Herbert A. Lassiter

A randomized study of a monoclonal antibody (pagibaximab) to prevent staphylococcal sepsis

BACKGROUND: Pagibaximab, a human chimeric monoclonal antibody developed against lipoteichoic acid, was effective against staphylococci preclinically and seemed safe and well tolerated in phase 1 studies. OBJECTIVE: To evaluate the clinical activity, pharmacokinetics, safety, and tolerability of weekly pagibaximab versus placebo infusions in very low birth weight neonates. PATIENTS AND METHODS: A phase 2, randomized, double-blind, placebo-controlled study was conducted at 10 NICUs. Patients with a birth weight of 700 to 1300 g and 2 to 5 days old were randomly assigned to receive 3 once-a-week pagibaximab (90 or 60 mg/kg) or placebo infusions. Blood was collected for pharmacokinetics, bacterial killing, and safety analyses. Adverse event and clinical outcome data were collected. RESULTS: Eighty-eight patients received pagibaximab at 90 (n = 22) or 60 (n = 20) mg/kg or placebo (n = 46). Groups were not different in demography, mortality, or morbidity. Pagibaximab demonstrated linear pharmacokinetics, a 14.5-day half-life, and nonimmunogenicity. Definite staphylococcal sepsis occurred in 0%, 20%, and 13% (P < .11) and nonstaphylococcal sepsis occurred in 0%, 10%, and 15% (P < .15) of patients in the 90 mg/kg, 60 mg/kg, and placebo groups, respectively. In all patients with staphylococcal sepsis, estimated or observed pagibaximab levels were <500 μg/mL (target level) at infection. CONCLUSIONS: Three once-a-week 90 or 60 mg/kg pagibaximab infusions, in high-risk neonates, seemed safe and well tolerated. No staphylococcal sepsis occurred in infants who received 90 mg/kg. Target levels were only consistently achieved after 2 to 3 doses. Dose optimization should enhance protection.

The administration of cobra venom factor reduces post-ischemic cerebral injury in adult and neonatal rats

The role of complement in post-ischemic cerebral injury is incompletely understood. Therefore, experiments were designed to test the effect of complement depletion on cerebral infarct volume in adult rats and cerebral atrophy in neonatal rats. Cerebral infarcts were induced in adult rats by transient filamentous occlusion of the right middle cerebral artery (MCAO). Cerebral atrophy was induced by subjecting 7-day-old rats to ligation of the right common carotid artery followed by 2.5h of hypoxia (8% O2). Forty-eight hours after MCAO, coronal sections of adult brains were obtained and stained with 2,3,5-triphenyl tetrazolium chloride. The infant rat brains were removed for analysis 6 weeks after the hypoxic-ischemic insult. Volumes of infarcts and normal hemispheric parenchyma were quantified by computer-based planimetry. Twenty-four hours prior to MCAO (adults) or hypoxia-ischemia (neonates), each animal received an i.p. injection of either 1 mcg/g body weight cobra venom factor (CVF; adult n=11; neonatal n=20) or normal saline (adult n=12; neonatal n=24). In the neonates, a second dose of CVF or saline was administered 2 days after hypoxia-ischemia. The administration of CVF significantly reduced: (1) post-ischemic cerebral infarct volume in the adults and (2) post-hypoxic-ischemic cerebral atrophy in the neonates. Therefore, complement activation augmented post-ischemic cerebral injury in adult and neonatal rats. Complement depletion induced by CVF significantly reduced post-ischemic cerebral infarct volume and atrophy in adult and neonatal rats.

The administration of complement component C9 enhances the survival of neonatal rats with Escherichia coli sepsis.

