Publications

As the volume, complexity, and cost of new medical technology increase, the need for evaluating benefits and risks becomes increasingly important. Once the formal requirements for Food and Drug Administration approval and insurance coverage are met, however, few systematic criteria are applied to ensure patient benefit. A more systematic policy approach regarding new technologies is needed, with input from balanced groups reviewing evidence of clinical outcomes data to determine patient benefit. This paper examines cardiac computed tomography angiography as a case study; it proposes procedures designed to ensure that the benefits of new technologies justify their costs.

Candida albicans is a species of fungus that typically resides in the gastrointestinal tracts of humans and other warm-blooded animals. It is also the most common human fungal pathogen, causing a variety of skin and soft tissue infections in healthy people and more virulent invasive and disseminated diseases in patients with compromised immune systems. How this microorganism manages to persist in healthy hosts but also to cause a spectrum of disease states in the immunocompromised host are questions of significant biological interest as well as major clinical and economic importance. In this review, we describe recent developments in population genetics, the mating process, and gene disruption technology that are providing much needed experimental insights into the biology of C. albicans.

In the prevailing model of HIV-1 trans-infection, dendritic cells (DCs) capture and internalize intact virions and transfer these virions to interacting T cells at the virological synapse. Here, we show that HIV-1 virions transmitted in trans from in vitro derived DCs to T cells principally originate from the surface of DCs. Selective neutralization of surface-bound virions abrogated trans-infection by monocyte-derived DCs and CD34-derived Langerhans cells. Under conditions mimicking antigen recognition by the interacting T cells, most transferred virions still derived from the cell surface, although a few were transferred from an internal compartment. Our findings suggest that attachment inhibitors could neutralize trans-infection of T cells by DCs in vivo.

PLS3 (phospholipid scramblase-3) is a new member of the family of phospholipid scramblases and transports CL (cardiolipin) from the inner to the outer mitochondrial membrane. In the present paper we examined whether changing the levels of functional PLS3 in HeLa cells altered de novo CL biosynthesis and its resynthesis. HeLa cells overexpressing PLS3 or expressing a disrupted PLS3 (F258V) or control were incubated with [1,3-3H]glycerol and radioactivity incorporated into CL was determined. CL biosynthesis from [1,3-3H]glycerol was increased 1.8-fold in PLS3 cells and 2.1-fold in F258V cells compared with control. This was due to a 64% (P<0.05) and 2.6-fold (P<0.05) elevation in CL synthase activity in PLS3 and F258V cells respectively, compared with control, and not due to changes in phosphatidylglycerolphosphate synthase activity. The increase in CL synthase activity in these cells was due to an increase in its mRNA expression. In contrast, resynthesis of CL from [1-14C]linoleic acid was reduced 52% (P<0.05) in PLS3 and 45% (P<0.05) in F258V cells compared with control and this was due to a reduction in mitochondrial monolysocardiolipin acyltransferase activity. Although protein levels of mitochondrial monolysocardiolipin acyltransferase were unaltered, activity and mRNA expression of endoplasmic reticulum monolysocardiolipin acyltransferase was upregulated in PLS3 and F258V cells compared with controls. These data indicate that the CL resynthesis in HeLa cells is sensitive to the mitochondrial localization of CL and not the level of the reacylating enzymes. Alterations in functional PLS3 levels in PLS3 or F258V cells did not affect the mitochondrial decarboxylation of phosphatidylserine to phosphatidylethanolamine indicating that the biosynthetic changes to CL were specific for this mitochondrial phospholipid. We hypothesize that the cardiolipin resynthesis machinery in the cell 'senses' altered levels of CL on mitochondrial membranes and that de novo CL biosynthesis is up-regulated in HeLa cells as a compensatory mechanism in response to altered movement of mitochondrial CL. The results identify PLS3 as a novel regulator of CL de novo biosynthesis and its resynthesis.

PURPOSE OF REVIEW

Melanocyte stimulating hormones (MSHs, melanocortins) have important roles in feeding and energy metabolism and in inflammation. Recent observations have uncovered major functions for these peptides, particularly gamma-MSH, in cardiovascular regulation and sodium metabolism.

