Immune cell subsets in necrotizing fasciitis: an immunohistochemical analysis.

Current concepts of the pathophysiology of necrotizing fasciitis (NF), a life-threatening infection of soft tissues associated with a toxic shock syndrome, emphasizes the role of bacterial superantigens as mediators of cytokine release by immune lymphocytes. In order to assess the cellular basis of immune activation, immunohistochemistry was applied to the analysis of inflammatory cell subsets in situ in 13 patients with NF. The percentage of inflammatory cells in skin and soft tissue was scored from 0 to 3+ (>50%). Substantial numbers of CD15+ polymorphonuclear leukocytes were present in 12 of 13 patients. CD3+ T-lymphocytes accounted for >10%, CD68+ macrophages for >50%, and Factor XIIIa+ mononuclear cells for >10% of the mononuclear cell infiltrates, respectively, in 10 of 13 patients, whereas CD1a+ cells were present in only 3 of 13 cases and accounted for <10% of mononuclear inflammatory cells. We conclude that immune lymphocytes and accessory immune cells are represented in substantial numbers in the early lesions of NF, and their presence supports current concepts with respect to the pathophysiology of this disorder.

An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry.

An algorithm for bacterial identification using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is being developed. This mass spectral fingerprint comparison algorithm is fully automated and statistically based, providing objective analysis of samples to be identified. Based on extraction of reference fingerprint ions from test spectra, this approach should lend itself well to real-world applications where samples are likely to be impure. This algorithm is illustrated using a blind study. In the study, MALDI-MS fingerprints for Bacillus atrophaeus ATCC 49337, Bacillus cereus ATCC 14579T, Escherichia coli ATCC 33694, Pantoea agglomerans ATCC 33243, and Pseudomonas putida F1 are collected and form a reference library. The identification of test samples containing one or more reference bacteria, potentially mixed with one species not in the library (Shewanella alga BrY), is performed by comparison to the reference library with a calculated degree of association. Out of 60 samples, no false positives are present, and the correct identification rate is 75%. Missed identifications are largely due to a weak B. cereus signal in the bacterial mixtures. Potential modifications to the algorithm are presented and result in a higher than 90% correct identification rate for the blind study data, suggesting that this approach has the potential for reliable and accurate automated data analysis of MALDI-MS.

Use of an internal control for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of bacteria.

A method to aid in the analysis of bacterial samples of unknown concentration by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is demonstrated. It is shown that in MALDI analysis of bacteria, the intensities of resulting peaks in spectra are sensitive to the microbial concentration. At the high and low ends of the concentration range, no signal can be obtained, leaving very concentrated or very dilute samples indistinguishable. The addition of cytochrome c as an internal control allows the differentiation of these concentrated and dilute samples. The presence of the internal control causes only a 20% to 30% decrease in signal intensity when the bacterial concentration is optimum. However, the signal quality is improved when the internal control is added to some low concentrations of bacteria.

Reproducibility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for replicate bacterial culture analysis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to demonstrate the reproducibility of bacterial spectra collected on different days. The reproducibility of analysis by MALDI-MS of intact Escherichia coli and Bacillus atrophaeus is presented as a replicate culture study in which spectra were collected on ten different occasions over a three-month period and by two different operators. The analysis resulted in the detection of specific biomarkers in the m/z 2000-20 000 range. Some of the peaks in the Escherichia coli spectra are identified by comparison with other published work. All of the spectra obtained are reproducible over the course of the experiment, but operator variability does exist. The Escherichia coli spectra show operator variability while the Bacillus atrophaeus spectra do not. This work demonstrates the utility of MALDI in obtaining consistent spectra from bacteria over a period of time.

Extracting and visualizing matrix-assisted laser desorption/ionization time-of-flight mass spectral fingerprints.

We have developed a method for constructing and extracting matrix-assisted laser desorption/ionization (MALDI) fingerprints. This method is fully automated and statistically based, allowing a large number of spectra to be analyzed at a time in an objective manner. This method can be used to extract the fingerprint of a particular analyte from a spectrum containing multiple analytes. Therefore, this method lends itself well to real-world applications where samples to be analyzed are likely to be impure. We illustrate this method on experimental results from a series of studies of E. coli and B. atrophaeus MALDI time-of-flight mass spectrometry (TOFMS) fingerprints.

Metaxin 1 interacts with metaxin 2, a novel related protein associated with the mammalian mitochondrial outer membrane.

A recently described protein, metaxin 1, serves as a component of a preprotein import complex in the outer membrane of the mammalian mitochondrion. A yeast two-hybrid screen with metaxin 1 as bait has now identified a novel protein, which we have termed metaxin 2, as a metaxin 1-binding protein. Metaxin 2 shares 29% identity with metaxin 1 at the amino acid level, but metaxin 2, unlike metaxin 1, lacks a C-terminal mitochondrial outer membrane signal-anchor domain. Two C. elegans hypothetical proteins, CelZC97.1 and CelF39B2.i, share high sequence similarity with metaxin 2 and metaxin 1, respectively, and likely represent the C. elegans orthologs. Affinity-purified antibodies against metaxin 2 were prepared against the recombinant protein produced in E. coli and were used to analyze the subcellular distribution of metaxin 2. In subcellular fractions of mouse liver, a 29 kD immunoreactive protein, consistent in size with the predicted translation product of metaxin 2 cDNA, was found solely in mitochondria. Alkali extraction of mitochondria indicated that metaxin 2 is peripherally associated with mitochondrial membranes. Metaxin 2 in intact mitochondria was susceptible to digestion with proteinase K, indicating that metaxin 2 is located on the cytosolic face of the mitochondrial outer membrane. Finally, baculoviruses encoding a His6-tagged metaxin 2 and an untagged metaxin 1 lacking its C-terminal transmembrane domain were produced and used separately or in combination to infect Sf21 insect cells. Metaxin 1 bound to a Ni2+-chelate affinity column only in the presence of metaxin 2, indicating that metaxin 1 and metaxin 2 interact when overexpressed in insect cells. These results suggest that metaxin 2 is bound to the cytosolic face of the mitochondrial outer membrane by means of its interaction with membrane-bound metaxin 1, and that this complex may play a role in protein import into mammalian mitochondria.