Human DCs (dendritic cells) express surface CD83 upon activation. Comparing the surface induction of CD83 with the upregulation of CD40, CD80 and CD86 during LPS (lipopolysaccharide)-induced DC maturation showed that CD83 induction occurred more rapidly. Despite the lack of CD83 on immature DCs, it was detected in these cells by Western blotting and flow cytometry. Indirect immunofluorescence revealed CD83 inside immature DCs in perinuclear regions. CD83 was absent on monocytes and macrophages, but it was detected inside these cells and found to be rapidly surface-expressed upon LPS-induced activation. Whereas CD83 expression on activated DCs was sustainable, its expression on monocytes and macrophages was transient. Optimal interleukin-4 co-stimulation during DC generation from monocytes was found to be essential for stable CD83 surface expression. CD83 was detected as 37 and 50 kDa forms in transfected 293T cells. Macrophages and immature DCs expressed the 37 kDa form, whereas mature DCs predominantly expressed the 50 kDa form. In monocytes, CD83 was detected as a 22 kDa detergent-insoluble form. The rapid CD83 surface induction on DCs and macrophages was blocked by brefeldin A, but not by cycloheximide, showing that fresh CD83 synthesis was not essential. Tunicamycin inhibited the expression of the 50 and 37 kDa CD83 forms, and also blocked CD83 surface expression on DCs and macrophages. PNGase F (peptide N-glycosidase F) digestion reduced the 37 and 50 kDa CD83 forms to 28 kDa. In summary, monocytes, macrophages and immature DCs contain preformed intracellular CD83, and its rapid surface expression upon activation is post-translationally regulated in a process involving glycosylation.

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Mannan-binding lectin (MBL), previously called 'mannan-binding protein' or MBP, is a plasma C-type lectin which, upon binding to carbohydrate structures on micro-organisms, activates the classical pathway of complement. Purification of MBL relies on its Ca(2+)-dependent affinity for carbohydrate, but existing methods are susceptible to contamination by anti-carbohydrate antibodies. In the present study a sequential-sugar-elution method has been developed which can achieve a preparation of virtually pure MBL and its associated serine protease (MBL-associated serine protease, MASP) by two steps of affinity chromatography. In further separation of MASP from MBL, it was found that activated MASP was associated with MBL independent of Ca2+. Since MBL was found to bind to underivatized Sepharose 4B, the MBL-MASP complex was purified using Sepharose 4B and protease inhibitors were included to purify the complex with MASP in its proenzyme form. Analysis of thus-purified MBL-MASP complex by gel filtration on a Sephacryl S-300 column at pH 7.8 showed that the proenzyme MASP was also associated with MBL independently of Ca2+, but that the complex could be disrupted at a low pH (5.0). Therefore the mechanism of MBL-MASP-mediated complement activation appears to be significantly different from the C1-mediated classical pathway.

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation betweenPlasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays forP. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive forP. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria.