/– mice had increased B cells and fewer neutrophils compared with Wt mice. Both Wt and IL-8r^–/– OVA/OVA mice recruited similar numbers of eosinophils to the BAL fluid and exhibited comparable degrees of pulmonary inflammation histologically. Both total and OVA-specific IgE levels were greater in multiply challenged IL-8r^–/– OVA/OVA mice than in Wt mice. Both the IL-8r^–/– OVA/OVA and OVA/PBS mice were significantly less responsive to methacholine than their respective Wt groups, but both Wt and IL-8r mice showed similar degrees of enhancement after multiple allergen challenge. The data demonstrate that the IL-8r modulates IgE production, airway responsiveness, and the composition of the cells (B cells and neutrophils) recruited to the airway lumen in response to antigen.

P arathyroid hormone (PTH) stimulates bone resorption by acting directly on osteoblasts/stromal cells and then indirectly to increase differentiation and function of osteoclasts. PTH acting on osteoblasts/stromal cells increases collagenase gene transcription and synthesis. To assess the role of collagenase in the bone resorptive actions of PTH, we used mice homozygous (r/r) for a targeted mutation (r) in Col1a1 that are resistant to collagenase cleavage of type I collagen. Human PTH(1–34) was injected subcutaneously over the hemicalvariae in wild-type (+/+) or r/r mice four times daily for three days. Osteoclast numbers, the size of the bone marrow spaces and periosteal proliferation were increased in calvariae from PTH-treated +/+ mice, whereas in r/r mice, PTH-induced bone resorption responses were minimal. The r/r mice were not resistant to other skeletal effects of PTH because abundant interstitial collagenase mRNA was detected in the calvarial periosteum of PTH-treated, but not vehicle-treated, r/r and +/+ mice. Calcemic responses, 0.5–10 hours after intraperitoneal injection of PTH, were blunted in r/r mice versus +/+ mice. Thus, collagenase cleavage of type I collagen is necessary for PTH induction of osteoclastic bone resorption.

T he kidneys and other nonreproductive organs vasodilate during early gestation; however, the “pregnancy hormones” responsible for the profound vasodilation of the renal circulation during pregnancy are unknown. We hypothesized that the ovarian hormone relaxin (RLX) contributes. Therefore, we tested whether the administration of RLX elicits renal vasodilation and hyperfiltration in conscious adult, intact female rats. After several days of treatment with either purified porcine RLX or recombinant human RLX 2 (rhRLX), effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) increased by 20%–40%. Comparable renal vasodilation and hyperfiltration was also observed in ovariectomized rats, suggesting that estrogen and progesterone are unnecessary for the renal response to rhRLX. The nitric oxide synthase inhibitor N^ω-nitro-L-arginine methyl ester completely abrogated the increase in ERPF and GFR elicited by chronic administration of purified porcine RLX. In contrast, the renal vasoconstrictory response to angiotensin II was attenuated by the RLX treatment. Short-term infusion of purified porcine RLX to conscious rats over several hours failed to increase ERPF and GFR. Plasma osmolality was consistently reduced by the chronic administration of both RLX preparations. In conclusion, the renal and osmoregulatory effects of chronic RLX administration to conscious rats resemble the physiological changes of pregnancy in several respects: (a) marked increases in ERPF and GFR with a mediatory role for nitric oxide; (b) attenuation of the renal circulatory response to angiotensin II; and (c) reduction in plasma osmolality.

P eanut allergy is a significant IgE-mediated health problem because of the increased prevalence, potential severity, and chronicity of the reaction. Following our characterization of the two peanut allergens Ara h 1 and Ara h 2, we have isolated a cDNA clone encoding a third peanut allergen, Ara h 3. The deduced amino acid sequence of Ara h 3 shows homology to 11S seed-storage proteins. The recombinant form of this protein was expressed in a bacterial system and was recognized by serum IgE from ∼45% of our peanut-allergic patient population. Serum IgE from these patients and overlapping, synthetic peptides were used to map the linear, IgE-binding epitopes of Ara h 3. Four epitopes, between 10 and 15 amino acids in length, were found within the primary sequence, with no obvious sequence motif shared by the peptides. One epitope is recognized by all Ara h 3–allergic patients. Mutational analysis of the epitopes revealed that single amino acid changes within these peptides could lead to a reduction or loss of IgE binding. By determining which amino acids are critical for IgE binding, it might be possible to alter the Ara h 3 cDNA to encode a protein with a reduced IgE-binding capacity. These results will enable the design of improved diagnostic and therapeutic approaches for food-hypersensitivity reactions.

