Biomedical subjects

Piet W Modderman

Publications and source records attributed to Piet W Modderman.

3 recordsLinked to original sources

PubMed2006

Variable mannose-binding lectin expression during postoperative acute-phase response.

BACKGROUND: Low plasma concentrations and genetic polymorphisms of mannan-binding lectin (MBL) have been associated with infectious disease complications during various conditions. The present study examined the nature and expression of MBL deficiency during a surgery-induced acute-phase response. METHODS: Blood was sampled from 20 consecutive patients before and 1, 3, 5, 7, and 10 days and 6 weeks after a uniform abdominal operation (transhiatal esophagectomy). Plasma concentrations of MBL, C-reactive protein (CRP), and secretory phospholipase A2 (sPLA2) were measured. Patients were classified as low- or high-level MBL producers by their preoperative concentration (<0.5 or > or = 0.5 micrograms/mL), and were cross-verified for actual MBL deficiency by nucleotide sequencing of both the MBL promoter and exon-1 alleles. RESULTS: Baseline plasma MBL concentrations correlated with maximal postoperative plasma concentrations (r = 0.88; p < 0.0001). This was not found for CRP and sPLA2 (r = 0.19 and r = 0.08, respectively). Alleles responsible for structural MBL variants were detected in 40% of patients and were associated with significantly reduced MBL concentrations (p = 0.005). The baseline cut-off value in plasma of 0.5 micrograms/mL clearly identified individuals with variant exon-1 alleles (sensitivity 100%, specificity 83%). CONCLUSIONS: Baseline MBL plasma concentrations are predictive of MBL expression during the acute-phase response. A baseline cut-off value of 0.5 micrograms/mL can be used to identify patients with variants in the exon-1 region of the MBL gene without the need for nucleotide sequencing. Clinical studies may use this easy and quick method to identify MBL deficient patients preoperatively, as they are conditionally at risk for infectious complications.

Acute-Phase Reaction↗

PubMed2004

Substitution of Pichia pastoris-derived recombinant proteins with mannose containing O- and N-linked glycans decreases specificity of diagnostic tests.

BACKGROUND: Recombinant proteins from Pichia pastoris need to be fully evaluated before used as diagnostic tools. OBJECTIVE: The objective of this study was to investigate whether glycosylation by P. pastoris interferes with the specificity of diagnostic tests. METHODS: An autoantigen involved in Wegener's disease (protease 3) and 2 major inhalant allergens from grass pollen (Dac g 5) and house dust mite (Der p 1) were produced as recombinant molecules in P. pastoris. O-linked glycans on Dac g 5 were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The immune reactivity of the recombinant proteins was compared to that of their natural counterparts by ELISA and a radio-allergosorbent test (RAST) as well as by ELISA and RAST inhibition. RESULTS: In contrast to the non-glycosylated natural allergen, recombinant Dac g 5 was shown to carry at least 2 small mannose-containing O-glycans. We showed that both these O-glycans and the N-linked glycans on recombinant protease 3 and recombinant Der p 1 were recognized in ELISA by IgG antibodies in sera of healthy individuals. These IgG responses were closely correlated. The natural autoantigen and allergens were not recognized by IgG antibodies from healthy subjects. The carbohydrate nature of the epitopes recognized by IgG on the recombinant proteins was confirmed by inhibition studies with mannose and yeast mannan. IgE recognition of yeast glycans was observed in 2 out of 9 positive sera from patients with allergic bronchopulmonary aspergillosis. CONCLUSION: Production of recombinant molecules in yeast (or moulds) can introduce IgG-binding glycans that negatively affect the specificity of diagnostic tests.

Allergens↗

PubMed2003

Recombinant human C1-inhibitor produced in Pichia pastoris has the same inhibitory capacity as plasma C1-inhibitor.

Therapeutic application of the serpin C1-inhibitor (C1-Inh) in inflammatory diseases like sepsis, acute myocardial infarction and vascular leakage syndrome seems promising, but large doses may be required. Therefore, a high-yield recombinant expression system for C1-Inh is very interesting. Earlier attempts to produce high levels of C1-Inh resulted in predominantly inactive C1-Inh. We describe the high yield expression of rhC1-Inh in Pichia pastoris, with 180 mg/l active C1-Inh at maximum. On average, 30 mg/l of 80-100% active C1-Inh was obtained. Progress curves were used to study the interaction with C1s, kallikrein, coagulation factor XIIa and XIa, and demonstrated that rhC1-Inh had the same inhibitory capacity as plasma C1-Inh. Structural integrity, as monitored via heat stability, was comparable despite differences in extent and nature of glycosylation. We conclude that the P. pastoris system is capable of high-level production of functionally and structurally intact human C1 inhibitor.

Cloning, Molecular↗