Human leukocyte antigen class II quantification by targeted mass spectrometry in dendritic-like cell lines and monocyte-derived dendritic cells.

The major histocompatibility complex II (HLA-II) facilitates the presentation of antigen-derived peptides to CD4+ T-cells. Antigen presentation is not only affected by peptide processing and intracellular trafficking, but also by mechanisms that govern HLA-II abundance such as gene expression, biosynthesis and degradation. Herein we describe a mass spectrometry (MS) based HLA-II-protein quantification method, applied to dendritic-like cells (KG-1 and MUTZ-3) and human monocyte-derived dendritic cells (DCs). This method monitors the proteotypic peptides VEHWGLDKPLLK, VEHWGLDQPLLK and VEHWGLDEPLLK, mapping to the α-chains HLA-DQA1, -DPA1 and -DRA1/DQA2, respectively. Total HLA-II was detected at 176 and 248 fmol per million unstimulated KG-1 and MUTZ-3 cells, respectively. In contrast, TNF- and LPS-induced MUTZ-3 cells showed a 50- and 200-fold increase, respectively, of total α-chain as measured by MS. HLA-II protein levels in unstimulated DCs varied significantly between donors ranging from ~ 4 to ~ 50 pmol per million DCs. Cell surface HLA-DR levels detected by flow cytometry increased 2- to 3-fold after DC activation with lipopolysaccharide (LPS), in contrast to a decrease or no change in total HLA α-chain as determined by MS. HLA-DRA1 was detected as the predominant variant, representing > 90% of total α-chain, followed by DPA1 and DQA1 at 3-7% and ≤ 1%, respectively.

Evaluation of a PCR-immunoassay technique for detection of Neisseria meningitidis in cerebrospinal fluid and peripheral blood.

A multiplex PCR-immunoassay for the rapid diagnosis of bacterial meningitis from cerebrospinal fluid (CSF) or peripheral blood was compared with conventional microbiological techniques used in the routine diagnostic laboratory. The multiplex PCR was designed to detect simultaneously a universal conserved sequence coding for bacterial 16S rRNA and the Neisseria meningitidis porA gene. The PCR-immunoassay had a detection limit of (3-5) x 10(2) cfu/ml (equivalent to 1-3 cfu/PCR) with spiked CSF or blood samples, compared with (3-5) x 10(3) cfu/ml for PCR followed by conventional agarose gel electrophoresis for detection of PCR products. Of 294 CSF samples from patients suspected on clinical grounds of suffering from meningitis, the PCR-immunoassay detected bacterial DNA in 29 CSF samples, 15 of which were also positive for N. meningitidis DNA. The 29 positive CSF samples comprised 25 samples that were also reported positive and four that were reported negative by conventional methodology; the latter four were all positive for N. meningitidis by the PCR-immunoassay. Of 173 peripheral blood samples examined, the PCR-immunoassay detected bacterial DNA in 18 samples, 14 of which were also positive for N. meningitidis DNA. In comparison, only 10 samples were reported positive for N. meningitidis by conventional methodology. All negative PCR-immunoassay results correlated with those obtained by conventional methodology for both CSF and blood samples. The sensitivity and speed of the PCR-immunoassay system indicated that it could be used as a routine diagnostic test for meningococcal meningitis, enabling a diagnosis to be made within 4 h of receipt of the specimen.

Use of an automated DNA analysis system (DARAS) for sequence-specific recognition of Neisseria meningitidis DNA.

OBJECTIVES: To combine use of the polymerase chain reaction (PCR) for rapid diagnosis of meningococcal meningitis with a novel automated detection system for sequence-specific recognition of PCR products. METHODS: DNA was extracted from cerebrospinal fluid (CSF) by a quick boil-lysis method, followed by PCR with primers specific for Neisseria meningitidis. Sequence-specific recognition of N. meningitidis DNA was performed with an automated DNA analysis system (DARAS) and the data were compared with results following agarose gel electrophoresis or conventional microbiological culture. RESULTS: The DARAS system had a sensitive detection limit of 102 meningococci/mL with spiked samples, compared with a detection limit of 104 meningococci/mL following agarose gel electrophoresis. When the system was used to examine 74 CSF samples, the 19 CSF samples positive for N. meningitidis by conventional microbiological methods were also all positive in the DARAS system and the 55 samples negative by DARAS for meningococci were also negative by conventional microbiological methods. CONCLUSION: The sensitivity and specificity of the DARAS system makes it a useful tool for the diagnosis of meningococcal meningitis. The system is user-friendly, requires minimal hands-on time and generates data in an informative numerical format.

