Quantitative immunohistochemical analysis of VIP-neurons in the rat visual cortex

A critical appraisal of quantitative immunohistochemistry of neuropeptides is presented defining the main criteria of selecting the type of immune-staining and preparation suitable for these investigations. As an example of meeting the established criteria, the immunohistochemical demonstration of vasoactive intestinal polypeptide (VIP)-containing neurons in the rat brain and the processing of VIP-immunostained preparations for computer-controlled image analysis are described.     

Structure–activity relationship of antiparasitic and cytotoxic indoloquinoline alkaloids,and their tricyclic and bicyclic analogues

Based on the indoloquinoline alkaloids cryptolepine (1), neocryptolepine (2), isocryptolepine (3) and isoneocryptolepine (4), used as lead compounds for new antimalarial agents, a series of tricyclic and bicyclic analogues, including carbolines, azaindoles, pyrroloquinolines and pyrroloisoquinolines was synthesized and biologically evaluated. None of the bicyclic compounds was significantly active against the chloroquine-resistant strain Plasmodium falciparum K1, in contrast to the tricyclic derivatives. The tricyclic compound 2-methyl-2H-pyrido[3,4-b]indole (9), or 2-methyl-β-carboline, showed the best in vitro activity, with an IC₅₀ value of 0.45 μM against P. falciparum K1, without apparent cytotoxicity against L6 cells (SI > 1000). However, this compound was not active in the Plasmodium berghei mouse model. Structure–activity relationships are discussed and compared with related naturally occurring compounds.     

Monitoring of lung tumour cell growth in artificial membranes

Morbidity of many tumour types is associated with invasion of tumour cells through the basement membrane and subsequent metastasis to vital organs. Tumour invasion is frequently detected late on as many patients present with advanced disease. The method of detecting invasion is through conventional histological staining techniques, which are time consuming and require processing of the sample. This can affect interpretation of the results. In this study, a new imaging technique, optical coherence tomography (OCT), was used to monitor lung tumour cell growth in two artificial membranes composed of either collagen type I or Matrigel. In parallel, standard histological section analysis was performed to validate the accuracy of the monitoring by OCT. Cross-sectional images from OCT revealed that lung tumour cells infiltrated only when low cell seeding density (5 x 10(5)) and low collagen concentration (1.5 mg/ml) were combined. The cells could be easily differentiated from the artificial membranes and appeared as either a brighter layer on the top of the membrane or brighter foci embedded within the darker membrane. These cell-membrane morphologies matched remarkably to the standard histological section images. Our results suggest that OCT has a great potential to become a useful tool for fast and robust imaging of cell growth in vivo and as a potential assessment of cell invasion.     

Hormonal regulation of [Ca(2+)](i) in periosteal-derived osteoblasts: effects of parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandin E(2)

The effects of hormonal modulators of osteoblast function, parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandins on [Ca(2+)](i) in periosteal-derived osteoblasts from rat femurs have been investigated. Our results show that application of parathyroid hormone PTH (10(-5) M) and prostaglandin E(2) (PGE(2)) (4 microM) result in a rapid heterogeneous elevation in [Ca(2+)](i) that, in the case of PTH, is dependent on both extracellular and intracellular sources of calcium. Variable responses to treatments have been found within populations of cells. The PGE(2) response is dose dependent. Treatment with 1,25(OH)(2)D(3) (10(-8) M) induces a brief (60-90 sec) elevation in [Ca(2+)](i) that is almost totally abolished in EGTA-buffered Ca(2+)-free medium. Interactive effects of multiple hormone treatments have been observed. Pretreatment with 1,25(OH)(2)D(3) results in near-total inhibition of the PTH and PGE(2) responses. In conclusion, modulation of [Ca(2+)](i) appears to play a role not only in the direct effects of osteotropic hormones on osteoblasts but also in the synergistic and antagonistic effects between circulating hormones.     

The Involvement of Immune Semaphorins in the Pathogenesis of Inflammatory Bowel Diseases (IBDs)

ResultsThe percentage (%) of T regulatory cells (Tregs) expressing sema3A in patients with active CD (64.5% ±14.49%) and active UC (49.8% ±16.45%) was significantly lower when compared to that of healthy controls (88.7% ±3.6%, p< 0.001 and p< 0.0001, respectively). This expression was seen to be in negative correlation with CD activity. Serum levels of Sema4A were significantly lower in patients with CD and UC when compared to that of controls (5.69±1.48ng\ml for CD, 5.26±1.23 ng/ml for UC patients vs 9.74±2.73ng/ml for normal controls, P<0.001). Sema4A was highly expressed in lymphocytes of the lamina propria of CD and UC patients but absent in patients with diverticulitis or in normal individuals.ConclusionsAltered % of Tregs expressing sema3A in patients with inflammatory bowel diseases (IBD) is partially responsible for their failure in preventing CD4+ effector T cell induced inflammation in IBD in peripheral blood. The increased expression of sema4A in bowel biopsies from CD and UC patients is suggestive of its central role in regulating local tissue inflammation in the bowel.     

