Decreased synthesis of serum carboxypeptidase N (SCPN) in familial SCPN deficiency

Serum carboxypeptidase N (SCPN) is the primary inactivator of the C3a, C4a, and C5a anaphylatoxins as well as an inactivator of bradykinin. Thus SCPN deficiency potentially could result in significant pathophysiologic consequences. Previous studies identified a deficient subject afflicted with frequent episodes of angioedema, and other family members also had SCPN deficiency. To delineate this abnormality further, the fractional catabolic rate (FRC) and enzyme synthesis were determined in three members of the afflicted kindred as well as in five normal persons following the infusion of homogeneous 125 I-SCPN. The mean FCR and synthesis rates for SCPN in the normal subjects were 1.3%/hr and 20,793 U/kg/hr, respectively. Reduced synthesis was concluded to be primarily responsible for the low SCPN levels in the afflicted kindred. The high FRC of SCPN discourages attempted maintenance therapy with infusions of enriched SCPN preparations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44847/1/10875_2004_Article_BF00915368.pd

Neutrophil Chemotactic Activity and C5a Following Systemic Activation of Complement in Rats

Using ELISA analysis, rat C5a was stimulated in serum from rats undergoing systemic activation of complement after intravenous infusion of purified cobra venom factor (CVF). Biological (neutrophil chemotactic) activity was also assessed. Serum levels of C5a were directly proportional to the amount of CVF infused. C5a and neutrophil chemotactic activity, peaked by 5 min, then plateaued. In vitro addition of anti-C5a to serum samples of CVF-infused rats totally abolished chemotactic activity, indicating that all biological activity could be ascribed to C5a. Blood neutrophils obtained from CVF-infused animals showed a significant upregulation of CD11b, the increase being reduced (38%) in animals pretreated with anti-C5a. These findings indicate that infusion of CVF into rats produces generation of C5a, all chemotactic activity in serum being related to C5a. The in vivo generation of C5a is, at least inpart, responsible for upregulation of CD11b on blood neutrophils.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44517/1/10753_2004_Article_426059.pd

Role of Chemotactic Factors in Neutrophil Activation After Thermal Injury in Rats

Acute thermal trauma is well known to produce evidence of a “systemic inflammatory response” in vivo, as manifested by evidence of complement activation, appearance in plasma of a variety of inflammatory factors, and development of multi-organ injury. The current studies were focused on acute thermal injury of rat skin and factors responsible for accompanying activation of blood neutrophils. Acute thermal injury of rat skin resulted in a time-dependent loss of L-selectin and up-regulation of Mac-1 (CD11b/CD18) on blood neutrophils, with no changes in LFA-1 (CD11a/CD18). The loss of L-selectin was prevented by blockade of C5a but not by blockade of the α-chemokine, macrophage inflammatory protein-2 (MIP-2). C5a, the α chemokines, MIP-2 and keratinocyte-derived cytokine (KC), and platelet activating factor (PAF) contributed to up-regulation of blood neutrophil Mac-1. Blocking interventions against these mediators also blunted the degree of neutropenia developing after thermal trauma. These data suggest that activation of blood neutrophils after thermal trauma is related to the role of several chemotactic mediators. These studies may provide clues regarding factors responsible for development of the “systemic inflammatory response syndrome” after thermal injury in the experimental model employed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44526/1/10753_2004_Article_408821.pd

Role of the second extracellular loop of human C3a receptor in agonist binding and receptor function

Producción CientíficaThe C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor with an unusually large second extra-cellular loop (e2 loop,;172 amino acids). To determinethe function of this unique structure, chimeric and de-letion mutants were prepared and analyzed in trans-fected RBL-2H3 cells. Whereas replacement of the C3aRN-terminal segment with that from the human C5a re-ceptor had minimal effect on C3a binding, substitutionof the e2 loop with a smaller e2 loop from the C5a recep-tor (C5aR) abolished binding of125I-C3a and C3a-stimu-lated calcium mobilization. However, as much as 65% ofthe e2 loop sequence (amino acids 198 –308) may be re-moved without affecting C3a binding or calcium re-sponses. The e2 loop sequences adjacent to the trans-membrane domains contain multiple aspartate residuesand are found to play an important role in C3a bindingbased on deletion mutagenesis. Replacement of five as-partate residues in the e2 loop with lysyl residues sig-nificantly compromised both the binding and functionalcapabilities of the C3a receptor mediated by intact C3aor by two C3a analog peptides. These data suggest atwo-site C3a-C3aR interaction model similar to that es-tablished for C5a/C5aR. The anionic residues near the Nand C termini of the C3aR e2 loop constitute a non-effector secondary interaction site with cationic resi-dues in the C-terminal helical region of C3a, whereas theC3a C-terminal sequence LGLAR engages the primaryeffector site in C3aR