Yow-Pin Lim

Correlation between mortality and the levels of inter-alpha inhibitors in the plasma of patients with severe sepsis.

Inter-alpha inhibitor protein (IalphaIp) is an endogenous serine protease inhibitor in human plasma. Circulating IalphaIp levels were lower in 51 patients with severe sepsis than in healthy volunteers. Mean levels were 688+/-295 mg/L in patients with severe sepsis who survived (n=32), 486+/-193 mg/L in patients with sepsis who died (n=19), and 872+/-234 mg/L in control subjects (n=25). IalphaIp levels were lower in patients with shock versus those without (540+/-246 [n=33] vs. 746+/-290 [n=18] mg/L; P=.0102). IalphaIp levels were inversely correlated with 28-day mortality rates and Acute Physiology and Chronic Health Evaluation II scores and directly correlated with antithrombin III, protein C, and protein S levels. The administration of IalphaIp (30 mg/kg body weight intravenously) increased the 50% lethal dose in mice by 100-fold after an intravenous challenge of Escherichia coli. Thus, human IalphaIp may be a useful predictive marker and potential therapeutic agent in sepsis.

Use of compact, porous units with immobilized ligands with high molecular masses in affinity chromatography and enzymatic conversion of substrates with high and low molecular masses

Different ligands with high molecular masses are immobilized on compact, porous separation units and used for affinity chromatography. In subsequent experiments different enzymes are immobilized and used for converting substrates with low and high molecular masses. Disk or tube with immobilized concanavalin A (ConA) are used as model systems for lectin affinity chromatography. The enzyme glucose oxidase is used as a standard protein to test the ConA units. Subsequently glycoproteins from plasma membranes of rat liver are separated, using units with immobilized ConA. The enzyme dipeptidyl peptidase i.v., which is used as a model protein in the experiments, is enriched about 40-fold in a single step, with a yield of over 90%. The results are only slightly better than those obtained with ConA when it is immobilized on bulk supports. The important improvement lies in the reduction of separation time to only 1 h. Experiments concerning the isolation of monoclonal antibodies against clotting factor VIII (FVIII) are carried out on disks, combining anion-exchange chromatography and protein A affinity chromatography as a model for multidimensional chromatography. Both IgG (bound to the protein A disk) and accompanying proteins (bound to the anion-exchange disk) from mouse ascites fluid are retarded and eluted separately. With the immobilized enzymes invertase and glucose oxidase (GOX) the corresponding substrates with low molecular masses, saccharose and glucose, are converted. It is shown that the amount of immobilized enzyme and the concentration of the substrate are responsible for the extent of the conversion, whereas the flow-rates used in the experiments have no effect at all. The influence of immobilization chemistry was investigated with GOX. Indirect immobilization with ConA as spacer proved to be the best alternative. With trypsin, immobilized on a disk, substrates with high molecular masses are digested in flow-through. For optimal digestion the proteins have to be denatured in the buffer for sodium dodecyl sulfate-polyacrlyamide gel electrophoresis prior to application. In contrast to the conversion of substrates with low molecular masses, flow-rates play an important part in conversion of substrates with high molecular masses. With lower flow-rates a higher degree of digestion is achieved.

Administration of human inter-α-inhibitors maintains hemodynamic stability and improves survival during sepsis

OBJECTIVES The major forms of human inter-alpha-inhibitor proteins circulating in the plasma are inter-alpha-inhibitor (IalphaI, containing one light peptide chain called bikunin and two heavy chains) and pre-alpha-inhibitor (PalphaI, containing one light and one heavy chain). Although it has been reported that a decrease in IalphaI/PalphaI is correlated with an increased mortality rate in septic patients, it remains unknown whether administration of IalphaI/PalphaI early after the onset of sepsis has any beneficial effects on the cardiovascular response and outcome of the septic animal. The aim of this study, therefore, was to determine whether IalphaI and PalphaI have any salutary effects on the depressed cardiovascular function, liver damage, and mortality rate after polymicrobial sepsis. DESIGN Prospective, controlled, randomized animal study. SETTING A university research laboratory. SUBJECTS Male adult rats were subjected to polymicrobial sepsis by cecal ligation and puncture or sham operation followed by the administration of normal saline (i.e., resuscitation). MEASUREMENTS AND MAIN RESULTS At 1 hr after cecal ligation and puncture, human IalphaI/PalphaI at a dose of 30 mg/kg body weight or vehicle (normal saline, 1 mL/rat) were infused intravenously over a period of 30 mins. At 20 hrs after cecal ligation and puncture (i.e., the late, hypodynamic stage of sepsis), cardiac output was measured by using a dye dilution technique, and blood samples were collected for assessing oxygen content. Oxygen delivery, consumption, and extraction ratio were determined. Plasma concentrations of liver enzymes alanine aminotransferase and aspartate aminotransferase as well as lactate and tumor necrosis factor-alpha also were measured. In additional animals, the necrotic cecum was excised at 20 hrs after cecal ligation and puncture with or without IalphaI/PalphaI treatment, and survival was monitored for 10 days thereafter. The results indicate that administration of human IalphaI/PalphaI early after the onset of sepsis maintained cardiac output and systemic oxygen delivery, whereas it increased oxygen consumption and extraction at 20 hrs after cecal ligation and puncture. The elevated concentrations of alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-alpha, and lactate were attenuated by IalphaI/PalphaI treatment. In addition, administration of human IalphaI/PalphaI improved the survival rate from 30% to 89% in septic animals at day 10 after cecal ligation and puncture and cecal excision. CONCLUSION Human IalphaI/PalphaI appears to be a useful agent for maintaining hemodynamic stability and improving survival during the progression of polymicrobial sepsis.

