Interleukin-17E, inducible nitric oxide synthase and arginase1 as new biomarkers in the identification of neutrophilic dermatoses.

BACKGROUND: Neutrophilic dermatoses (ND) are a heterogeneous group of diseases, but can often have a relatively similar histological appearance. AIM: To identify a combination of biomarkers allowing a better differentiation of ND types. METHODS: Biopsies were obtained from normal human skin (NS; n = 4), chronic plaque-type psoriasis (PsO; n = 7), paradoxical psoriasis (PP; n = 8), generalized pustular psoriasis (GPP; n = 9), subcorneal pustular dermatosis of Sneddon-Wilkinson (SPD; n = 3), acute generalized exanthematous pustulosis (AGEP; n = 3), hidradenitis suppurativa (HS; n = 7), Sweet syndrome (SS; n = 8) and pyoderma gangrenosum (PG; n = 8). Samples were analysed by immunofluorescence using three biomarkers, interleukin (IL)-17E, inducible nitric oxide synthase (iNOS) and arginase1, each one in combination with two cell markers, myeloperoxidase (MPO) and CD68, which allow the identification of neutrophils and macrophages, respectively. RESULTS: We found that SS is characterized by high expression of IL-17E and iNOS in the epidermis, while PG exhibits low expression. The density of the neutrophil infiltrate helps to differentiate PP (high-density infiltrate) from PsO (low-density infiltrate). High expression of arginase1 in the granular layer of the epidermis is a hallmark of SPD. Finally, mature neutrophils and proinflammatory macrophages are readily detectable in PP, SPD and PG, whereas immature neutrophils and anti-inflammatory macrophages are more frequent in GPP, AGEP, HS and SS. CONCLUSIONS: The analysis of ND by immunofluorescence using IL-17E, iNOS and arginase1 in combination with MPO and CD68 allows for characterization of differential expression patterns in the epidermis as well as the determination of the polarization status of the dermal neutrophils and macrophages. The appropriate markers may help in the differentiation of ND in clinical practice.

Application of mid-infrared (MIR) microscopy imaging for discrimination between follicular hyperplasia and follicular lymphoma in transgenic mice.

Mid-infrared (MIR) microscopy imaging is a vibrational spectroscopic technique that uses infrared radiation to image molecules of interest in thin tissue sections. A major advantage of this technology is the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue. Therefore, this technology has become an essential tool for the detection and characterization of the molecular components of many biological processes. Using this method, it is possible to investigate the spatial distribution of proteins and small molecules within biological systems by in situ analysis. In this study, we have evaluated the potential of mid-infrared microscopy imaging to study biochemical changes which distinguish between reactive lymphadenopathy and cancer in genetically modified mice with different phenotypes. We were able to demonstrate that MIR microscopy imaging and multivariate image analyses of different mouse genotypes correlated well with the morphological tissue features derived from HE staining. Using principal component analyses, we were also able to distinguish spectral clusters from different phenotype samples, particularly from reactive lymphadenopathy (follicular hyperplasia) and cancer (follicular lymphoma).

Suitability of Fourier transform infrared microscopy for the diagnosis of cystic echinococcosis in human tissue sections.

Cystic echinococcosis (CE) is a global health concern caused by cestodes, posing diagnostic challenges due to nonspecific symptoms and inconclusive radiographic results. Diagnosis relies on histopathological evaluation of affected tissue, demanding comprehensive tools. In this retrospective case study, Fourier transform infrared microscopy was explored for detecting and identifying CE through biochemical changes in human tissue sections. Tissue samples from 11 confirmed CE patients were analyzed. Archived FFPE blocks were cut and stained, and then CE-positive unstained sections were examined using Fourier transform infrared microscopy post-deparaffinization. Results revealed the method's ability to distinguish echinococcus elements from human tissue, irrespective of organ type. This research showcases the potential of mid-infrared microscopy as a valuable diagnostic tool for CE, offering promise in enhancing diagnostic precision in the face of the disease's complexities.

