Chemoselective and enantioselective analysis of proteinogenic amino acids utilizing N-derivatization and 1-D enantioselective anion-exchange chromatography in combination with tandem mass spectrometric detection.

D-Amino acid analysis in biological samples still poses a challenge to analytical chemists. In higher developed species trace amounts of d-amino acids have to be detected in vast excesses of the corresponding L-enantiomers. This method utilizes an easy-to-carry-out derivatization step on the amino group with an iron ferrocenyl propionate hydroxy succinimide ester followed by one-dimensional enantioselective anion exchange chromatography with cinchona alkaloid based chiral stationary phases (CSPs). MS detection is carried out in the highly sensitive SRM (selected reaction monitoring) mode, which allows a chemoselective differentiation of amino acid derivatives as well as their enantioselective separation in one step. Application of this method allows LOD (limits of detection) in the low µmol L(-1) range and baseline enantioseparation for all proteinogenic amino acids except for Pro, Arg and His. The D-enantiomers of isomeric Leu and Ile were separated chromatographically and pose an example for the complementary selectivities of LC and MS. A successful application of this procedure to unprocessed human urine indicated the eligibility to analyse biological samples.

Small-molecule inhibitors of NADPH oxidase 4.

NOX enzymes are the major contributors in many oxidative damage related diseases. Unfortunately, at present no specific NOX inhibitor is available. Here, we describe the discovery and development of novel NOX4 inhibitors. Compound libraries were tested in a cell-based assay as a primary screen, monitoring H2O2 production. Twenty-four compounds inhibited Nox4 activity with low-micromolar IC(50) values of which three were selected for further drug development.

IL-1-induced inflammation promotes development of leishmaniasis in susceptible BALB/c mice.

The role of host-derived IL-1 on the course of Leishmania major infection in susceptible BALB/c mice was assessed. Manifestations of the disease were more severe in mice deficient in the physiological inhibitor of IL-1, the IL-1 receptor antagonist (IL-1Ra) in comparison with control mice. In mice lacking one of the IL-1 genes (IL-1alpha or IL-1beta), there was delayed development of the disease and more attenuated systemic inflammatory responses. IL-1alpha-deficient mice were slightly more resistant to L. major infection compared with IL-1beta-knockout mice. During disease progression in IL-1Ra KO and control mice, myeloid-derived suppressor cells invaded the spleen, concomitant to suppression of T cell-mediated immunity and expression of systemic high levels of pro-inflammatory cytokines. In IL-1-deficient mice, T(h)1 responses were still apparent, even at late stages of the disease. Thus, dose-dependent effects of IL-1 were shown to influence the pathogenesis of murine leishamaniasis in susceptible BALB/c mice. Physiological and supra-physiological levels of IL-1 in the microenvironment promoted an exacerbated form of disease, whereas sub-physiological doses of IL-1 induced a less progressive disease. Thus, manipulation of IL-1 levels in the host, using the IL-1Ra or specific antibodies, has the potential to alleviate symptoms of visceral manifestations of leishmaniasis.

The role of macrophage-derived IL-1 in induction and maintenance of angiogenesis.

Inflammation and angiogenesis are pivotal processes in the progression of many diseases, including malignancies. A hypoxic microenvironment often results in a milieu of proinflammatory and proangiogenic cytokines produced by infiltrating cells. We assessed the role of macrophage-derived hypoxia-associated cytokines in promoting inflammation and angiogenesis. Supernatants of macrophages, stimulated under hypoxia with or without an inflammatory stimulus (LPS), promoted angiogenesis when incorporated into Matrigel plugs. However, neutralization of IL-1 in the supernatants, particularly IL-1beta, completely abrogated cell infiltration and angiogenesis in Matrigel plugs and reduced vascular endothelial growth factor (VEGF) levels by 85%. Similarly, supernatants from macrophages of IL-1beta knockout mice did not induce inflammatory or angiogenic responses. The importance of IL-1 signaling in the host was demonstrated by the dramatic reduction of inflammatory and angiogenic responses in Matrigel plugs that contained macrophage supernatants from control mice which had been implanted in IL-1 receptor type I knockout mice. Myeloid cells infiltrating into Matrigel plugs were of bone marrow origin and represented the major source of IL-1 and other cytokines/chemokines in the plugs. Cells of endothelial lineage were the main source of VEGF and were recruited mainly from neighboring tissues, rather than from the bone marrow. Using the aortic ring sprouting assay, it was shown that in this experimental system, IL-1 does not directly activate endothelial cell migration, proliferation and organization into blood vessel-like structures, but rather activates infiltrating cells to produce endothelial cell activating factors, such as VEGF. Thus, targeting IL-1beta has the potential to inhibit angiogenesis in pathological situations and may be of considerable clinical value.

Characterisation of mixed lithium dodecyl sulphate/lithium perfluorooctanesulphonate pseudo-stationary phases in MEKC.