Longitudinal studies of inter-alpha inhibitor proteins in severely septic patients: a potential clinical marker and mediator of severe sepsis.

Objective:To determine the clinical relevance and prognostic significance of serial measurement of inter-alpha inhibitor proteins (I&agr;Ip) in severely septic patients. Design:A laboratory-based study of serial plasma samples over the first 5 days of severe sepsis from a prospective clinical trial. Setting:Small business and academic medical center research laboratories. Patients:Two hundred sixty-six patients with severe sepsis from a multiple-center phase III clinical trial. Interventions:None. Measurements and Main Results:Inter-alpha inhibitor proteins serve as endogenous serine protease inhibitors in human plasma. The levels of I&agr;Ip were markedly reduced to a mean value of 290 ± 15 &mgr;g/mL at the onset of severe sepsis compared with normal plasma levels (617 ± 197 &mgr;g/mL). Failure of I&agr;Ip levels to recover over the first 5 days of sepsis was associated with an unfavorable outcome (p < .001). I&agr;Ip levels were inversely correlated with interleukin-6 levels at study entry and over the first 5 days of management of severe sepsis. I&agr;Ip levels were significantly lower in women, with increased age, in the presence of multiple organ failure and in patients with intra-abdominal sources of sepsis. Conclusions:Inter-alpha inhibitor proteins are markedly reduced in severe sepsis, and failure of recovery of I&agr;Ip levels over the course of sepsis is associated with an unfavorable outcome.

Inter-α inhibitor proteins in infants and decreased levels in neonatal sepsis

Objective Adjunctive tests are needed to predict sepsis in the newborn and to lower the rate or duration of unnecessary antibiotic use. We evaluated the normal Inter-α inhibitor protein (IaIp) values in infants and the association of plasma levels of IaIp with sepsis in term and preterm newborns. Methods Plasma IaIp levels were measured by enzyme-linked immunosorbent assay in samples from 135 newborn infants at a wide range of gestational ages (24–42 weeks). IaIp levels were also determined in 19 infants undergoing prospective evaluation for sepsis. ResultsIaIp levels in umbilical cord blood and circulating peripheral blood of healthy newborn infants (525±66 mg/L) were not significantly different from the level in healthy adults (691±80 mg/L). IaIp levels were similar in infants between 24 and 42 weeks gestational age. There was a significant reduction in IaIp levels in infants with sepsis compared with nonseptic controls (169±126 mg/L vs 613±286 mg/L, P<.0001). ConclusionsIaIp levels in the blood of newborns are independent of gestational age and similar to adults. IaIp levels are significantly reduced in infants with bacterial sepsis and might serve as an adjunctive diagnostic marker to allow prospective reduction of antibiotic use.

Degradation products of factor VIII which can lead to increased immunogenicity.

The biochemical and immunochemical aspects of the development of inhibitors with a plasma–derived, double–virus inactivated factor VIII (FVIII) concentrate (marketed as Octavi SDPlus in Germany and Bisinact in Belgium) are described. A total of 12 cases of inhibitor formation (predominantly type II) were reported in Germany, 8 in Belgium but none in Portugal. Initially, the only difference between the non–pasteurised, SD virus–inactivated product Octavi and the pasteurised product Octavi SDPlus appeared to be pasteurisation, though subsequently, the quality of source material for the product was found to differ in different countries. Separation studies revealed the presence of a 40 kDa peptide fragment in some batches. It was subsequently shown that there was a strong correlation between inhibitor development and batches containing the 40 kDa marker, and a relationship between elevated markers of coagulation activation (FPA in particular) and the occurrence of the 40 kDa marker. Further work revealed that analytical methods commonly used for quality control were not suitable to highlight batch–to–batch differences. It was concluded that inhibitor potential (neoantigenicity) in Octavi SDPlus arose due to two effects; degradation of FVIII already present in source material; and heating of unstable FVIII degradation products. In this case, inhibitors were not caused by the overall production process, nor by GMP failures. The problem of inhibitor potential can be avoided if appropriate preventive measures are taken. Further work is needed to prove non–neoantigenicity and to reinforce the scientific findings, and to characterise pilot batches.

