Soluble recombinant neutral endopeptidase (CD10) as a potential antiinflammatory agent.

Several endogenous peptides, including bradykinin and substance P, have potent inflammatory effects in the joint. Levels of these peptides are regulated by plasma and cell-associated peptide degrading enzymes. One of these peptidases, neutral endopeptidase-24.11 (NEP-24.11), is expressed constitutively and in high density on human synovial cells and is presumed to play a critical role in local regulation of peptide levels in the joint. We examined the role of endogenous NEP-24.11 in regulating bradykinin-mediated effects in an articular model, and investigated the ability of soluble, recombinant human NEP-24.11 to augment the effects of the endogenous enzyme. Our studies demonstrate that endogenous synovial NEP-24.11 does not significantly modulate inflammatory peptide effects on cells when competing with colocalizing peptide receptors expressed in high density. Administration of excess, soluble recombinant NEP-24.11 can overcome this problem, however. Furthermore, the activity of the recombinant enzyme was not compromised in the presence of oxidants or inflammatory joint fluids. Recombinant NEP-24.11 holds promise as a novel therapeutic strategy for the treatment of inflammatory arthritis.

Influence of the cytosolic carboxyl termini of human B1 and B2 kinin receptors on receptor sequestration, ligand internalization, and signal transduction.

To determine the role of the cytoplasmic carboxyl termini of human B1 and B2 kinin receptors (B1KR and B2KR, respectively) in the internalization of their respective ligands, des-Arg10-kallidin and bradykinin (BK), both wild type receptors, as well as truncated B2KRs, a mutated B2KR, and chimeric receptors were stably expressed in Chinese hamster ovary cells. Incubation of [3H]BK at 37 degrees C with cells expressing wild type B2KR resulted in pronounced and rapid ligand internalization ( approximately 80% after 10 min). By contrast, incubation of 3H-labeled des-Arg10-kallidin with cells expressing B1KR resulted in a modest, slow internalization of the ligand (<20% after 10 min). Replacement, from Cys324, of the cytoplasmic carboxyl terminus of the B2KR with that of the B1KR from Cys330 (both Cys residues are putative palmitoylation sites) greatly reduced ligand internalization ( approximately 40% after 10 min) without significantly altering Kd or ligand-induced signal activation. By marked contrast, the corresponding replacement, of the sequence from Cys330 of the cytoplasmic carboxyl terminus of the B1KR with the segment of the B2KR, led to a striking increase of ligand internalization ( approximately 75% within 10 min) without altering Kd or ligand-induced signal activation. Truncation of the B2KR to within three amino acids of Cys324 (truncation at Gly327) led to strongly reduced ligand internalization ( approximately 40% after 10 min). Truncation of the B2KR up to Lys315 almost completely abolished internalization of [3H]BK (10% after 10 min). This additional reduction is apparently not caused by the loss of the potential palmitoylation site at Cys324, since a B2KR with a point mutation of Cys324 to Ala internalized [3H]BK as rapidly as the wild type B2KR. From these results we conclude that the cytoplasmic carboxyl terminus of the human B2KR contains sequences that are necessary and sufficient to permit rapid ligand-induced sequestration of human kinin receptors and internalization of their agonists.

The value of IDPN as a supplemental therapy when elderly patients fail to thrive--two case studies.

On the last day of his life, H.J. came into the dialysis unit smiling and joking. He spent the time watching a movie and visiting with visitors and staff. He reported feeling better than he had in a long time. His predicted 3-day death watch had become nearly 5 months of improved quality of life and precious time spent with his large extended family. He frequently expressed gratitude for the chance to prolong those few days into such an unexpected extension. He and his family attributed this gift to the nursing care and encouragement he received in the dialysis unit. Everyone was aware that the IDPN therapy, along with improvement in his own daily nutritional intake, was a critical element. Later that night, H.J. had a cardiac arrest, and he died peacefully at home with his family. There is no doubt that without the financial resources available to him to pay for his IDPN, the outcome would not have been the same. As it was, all the intended patient outcomes were achieved.

