Stereoselectivity in β-cyclodextrin complexation of 1,4-dihydropyridine derivatives

Structure–interaction relationships, stereoselectivity, and solubility enhancement in inclusion compexation of β-cyclodextrins (CDs) with some racemic and enantiomerically pure 1,4-dihydropyridine derivatives (DHPs) were investigated. 1:1 and 1:2 (mole ratio) complexes were prepared and characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), MS-FAB spectrometry, ¹H-NMR spectroscopy, water and phase solubility. The solubility studies have revealed different complexation equilibria for optically pure DHP enantiomers, and corresponding racemic mixtures in water solutions. By means of ¹H-NMR chemical shift measurements, the inclusion of aromatic fragments of racemic and enantiomerically pure DHP molecules within the cavities of different CDs was elucidated. Considerable stereoselectivity in complexation interactions was observed. The results indicate the potential use of cyclodextrins as chiral selectors for enantiomeric resolution of 1,4-DHP calcium antagonists. © 1993 Wiley-Liss, Inc.     

Distinctive probiotic features share common TLR2‐dependent signalling in intestinal epithelial cells

The underlying mechanisms of probiotics and postbiotics are not well understood, but it is known that both affect the adaptive and innate immune responses. In addition, there is a growing concept that some probiotic strains have common core mechanisms that provide certain health benefits. Here, we aimed to elucidate the signalization of the probiotic bacterial strains Lactobacillus paragasseri K7, Limosilactobacillus fermentum L930BB, Bifidobacterium animalis subsp. animalis IM386 and Lactiplantibacillus plantarum WCFS1. We showed in in vitro experiments that the tested probiotics exhibit common TLR2‐ and TLR10‐dependent downstream signalling cascades involving inhibition of NF‐κB signal transduction. Under inflammatory conditions, the probiotics activated phosphatidylinositol 3‐kinase (PI3K)/Akt anti‐apoptotic pathways and protein kinase C (PKC)‐dependent pathways, which led to regulation of the actin cytoskeleton and tight junctions. These pathways contribute to the regeneration of the intestinal epithelium and modulation of the mucosal immune system, which, together with the inhibition of canonical TLR signalling, promote general immune tolerance. With this study we identified shared probiotic mechanisms and were the first to pinpoint the role of anti‐inflammatory probiotic signalling through TLR10.     

Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates

?. P. Marin?ek, N. Nolde, I. Kardum‐Skelin, R. Nizzoli, B. Önal, T. Rezanko, E. Tani, K. T. Ostovi?, P. Vielh, F. Schmitt and G. Kocjan
Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates Objectives: To collect data on the variability of immunocytochemical (ICC) procedures used to detect oestrogen/progesterone receptors (ER/PR) on cytological material; to test the reproducibility of results; and to identify the crucial points in the ICC procedures that affect the result. Methods: Ten laboratories from eight countries participated in a two‐part study. In the first part, one of the participants (the coordinator) prepared and distributed cytospins from a fine needle aspirate of a primary breast carcinoma. Laboratories performed ICC staining for ER/PR according to their own methods on the test slides and in‐house positive controls. Slides were returned to the coordinator together with information on the preparation of positive control slides and the ICC methodology used. In the second part, obligatory methods of fixation and antigen retrieval were specified. Evaluation of results included grading the number of positive cells, staining intensity, background staining, cytoplasmic staining, sample condition and cellularity. Participants evaluated their own results, which were subsequently evaluated by the coordinator. Results: There was great variability in the preparation of slides for in‐house controls and ICC methodology. The outcome of ICC staining of in‐house control slides was excellent in two laboratories, adequate in three, sub‐optimal in four and inadequate in one. Only six obtained a positive reaction on the test slides and not all were of a high quality. Results of the second run were greatly improved in terms of cellularity of in‐house positive control slides, and scores for the percentage of stained cells and staining intensity of control and test slides. Cytospins and monolayer (ThinPrep^®) preparations were superior to direct smears; methods of fixation and antigen retrieval were the key points in the staining process. Conclusions: Our experience points to the need for guidelines for hormonal receptor determination and external quality control on cytological material, in order for cytological methods to be used in routine clinical practice with a suitable degree of confidence.     