ALTERED LEVELS AND MOLECULAR FORMS OF GRANZYME K IN PLASMA FROM SEPTIC PATIENTS

Granzyme K (GrK) is a member of a highly conserved group of potent serine proteases specifically found in the secretory granules of cytotoxic T lymphocytes and natural killer cells. Based on the report indicating that inter-alpha inhibitor proteins are the physiological inhibitors of GrK and on previous findings that showed a significant decrease in plasma inter-alpha inhibitor proteins in patients with sepsis, it was our aim to determine whether increased levels of uninhibited GrK would contribute to the development of sepsis. To test this hypothesis, a competitive enzyme-linked immunosorbent assay system was developed; and the levels of GrK were measured in plasma samples obtained from healthy controls and 2 sets of patients with sepsis: patients admitted to the emergency department with a putative diagnosis of sepsis and patients with severe sepsis enrolled in a clinical trial. In addition, the molecular form(s) of GrK present in these samples was analyzed by Western blot. The levels of GrK were significantly increased in emergency department patients compared with healthy controls and significantly decreased in patients with severe sepsis enrolled in a clinical trial compared with healthy controls. GrK was detected as high-molecular-weight protein complexes in healthy controls but as complexes of lower molecular weight in the septic patients. The decrease in complex size correlated with the appearance of a band at 26 kDa similar to the size of free GrK. Our results indicate that plasma levels of GrK could serve as a useful diagnostic marker to stage sepsis, permitting better classification of septic patients and enabling targeting of specific treatments, and may play a functional role in the development of sepsis.

Application of high-performance membrane chromatography for separation of annexins from the plasma membranes of liver and isolation of monospecific polyclonal antibodies

The separation of annexins, calcium-binding plasma membrane-associated proteins from rat liver and Morris hepatoma 7777 by high-performance membrane chromatography (HPMC) is described. The annexins with low molecular masses, CBP 33 and CBP 35, and the annexin with a high molecular mass, CBP 65/67, can be separated within 10 min from one another by anion-exchange HPMC under non-denaturing conditions. The separation devices used consist of compact, porous disks (QuickDisk) on the one hand and of bundled membranes made of cellulose fibers (MemSep) on the other. Both have been found to be equally well suited for this separation. The annexins obtained in this way are subsequently bound to epoxy-activated porons disks and used for the separation of monospecific polyclonal antibodies against the annexin CBP 65/67.

Inter-Alpha Inhibitor Protein Level in Neonates Predicts Necrotizing Enterocolitis

OBJECTIVES To compare inter-alpha inhibitor protein (IaIp) levels in neonates with proven necrotizing enterocolitis (NEC) and neonates with other, nonspecific abdominal disorders. STUDY DESIGN This was a prospective observational study of neonates in the neonatal intensive care unit. NEC was diagnosed according to Bells staging criteria. The nNeonates in the control group had a nonspecific abdominal disorder, but no radiographic evidence of NEC and no disease progression. All neonates with radiographically confirmed NEC were included. Plasma IaIp levels were quantitated by enzyme-linked immunosorbent assay. RESULTS Seventeen neonates had confirmed NEC, and 34 neonates had nonspecific abdominal disorders that improved rapidly. Gestational age, postnatal age, weight, sex, maternal obstetric variables, rupture of membranes, and mode of delivery did not differ between the two groups. Mean IaIp level was significantly lower in the NEC group compared with the control group (137 ± 38 mg/L; 95% confidence interval [CI], 118-157 mg/L vs 258 ± 53 mg/L; 95% CI, 238-277 mg/L; P <.0001). CONCLUSIONS The finding of significantly lower IaIp levels in neonates with NEC suggests that IaIp might be a useful, sensitive biomarker, allowing initiation of appropriate therapy and reducing antibiotic overuse in neonates with suspected but unproven NEC. Administration of IaIp may significantly reduce the severity of systemic inflammation and associated tissue injury.

The Role of Inter-Alpha Inhibitor Proteins in the Diagnosis of Neonatal Sepsis

We evaluated Inter-alpha inhibitor proteins (IaIp) as a diagnostic marker in neonatal sepsis. Samples were collected from 573 neonates who were examined for suspected sepsis. IaIp level was significantly lower in the septic group (121+/-71 mg/L) than in the non-septic group (322+/-91 mg/L). The optimal cutoff value with the receiver operating characteristic curve was <or= 177 mg/L (sensitivity, 89.5%; specificity, 99%; positive predictive value, 95%; negative predictive value, 98%) with area under the curve of 0.94. IaIp is a more reliable diagnostic marker for neonatal sepsis than other available tests.