Infection of primary dermal microvascular endothelial cells by Kaposi's sarcoma-associated herpesvirus.

OBJECTIVE: To develop an in vitro model for infection of primary human cells with Kaposi's sarcoma (KS) herpesvirus (KSHV). DESIGN: The recent identification of a herpesvirus associated with KS, its successful isolation in vitro, and its complete DNA sequencing facilitates experiments on the pathogenesis of AIDS-related KS. Completed studies demonstrate that the endothelial cells lining the vascular slits in KS lesions are productively infected with KSHV and may be the principal site of virus replication. We have designed a model system to study the infection of primary human cells with KSHV. METHODS: A coculture technique was used with KS cells (KS-1) and primary dermal microvascular endothelial cells. RESULTS: We detected increasing viral DNA concentrations as well as viral mRNA suggesting that a productive virus infection occurs in the target cells. Infection of these cells is dose- and time-dependent and is inhibited by lobucavir, foscarnet and 9-(2-phosphomethoxyethyl) adenine. With a modification of the model, KSHV can be serially passaged in primary cells in excess of 16 passages. CONCLUSIONS: This novel model assay system makes new studies on the role of KSHV and KSHV-induced cellular products on the pathogenesis of KS possible. It also provides a high volume screening method to detect agents that inhibit KSHV infection of primary endothelial cells.

Gallinacins: cysteine-rich antimicrobial peptides of chicken leukocytes.

We purified three homologous antimicrobial peptides ('gallinacins') from chicken leukocytes, examined their antimicrobial activity in vitro, and established their primary sequences by a combination of gas phase microsequencing and on-line LC-ESI-MS analysis of endo- and exoprotease peptide digests. The peptides contained 36-39 amino acid residues, were relatively cationic due to their numerous lysine and arginine residues, and each contained 3 intramolecular cystine disulfide bonds. Gallinacins showed primary sequence homology to the recently delineated beta-defensin family, heretofore found only in the respiratory epithelial cells and neutrophils of cattle, suggesting that beta-defensins originated at least 250 million years ago, before avian and mammalian lineages diverged. The 9 invariant residues (6 cysteines, 2 glycines and 1 proline) common to avian gallinacins and bovine beta-defensins are likely to constitute the essential primary structural motif of this ancient family of host-defense peptides.

Plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis.

We measured concentrations of defensins (human neutrophil peptides) in the plasma of healthy volunteers and patients with sepsis and meningitis. When a sensitive enzyme immunoassay was used, defensins were detected in plasma samples from 13 of 24 healthy blood donors, with a mean +/- SD of 42 +/- 53 ng/ml. Defensin levels in plasma samples from seven patients with sepsis at the onset of disease ranged from 900 ng/ml to 170,000 ng/ml. In 10 patients with meningitis in the initial phase of disease, plasma defensin concentrations ranged from 120 ng/ml to 910 ng/ml. Defensin concentrations in the plasma of both patient groups were significantly higher than those in healthy blood donors (p << 0.01), and patients with sepsis had higher defensin levels than patients with meningitis (p < 0.01). Defensin levels were significantly (p < 0.01) lower after the beginning of specific antibiotic therapy. Defensin concentrations in the plasma of patients with sepsis and meningitis correlated only weakly (r = 0.38) with blood neutrophil count. In vitro studies of defensin added to plasma indicated that all defensin was bound to plasma proteins. At high concentrations (1000 micrograms/ml), defensins caused precipitation of plasma proteins. Because plasma defensin levels may reflect neutrophil activation at sites of infection and inflammation, studies of the clinical utility of defensin ELISA are indicated.

Protegrins: leukocyte antimicrobial peptides that combine features of corticostatic defensins and tachyplesins.

Porcine leukocytes contained three homologous peptides, PG-1, 2 and 3, that manifested potent microbicidal activity against Escherichia coli, Listeria monocytogenes and Candida albicans in vitro. The peptides ('protegrins') were composed of 16 (PG-2) or 18 amino acid residues, and, like tachyplesins (broad-spectrum antibiotic peptides of horseshoe crab hemocytes), they contained two intramolecular cystine disulfide bonds. Considerably smaller than defensins, protegrins nevertheless showed substantial homology to them, especially to the 'corticostatic' rabbit defensin, NP-3a. The relatively simple structure of protegrins should provide useful prototypes for constructing congeners with selectively enhanced host defense activities.