Issue published November 15, 2003

L aboratory animals play a crucial role in biomedical research – indeed many advances now incorporated into human health care would have been impossible without them. Informed and well-trained scientists have the privilege, but not the automatic right, to use animals as experimental subjects. This privilege must not be abused.

P seudomonas aeruginosa has two complete quorum-sensing systems. Both of these systems have been shown to be important for Pseudomonas virulence in multiple models of infection. Thus, these systems provide unique targets for novel antimicrobial drugs.

U nequivocal direct observations have established that the bacteria that cause device-related and other chronic infections grow in matrix-enclosed biofilms. The diagnostic and therapeutic strategies that have served us so well in the partial eradication of acute epidemic bacterial diseases have not yielded accurate data or favorable outcomes when applied to these biofilm diseases. We discuss the potential benefits of the application of the new methods and concepts developed by biofilm science and engineering to the clinical management of infectious diseases.

H ematopoietic stem cell (HSC) gene therapy can potentially cure a variety of human hematopoietic diseases, such as sickle cell disease. Selection and expansion of gene-corrected HSCs has now been accomplished for the first time using HSC from large animals — dogs and humans — with a novel drug-resistance gene, MGMT, which is not expressed in normal HSCs (see the related articles beginning on pages 1561 and 1581). Highly efficient lentiviral transfer and expression of MGMT into relatively few HSCs led to repopulation of most of the hematopoietic compartment with gene-corrected cells following suitable drug treatment. This selection system may be useful in human clinical trials to permit gene therapy in autologous and allogeneic bone marrow transplantation settings.

S ystemic lupus erythematosus (SLE) is a prototypical systemic autoimmune disease characterized by the production of pathogenic autoantibodies. A new study demonstrates that passive antibody specific for the TNF family member, CD154, ameliorates disease by reducing levels of self-reactive antibody in the serum. This study demonstrates a substantial potential for anti-CD154 antibody in the treatment of humoral autoimmunity.

E ver more unexpected roles for the LDL receptor gene family in a variety of cellular signaling pathways continue to emerge. Three recent studies now add another function to this collection. By interacting with active tissue-type plasminogen activator, LDL receptor–related protein appears to control permeability of the blood-brain barrier, vascular tone, and the expression of MMPs. All of these parameters impact upon postischemic infarct size following stroke. These novel findings are discussed in the context of known mechanisms of signaling by the LDL receptor family.

T ubulointerstitial fibrosis is the final common result of a variety of progressive injuries leading to chronic renal failure. Transforming growth factor-β (TGF-β) is reportedly upregulated in response to injurious stimuli such as unilateral ureteral obstruction (UUO), causing renal fibrosis associated with epithelial-mesenchymal transition (EMT) of the renal tubules and synthesis of extracellular matrix. We now show that mice lacking Smad3 (Smad3^ex8/ex8), a key signaling intermediate downstream of the TGF-β receptors, are protected against tubulointerstitial fibrosis following UUO as evidenced by blocking of EMT and abrogation of monocyte influx and collagen accumulation. Culture of primary renal tubular epithelial cells from wild-type or Smad3-null mice confirms that the Smad3 pathway is essential for TGF-β1–induced EMT and autoinduction of TGF-β1. Moreover, mechanical stretch of the cultured epithelial cells, mimicking renal tubular distention due to accumulation of urine after UUO, induces EMT following Smad3-mediated upregulation of TGF-β1. Exogenous bone marrow monocytes accelerate EMT of the cultured epithelial cells and renal tubules in the obstructed kidney after UUO dependent on Smad3 signaling. Together the data demonstrate that the Smad3 pathway is central to the pathogenesis of interstitial fibrosis and suggest that inhibitors of this pathway may have clinical application in the treatment of obstructive nephropathy.

C C chemokine ligand 21 (CCL21)/secondary lymphoid chemokine (SLC), a ligand for CC chemokine receptor 7 (CCR7), has been demonstrated to play a vital role in the homing and localization of immune cells to lymphoid tissues, but its role in nonlymphoid tissues largely remains undefined. Here, we provide evidence that CCL21 in lymphoid and nonlymphoid tissues is differentially regulated by lymphotoxin-dependent (LT-dependent) and -independent mechanisms, respectively. This differential regulation is due to the selective regulation of the CCL21-Ser/CCL21a but not the CCL21-Leu/CCL21b gene by the LT and noncanonical NF-κB pathways. This alternate pathway, not dependent on LT or lymphocytes, leading to constitutive expression of CCL21 in nonlymphoid tissues, is critical for the initial recruitment of T lymphocytes to peripheral effector sites. CCL21 expression is subsequently further enhanced in a LT-dependent fashion following airway challenge, potentially facilitating a positive feedback loop to attract additional CCR7⁺ effector cells. These findings establish an essential role for CCL21 in the recruitment of effector T cells to peripheral tissues and suggest that LT-dependent and -independent regulation of CCL21 plays a role in balancing the central and peripheral immune responses between lymphoid and nonlymphoid tissues.