Deep learning analysis of mid-infrared microscopic imaging data for the diagnosis and classification of human lymphomas.

The present study presents an alternative analytical workflow that combines mid-infrared (MIR) microscopic imaging and deep learning to diagnose human lymphoma and differentiate between small and large cell lymphoma. We could show that using a deep learning approach to analyze MIR hyperspectral data obtained from benign and malignant lymph node pathology results in high accuracy for correct classification, learning the distinct region of 3900 to 850 cm-1 . The accuracy is above 95% for every pair of malignant lymphoid tissue and still above 90% for the distinction between benign and malignant lymphoid tissue for binary classification. These results demonstrate that a preliminary diagnosis and subtyping of human lymphoma could be streamlined by applying a deep learning approach to analyze MIR spectroscopic data.

Clinical infrared microscopic imaging: An overview.

New developments in Mid-infrared microscopic imaging instrumentation and data analysis have turned this method into a conventional technique. This imaging method offers a global analysis of samples, with a resolution close to the cellular level enabling the acquisition of local molecular expression profiles. It is possible to get chemo-morphological information about the tissue status, which represents an essential benefit for future analytical interpretation of pathological changes of tissue. In this review, we give an overview of Mid-infrared microscopic imaging and its applications in clinical research.

RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae.

RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.

Post-mortem interval estimation of human skeletal remains by micro-computed tomography, mid-infrared microscopic imaging and energy dispersive X-ray mapping.

In this study different state-of-the-art visualization methods such as micro-computed tomography (micro-CT), mid-infrared (MIR) microscopic imaging and energy dispersive X-ray (EDS) mapping were evaluated to study human skeletal remains for the determination of the post-mortem interval (PMI). PMI specific features were identified and visualized by overlaying molecular imaging data and morphological tissue structures generated by radiological techniques and microscopic images gained from confocal microscopy (Infinite Focus (IFM)). In this way, a more distinct picture concerning processes during the PMI as well as a more realistic approximation of the PMI were achieved. It could be demonstrated that the gained result in combination with multivariate data analysis can be used to predict the Ca/C ratio and bone volume (BV) over total volume (TV) for PMI estimation. Statistical limitation of this study is the small sample size, and future work will be based on more specimens to develop a screening tool for PMI based on the outcome of this multidimensional approach.

Overexpression of tubulin in yeast: differences in subunit association.

Microtubules are cytoskeletal organelles composed principally of polymerized alpha beta-tubulin heterodimers. The specific roles and the detailed structures of the individual alpha- and beta-tubulin subunits have not been established, since the conditions necessary for separating the heterodimer result in loss of the subunits' ability to repolymerize. We have overcome this obstacle by constructing plasmids which allow regulated overexpression of individual tubulin subunits in the yeast Saccharomyces cerevisiae under control of the galactose promoter. Overproduction was monitored with alpha- and beta-tubulin-specific antibodies using immunoblotting of cell extracts, and the state of association of the individual subunits in vivo was determined by immunofluorescence microscopy. Cells overproducing only beta-tubulin accumulated fibrous structures associated with the cell membrane, whereas cells overproducing only alpha-tubulin displayed a diffuse signal throughout the cytoplasm. Cells simultaneously overexpressing alpha- and beta-tubulin subunits accumulated membrane-associated, filamentous arrays in which both subunits were incorporated. When cells with the fibrous tubulin-containing structures are treated with zymolyase, a yeast cell wall disrupting enzyme, the fibers appear to splay apart, suggesting that the immunofluorescent rings represent bundles of fibers. Cells overproducing beta-tubulin alone or both alpha- and beta-tubulin were examined at various times after galactose induction, and significant differences were found in the tubulin association state prior to the formation of fibers. For alpha beta-tubulin, fibers form directly from a nuclear structure, whereas beta-tubulin alone first accumulates in the cytoplasm. The differences in patterns of tubulin accumulation and assembly presumably reflect a difference in the intrinsic association properties of the alpha- and beta-subunits.

