Unveiling the bioactive potential of the essential oil of a Portuguese endemism, Santolina impressa.

ETHNOPHARMACOLOGICAL RELEVANCE: Santolina species are widely used in traditional medicine in the Mediterranean region for their anti-inflammatory, antimicrobial, antispasmodic, digestive, and analgesic properties. S. impressa, a Portuguese endemism, is traditionally recognized for its beneficial anti-inflammatory properties in several gastrointestinal affections and is also used in oropharyngeal infections. AIM OF THE STUDY: The present study aims to characterize the essential oil of S. impressa growing in Portugal and validate its traditional uses by assessing the anti-inflammatory potential of its essential oil at concentrations without toxicity. The antifungal properties of the oil are also addressed, as well as, the putative mechanism of action underlying these effects. MATERIAL AND METHODS: The essential oil was obtained in accordance with the European Pharmacopoeia and characterized by GC and GC-MS. The anti-inflammatory potential of the oil was assessed on LPS-stimulated macrophages, through the production of nitric oxide (NO) using the Griess reaction. Putative mechanisms of action included the role of the oil as a NO scavenger, as well as its effect on the expression of two key pro-inflammatory enzymes, iNOS and COX-2 by Western blot analysis. The antifungal effect of the oil was evaluated according to the CLSI guidelines on several yeast and filamentous strains and on two major virulence factors in Candida albicans, namely germ tubes and biofilms. Ultrastructural modifications on dermatophytes were also unveiled by transmission electron microscopy. RESULTS: S. impressa essential oil was primarily characterized by the presence of monoterpene hydrocarbons and oxygenated monoterpenes, being the main compounds ß-pinene (22.5%), 1,8-cineole (10.0%), limonene (9.1%), camphor (8.1%) and ß-phellandrene (8.0%). A significant decrease (ca 60.0%) in nitrite levels was observed in LPS-stimulated macrophages treated with the oil without affecting cell viability. This effect could be explained by a great reduction on iNOS expression (85.0% inhibition), thus underpinning the anti-inflammatory potential of the oil. The oil also showed a fungicidal effect, being more active against Cryptococcus neoformans, Epidermophyton floccosum and Trichophytum rubrum. For these dermatophytes, significant ultrastructural modifications in cell wall structure were detected. Strikingly, for C. albicans, the oil showed a significant anti-infective potential (at 0.07 mg/mL for germ tube inhibition and 0.02 mg/mL for biofilm disruption) before fungal growth inhibition occurred. CONCLUSIONS: Our results validate the main traditional use ascribed to S. impressa, namely its anti-inflammatory effect. In addition, an antifungal potential is pointed out, thus corroborating the antimicrobial uses and adding new value to an endemic species poorly recognized by the industry.

Assessment of messenger RNA (mRNA) of Mycobacterium tuberculosis as a marker of cure in patients with pulmonary tuberculosis.

AIMS: To analyse the performance of RT-qPCR using 85B mRNA in the diagnosis of Mycobacterium tuberculosis infection and in the assessment of the response to treatment for pulmonary tuberculosis (TB). METHODS AND RESULTS: Ninety-eight patients with signs of pulmonary TB were selected: 56 were considered infected with Myco. tuberculosis and they had positive cultures or evident clinical response to anti-TB treatment. Patients with pulmonary tuberculosis were evaluated by culture and RT-qPCR for a 30-day specific treatment. It was found that both tests demonstrated a decline in viable bacilli at 15 and 30 days after the beginning of the therapy in most of the patients. The quantification of the 85B mRNA target was performed in 52 patients who had initially shown positive results by RT-qPCR and who were followed on the days 15 and 30 after the specific treatment. Thus 85B mRNA was detectable in sputum samples in 52 patients with a confirmed diagnosis of pulmonary tuberculosis on day 0. During the specific treatment the 85B mRNA was detectable in 13 patients on day 15 and in only three patients on day 30. CONCLUSIONS: Mycobacterium tuberculosis mRNA in the sputum is a useful prognostic marker and its quantification, an early and reliable indicator for monitoring response to treatment, drug resistance, re-infection and relapse. SIGNIFICANCE AND IMPACT OF THE STUDY: RT-qPCR is a tool that can be used in clinical and therapeutic monitoring as an indicator of bacterial resistance and indicator of the period of transmissibility of Myco. tuberculosis in patients with pulmonary TB undergoing treatment.