To determine the significance of neonatal C9 deficiency, an animal model was developed in the rat. By rocket immunoelectrophoresis, the concentration of C9 in pooled adult rat serum was 224 ± 7.2 μg/mL. In contrast, the concentration of C9 in pooled serum from 1-d-old rats was only 43 ± 3.8 μg/mL and increased during the first 3 wk of life to 170 ± 20μg/mL. Similarly, the capacities of neonatal rat serum to kill two pathogenic strains of Escherichia coli and to lyse sensitized sheep erythrocytes were diminished compared with adult serum but increased during the first 3 wk of life. Supplemental human C9 significantly enhanced the bactericidal and hemolytic activity of neonatal rat serum. The capacity of neonatal rats to survive after the intrapulmonary injection of E. coli was positively correlated with the serum C9 concentration, bactericidal activity, and hemolvtic activity. In 2-d-old rats infected with E. coli, the intraperitoneal administration of human C9 significantly enhanced survival and also enhanced the protective effect of intraperitoneal human IgG antibodies. The data indicate that C9 deficiency predisposed neonatal rats to invasion by E. coli. The neonatal rat appears to be a suitable model with which to investigate the significance of C9 deficiency.

Supplemental Complement Component C9 Enhances the Capacity of Neonatal Serum to Kill Multiple Isolates of Pathogenic Escherichia coli

ABSTRACT: Previous studies demonstrated that, compared with adult serum, neonatal serum contained a diminished concentration of complement component C9 and that supplemental C9 enhanced the capacity of neonatal serum to kill an isolate of Escherichia coli. Therefore, experiments were designed to determine the mechanisms by which supplemental C9 enhances the bactericidal capacity of neonatal serum and to determine whether supplemental C9 enhances the capacity of neonatal serum to kill several different pathogenic strains of E. coli. A radiobinding assay and immunogold electron microscopy using a monoclonal anti-C9 antibody revealed that, compared with 40% adult serum, neonatal serum deposited a diminished quantity of C9 onto E. coli O7w:K1:NM. Supplemental C9 (75 mg/L) significantly enhanced the quantity of C9 deposited by the neonatal serum. Treatment with 10 mM MgEGTA (a mixture of 100 mM MgCl2 and 100 mM EGTA that blocks activation of the classic complement pathway but leaves the alternative pathway intact) abolished the capacity of neonatal serum to deposit C9 and to kill the bacteria. Supplemental C9 enhanced the capacity of neonatal serum to kill eight different blood isolates of E. coli. Therefore, supplemental C9 enhanced the capacity of neonatal serum to kill E. coli by increasing the total quantity of C9 deposited via activation of the classic complement pathway. Neonatal serum contained sufficient quantities of classic pathway components, other than C9, to deposit the supplemental C9 onto E. coli and to enhance bacterial killing. The bactericidal activity of neonatal serum against multiple isolates of pathogenic E. coli was increased after C9 supplementation. We speculate that C9 deficiency may be one of the defects in antibacterial host defense that predisposes neonates to the acquisition of E. coli sepsis.

Complement component 9 activation, consumption, and neuronal deposition in the post-hypoxic-ischemic central nervous system of human newborn infants.

The role of complement in neonatal hypoxic-ischemic brain injury is not known. Therefore, cerebral spinal fluid (CSF) and post-mortem cerebral tissue were analyzed to determine whether complement is activated and complement component 9 (C9) is deposited on neurons in the central nervous systems (CNS) of newborn infants who developed moderate to severe hypoxic-ischemic encephalopathy (HIE). Control CSF samples were obtained during routine evaluation for possible sepsis from infants who were not depressed at birth. In ELISA assays of CSF obtained from 16 infants with HIE, compared to CSF from 7 control infants, the mean concentration of terminal complement complexes was elevated and the mean C9 concentration was diminished. Immunofluorescence microscopy of post-mortem frozen brain tissue obtained from two infants who expired at 4-5 days of life after severe HIE revealed that activated C9 was deposited on cells in all lobes. Double label immunofluorescence microscopy demonstrated that nearly all of the C9-positive cells were neurons and essentially all of the neurons were C9-positive. Immunoperoxidase immunohistochemistry of formalin-fixed tissue also confirmed the presence of many C9-positive cells, particularly in the hippocampus. The C9-positive cells usually manifested morphology consistent with neurons, most of which contained fragmented nuclei. In summary, complement was activated in the CNS of newborn infants who developed moderate to severe HIE. C9 was deposited on neurons, including morphologically apoptotic neurons. Further investigations into a possible role of complement in the pathogenesis of neonatal hypoxic-ischemic cerebral injury are warranted.