RECENT FINDINGS

Both alpha- and gamma-MSH acutely elevate blood pressure and heart rate through central stimulation of sympathetic nervous outflow. This action of alpha-MSH is mediated by the melanocortin 4 receptor (MC4R), whereas sympathetic nervous stimulation by gamma-MSH does not involve its receptor MC3R but rather is likely due to activation of a sodium channel in the central nervous system. In contrast, gamma-MSH deficiency in rodents, or disruption of MC3R, leads to marked salt-sensitive hypertension, again through a central mechanism: a small dose of exogenous peptide delivered into the cerebroventricular system of mice with gamma-MSH deficiency restores blood pressure to normal. This salt-sensitive hypertension is accompanied by the development of insulin resistance; the mechanism linking these two consequences of a high-salt diet is not yet known but may involve activation of the sympathetic nervous system.

SUMMARY

The study of MSH peptides in blood pressure regulation offers a new opportunity to gain insight into the mechanisms underlying salt sensitivity and its link to insulin resistance, and to new therapies.

The upper airway occupies a sentinel position with respect to the physical and chemical qualities of the inspired atmosphere. Responses of the upper airway can be acute or chronic, as well as primary (sensory) or secondary (physiologic). Olfaction and sensory irritation are cofactors in the perception of air quality. Secondary reflex responses to airborne irritants may include blockage (airflow obstruction), secretion (with or without associated inflammation), and alterations in mucociliary clearance. Of the above end points, obstruction has been documented in response to a variety of agents, including acetic acid vapor, ammonia, Cl2, ETS, mixed VOCs, vapors from carbonless copy paper, and (variably) SO2. Alterations in mucociliary clearance have been variably observed with SO2 and ETS exposure. A neutrophilic inflammatory response has been documented after acute exposure to either ozone or VOCs, and metaplastic mucosal changes after prolonged exposures to photochemical mixed air pollutants. Augmented reactivity to irritants is a phenotypic characteristic of both nonallergic and allergic rhinitis; however, understanding of underlying mechanisms remains elusive (75-78). Differential physiologic responsiveness to environmental irritant stimuli has been documented by allergic rhinitis status for acetic acid and Cl2 (objectively) and for mixed VOCs (subjectively only). Differential responsiveness by nonallergic rhinitis status has, to our knowledge, been documented for paper dust only, although a somewhat wider array of pollutants (including ETS and carbonless copy paper) has been studied in groups differing by self-reported pollutant reactivity. Interestingly, although the congestive response to allergens and irritants is similar, the underlying mechanisms appear to differ, with neither mast cell degranulation nor cholinergic parasympathetic reflexes appearing critical to the response (Fig. 3). Although neuropeptide release does not accompany Cl2-induced nasal obstruction, in one model system (hypertonic saline challenge), substance P release accompanied augmented secretions (80,81). In yet another hypertonic model (dry mannitol powder challenge), arachidonic acid metabolites characteristic of epithelial cell activation accompanied nasal obstruction (82). The relevance of these model systems to environmentally realistic (airborne) irritants remains unclear at this time. Overall, nonallergic rhinitis has received considerably less attention than has allergic rhinitis in the context of descriptive, pathophysiologic, and intervention studies. This statement applies equally in the context of environmental nonallergic rhinitis. As is hopefully evident from the above discussion, many potential research questions in this area remain to be addressed.

Each of the 24 known integrin subunits has now been inactivated in mice, and a growing number of conditional null lines are becoming available. Lines of mice expressing null mutations in integrin subunit genes have taught us a great deal about the remarkably diverse functions that integrins perform in vivo in mammals. Thorough evaluation of the phenotypes manifested by these lines has also revealed a number of previously unexpected integrin ligands and signaling partners. In this article, we review approaches that can contribute to optimal use of this valuable resource.