Serum RANKL levels associate with anti- citrullinated protein antibodies in early untreated rheumatoid arthritis and are modulated following methotrexate

IntroductionReceptor activator of nuclear factor kappa B ligand (RANKL) is a key regulator of bone metabolism. Anti-citrullinated protein antibodies (ACPA) have been suggested to cause bone destruction by osteoclast activation. We investigated the relationship between RANKL and ACPA in patients with early untreated rheumatoid arthritis (RA).MethodsPatients with newly diagnosed untreated RA (n = 183) were analyzed at baseline and 3 months after initiating methotrexate (MTX) treatment. Serum RANKL (total RANKL), ACPA (anti-CCP2) and ACPA specificities (anti-citrullinated (cit)-vimentin, anti-cit-enolase and anti-cit-fibrinogen) were determined by enzyme-linked immunosorbent assay (ELISA). Synovial RANKL expression was evaluated by immunohistochemistry in a small group of patients (n = 15). The relationship between anti-cit-vim antibodies and bone destruction was further validated in 1116 RA patients included in the EIRA cohort. Pearson’s chi-square test, Wilcoxon rank sum test, Wilcoxon signed rank test and linear regression models were used.ResultsSerum RANKL concentration was significantly higher (p <0.05) in ACPA-positive (median: 689 pmol/L, IQR 342–1253) compared with ACPA-negative (median: 159 pmol/L, IQR 96–243) patients and this difference was also seen for synovial RANKL expression. Serum RANKL associated with ACPA (p <0.05) and bone erosions in rheumatoid factor (RF)-negative patients (n = 59). Among ACPA specificites, anti-cit-vimentin (amino acids 60–75) was associated with higher RANKL concentration and higher prevalence of bone erosion (p <0.05). Significant reductions in both serum RANKL and ACPA levels were observed after 3 months of MTX treatment (p <0.05).ConclusionsRANKL was elevated in ACPA-positive and in anti-cit-vimentin-positive patients with early untreated RA and associated with bone erosions. These findings give further support for an early direct pathogenic link between ACPA and bone destruction in RA.     

Fine-Scale Genetic Structure in the United Arab Emirates Reflects Endogamous and Consanguineous Culture,Population History,and Geography

The indigenous population of the United Arab Emirates (UAE) has a unique demographic and cultural history. Its tradition of endogamy and consanguinity is expected to produce genetic homogeneity and partitioning of gene pools while population movements and intercontinental trade are likely to have contributed to genetic diversity. Emiratis and neighboring populations of the Middle East have been underrepresented in the population genetics literature with few studies covering the broader genetic history of the Arabian Peninsula. Here, we genotyped 1,198 individuals from the seven Emirates using 1.7 million markers and by employing haplotype-based algorithms and admixture analyses, we reveal the fine-scale genetic structure of the Emirati population. Shared ancestry and gene flow with neighboring populations display their unique geographic position while increased intra- versus inter-Emirati kinship and sharing of uniparental haplogroups, reflect the endogamous and consanguineous cultural traditions of the Emirates and their tribes.     

Do plant functional types based on leaf dry matter content allow characterizing native grass species and grasslands for herbage growth pattern?

Few studies have focused on vegetation characteristics of importance to feeding domestic herbivores, mainly the seasonal pattern of herbage growth at spring. Our objective is to establish and to evaluate a simple method of ranking grassland communities for these characteristics. We combined approaches at plant species level (comparison of grass species growing in a pure stand) and plant community level (comparison of grasslands differing mainly in their nutrient availability). Firstly, we ask if the ranking of species by leaf dry matter content (LDMC), a functional parameter used to assess the plant strategy for resource acquisition and use, is consistent with a classification of the species using three plant features that determine plant growth pattern at spring (beginning and ending of stem elongation, leaf lifespan). Secondly, for three networks of natural grasslands, we test whether there is consistency when ranking them by their dominant plant functional type (PFT A, B or C) established previously at species level, and by the three agronomic characteristics. For species growing in pure stands, there was a significant effect of PFT for the three plant features. For species having a low LDMC (A and B PFT), there were earlier stem elongation in the season, earlier flowering and shorter leaf lifespan. The opposite was observed for species having a high LDMC (C and D PFT). For grassland communities dominated by A-PFT, the ceiling yield for leaves and stems occurred earlier in spring than for those dominated by C-PFT. Results were consistent at plant and community levels. Scaling up from plant to community was well mediated by PFT. Plant features which characterize species for resource acquisition and use are consistent with herbage growth patterns at plant community level. These results show that herbage growth pattern and composition depend on PFTs and that knowing the PFT dominance is of great importance to plan the use of grasslands. We can expect to use the PFT approach to perform vegetation diagnosis at field level when the objective is to rank grassland communities for their agronomic characteristics. Fernando L. F. de Quadros was supported by CAPES and CNPq (BP-2).