Antifungal, antioxidant and anti-inflammatory activities of Oenanthe crocata L. essential oil.

The present study reports the chemical composition, antifungal, antioxidant and anti-inflammatory properties as well as the cytotoxicity of Oenanthe crocata essential oil and one of its main compounds. The essential oil was obtained from the aerial parts of the plant by hydrodistillation and analysed by GC and GC/MS. The oil was predominantly composed of monoterpene hydrocarbons (85.8%), being the main compounds trans-ß-ocimene (31.3%), sabinene (29.0%) and cis-ß-ocimene (12.3%). For the antifungal activity, the minimal inhibitory and minimal lethal concentrations (MICs and MLCs) were determined. The oil was particularly active against dermatophytes and Cryptococcus neoformans, with MIC values ranging from 0.08 to 0.16 µL/mL. Regarding the anti-inflammatory activity, both the oil and sabinene demonstrated strong anti-inflammatory activity through nitric oxide (NO) production inhibition in lipopolysaccharide (LPS) plus interferon gamma (IFN-γ)-triggered macrophages. Furthermore, the essential oil showed a potent NO scavenging effect and inhibited inducible NO synthase expression. Interestingly, and although we detected a cytotoxic effect in macrophages and keratinocytes for the highest concentrations tested of the oil and sabinene, we also disclosed bioactive and safe concentrations to be further explored for therapeutic proposes. Taking together, these results support the use of the oil and sabinene for the management of dermatophytosis and/or inflammatory-related diseases.

Lavandula luisieri essential oil as a source of antifungal drugs.

This work reports the antifungal activity of Lavandula luisieri essential oils against yeast, dermatophyte and Aspergillus strains responsible for human infections and food contamination. The oil's cytotoxicity and its effect on the yeast-mycelium transition in Candida albicans, an important virulence factor, were also evaluated. Analyses by GC and GC/MS showed a peculiar composition of irregular monoterpenes. Significant differences between the samples occurred in the amounts of 1,8-cineole, fenchone and trans-α-necrodyl acetate. The oil with higher amounts of irregular monoterpenes was the most effective. The influence of the oils on the dimorphic transition in C. albicans was also studied through the germ tube inhibition assay. Filamentation was completely inhibited at concentrations sixteen times lower than the minimal inhibitory concentration. The results support the use of L. luiseiri essential oils in the development of new phytopharmaceuticals and food preservatives and emphasise its antifungal properties at concentrations not cytotoxic or with very low detrimental effects on mammalian cells.

Antifungal activity of phenolic-rich Lavandula multifida L. essential oil.

This study evaluates the antifungal activity and mechanism of action of a new chemotype of Lavandula multifida from Portugal. The essential oil was analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS), and the minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) of the oil and its major compounds were determined against several pathogenic fungi responsible for candidosis, meningitis, dermatophytosis, and aspergillosis. The influence of the oil on the dimorphic transition in Candida albicans was also studied, as well as propidium iodide (PI) and FUN-1 staining of C. albicans cells by flow cytometry. The essential oil was characterized by high contents of monoterpenes, with carvacrol and cis-ß-ocimene being the main constituents. The oil was more effective against dermatophytes and Cryptococcus neoformans, with MIC and MLC values of 0.16 µL/mL and 0.32 µL/mL, respectively. The oil was further shown to completely inhibit filamentation in C. albicans at concentrations below the respective MIC (0.08 µL/mL), with cis-ß-ocimene being the main compound responsible for this inhibition (0.02 µL/mL). The flow cytometry results suggest a mechanism of action ultimately leading to cytoplasmic membrane disruption and cell death. L. multifida essential oil may be useful in complementary therapy to treat disseminated candidosis, since the inhibition of filamentation alone appears to be sufficient to treat this type of infection.

Targeting of adenovirus to endothelial cells by a bispecific single-chain diabody directed against the adenovirus fiber knob domain and human endoglin (CD105).

