Margaret K. Hostetter

Pathophysiology of chronic tubulo-interstitial disease in rats. Interactions of dietary acid load, ammonia, and complement component C3.

The human end-stage kidney and its experimental analogue, the remnant kidney in the rat, exhibit widespread tubulo-interstitial disease. We investigated whether the pathogenesis of such tubulo-interstitial injury is dependent upon adaptive changes in tubular function and, in particular, in ammonia production when renal mass is reduced. Dietary acid load was reduced in 1 3/4-nephrectomized rats by dietary supplementation with sodium bicarbonate (NaHCO3), while control rats, paired for serum creatinine after 1 3/4 nephrectomy, were supplemented with equimolar sodium chloride. After 4-6 wk, NaHCO3-supplemented rats demonstrated less impairment of tubular function as measured by urinary excretory rates for total protein and low molecular weight protein and higher transport maximum for para-aminohippurate per unit glomerular filtration rate, less histologic evidence of tubulo-interstitial damage, less deposition of complement components C3 and C5b-9, and a lower renal vein total ammonia concentration. Such differences in tubular function could not be accounted for simply on the basis of systemic alkalinization, and differences in tubular injury could not be ascribed to differences in glomerular function. Because nitrogen nucleophiles such as ammonia react with C3 to form a convertase for the alternative complement pathway, and because increased tissue levels of ammonia are associated with increased tubulo-interstitial injury, we propose that augmented intrarenal levels of ammonia are injurious because of activation of the alternative complement pathway. Chemotactic and cytolytic complement components are thereby generated, leading to tubulo-interstitial inflammation. Thus, alkali supplementation reduces chronic tubulo-interstitial disease in the remnant kidney of the rat, and we propose that this results, at least in part, from reduction in cortical ammonia and its interaction with the alternative complement pathway.

Handicaps to Host Defense: Effects of Hyperglycemia on C3 and Candida albicans

The hyperglycemic patient remains persistently at risk for infectious complications. Whether ascribable to diabetes mellitus, to the administration of glucocorticoids, or to the infusion of hyperalimentation fluids, hyperglycemia may impair several mechanisms of humoral host defense, including such varied neutrophil functions as adhesion, chemotaxis, and phagocytosis. In addition, binding of glucose to the biochemically active site of the third component of complement C3 inhibits the attachment of this protein to the microbial surface and thereby impairs opsonization. Last, several pathogens frequently encountered in hyperglycemic patients possess unique mechanisms of virulence that flourish in the hyperglycemic environment. Most notable in this regard is the yeast Candida albicans, which expresses a glucose-inducible protein that is structurally and functionally homologous to a complement receptor on mammalian phagocytes. This protein promotes adhesion in the yeast and subverts phagocytosis by the host. Thus, hyperglycemia serves as a central mechanism in the predisposition of hyperglycemic patients to infection.

Adhesins and ligands involved in the interaction of Candida spp. with epithelial and endothelial surfaces

Adhesion of candidal species to the epithelium of the gastrointestinal or genitourinary tract stands as a critical first step in the pathogenesis of candidal infection. After colonization and replication at mucosal surfaces, Candida albicans and other pathogenic species may penetrate the mucosal barrier, enter the vascular tree, and disseminate hematogenously. The consequences of this pathogenic cascade evoke considerable morbidity and mortality, especially among immunocompromised patients. Thus, interactions of C. albicans and other candidal species with epithelium and endothelium may lead to serious consequences for the human host. This review evaluates candidate candidal adhesions for epithelial and endothelial surfaces, with emphasis on the specificity of the interaction, the inhibitors that have been employed, and the ligands that have been identified on mammalian cells or matrices. Three types of interactions are described: protein-protein interactions, lectin-like interactions, and incompletely defined interactions in which the adhesive ligand is as yet unidentified. Special attention is given to the roles of integrin-like proteins. Differences in the mechanisms of candidal attachment to epithelium and endothelium are delineated. Last, on the basis of the available literature, avenues of potentially fruitful investigation are proposed. Images