Prevalence of the Helicobacter pylori babA2 gene and correlation with the degree of gastritis in infected Slovenian children

The aims of our study were to determine the prevalence of the babA2 gene within Helicobacter pylori strains circulating in the Slovenian pediatric population, to further clarify its significance in causing inflammation of gastric mucosa in children and to verify whether cagA, vacA, iceA and babA genes work independently or synergistically in causing gastritis. A total of 163 H. pylori isolates obtained from the same number of children were tested for the presence of cagA, vacA and iceA genes using previously established methods, while the babA2 gene was determined using novel polymerase chain reaction assay targeting a 139-bp fragment of the central region of babA2. The babA2 gene was detected in 47.9 % of H. pylori samples. The presence of the babA2 gene was strongly associated with cagA, vacA s1 and vacA m1 genotype. The babA2 status correlated positively with bacterial density score, activity of inflammation and chronic inflammation of gastric mucosa. No significant correlation was found between the babA2 status and the presence of atrophy or intestinal metaplasia. In addition, the activity of gastric inflammation and density score were significantly associated with the coexpression of the cagA, vacA s1, vacA m1 and babA2 genes. The study, which included the largest number of pediatric H. pylori samples to date, confirmed that babA2 gene plays an important role in the pathogenesis of H. pylori gastritis in children. Furthermore, our results suggest that babA2, cagA and vacA s1 and m1 gene products may work synergistically in worsening the inflammation of gastric mucosa.     

C-myc expression in the microvessels of medulloblastoma

The increased expression of c-myc is related to neoplastic transformation and angiogenesis. Therefore, the assessment of expression of c-myc in endothelial cells and neovascularization could help to determine the biological behavior of the tumor. We analyzed neovascularization and c-myc expression in 36 medulloblastoma specimens. The results were shown by determining immunohistochemical staining index (ISI), the sum of staining intensity (SI) and the percentage of positive cells (PPC) in the blood vessels endothelium of the tumor. We also performed the microvessel count (MVC) in 10 high-power fields (400X) with the most prominent vascularization and expressed it as microvessel density per mm2 (MVD). C-myc immunostaining intensity index in blood vessel endothelium is grouped into four groups, 0--no reaction, I-weak reaction (ISI = 1 or 2), II--moderate reaction (ISI = 3 or 4), III--strong reaction (ISI = 5 or 6). Statistically significant differences (p = 0.0214) have been found between groups 0 and 1 compared to groups 2 and 3. A higher percentage of positive cells has been found in male patients than in female ones (p = 0.0483). C-myc PPC 0 or 1 has on the average smaller density of blood vessels per mm2 than c-myc PPC 2 or 3, but the difference is not statistically significant. C-myc ISI 0 or 1 has, on the average, smaller density of blood vessels per mm2 than c-myc ISI 2 or 3, but the difference is not statistically significant. We concluded that c-myc staining intensity was associated with higher microvessels density.     

Clonality analysis of archival cervical smears. Correlation of monoclonality with grade and clinical behavior of cervical intraepithelial neoplasia

OBJECTIVE: To establish a polymerase chain reaction (PCR)-based clonality assay for archival cervical smears and examine its value in the detection of cervical intraepithelial neoplasia (CIN) and prediction of its clinical behavior. STUDY DESIGN: Dyskaryotic cells were microdissected from archival cervical smears of 33 cases and subjected to PCR-based clonality analysis of the androgen receptor gene. High-risk HPV subtypes were screened by PCR. RESULTS: Monoclonal patterns were found in 9/9 CIN 3 and 15/21 CIN 2, while polyclonal patterns were observed in the remaining 6 CIN 2 and 3/3 CIN 1. All patients with monoclonal CIN lesions, including 15 CIN 2, showed recurrence of the disease despite treatment. The original CIN 2 and recurrent CIN lesion in each of the 6 examined cases showed the same monoclonal pattern, suggesting a clonal link. In contrast, the patients with polyclonal CIN 1 or 2 became negative and remained disease free. High-risk HPV subtypes were found in all monoclonal CIN lesions, including 9 CIN 3 and 15 CIN 2, and in 4/6 polyclonal CIN 2 but not in CIN 1 lesions. CONCLUSION: Clonality analysis of cervical smears is potentially valuable in the identification of true neoplastic cells and prediction of clinical behavior of CIN 2 lesions.