T o determine the role of CD154-CD40 interactions in the B cell overactivity exhibited by patients with active systemic lupus erythematosus (SLE), CD19⁺ peripheral B cells were examined before and after treatment with humanized anti-CD154 mAb (BG9588, 5c8). Before treatment, SLE patients manifested activated B cells that expressed CD154, CD69, CD38, CD5, and CD27. Cells expressing CD38, CD5, or CD27 disappeared from the periphery during treatment with anti-CD154 mAb, and cells expressing CD69 and CD154 disappeared from the periphery during the post-treatment period. Before treatment, active-SLE patients had circulating CD38^bright Ig-secreting cells that were not found in normal individuals. Disappearance of this plasma cell subset during treatment was associated with decreases in anti–double-stranded DNA (anti-dsDNA) Ab levels, proteinuria, and SLE disease activity index. Consistent with this finding, peripheral B cells cultured in vitro spontaneously proliferated and secreted Ig in a manner that was inhibited by anti-CD154 mAb. Finally, the CD38^+/++IgD⁺, CD38⁺⁺⁺, and CD38⁺IgD^– B cell subsets present in the peripheral blood also disappeared following treatment with humanized anti-CD154. Together, these results indicate that patients with active lupus nephritis exhibit abnormalities in the peripheral B cell compartment that are consistent with intensive germinal center activity, are driven via CD154-CD40 interactions, and may reflect or contribute to the propensity of these patients to produce autoantibodies.

T he insulin receptor substrate-2 (Irs2) branch of the insulin/IGF signaling system coordinates peripheral insulin action and pancreatic β cell function, so mice lacking Irs2 display similarities to humans with type 2 diabetes. Here we show that β cell–specific expression of Irs2 at a low or a high level delivered a graded physiologic response that promoted β cell growth, survival, and insulin secretion that prevented diabetes in Irs2^–/– mice, obese mice, and streptozotocin-treated mice; and that upon transplantation, the transgenic islets cured diabetes more effectively than WT islets. Thus, pharmacological approaches that promote Irs2 expression in β cells, especially specific cAMP agonists, could be rational treatments for β cell failure and diabetes.

T he regulation of cerebrovascular permeability is critical for normal brain homeostasis, and the “breakdown” of the blood-brain barrier (BBB) is associated with the development of vasogenic edema and intracranial hypertension in a number of neurological disorders. In this study we demonstrate that an increase in endogenous tissue-type plasminogen activator (tPA) activity in the perivascular tissue following cerebral ischemia induces opening of the BBB via a mechanism that is independent of both plasminogen (Plg) and MMP-9. We also show that injection of tPA into the cerebrospinal fluid in the absence of ischemia results in a rapid dose-dependent increase in vascular permeability. This activity is not seen with urokinase-type Plg activator (uPA) but is induced in Plg^–/– mice, confirming that the effect is Plg-independent. However, the activity is blocked by antibodies to the LDL receptor–related protein (LRP) and by the LRP antagonist, receptor-associated protein (RAP), suggesting a receptor-mediated process. Together these studies demonstrate that tPA is both necessary and sufficient to directly increase vascular permeability in the early stages of BBB opening, and suggest that this occurs through a receptor-mediated cell signaling event and not through generalized degradation of the vascular basement membrane.

A lthough capillary barrier deterioration underlies major inflammatory lung pathology, barrier-enhancing strategies are not available. To consider hyperosmolar therapy as a possible strategy, we gave 15-minute infusions of hyperosmolar sucrose in lung venular capillaries imaged in real time. Surprisingly, this treatment enhanced the capillary barrier, as indicated by quantification of the capillary hydraulic conductivity. The barrier enhancement was sufficient to block the injurious effects of thrombin, TNF-α, and H₂O₂ in single capillaries, and of intratracheal acid instillation in the whole lung. Capillary immunofluorescence indicated that the hyperosmolar infusion markedly augmented actin filament formation and E-cadherin expression at the endothelial cell periphery. The actin-depolymerizing agent latrunculin B abrogated the hyperosmolar barrier enhancement as well as the actin filament formation, suggesting a role for actin in the barrier response. Furthermore, hyperosmolar infusion blocked TNF-α–induced P-selectin expression in an actin-dependent manner. Our results provide the first evidence to our knowledge that in lung capillaries, hyperosmolarity remodels the endothelial barrier and the actin cytoskeleton to enhance barrier properties and block proinflammatory secretory processes. Hyperosmolar therapy may be beneficial in lung inflammatory disease.