Complement inhibition does not reduce post-hypoxic-ischemic cerebral injury in 21-day-old rats

Hypoxic-ischemic (HI) cerebral injury frequently follows resuscitation and is a recognized cause of permanent long-term neurologic disability in children. Complement activation has been shown to participate in post-ischemic injury to a variety of tissues and organs. To test the hypothesis that complement activation participates in post-HI cerebral injury in immature rats, 21-day-old rats were subjected to right common carotid artery ligation and 8% O(2). This combination of ischemia and hypoxia resulted in the development of significant neuronal loss, edema, and atrophy in the right cerebral hemisphere. However, intraperitoneal administration of the complement inhibitors soluble complement receptor type 1 or cobra venom factor did not reduce the neuronal loss, edema, or atrophy. Therefore, complement activation did not contribute significantly to the cerebral injury observed in this immature rat model.

Comparative effects of cytokines and cytokine combinations on complement component C3 secretion by HepG2 cells.

The mechanisms that control complement protein synthesis are incompletely understood. Recent evidence suggests that cytokines are involved in the regulation of hepatic synthesis of circulating complement components. Therefore, we compared the effects of human recombinant IL-1alpha, IL-1beta, IL-6, IFN-gamma, and TNF-alpha individually or in combination, on HepG2 secretion of complement component C3, the major opsonic protein of the complement system. HepG2 cells were incubated with each cytokine alone and with various combinations of the cytokines. At 24, 48, 72, and 96 h of incubation, the C3 and albumin secreted by the HepG2 cells were quantified by a sandwich ELISA. IL-1alpha and IFN-gamma significantly enhanced C3 secretion by the cells (P<0.02 vs. control cells). IL-1beta when combined with either IL-6 or IFN-gamma also increased C3 secretion (P<0.03 vs. control cells). The stimulatory effect on HepG2 cells by the IL-1beta/IL-6 combination was synergistic. With the exception of IL-1alpha, which increased albumin secretion, HepG2 secretion of albumin was not affected by incubation with individual cytokines or the cytokine combinations. Therefore, IL-1alpha, IFN-gamma, and the combination of IL-1beta with IL-6 or IFN-gamma specifically enhanced C3 secretion by HepG2 cells. The greatest magnitude of C3 secretion was induced by the combination of IL-1beta and IL-6.

Effects of endotoxin administration and cerebral hypoxia-ischemia on complement activity and local transcriptional regulation in neonatal rats.

It is not known whether up-regulation of complement components, either circulating or locally synthesized, contributes to an increased susceptibility to neonatal hypoxic-ischemic (HI) cerebral injury. Therefore, we tested the hypothesis that in neonatal rats subjected to a unilateral HI cerebral insult, prior administration of E. coli lipopolysaccharide (LPS) augments (1) complement-mediated serum hemolytic activity, and (2) C3 mRNA and C9 mRNA levels in hepatic and cerebral tissue. Pregnant rats were injected subcutaneously with sterile normal saline (NS) or 500 microg/kg of LPS on gestational days 18 and 19. Following birth, the pups received intraperitoneal injections of NS or 250 microg/kg of LPS on postnatal days 3 and 5. On postnatal day 7, each animal was subjected to ligation of the right common carotid artery followed by 2.5h of hypoxia (8% O(2)). At 3, 6,18, 24 and 48 h after hypoxia, the complement-mediated hemolytic activity of pooled serum was measured. Hepatic and cerebral C3 mRNA and C9 mRNA were quantified by qRT-PCR at 3, 6, and 18 h after HI. Serum hemolytic activity, hepatic C3 mRNA, and hepatic C9 mRNA were up-regulated after cerebral HI. LPS administration potentiated the effect of HI on serum hemolytic activity and increased cerebral C3 mRNA levels. Cerebral C9 mRNA was not detected and was not affected by HI, with or without the prior LPS administration. These observations support the theory that previously reported C9-mediated neurotoxicity following cerebral HI is induced by circulating, rather than locally synthesized C9.