Characterization of in vitro constructed IS30-flanked transposons.

In order to facilitate functional studies on the mobile genetic element IS30, a resident of the Escherichia coli chromosome, transposon structures with two copies of IS30 flanking the chloramphenicol-resistance gene cat were constructed in vitro. Transposons containing IS30 as direct repeats (Tn2700 and Tn2702) transpose from multicopy plasmids into the genome of phage P1-15, thus giving rise to special transduction for cat with frequencies between 10(-5) and 10(-8)/plaque-forming unit. In contrast, transposon structures with IS30 in inverted repeat (Tn2701 and Tn2703) showed no detectable (less than 10(-9] transposition activity in vivo. By restriction analysis, two insertion sites of Tn2700 and Tn2702 on the phage P1-15 genome were indistinguishable from those observed earlier with a single copy of the IS30 element. These two insertion sites were used several times independently by Tn2700 and Tn2702. This confirms the non-random target selection by the element and it indicates that transposition of Tn2700 and Tn2702 follows the same rules as that of IS30.

Mutations in the carboxy-terminal part of IS30 transposase affect the formation and dissolution of (IS30)2 dimer.

The transposase of IS30 catalyses different transpositional rearrangements via the dimer (IS30)2 intermediate structure. Mutation analysis provides evidence that the C-terminal part of IS30 transposase is required for the formation and dissolution of (IS30)2 dimer. C-terminal mutants are also defective in transpositional fusion; however, this deficiency can be 'suppressed' by addition of the final product of site-specific dimerisation, the core (IS30)2 intermediate structure. The transposase part studied shows significant homologies in three highly conserved regions to proteins of IS30-related mobile elements.

A carcinogenicity study of instant coffee in Swiss mice.

Commercially available regular instant coffee was given in the diet to barrier-maintained, specified pathogen-free Swiss mice for 2 yr. Groups of 150 males and 150 females were fed diets containing 10, 25 or 50 g instant coffee powder/kg. The animals had already been exposed to coffee in utero. Coffee increased the energy expenditure of the animals as shown by increased daily calorific intake and depressed growth. The overall tumour incidence was inversely correlated to the coffee intake, and no unusual tumour or site of origin was found. The most frequent neoplasms were lymphosarcomas, bronchiolo-alveolar adenomas and adenocarcinomas, as well as hepatocellular adenomas. The incidence of total neoplasms (benign and malignant) decreased from 70.6 and 56.8% in control males and females, respectively, to 34.8 and 36.2%, respectively, in the high-dose group. This decrease, which was essentially due to a reduction in the number of lymphosarcomas and hepatocellular adenomas, was associated with a slower growth rate. The number of leiomyomas in the uterus was slightly increased due to coffee intake as shown by the analysis of positive trend (P less than or equal to 0.05). However, the incidence of this benign tumour was very low; 2.72% of mice affected in the high-dose group, 1.37% in the low-dose group and 0% in the control and medium-dose groups. From this study it is concluded that instant coffee did not increase the incidence of malignant neoplasms in mice when fed at dietary levels of up to 5% for 2 yr.

Haemodynamic effects of bradykinin in rats.

The intravenous infusion of bradykinin (20 micrograms/kg X min) induced an initial fall in blood pressure and total peripheral resistance in anaesthetized rats. These changes were accompanied with an increase in cardiac output and stroke volume. These effects were transient and after 20 min of infusion blood pressure and total peripheral resistance were much less decreased than initially. Cardiac output and stroke volume had completely normalized. Lower doses of bradykinin (1 microgram/kg X min), which did not affect blood pressure, induced a reduction in mesenteric and renal vascular resistance. After higher doses (4 or 20 micrograms/kg X min) mesenteric vascular resistance was further decreased but renal vascular resistance was unchanged or markedly increased. This renal vasoconstrictor response was almost absent in rats which were pretreated with indomethacin (4 mg/kg i.v.). Hence the increase in renal vascular resistance was most probably due to the stimulation of the synthesis of prostaglandins, which act as renal vasoconstrictors in the rat.