The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults.

To assess changes since the mid-1970s, we reviewed 843 episodes of positive blood cultures in 707 patients with septicemia. The five most common pathogens were Staphylococcus aureus, Escherichia coli, coagulase-negative staphylococci (CNS), Klebsiella pneumoniae, and Enterococcus species. Although CNS were isolated most often, only 12.4% were clinically significant. Half of all episodes were nosocomial, and a quarter had no recognized source. Leading identifiable sources included intravenous catheters, the respiratory and genitourinary tracts, and intraabdominal foci. Septicemia-associated mortality was 17.5%. Patients who received appropriate antimicrobial therapy throughout the course of infection had the lowest mortality (13.3%). Multivariate analysis showed that age (relative risk [RR], 1.80), microorganism (RR, 2.27), source of infection (RR, 2.86), predisposing factors (RR, 1.98), blood pressure (RR, 2.29), body temperature (RR, 2.04), and therapy (RR, 2.72) independently influenced outcome. Bloodstream infections in the 1990s are notable for the increased importance of CNS as both contaminants and pathogens, the proportionate increase in fungi and decrease in anaerobes as pathogens, the emergence of Mycobacterium avium complex as an important cause of bacteremia in patients with advanced human immunodeficiency virus infection, and the reduction in mortality associated with infection.

Mechanisms of prostanoid synthesis in human synovial cells: cytokine-peptide synergism.

Bradykinin (BK)2 and interleukin-1 (IL-1) interact synergistically to stimulate prostaglandin synthesis in human synovial fibroblast-like cells. The effect of BK is rapid and correlates with its capacity to elevate cytosolic levels of calcium ([Ca2+]i), while IL-1's effect is slow and s dependent upon de novo protein synthesis. The mechanism of this synergistic interaction was investigated. In the basal state, high levels of arachidonic acid (AA) were spontaneously released from synovial cells but near absent levels of cyclooxygenase activity prevented metabolism of AA to prostanoid. BK was a potent stimulus for elevating AA, but not prostaglandins, above basal levels. IL-1, in contrast, increased prostaglandins but not AA, above basal levels. IL-1 treatment was not associated with a loss or redistribution of AA among phospholipid classes. These results are consistent with high basal phospholipase activity in synovial cells and demonstrate the ability of BK, presumably via its ability to raise [Ca2+]i, to further elevate this activity(ies). Metabolism of AA to prostanoid is minimal in resting and BK-stimulated synovial cells, however, without the concomitant induction of cyclooxygenase activity by IL-1. These studies clarify the different, but synergistic, mechanisms of action of a peptide and cytokine in stimulating prostanoid synthesis in synovial cells. In addition, these data extend the results of previous investigations in demonstrating that basal phospholipase activity provides sufficient AA substrate for IL-1 induced prostanoid synthesis without invoking the concomitant induction of phospholipase activity by IL-1.

Resistant enterococci: a prospective study of prevalence, incidence, and factors associated with colonization in a university hospital.