T o maintain the integrity of the vascular barrier, endothelial cells (EC) are resistant to cell death. The molecular basis of this resistance may be explained by the function of antiapoptotic genes such as bcl family members. Overexpression of Bcl-2 or Bcl-X_L protects EC from tumor necrosis factor (TNF)–mediated apoptosis. In addition, Bcl-2 or Bcl-X_L inhibits activation of NF-κB and thus upregulation of proinflammatory genes. Bcl-2–mediated inhibition of NF-κB in EC occurs upstream of IκBα degradation without affecting p65-mediated transactivation. Overexpression of bcl genes in EC does not affect other transcription factors. Using deletion mutants of Bcl-2, the NF-κB inhibitory function of Bcl-2 was mapped to bcl homology domains BH2 and BH4, whereas all BH domains were required for the antiapoptotic function. These data suggest that Bcl-2 and Bcl-X_L belong to a cytoprotective response that counteracts proapoptotic and proinflammatory insults and restores the physiological anti-inflammatory phenotype to the EC. By inhibiting NF-κB without sensitizing the cells (as with IκBα) to TNF-mediated apoptosis, Bcl-2 and Bcl-X_L are prime candidates for genetic engineering of EC in pathological conditions where EC loss and unfettered activation are undesirable.

T he mammalian lung expresses water channel aquaporin-1 (AQP1) in microvascular endothelia and aquaporin-4 (AQP4) in airway epithelia. To test whether these water channels facilitate fluid movement between airspace, interstitial, and capillary compartments, we measured passive and active fluid transport in AQP1 and AQP4 knockout mice. Airspace–capillary osmotic water permeability (P_f) was measured in isolated perfused lungs by a pleural surface fluorescence method. P_f was remarkably reduced in AQP1 (–/–) mice (measured in cm/s × 0.001, SE, n = 5–10: 17 ± 2 [+/+]; 6.6 ± 0.6 AQP1 [+/–]; 1.7 ± 0.3 AQP1 [–/–]; 12 ± 1 AQP4 [–/–]). Microvascular endothelial water permeability, measured by a related pleural surface fluorescence method in which the airspace was filled with inert perfluorocarbon, was reduced more than 10-fold in AQP1 (–/–) vs. (+/+) mice. Hydrostatically induced lung interstitial and alveolar edema was measured by a gravimetric method and by direct measurement of extravascular lung water. Both approaches indicated a more than twofold reduction in lung water accumulation in AQP1 (–/–) vs. (+/+) mice in response to a 5- to 10-cm H₂O increase in pulmonary artery pressure for five minutes. Active, near-isosmolar alveolar fluid absorption (J_v) was measured in in situ perfused lungs using ¹²⁵I-albumin as an airspace fluid volume marker. J_v (measured in percent fluid uptake at 30 min, n = 5) in (+/+) mice was 6.0 ± 0.6 (37°C), increased to 16 ± 1 by β-agonists, and inhibited to less than 2.0 by amiloride, ouabain, or cooling to 23°C. J_v (with isoproterenol) was not affected by aquaporin deletion (18.9 ± 2.2 [+/+]; 16.4 ± 1.5 AQP1 [–/–]; 16.3 ± 1.7 AQP4 [–/–]). These results indicate that osmotically driven water transport across microvessels in adult lung occurs by a transcellular route through AQP1 water channels and that the microvascular endothelium is a significant barrier for airspace–capillary osmotic water transport. AQP1 facilitates hydrostatically driven lung edema but is not required for active near-isosmolar absorption of alveolar fluid.