Lipophilicity and methylene selectivity of mixed pseudo-stationary phases (PSPs) (containing lithium dodecyl sulphate (LDS) and lithium perfluorooctanesulphonate (LiPFOS) in different molar ratios) applied in MEKC have been investigated. Micellar proportion (t(prop,mic), a quantity expressing that how much time is spent by the analyte in the micellar phase related to its whole migration time), CLOGP(50) value (showing the value of hydrophobicity of a molecule spending exactly 50% of its migration time in the PSP) and methylene selectivity have been determined for different LDS/LiPFOS mixed phases. Values of the above-mentioned parameters have been determined for analytes with different chemical structures (alkylbenzene and alkylphenone homologous series, alcohols). Good linear correlation was obtained between either the micellar proportion, CLOGP(50), or methylene selectivity and the phase composition for the mixed phases. Lipophilicity and methylene selectivity of the mixed LDS/LiPFOS PSPs can be calculated and can continuously be changed by mixing the two single phases (LDS and LiPFOS) in the appropriate (and calculable) portion.

Expression of p53 in the evolution of squamous cell carcinoma: correlation with the histology of the lesion.

BACKGROUND: The evolution of squamous cell carcinoma (SCC) on sun-exposed areas is a multistep process triggered by ultraviolet radiation (UVR), in which precursor lesions exist. However, the exact classification of the various lesions in this process, mainly solar keratosis (SK), is still disputed, and its pathogenesis requires further clarification. OBJECTIVE: To further elucidate the evolution of SCC on sun-damaged skin by correlating the levels of p53 protein expression, a parameter that reflects UVR damage to cells, and the morphology of the lesions that develop on sun-exposed areas. METHODS: Biopsy specimens from normal skin (n = 4), normal skin with various degrees of solar elastosis (SE) (n = 16), various degrees of SK (n = 17) and SCCs from sun-exposed (n = 12) and sun-protected (n = 7) areas were stained with anti-p53 antibodies. A semiquantitative evaluation of the degree of staining was performed and correlated with the histological features. RESULTS: Nuclear staining in keratinocytes was observed already in normal skin with mild SE and was increased gradually to its highest level of expression in advanced SK. It was also expressed in SCCs, but to a lesser degree. Statistical analysis revealed association between the morphology of the lesion and the level of p53 expression (P < .01); it also showed that in general the level of p53 is correlated with the histology of the lesion (P < .001). Furthermore, with regard to p53 expression, two groups of lesions exist: one showing a low level of expression of p53 that includes normal skin, skin with various degrees of SE and SCC from sun-protected areas, and a second group showing a high level of expression that includes SK and SCC occurring on sun-damaged skin. LIMITATION: This is an immunohistochemical study of relatively few cases and in which the antibody detects all types of p53 protein. CONCLUSIONS: This study furnishes further evidence that the development of SCC on sun-damaged skin is a gradual process not only morphologically but also on the molecular level. The process starts already in normal-appearing epidermis with SE. In that respect, SK should be regarded as a part of the continuum in the development of SCC, analogous to the situation in other epithelia. The molecular events involved in the development of SCC on sun-exposed areas may be different from those involving the development of SCC on sun-protected areas.

Interleukin-1beta-driven inflammation promotes the development and invasiveness of chemical carcinogen-induced tumors.

The role of microenvironment interleukin 1 (IL-1) on 3-methylcholanthrene (3-MCA)-induced carcinogenesis was assessed in IL-1-deficient mice, i.e., IL-1beta(-/-), IL-1alpha(-/-), IL-1alpha/beta(-/-) (double knockout), and mice deficient in the naturally occurring inhibitor of IL-1, the IL-1 receptor antagonist (IL-1Ra). Tumors developed in all wild-type (WT) mice, whereas in IL-1beta-deficient mice, tumors developed slower and only in some of the mice. In IL-1Ra-deficient mice, tumor development was the most rapid. Tumor incidence was similar in WT and IL-1alpha-deficient mice. Histologic analyses revealed fibrotic structures forming a capsule surrounding droplets of the carcinogen in olive oil, resembling foreign body-like granulomas, which appeared 10 days after injection of 3-MCA and persisted until the development of local tumors. A sparse leukocyte infiltrate was found at the site of carcinogen injection in IL-1beta-deficient mice, whereas in IL-1Ra-deficient mice, a dense neutrophilic infiltrate was observed. Treatment of IL-1Ra-deficient mice with recombinant IL-1Ra but not with an inhibitor of tumor necrosis factor abrogated the early leukocytic infiltrate. The late leukocyte infiltrate (day 70), which was dominated by macrophages, was also apparent in WT and IL-1alpha-deficient mice, but was nearly absent in IL-1beta-deficient mice. Fibrosarcoma cell lines, established from 3-MCA-induced tumors from IL-1Ra-deficient mice, were more aggressive and metastatic than lines from WT mice; cell lines from IL-1-deficient mice were the least invasive. These observations show the crucial role of microenvironment-derived IL-1beta, rather than IL-1alpha, in chemical carcinogenesis and in determining the invasive potential of malignant cells.

Pseudorheumatoid nodules in adults: a juxta-articular form of nodular granuloma annulare.