OBJECTIVE: To determine the prevalence of gastrointestinal tract colonization with antibiotic-resistant enterococci at ward entry and to study the incidence and risk factors for nosocomial acquisition of colonization with resistant enterococci. DESIGN: A prospective cohort study conducted between February 1 and March 15, 1993. METHODS: Rectal cultures were obtained within 24 hours of admission or transfer onto the study wards and repeated at weekly intervals and at the time of discharge. Patients harboring antibiotic-resistant enterococci at the time of admission or after admission were compared to patients who were not colonized with these organisms. Clinical and epidemiologic risk factors for colonization were abstracted prospectively by daily chart review. Following a univariate analysis of risk factors associated with colonization, a multivariate statistical analysis using three separate models was done. SETTING: A 1,125-bed, tertiary-care teaching hospital in North Carolina. PATIENTS: A total of 350 patients admitted to two general medical wards and the medical intensive care unit during the study period. RESULTS: Antibiotic-resistant enterococci were isolated from 52 patients: 19 were colonized at admission to the study, and 33 later acquired resistant strains. At the time of admission, 5.4% of the patients were colonized with ampicillin-resistant enterococci (ARE), including 1.1% that were colonized with vancomycin-resistant enterococci. Prior hospitalization was associated with colonization with ARE at admission (P = .01). Independent risk factors for nosocomial acquisition of ARE included treatment with more than three antibiotics, empiric use of antibiotics, use of third-generation cephalosporins, and the use of enteral tube feedings. Antibiotics used prophylactically were not associated with resistant enterococcal colonization. CONCLUSIONS: Our data help to elucidate the epidemiology of gastrointestinal tract colonization with resistant enterococci. We hypothesize that surveillance and control programs will be more likely to succeed if targeted at patients receiving more than three antibiotics, empiric antibiotics, and enteral tube feedings (Infect Control and Hosp Epidemiol 1996;17:36-41).

Type VI collagen-specific messenger RNA is expressed constitutively by cultured human synovial fibroblasts and is suppressed by interleukin-1.

OBJECTIVE: Type VI collagen is a prominent constituent of the synovial extracellular matrix. The cellular source of this matrix protein and the identity of local factor sin synovium that may regulate its expression have not been delineated, however. We examined the capacity of human fibroblast-like synovial cells to synthesize type VI collagen as well as the effect of interleukin-1 (IL-1) on this expression. METHODS: RNA was extracted from cultured human synovial cells derived from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Northern blots were analyzed using sequence-specific probes, and steady-state messenger RNA (mRNA) levels of the 3 alpha (VI) procollagen chains were measured. The effect of IL-1 treatment on these levels was determined. RESULTS: Abundant expression of 3 characteristic mRNA transcripts, corresponding to the alpha 1 (4.2-kb), alpha 2 (3.5-kb), and alpha 3 (8.5-kb) chains of type VI procollagen, was observed in untreated cells derived from RA and OA patients. IL-1 treatment consistently suppressed steady-state mRNA levels for all 3 alpha (VI) procollagen chains in a time- and dose-dependent manner. Tumor necrosis factor alpha induced a response similar to that of IL-1, while IL-2 was ineffective in this regard. Indomethacin partially restored alpha (VI) mRNA expression in IL-1--treated cells. CONCLUSION: These studies provide novel data demonstrating abundant steady-state levels of mRNA transcripts coding for all 3 type VI procollagen polypeptides in human synovial fibroblast-like cells, as well as coordinated down-regulation of these transcripts by IL-1. Local production of IL-1 may thus constitute an important means in vivo of regulating the production of type VI collagen.

Characterization of kinin receptors on human synovial cells and upregulation of receptor number by interleukin-1.

Bradykinin has been implicated in the pathogenesis of inflammatory arthritis by virtue of its potent proinflammatory properties. We have previously shown bradykinin to be a potent stimulus for the release of prostanoids from interleukin-1 (IL-1)-treated, but not untreated, human synovial cells. We hypothesize that one mechanism by which IL-1 induces responsiveness to bradykinin is by upregulation of number or affinity of kinin receptors on human synovial cells. We performed [3H]bradykinin binding studies in intact human synovial tissue and in cultured human synovial cells. Specific, saturable [3H]bradykinin binding sites in intact synovia were identified by autoradiographic localization and were present in much higher density in rheumatoid, than in osteoarthritis, synovia. In untreated human synovial cells in culture, a single (B2) class of kinin binding sites with a Kd of 2.3 nM and Bmax of 58 +/- 9 fmol/10(6) cells was demonstrated. In matched experiments, IL-1 treatment enhanced specific [3H]bradykinin binding 1.5- to 2.0-fold above that observed in untreated cells. This enhancement was attributable to an increase in Bmax (53 +/- 4 vs. 105 +/- 24 fmol/10(6) cells in untreated and IL-1-treated cells, respectively), rather than an alteration in Kd (1.7 and 1.4 nM, respectively). The potencies of a series of kinin analogs and antagonists and unrelated peptides in displacing [3H]bradykinin from IL-1-treated cells correlated well with their abilities to induce prostanoid release. These studies provide novel information regarding the nature of kinin receptors in intact human synovia and in cultured human synovial cells, their regulation by IL-1 and their role in IL-1-treated cells in kinin-mediated prostaglandin E2 production.