G ranulocyte-macrophage colony-stimulating factor (GM-CSF) gene-targeted mice (GM^–/–) cleared group B streptococcus (GBS) from the lungs more slowly than wild-type mice. Expression of GM-CSF in the respiratory epithelium of GM^–/– mice improved bacterial clearance to levels greater than that in wild-type GM^+/+ mice. Acute aerosolization of GM-CSF to GM^+/+ mice significantly enhanced clearance of GBS at 24 hours. GBS infection was associated with increased neutrophilic infiltration in lungs of GM^–/– mice, while macrophage infiltrates predominated in wild-type mice, suggesting an abnormality in macrophage clearance of bacteria in the absence of GM-CSF. While phagocytosis of GBS was unaltered, production of superoxide radicals and hydrogen peroxide was markedly deficient in macrophages from GM^–/– mice. Lipid peroxidation, assessed by measuring the isoprostane 8-iso-PGF_2α, was decreased in the lungs of GM^–/– mice. GM-CSF plays an important role in GBS clearance in vivo, mediated in part by its role in enhancing superoxide and hydrogen peroxide production and bacterial killing by alveolar macrophages.

B oth chlamydial and human heat shock protein 60s (HSP 60), which colocalize in human atheroma, may contribute to inflammation during atherogenesis. We tested the hypothesis that chlamydial or human HSP 60 activates human endothelial cells (ECs), smooth muscle cells (SMCs), and monocyte-derived macrophages. We examined the expression of adhesion molecules such as endothelial-leukocyte adhesion molecule-1 (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), and the production of the proinflammatory cytokine interleukin-6 (IL-6). We also tested whether either HSP 60 induces nuclear factor-κB (NF-κB), which contributes to the gene expression of these molecules. Either chlamydial or human HSP 60 induced E-selectin, ICAM-1, and VCAM-1 expression on ECs similar to levels induced by Escherichia coli lipopolysaccharide (LPS). Each HSP 60 also significantly induced IL-6 production by ECs, SMCs, and macrophages to an extent similar to that induced by E. coli LPS, as assessed by enzyme-linked immunosorbent assay (ELISA). In ECs, either HSP 60 triggered activation of NF-κB complexes containing p65 and p50 Rel proteins. Heat treatment abolished all these effects, but did not alter the ability of E. coli LPS to induce these functions. Chlamydial and human HSP 60s therefore activate human vascular cell functions relevant to atherogenesis and lesional complications. These findings help to elucidate the mechanisms by which a chronic asymptomatic chlamydial infection might contribute to the pathophysiology of atheroma.

P rimary fibroblasts are not efficiently transduced by subgroup C adenovirus (Ad) vectors because they express low levels of the high-affinity Coxsackie virus and adenovirus receptor (CAR). In the present study, we have used primary human dermal fibroblasts as a model to explore strategies by which Ad vectors can be designed to enter cells deficient in CAR. Using an Ad vector expressing the human CAR cDNA (AdCAR) at high multiplicity of infection, primary fibroblasts were converted from being CAR deficient to CAR sufficient. Efficiency of subsequent gene transfer by standard Ad5-based vectors and Ad5-based vectors with alterations in penton and fiber was evaluated. Marked enhancement of binding and transgene expression by standard Ad5 vectors was achieved in CAR-sufficient fibroblasts. Expression by AdΔRGDβgal, an Ad5-based vector lacking the arginine-glycine-aspartate (RGD) α_V integrin recognition site from its penton base, was achieved in CAR-sufficient, but not CAR-deficient, cells. Fiber-altered Ad5-based vectors, including (a) AdF(pK7)βgal (bearing seven lysines on the end of fiber) (b) AdF(RGD)βgal (bearing a high-affinity RGD sequence on the end of fiber), and (c) AdF9sK βgal (bearing a short fiber and Ad9 knob), demonstrated enhanced gene transfer in CAR-deficient fibroblasts, with no further enhancement in CAR-sufficient fibroblasts. Together, these observations demonstrate that CAR deficiency on Ad targets can be circumvented either by supplying CAR or by modifying the Ad fiber to bind to other cell-surface receptors.