The value of serum magnesium estimations in the diagnosis of acute perioperative myocardial infarction after coronary artery surgery.

Serum magnesium levels fall significantly after acute myocardial infarction. Even greater decreases have been found in all patients undergoing aorto-coronary bypass surgery. This is probably related to the use of cardiopulmonary bypass. No significant difference in serum magnesium levels was found between those patients who had a well documented peri-operative infarct and those who had an uncomplicated course. Thus determination of serum magnesium levels if of no use in the diagnosis of peri-operative myocardial infarction.

Evidence against the reported antithiamine effect of caffeic and chlorogenic acids.

Contrary to earlier literature reports, chlorogenic acid (I) is shown to have little or no deleterious effect on thiamine (II) when the 2 compounds are codissolved in aqueous solution. Evidence from nmr spectroscopy and from t. l. c. determination demonstrate that when II is heated at 60 degrees C and at pH 7.8 for 24h, about 10% is destroyed. This result is the same whether I is present or not. Incubating II with I or with caffeic acid (III) in a phosphate buffer at pH 7 or in thiamine assay medium at pH 6 for up to 24h at 37 degrees C did not reduce the growth activity of II or Lactobacillus viridescens. We conclude that I and III are not antithiamine agents.

The influence of food intake on the development of diethylnitrosamine-induced liver tumours in mice.

The aim of this study was to determine whether a dietary restriction, which significantly decreases body weight gain, also influences the development of diethylnitrosamine (DEN)-induced liver tumours in Swiss mice. At birth, mice were injected i.p. with a single dose of DEN (0.4 mumol/g body wt); negative control mice were sham injected. After weaning the animals received a stock diet either ad libitum (control group) or at 30% restriction (restricted group). A positive control group was fed ad libitum and received phenobarbital (500 p.p.m.) in their drinking water. At 12 weeks of age, glucose-6-phosphatase (G6Pase)-deficient foci were present in the liver of 80% of the control animals but only in 32% of the restricted group. Quantitatively, restricted animals had fewer foci per unit volume liver and these were smaller than in the control animals. By 36 weeks of age, hepatocarcinomas were seen in 100% of the control mice while in the restricted group there were no such malignant lesions and only 32% showed adenomas. The results clearly show that restriction of food intake inhibits promotion and progression of induced liver tumours. Amongst other uses, this model permits the study of the effect of dietary restriction on liver tumours, at an early stage.

Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements.

Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements. The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)2 structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA- bacteria, replicated copies of the introduced (IS30)2 structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)2 are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)2, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS30)2 intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)2 may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)2. Evolutionary implications of these findings are discussed.

The N-terminal domain of the insertion sequence 30 transposase interacts specifically with the terminal inverted repeats of the element.

The gene for the insertion sequence (IS) 30 transposase is placed under the control of the tac promoter, and large quantities of transposase are expressed upon induction. The resulting protein precipitates inside the Escherichia coli cells in the form of inclusion bodies which, upon cell lysis, cannot be dissolved under nondenaturing conditions. In contrast, the N-terminal third of the transposase, a 17-kDa protein produced by a truncated gene, can be purified and is able to interact site specifically with the ends of the IS30 element. In DNase I footprint experiments, regions of 26 nucleotides on one DNA strand and 19 nucleotides on the other strand at either end of the element are protected from nuclease digestion. It is concluded that a functional DNA-binding domain can be formed by expression of only one-third of the complete IS30 transposase. Sequence comparison shows a homology of the IS30 ends to the ends of IS4351 and to the L1 end of bacteriophage Mu.