An improved method for determining the microbial inhibitory activity of serum and its application to the study of patients with leukemia.

Numerous investigators have demonstrated that derangements in serum transferrin and iron can contribute to susceptibility to infection, but the complexity and imprecision of assays have impeded both research and development of clinical testing in this area. This article describes an automated assay for measuring the microbial inhibitory activity of transferrin in serum and its use in patients with acute myelogenous leukemia (AML) and in normal controls. The assay measured the ability of heat-inactivated serum to inhibit the growth of an antibiotic-resistant strain of Pseudomonas aeruginosa. The serum dilutions were prepared in a special low iron chemically defined broth. An inhibition index, the reciprocal of the serum dilution producing 50% inhibition of bacterial growth when compared with the growth in broth alone, was determined. The results showed the serum from the patients with leukemia had a significantly lower inhibition index than that of controls (16 +/- 11 vs. 35 +/- 13, P less than 0.01). In addition, they had higher serum iron levels (162 +/- 65 vs. 75 +/- 27, P less than 0.01), lower serum transferrin levels (231 +/- 65 vs. 309 +/- 71, P less than 0.01), and higher percentage saturation of transferrin with iron (59 +/- 21 vs. 20 +/- 8, P less than 0.01) than did controls. Because the assay uses equipment available in many clinical laboratories, it could be developed for routine use as an index of susceptibility to infection in selected patients.

Transferrin in disease II: defects in the regulation of transferrin saturation with iron contribute to susceptibility to infection.

Patients with leukemia were found to have a high percentage of saturation of their serum transferrin with iron to an extent only rarely observed with other malignancies. This was associated with a reduced ability of their serum to inhibit the growth of a test strain of Pseudomonas aeruginosa. Serum iron, transferrin, and related parameters were measured serially in patients undergoing bone marrow transplantation for leukemia or aplastic anemia. It was found that a high proportion of these patients also have a high saturation of their transferrin with iron. This was related to three distinct physiologic deficits: a low level of serum transferrin; a high level of iron; and an inability to reduce the level of serum iron during infection. Three of six patients who were unable to reduce their serum during fever and infection subsequently died of sepsis. These data support the hypothesis that derangements in nonspecific serologic defense mechanisms involving iron contribute to susceptibility to infection in patients with leukemia undergoing bone marrow transplantation.

Clostridium difficile. Colonization and toxin production in a cohort of patients with malignant hematologic disorders.

We examined 45 (80%) of 56 consecutive adult patients with malignant hematologic disorders who were hospitalized during a 15-week period at Emory University Hospital, Atlanta. Stool samples for Clostridium difficile culture and cytotoxin assay were obtained on admission and then weekly during each patient's hospitalization. On admission, four patients had detectable C difficile in their stool samples, which was associated with prior antimicrobial use but not with prior cancer chemotherapy. One of the four patients with positive stool samples also had toxin present in the stool sample and was the only one with diarrhea. Eight (36%) of 22 patients hospitalized for one or more weeks had C difficile isolated from at least one stool specimen. The positive cultures showed no clustering in time, and no risk factors were identified for colonization. Only seven of 15 culture-positive stool samples and three of seven toxin-positive samples were associated with diarrhea.