Molecular epidemiology of aminoglycoside resistance in Acinetobacter spp.

Most aminoglycoside resistance in Acinetobacter spp. involves production of aminoglycoside-modifying enzymes. Previous studies have shown that the genes encoding these enzymes can be present on plasmids, transposons or within integron-type structures. To determine whether particular mechanisms of aminoglycoside resistance have developed in strains from specific geographical locations (with subsequent clonal spread), or whether common mechanisms have been acquired by genotypically distinct clinical isolates of Acinetobacter spp. throughout the world, a genotypically heterogeneous collection of 24 multiresistant clinical isolates of Acinetobacter spp. from 15 hospitals in 11 countries worldwide was studied. All were resistant to two or more aminoglycoside antibiotics. The full aminoglycoside resistance profile was determined for each isolate, allowing a putative enzyme content to be inferred, with subsequent confirmation of enzyme content and genetic location by polymerase chain reaction (PCR) and hybridisation techniques. All produced at least one aminoglycoside-modifying enzyme, most commonly AAC(3)-I and ANT(3'')-I in various combinations. Other enzymes found were AAC(3)-II, AAC(6')-I, ANT(2''), APH(3')-I and APH(3')-VI. None was confined to strains from a particular geographical area. Nine isolates transferred resistance mediated by AAC(3)-I, ANT(2'')-I, APH(3')-I or APH(3)'-VI by conjugation to a sensitive strain of A. baumannii, but most resistance was non-transferable. PCR mapping revealed an integron location in six isolates for the aac(3)-Ia gene and in three isolates for the ant(3'')-Ia gene. Overall, the study demonstrated that similar aminoglycoside-modifying enzymes are found in unrelated isolates of Acinetobacter spp., and that particular genes are not restricted to specific areas of the world. The demonstration of certain genes on plasmids and integrons emphasises the probable importance of these structures in the dissemination of certain types of aminoglycoside resistance in Acinetobacter spp.

Direct comparison of two commercially available computer programs for analysing DNA fingerprinting gels.

Randomly amplified polymorphic DNA (RAPD) fingerprints were generated with M13 and DAF4 primers for 25 isolates of Acinetobacter spp. obtained from 16 different hospitals situated in 12 countries. The overall robustness of the algorithms and the reproducibility of the cluster analysis results generated by two commercially available computer programs (GelCompar and DENDRON) for analysing DNA fingerprinting gels were tested by examining the same set of fingerprinting data independently in two laboratories with the different software packages. Both programs were efficient at recognising and grouping strains with closely similar RAPD fingerprints, i.e., strains which might be expected to have a close epidemiological or evolutionary relationship. However, the relationships suggested for less closely related strains showed considerable variation in terms of the overall similarity or percentage correlation values suggested by the programs. It was concluded that both programs were useful tools for indicating close genotypic relationships between individual strains, but that epidemiological conclusions based on the similarity or correlation values (or the dendrograms derived from them) obtained for less closely related strains should be treated with considerable caution.

Cytokine rescue from glucocorticoid induced apoptosis in T cells is mediated through inhibition of IkappaBalpha.

We previously reported that dexamethasone (DEX), a synthetic glucocorticoid, causes apoptosis in mature Th cell lines, and that this induction of cell death is prevented by specific cytokines, namely, by IL-2 in Th1 cells and by IL-4 in Th2 cells. We now show that this differential rescue by specific cytokines in Th cells correlates with the level of IkappaBalpha that is regulated by DEX and cytokines. In both cell types the cellular levels of IkappaBalpha mRNA and protein were evaluated by DEX treatment. Interestingly, the DEX-mediated IkappaBalpha induction was completely inhibited by IL-2, but not IL-4, in Th1 cells, while the reverse profile was seen in Th2 cells. In both cell types, the cytokine that inhibits the induction of IkappaBalpha by DEX, also rescues these cells from DEX-induced apoptosis, although the rescue cytokine is different in Th1 and Th2 cells. Our results imply that T cells need to maintain a certain level of NF-kappaB transcriptional activity in order to survive; up- or down-regulation of nuclear NF kappaB through modulation of IkappaBalpha expression by cytokines or DEX may lead to cell survival or cell death, respectively.