The transforming growth factor (TGF)-beta-inducible integrin alpha v beta6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-beta. Herein, we show that alpha v beta6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of alpha v beta6 in renal disease, we studied the effects of function-blocking alpha v beta6 monoclonal antibodies (mAbs) and genetic ablation of the beta6 subunit on kidney fibrosis in Col4A3-/- mice, a mouse model of Alport syndrome. Expression of alpha v beta6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with alpha v beta6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in beta6-deficient Alport mice. Transcript profiling of kidney tissues showed that alpha v beta6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-beta RII treatment, suggesting shared regulatory functions of alpha v beta6 and TGF-beta. These findings demonstrate that alpha v beta6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target.

Interleukin 4 receptor alpha (IL-4Ralpha) is essential for effective clearance of gastrointestinal nematode infections. Smooth muscle cells are considered to play a role in the type 2 immune response-driven expulsion of gastrointestinal nematodes. Previous studies have shown in vitro that signal transducer and activator of transcription 6 signaling in response to parasitic nematode infection significantly increases smooth muscle cell contractility. Inhibition of the IL-4Ralpha pathway inhibits this response. How this response manifests itself in vivo is unknown. In this study, smooth muscle cell IL-4Ralpha-deficient mice (SM-MHC(Cre)IL-4Ralpha(-/lox)) were generated and characterized to uncover any role for IL-4/IL-13 in this non-immune cell type in response to Nippostrongylus brasiliensis infection. IL-4Ralpha was absent from alpha-actin-positive smooth muscle cells, while other cell types showed normal IL-4Ralpha expression, thus demonstrating efficient cell-type-specific deletion of the IL-4Ralpha gene. N. brasiliensis-infected SM-MHC(Cre)IL-4Ralpha(-/lox) mice showed delayed ability to resolve infection with significantly prolonged fecal egg recovery and delayed worm expulsion. The delayed expulsion was related to a delayed intestinal goblet cell hyperplasia, reduced T helper 2 cytokine production in the mesenteric lymph node, and reduced M3 muscarinic receptor expression during infection. Together, these results demonstrate that in vivo IL-4Ralpha-responsive smooth muscle cells are beneficial for N. brasiliensis expulsion by coordinating T helper 2 cytokine responses, goblet hyperplasia, and acetylcholine responsiveness, which drive smooth muscle cell contractions.

Recent investigations point to an important role for peptidases in regulating transcellular ion transport by the epithelial Na(+) channel, ENaC. Several peptidases, including furins and proteasomal hydrolases, modulate ENaC maturation and disposal. More idiosyncratically, apical Na(+) transport by ENaC in polarized epithelia of kidney, airway, and gut is stimulated constitutively by one or more trypsin-family serine peptidases, as revealed by inhibition of amiloride-sensitive Na(+) transport by broad-spectrum antipeptidases, including aprotinin and bikunin/SPINT2. In vitro, the transporting activity of aprotinin-suppressed ENaC can be restored by exposure to trypsin. The prototypical channel-activating peptidase (CAP) is a type 1 membrane-anchored tryptic peptidase first identified in Xenopus kidney cells. Frog CAP1 strongly upregulates Na(+) transport when coexpressed with ENaC in oocytes. The amphibian enzyme's apparent mammalian orthologue is prostasin, otherwise known as CAP1, which is coexpressed with ENaC in a variety of epithelia. In airway cells, prostasin is the major basal regulator of ENaC activity, as suggested by inhibition and knockdown experiments. Other candidate regulators of mature ENaC include CAP2/TMPRSS4 and CAP3/matriptase (also known as membrane-type serine protease 1/ST14). Mammalian CAPs are potential targets for treatment of ENaC-mediated Na(+) hyperabsorption by the airway in cystic fibrosis (CF) and by the kidney in hypertension. CAPs can be important for mammalian development, as indicated by embryonic lethality in mice with null mutations of CAP1/prostasin. Mice with selectively knocked out expression of CAP1/prostasin in the epidermis and mice with globally knocked out expression of CAP3/matriptase exhibit phenotypically similar defects in skin barrier function and neonatal death from dehydration. In rats, transgenic overexpression of human prostasin disturbs salt balance and causes hypertension. Thus, several converging lines of evidence indicate that ENaC function is regulated by peptidases, and that such regulation is critical for embryonic development and adult function of organs such as skin, kidney, and lung.