W e have previously described the only reported case of human proprotein convertase 1 (PC1) deficiency, in a female (Subject A) with obesity, hypogonadism, hypoadrenalism, and reactive hypoglycemia. We now report the second case of human PC1 deficiency (Subject B), also due to compound heterozygosity for novel missense and nonsense mutations. While both subjects shared the phenotypes of obesity, hypoadrenalism, reactive hypoglycemia, and elevated circulating levels of certain prohormones, the clinical presentation of Subject B was dominated by severe refractory neonatal diarrhea, malabsorptive in type. Subsequent investigation of Subject A revealed marked small-intestinal absorptive dysfunction, which was not previously clinically suspected. We postulate that PC1, presumably in the enteroendocrine cells, is essential for the normal absorptive function of the human small intestine. The differences in the nature and severity of presentation between the two cases cannot readily be explained on the basis of allelic heterogeneity, as the nonsense and missense mutations from both subjects had comparably severe effects on the catalytic activity of PC1. Despite Subject A’s negligible PC1 activity, some mature ACTH and glucagon-like peptide 1^7-36amide were detectable in her plasma, suggesting that the production of these hormones, at least in humans, does not have an absolute dependence on PC1. The presence of severe obesity and the absence of growth retardation in both subjects contrast markedly with the phenotype of mice lacking PC1 and suggest that the precise physiological repertoire of this enzyme may vary between mammalian species.

I nfusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O⁶-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O⁶-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU–conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O⁶-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus–transduced human SRCs following BG/BCNU treatment.

P soriasis is a chronic inflammatory disease of the skin characterized by epidermal hyperplasia, dermal angiogenesis, infiltration of activated T cells, and increased cytokine levels. One of these cytokines, IL-15, triggers inflammatory cell recruitment, angiogenesis, and production of other inflammatory cytokines, including IFN-γ, TNF-α, and IL-17, which are all upregulated in psoriatic lesions. To investigate the role of IL-15 in psoriasis, we generated mAb’s using human immunoglobulin-transgenic mice. One of the IL-15–specific antibodies we generated, 146B7, did not compete with IL-15 for binding to its receptor but potently interfered with the assembly of the IL-15 receptor α, β, γ complex. This antibody effectively blocked IL-15–induced T cell proliferation and monocyte TNF-α release in vitro. In a human psoriasis xenograft model, antibody 146B7 reduced the severity of psoriasis, as measured by epidermal thickness, grade of parakeratosis, and numbers of inflammatory cells and cycling keratinocytes. These results obtained with this IL-15–specific mAb support an important role for IL-15 in the pathogenesis of psoriasis.

C linical application of gene therapy for genetic and malignant diseases has been limited by inefficient stem cell gene transfer. Here we studied in a clinically relevant canine model whether genetic chemoprotection mediated by a mutant of the DNA-repair enzyme methylguanine methyltransferase could circumvent this limitation. We hypothesized that genetic chemoprotection might also be used to enhance allogeneic stem cell transplantation, and thus we evaluated methylguanine methyltransferase–mediated chemoprotection in an allogeneic setting. We demonstrate that gene-modified allogeneic canine CD34⁺ cells can engraft even after low-dose total body irradiation conditioning. We also show that cytotoxic drug treatment produced a significant and sustained multilineage increase in gene-modified repopulating cells. Marking in granulocytes rose to levels of up to 98%, the highest in vivo marking reported to date to our knowledge in any large-animal or human study. Increases in transgene-expressing cells after in vivo selection provided protection from chemotherapy-induced myelosuppression, and proviral integration site analysis demonstrated the protection of multiple repopulating clones. Drug treatment also resulted in an increase in donor chimerism. These data demonstrate that durable, therapeutically relevant in vivo selection and chemoprotection of gene-modified cells can be achieved in a large-animal model and suggest that chemoprotection can also be used to enhance allogeneic stem cell transplantation.

B asic and clinical observations suggest that thrombosis and inflammation are closely related. Here we addressed the role played by TNF-α in thrombus formation and growth in an in vivo mouse model. Using intravital microscopy, we show that systemic administration of TNF-α at doses found in sepsis transiently inhibits thrombus formation and delays arterial occlusion upon vascular injury. These results were reflected in a prolonged bleeding time. Platelets isolated from the TNF-α–treated mice showed a marked decrease in fibrinogen binding and P-selectin expression as well as reduced platelet aggregation in response to various agonists. In contrast, in vitro treatment of platelets with TNF-α did not affect their function. TNF receptor 1– and 2–deficient mice exhibited normal thrombogenesis in the presence of TNF-α. Additionally, the inhibitory effect of TNF-α was lost either after treatment with N^G-monomethyl-L-arginine, an inhibitor of NO production, or in mice deficient for iNOS. These results indicate that under inflammatory conditions, when leukocytes need free passage to transmigrate into tissues, TNF-α decreases platelet activation and inhibits thrombi formation. This effect is not exerted directly on platelets but mediated through the rapid generation of NO in the vessel wall.