Pseudorheumatoid nodules are considered a deep form of granuloma annulare. Most cases are described in children, occur mainly on the lower legs and scalp, and have favorable prognosis. Their appearance in adults is rare. In this series, fourteen women with pseudorheumatoid nodules were studied. The average age of onset was 36 years old. Lesions consisted of erythematous, violaceous, or skin-colored nodules located mainly on the small joints of the hands. None of the patients developed collagen vascular disease. Persistence was common. Biopsy specimens showed deep dermal nodules composed of epithelioid granulomata separated by thickened collagen bundles. In some areas eosinophilic material was surrounded by histiocytes in a palisaded array. Granuloma annulare was present at the periphery of eight cases. Special stains revealed that most of the eosinophilic material was collagen and mucin was present in eleven cases. In sum these findings demonstrate that pseudorheumatoid nodules in adults are a distinct clinical and pathologic entity, which may be mistaken for rheumatoid nodules. They are probably a juxta-articular variant of granuloma annulare.

Expression of e-cadherin and beta-catenin in cutaneous squamous cell carcinoma and its precursors.

The E-cadherin-beta-catenin complex regulates the architectural integrity of epithelia by mediating intercellular adhesion. Down-regulation of its expression may contribute to invasion and metastatic behavior of carcinoma cells. Several studies demonstrated an abnormal expression of E-cadherin, beta-catenin, or both in various carcinomas, including non-melanoma skin cancer. The aim of the present study was to investigate the involvement of E-cadherin-catenin adhesion system in the progression of human cutaneous squamous cell carcinoma (SCC). For that purpose, sections from normal skin, skin showing solar elastosis (SE), solar keratosis (SK), and SCC were stained with monoclonal antibodies against E-cadherin and beta-catenin. Evaluation of the staining results was performed using a semi-quantitative method in which pattern and intensity of staining, percentage of positive cells, and cytoplasmic staining were evaluated. Normal skin and skin showing mild and moderate solar elastosis strongly expressed membranous E-cadherin and beta-catenin. E-cadherin expression was progressively reduced in the epidermis of skin with severe solar elastosis through solar keratosis to SCC. The same phenomenon was observed for beta-catenin starting from solar keratosis. In some cases of SCC, additional cytoplasmic staining was observed. We found no correlation between E-cadherin and beta-catenin expression and tumor differentiation or between SCC from sun-exposed and sun-protected skin. Statistical analysis revealed correlation between expression of both E-cadherin and beta-catenin and the morphology of the lesion. These results support a gradual evolution from severely sun-damaged skin to SCC, not only on a morphologic level, but also at the molecular level.

The significance of routine duodenal biopsies in pediatric patients undergoing upper intestinal endoscopy.

GOALS: To determine the significance of performing routine duodenal biopsies during upper intestinal endoscopy in a pediatric population and to evaluate their contribution to the overall diagnosis. BACKGROUND: Performing duodenal biopsy during every upper endoscopy regardless of the indication for endoscopy and the macroscopic findings, is a controversial topic. Advocates of performing routine biopsies argue that unexpected pathology such as villous atrophy, may have significant clinical implications. Opponents argue that the yield of performing a biopsy on an apparently normal mucosa is low. STUDY: Duodenal biopsies, routinely taken from 201 pediatric patients during upper endoscopy over a 26-month period were retrospectively reviewed. Duodenal biopsies taken during this period for suspected mucosal lesions were not included in the analysis. Indications for endoscopy included suspected peptic disease, gastroesophageal reflux, unexplained vomiting, abdominal pain, iron deficiency anemia and Crohn disease. RESULTS: Of the 201 sets of biopsies reviewed, 159 (79.1%) were normal, 7 had insufficient material for evaluation and 35 (17.4%) carried abnormalities that included: 10 Giardia lamblia (4.9%), 13 mild chronic inflammation (6.5%), and 8 increased intraepithelial lymphocytes (3.9%). Two biopsies showed mixed acute and chronic inflammation, 1 showed lymphatic dilatation and 1 had a mild mucosal lesion. The risk for microscopic pathology in the duodenum was higher when Helicobacter pylori was present in the gastric biopsy (25.98% vs. 12.16% P < 0.02). The negative predictive value of a normal appearing duodenal mucosa was 81.5%, implying that a normal appearing mucosa does not rule out pathology. No complications were encountered in our series. CONCLUSION: We suggest that the inclusion of routine duodenal biopsies as part of upper endoscopy in pediatric patients should be considered favorably. This practice may yield additional pathologic findings that otherwise could have been missed. It should be done regardless of the indication for endoscopy or the gross appearance of the mucosa. This practice does not increase the risk of the procedure.

Recurrent polymorphous sweat gland carcinoma of the skin.

Polymorphous sweat gland carcinoma is an unusual, recently described variant of low-grade malignant adnexal neoplasm of the skin characterized by a prolonged clinical course and predilection for the extremities. We describe a case of recurrent polymorphous sweat gland carcinoma in a 56-year-old man who presented with multiple large skin nodules distributed along the flexor surface of his left arm. The lesions were treated by surgical excision; multiple local recurrences, as well as the development of new lesions, were observed over a period of 5 years. No distant metastases have been observed so far. The clinical differential diagnosis and management of these unusual lesions are discussed.