Mutant analysis was used to identify Moraxella catarrhalis gene products necessary for biofilm development in a crystal violet-based assay involving 24-well tissue culture plates. The wild-type M. catarrhalis strains that formed the most extensive biofilms in this system proved to be refractory to transposon mutagenesis, so an M. catarrhalis strain was constructed that was both able to form biofilms in vitro and amenable to transposon mutagenesis. Chromosomal DNA from the biofilm-positive strain O46E was used to transform the biofilm-negative strain O35E; transformants able to form biofilms were identified and subjected to transposon-mediated mutagenesis. Biofilmnegative mutants of these transformants were shown to have a transposon insertion in the uspA1 gene. Nucleotide sequence analysis revealed that the biofilm-positive transformant T14 contained a hybrid O46E-O35E uspA1 gene, with the N-terminal 155 amino acids being derived from the O46E UspA1 protein. Transformant T14 was also shown to be unable to express the Hag protein, which normally extends from the surface of the M. catarrhalis cell. Introduction of a wild-type O35E hag gene into T14 eliminated its ability to form a biofilm. When the hybrid O46E-O35E uspA1 gene from T14 was used to replace the uspA1 gene of O35E, this transformant strain did not form a biofilm. However, inactivation of the hag gene did allow biofilm formation by strain O35E expressing the hybrid O46E-O35E uspA1 gene product. The Hag protein was shown to have an inhibitory or negative effect on biofilm formation by these M. catarrhalis strains in the crystal violet-based assay.Moraxella catarrhalis is an important cause of otitis media in infants and very young children (9, 21). In adults with chronic obstructive pulmonary disease, M. catarrhalis is known to be a significant cause of infectious exacerbations (28, 38). In addition, M. catarrhalis has been shown to be an infrequent cause of several other diseases including pneumonia and sinusitis (for a review, see reference 28).The ability of this organism to colonize the mucosal surface of the nasopharynx is key to its ability to cause disease in other anatomic regions, because this colonization event provides a foothold for M. catarrhalis in its human host. In fact, nasopharyngeal colonization with M. catarrhalis is common throughout infancy, and a high rate of colonization with this organism is associated with an increased risk of otitis media (10). The mechanism(s) essential for colonization of the nasopharyngeal mucosa by M. catarrhalis has not been determined conclusively, although a number of M. catarrhalis gene products that may be involved in this process have been identified in the past few years. The UspA1 and UspA2H proteins have both been shown to function as adhesins for human epithelial cells in vitro (22); more recently, the Hag (MID) protein was shown to bind to both A549 human lung cells (11,18) and primary cultures of human middle ear epithelial cells (18). The M. catarrhalis OmpCD protein has been shown to bind bot...

Purpose:We have demonstrated previously that a mixture of three anti-Her-2 monoclonal antibodies (MAbs) that bind to different epitopes on the extracellular domain of Her-2 expressed on a human breast cancer cell line has more potent antitumor activity than the individual MAbs both in vitro and in xenografted severe combined immunodeficient mice. Because the activity of Herceptin is Fc dependent, we determined whether this would also be the case when a mixture of these three anti-Her-2 MAbs was used.Experimental Design: IgG and highly purified F(ab) 2 fragments of the anti-Her-2 MAbs and Herceptin were prepared and evaluated for their ability to induce cell death, inhibit vascular endothelial growth factor secretion, and mediate antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity in vitro. They were also compared for their abilities to induce regression of large BT474 tumors in severe combined immunodeficient mice.Results: All of the F(ab) 2 fragments were >95% pure and, as expected, did not mediate antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity in vitro. The in vitro antiproliferative and proapoptotic effects of the IgGs and F(ab) 2 fragments were similar. In contrast, the IgGs had significant antitumor activity in vivo, whereas their F(ab) 2 fragments were only marginally effective even at 5-fold higher doses to offset their shorter half-lives.Conclusions: These results confirm the importance of the Fc portion of Herceptin for optimal in vivo activity and demonstrate that even a mixture of three anti-Her-2 MAbs that are highly effective at inducing cell death in vitro requires Fc-mediated effector function for optimal in vivo activity.

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable Haemophilus influenzae (NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobinhaptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic hgbA hgbB hgbC mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the hxuC gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by H. influenzae.