The use of adenoviruses for antivascular cancer gene therapy is limited by their low transduction efficiency for endothelial cells. We have developed a recombinant bispecific antibody as a molecular bridge, linking the adenovirus capsid to the endothelial cell surface protein endoglin, for vascular targeting of adenoviruses. Endoglin (CD105), a component of the transforming growth factor beta receptor complex, represents a promising target for antivascular cancer therapy. Endoglin is expressed predominantly on endothelial cells and is upregulated in angiogenic areas of tumors. We isolated single-chain Fv fragments directed against human endoglin from a human semisynthetic antibody library. One of the isolated scFv fragments (scFv C4) bound specifically to various proliferating primary endothelial cells or cell lines including HUVEC, HDMEC, HMVEC, and HMEC. ScFv C4 was therefore used to construct a bispecific single-chain diabody directed against endoglin and the adenovirus fiber knob domain (scDb EDG-Ad). This bispecific molecule mediated enhanced and selective adenovirus transduction of HUVECs, which was independent from binding to the coxsackievirus and adenovirus receptor (CAR) and alpha(v)-integrins. Thus, adenovirus infection was redirected to a new cellular receptor (CD105) and cell entry pathway. These results demonstrate the utility of bispecific single-chain diabodies, which can be produced in large quantities in bacteria, for the retargeting of adenoviruses in cancer gene therapy.

Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells.

CD14-positive monocytes obtained from human peripheral blood were cultured with GM-CSF and IL-4. During the early culture phase immature dendritic cells (DCs) developed which not only expressed CD1a, HLA-DR and CD86, but also expressed the endothelial cell markers von Willebrand factor (vWF), VE-cadherin and VEGF receptors Flt-1 and Flt-4. Further maturation of DCs was achieved by prolonged cultivation with TNFalpha. These cells showed typical DC morphology and like professional antigen-presenting cells (APCs) expressed CD83 and high levels of HLA-DR and CD86. However, if immature DCs were grown with VEGF, bFGF and IGF-1 on fibronectin/vitronectin-coated culture dishes, a marked change in morphology into caudated or oval cells occurred. In the presence of these angiogenic growth factors the cultured cells developed into endothelial-like cells (ELCs), characterized by increased expression of vWF, KDR and Flt-4 and a disappearance of CD1a and CD83. Addition of IL-4 and Oncostatin M also increased VE-cadherin expression, and the loosely adherent cells formed clusters, cobblestones and network-like structures. vWF- expressing ELCs mainly originated from CD1a-positive cells, and VEGF was responsible for the decrease in the expression of the DC markers CD1a and CD83. In mixed leukocyte cultures, mature DCs were more potent APCs than ELCs. Moreover, Ac-LDL uptake, and the formation of tubular structures on a plasma matrix was restricted to ELCs. These results suggest that in the presence of specific cytokines immature DCs have the potential to differentiate along different lineages, i.e. into a cell type resembling ELCs.

Endothelial-like cells derived from human CD14 positive monocytes.

In the present study, we show that endothelial-like cells (ELCs) can develop from human CD14-positive mononuclear cells (CD14 cells) in the presence of angiogenic growth factors. The CD14 cells became loosely adherent within 24 h of culture and subsequently underwent a distinct process of morphological transformation to caudated or oval cells with eccentric nuclei. After 1 week in culture the cells showed a clear expression of endothelial cell markers, including von Willebrand factor (vWF), CD144 (VE-cadherin), CD105 (endoglin), acetylated low-density lipoprotein (AC-LDL)-receptor, CD36 (thrombospondin receptor), FLT-1, which is vascular endothelial cell growth factor (VEGF) receptor-1, and, to a weaker extent, KDR (VEGF receptor-2). Furthermore, in these cells structures resembling Weibel-Palade bodies at different storage stages were identified by electron microscopy, and upon culturing on three-dimensional fibrin gels the cells build network-like structures. In addition, cell proliferation and vWF expression was stimulated by VEGF, and the endothelial cell adhesion molecules CD54 (ICAM-1), and CD106 (VCAM-1) became transiently inducible by tumor necrosis factor-alpha (TNF-alpha). In contrast, the dendritic markers CD1a, and CD83 were not expressed to any significant extent. The expression of CD68, CD80 (B7-1), CD86 (B7-2), HLA-DR and CD36 may also suggest that ELCs might be related to macrophages, sinus lining or microvascular endothelial cells. Taken together, our observations indicate that ELCs can differentiate from cells of the monocytic lineage, suggesting a closer relationship between the monocyte/macrophage- and the endothelial cell systems than previously supposed.