The role of complement in neurodevelopmental impairment following neonatal hypoxic-ischemic encephalopathy.

Evidence has accumulated implicating complement activation in the pathogenesis of acute post-hypoxic-ischemic cerebral injury in infants who develop hypoxic-ischemic encephalopathy (HIE). However, the relationship between complement activation and subsequent neurological impairment is not known. We tested the hypothesis that in human neonates, post-hypoxic-ischemic complement activation within the central nervous system is positively associated with the acquisition of subsequent neurodevelopmental abnormalities. This prospective study included 18 full-term infants diagnosed with HIE following resuscitation at birth and seven control infants. Cerebrospinal fluid (CSF) samples were obtained from all infants in the first 24 hours of life as part of routine investigations to exclude sepsis and meningitis. Concentrations of terminal complement complexes (TCC), complement component 9 (C9), and albumin were quantified by enzyme-linked immunosorbent assay in all CSF samples. Neurological examination and Denver Developmental Screening Test II were performed at 6 and 12 months of life. Of the 18 HIE subjects, nine died, six survived with significant neurological impairment, and three had normal neurological outcomes. In the CSF of the 15 HIE infants who died or survived with abnormal outcomes, the mean concentration of TCC was increased compared with controls (p = 0.026) and the mean C9 concentration appeared to be decreased but the difference was not statistically significant (p = 0.056). Similar to the TCC concentration, the concentration of albumin in the CSF was significantly increased in infants with abnormal outcomes (p = 0.005). This study indicates that complement activation following resuscitation at birth, as manifested by increased TCC in the CNS, is positively correlated with the combination of the development of subsequent neurological sequelae and death. Further study incorporating larger sample sizes will be required to confirm this association. This step is essential before clinical trials of complement inhibitors can be justified in human neonates who suffer birth asphyxia.

Nonspecific Human IgG Reduces Survival in Neonatal Rats Infected with Escherichia coli

Background: Human intravenous IgG (IVIG) containing specific antibodies protects neonatal rats from septic death. However, IVIG has immunosuppressive properties and clinical trials of IVIG in neonates at risk for sepsis have yielded conflicting results. Hypothesis: This study was designed to test the hypothesis that nonspecific antibodies in IVIG reduce survival in neonatal rats infected with Escherichia coli. Methods: Specific antibodies were adsorbed from IVIG with E coli to produce IVIG/anti‐E coli −. After transthoracic administration of E coli, survival was determined in neonatal rats injected intraperitoneally with phosphate‐buffered saline, IVIG/anti‐E coli − (500 mg/kg) or IVIG containing anti‐E coli antibodies (IVIG/anti‐E coli +). Complement‐mediated hemolytic activity of neonatal rat serum was quantified using sensitized sheep erythrocytes. Results: Compared with placebo, intraperitoneal IVIG/anti‐E coli − reduced neonatal survival after E coli infection. In contrast, IVIG/anti‐E coli + protected infected animals. Both IVIG/anti‐E coli − and IVIG/anti‐E coli + impaired the complement‐mediated hemolytic activity of neonatal rat serum. Conclusions: IVIG contained (1) nonspecific antibodies that reduced survival in neonatal rats infected with E coli and (2) protective anti‐E coli antibodies that enhanced survival in neonatal rats infected with E coli. We speculate that in clinical trials of IVIG to treat or prevent neonatal sepsis, inconsistent results may be caused, in part, by lot‐to‐lot variations in the ratio of immunosuppressive, nonspecific antibodies to protective, specific antibodies.