Medical Evaluation of Internationally Adopted Children

BACKGROUND Despite many reports of medical illness in children adopted from abroad, there are currently no accepted guidelines for medical evaluation of this population. METHODS Two hundred ninety-three children adopted from 15 countries (mean age, 14.0 months; 55 percent girls) were evaluated by history taking, physical examination, and screening tests for hepatitis B virus (HBV), human immunodeficiency virus type 1, tuberculin reactivity, intestinal parasites, syphilis, excretion of cytomegalovirus, renal disease, and anemia. All but four were seen within one month of their arrival in the United States. RESULTS Fifty-seven percent of the children (168 of 293) were found to have at least one important medical condition. Eighty-one percent of the diagnoses were established by screening test, rather than by history taking or physical examination. Infectious diseases made up the majority of the medical conditions (73 percent). Serologic testing for hepatitis B surface antigen was positive in 5 percent of the children. Characteristics associated with the acquisition of HBV infection included arrival within the first three years of the study (P = 0.017), Asian origin (P = 0.011), and receipt of a blood transfusion abroad (P = 0.008). Ten children (3 percent) had positive Mantoux skin tests, and four of these had active pulmonary tuberculosis. Tuberculin reactivity was significantly associated with older age (P less than 0.001) and lower weight (P = 0.037). Intestinal parasites were isolated from 14 percent of the international adoptees. Non-Korean adoptees were 16 times more likely to be harboring at least one intestinal parasite than were Korean adoptees (P = 0.005). CONCLUSIONS Directed screening tests should be a routine component of the medical evaluation of all children adopted from abroad, regardless of age, sex, or country of origin.

Hypokalemic nephropathy in the rat. Role of ammonia in chronic tubular injury.

Chronic potassium deficiency results in progressive tubulointerstitial injury, associated with augmented renal ammoniagenesis. We investigated the role of elevated renal ammonia levels and the interaction of ammonia with the complement system in this injury. Potassium deficiency was induced in rats by feeding a low potassium diet. Experimental animals received 150 mM NaHCO3 or equimolar NaCl, as drinking water. After 3 wk, NaHCO3 supplemented rats demonstrated decreased ammonia production, less renal hypertrophy, less histologic evidence of injury, and less proteinuria. In in vitro studies on normal cortical tubular fragments, the addition of ammonia to serum in concentrations comparable to renal cortical levels in potassium-deficient animals significantly increased tubular deposition of C3 as quantitated by a radiolabeled antibody binding technique. Thus, alkali supplementation reduced chronic tubulointerstitial disease in a rat model of hypokalemic nephropathy. We propose that increased cortical ammonia levels contribute to hypokalemic nephropathy through ammonia-mediated activation of the alternative complement pathway.

Molecular mimicry in Candida albicans. Role of an integrin analogue in adhesion of the yeast to human endothelium.

Hematogenous infection with the yeast Candida albicans now occurs with increasing frequency in the neonate, the immunocompromised patient, and the hyperglycemic or hyperalimented host. Yeast-phase C. albicans expresses a protein that is antigenically and structurally related to CD11b/CD18, a member of the beta 2 integrins and a well-characterized adhesin for mammalian neutrophils. Both the neutrophil protein and its analogue in C. albicans have an identical affinity for the C3 ligand iC3b, and both proteins are significantly increased in expression at 37 degrees C. Given these several similarities, we therefore studied the role of the integrin analogue on C. albicans in the adhesion of the yeast to human umbilical vein endothelium (HUVE). After growth of C. albicans in 20 mM D-glucose, as opposed to 20 mM L-glutamate, flow cytometric analysis with monoclonal antibodies recognizing the alpha-subunit of CD11b/CD18 demonstrated a 25.0% increase in mean channel fluorescence (range 18.4-31.8%), as well as an increased percentage of yeasts fluorescing (P less than 0.02). This increased intensity of fluorescence, which corresponds to increased expression of the integrin analogue, also correlated with a significant increase of 30-80% in adhesion of glucose-grown C. albicans to HUVE (P less than 0.02). Blockade of the integrin analogue on C. albicans by monoclonal antibodies recognizing adhesive epitopes on neutrophil CD11b/CD18 inhibited glucose-enhanced adhesion of C. albicans to HUVE. Incubation of glucose-grown C. albicans with saturating concentrations of purified human iC3b, the ligand for CD11b/CD18, reduced adhesion of the yeast to HUVE by 49.7%, whereas BSA in equimolar concentration had no effect (P less than 0.001). These results identify a glucose-responsive integrin analogue on C. albicans as one of possibly several cellular structures that mediate adhesion of the yeast to human endothelium.

Recombinant PhpA Protein, a Unique Histidine Motif-Containing Protein from Streptococcus pneumoniae, Protects Mice against Intranasal Pneumococcal Challenge

ABSTRACT The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the current conjugate vaccine may still cause pneumococcal disease. To address these serotypes and the remaining otitis media due to Streptococcus pneumoniae, we have been evaluating antigenically conserved proteins from S. pneumoniae as vaccine candidates. A previous report identified a 20-kDa protein with putative human complement C3-proteolytic activity. By utilizing the publicly released pneumococcal genomic sequences, we found the gene encoding the 20-kDa protein to be part of a putative open reading frame of approximately 2,400 bp. We recombinantly expressed a 79-kDa fragment (rPhpA-79) that contains a repeated HxxHxH motif and evaluated it for vaccine potential. The antibodies elicited by the purified rPhpA-79 protein were cross-reactive to proteins from multiple strains of S. pneumoniae and were against surface-exposed epitopes. Immunization with rPhpA-79 protein adjuvanted with monophosphoryl lipid A (for subcutaneous immunization) or a mutant cholera toxin, CT-E29H (for intranasal immunization), protected CBA/N mice against death and bacteremia, as well as reduced nasopharyngeal colonization, following intranasal challenge with a heterologous pneumococcal strain. In contrast, immunization with the 20-kDa portion of the PhpA protein did not protect mice. These results suggest that rPhpA-79 is a potential candidate for use as a vaccine against pneumococcal systemic disease and otitis media.

C3 as Substrate for Adhesion of Streptococcus pneumoniae

The ability of choline-binding protein A (CbpA) of Streptococcus pneumoniae to bind the third component of complement (C3) suggests possible interactions with opsonic C3 in the bloodstream or with C3 secreted by epithelial cells. The latter possibility was investigated by measuring C3 in supernatants of resting and cytokine-activated monolayers of type II pulmonary epithelial cells (A549 cells). Expression of C3 on the epithelial cell surface was confirmed by immunofluorescence. Epithelially produced C3 bound to CbpA, as determined by Western blot test. cbpa(-) mutants and lysates therefrom failed to bind C3, were completely deficient in adhesion to a matrix in which C3 was the sole substrate, and demonstrated a moderate yet significant decrease in adhesion to type II pulmonary epithelial cells. These results confirm the interaction of the pneumococcal protein CbpA and its substrate, C3, in 2 in vitro models of adhesion.

Increased ammoniagenesis as a determinant of progressive renal injury

Loss of renal mass evokes increased ammoniagenesis in surviving nephrons, which in turn enables net acid excretion by the kidney. However, this compensatory increase in ammonia production in surviving nephrons triggers the alternative complement pathway, thereby instigating progressive tubulointerstitial injury. Ammonia has recently been identified as a stimulus to renal growth. Enhanced renal growth may serve as a forerunner for renal injury. The growth-promoting properties of ammonia may provide another mechanism through which augmented ammoniagenesis may underlie the enhancement of renal growth and injury observed in such models as the remnant kidney, hypokalemic nephropathy, high protein feeding, experimental diabetes nephropathy, and dietary deficiency of antioxidants.

Distinct mechanisms of epithelial adhesion for Candida albicans and Candida tropicalis. Identification of the participating ligands and development of inhibitory peptides.

The yeast Candida albicans is the leading cause of disseminated fungal infection in neonates, immunocompromised hosts, diabetics, and postoperative patients; Candida tropicalis is the second most frequent isolate. Because the integrin analogue in C. albicans shares antigenic, structural, and functional homologies with the beta 2-integrin subunits alpha M and alpha X, we investigated the role of integrin analogues in epithelial adhesion of C. albicans and C. tropicalis. On flow cytometry with the monoclonal antibody (mAb) OKM1, surface fluorescence was highest for C. albicans and significantly reduced for C. tropicalis (P < 0.001). However, adhesion to the human epithelial cell line HeLa S3 did not differ for these two candidal species: specific adhesion was highest for C. albicans at 44.0 +/- 1.8%, and only slightly lower for C. tropicalis at 38.8 +/- 3.6% (P = NS). The disparity between expression of the integrin analogue and epithelial adhesion suggested distinct mechanisms for this process in C. albicans versus C. tropicalis. Preincubation of C. albicans with anti-alpha M mAbs, with purified iC3b (the RGD ligand for the integrin analogue), or with 9-15-mer RGD peptides from iC3b all inhibited epithelial adhesion significantly (P < 0.001-0.04). Purified fibronectin or fibronectin-RGD peptides failed to block C. albicans adhesion. In contrast, epithelial adhesion of C. tropicalis was significantly inhibited by purified fibronectin and its RGD peptides (P < or = 0.021), but not by iC3b nor the iC3b-RGD peptides. Both iC3b and fibronectin were identified on the surface of epithelial cells after growth in serum-free medium. A polyclonal antibody to C3 inhibited C. albicans adhesion while a control antibody to fibronectin was ineffective; the converse was true for C. tropicalis. These results indicate that the pathogenic yeasts C. albicans and C. tropicalis recognize distinct RGD ligands present at the surface of the epithelial cell and that these interactions can be differentially inhibited by defined RGD peptides containing appropriate flanking sequences.