Preparation and Comparison of Effects of Different Herbal Oil Ointments as Wound-Healing Agents.

Wound healing is a complex process and some agents have been reported to accelerate it. The aim of this study was to evaluate the healing effect of Eucerin-based ointments of lemon, sesame and olive oils on infected full-thickness wounds in rats. Wounds were created on the dorsal surface of Male Albino Wistar rats (n = 12). Wounds were treated with an Eucerin-based ointment containing either of lemon, sesame or olive oils (33% w/w) twice a day for 14 days. Histopathology results showed that contraction of wounds treated with lemon and sesame oils was higher than in the olive oil and control groups on days 10 and 14. In the lemon- and sesame-oil treated groups, on day 14, 50% of rat lesions were completely healed. Total number of inflammatory cells in lemon oil treatment group was significantly smaller than that of others on day 14 (p < 0.001). Also, thickness of the epidermal layer and rejuvenation of the hair follicles and other skin appendages was normal in lemon and sesame oil treated groups. The lemon and sesame oil ointments accelerated the healing process of wounds in macroscopic, morphological and morphmetrical analyses. Therefore, lemon and sesame oil ointments could be considered as alternative dressings for infected full-thickness wounds because of improved wound healing characteristics.

Identification of common vaginal Lactobacilli immunoreactive proteins by immunoproteomic techniques.

Lactobacilli are considered as the most important microorganisms in regulating immune system and maintaining vaginal health. The uses and benefits of Lactobacilli as probiotics, particularly the regulation of immune system, are dependent on the strain used and a comprehensive understanding of their effects on the host. Several factors have been identified in Lactobacilli that influence the immune response, such as exopolysaccharides and proteins. The current study was designed to investigate the serum immunoreactivity of healthy women against common vaginal Lactobacilli immunoreactive proteins. Three common vaginal Lactobacillus strains (L. crispatus L1, L. gasseri L9, and L. fermentum L2) were compared for immune response. The ELISA results showed that the levels of total immunoglobulin (Ig-total) antibody for L. crispatus L1, L. fermentum L2, and L. gasseri L9 were 47%, 45% and 29%, respectively. Regarding the lower prevalence of L. fermentum L2 in comparison with the other two strains, the approximately equal levels of Ig-total compared to L. crispatus L1 and more than L. gasseri L9 indicate that L. fermentum L2 has the greater antigenicity ability. Accordingly, the immunoreactive proteins of L. fermentum L2 were identified using MALDI-TOF-MS detected by SDS-PAGE and Western blotting. These proteins included 30s ribosomal protein S4 and 50s ribosomal protein L5. Antigenic epitopes on the 3D structure of these proteins was also predicted using bioinformatics analysis. The presence of antibody in serum of healthy pre-menopausal women indicates that Lactobacilli (normal flora) proteins can stimulate host immune response. Purification and further studies of the proteins may allow their potential use as an adjuvant to improve the efficacy of vaccines.

Immunosuppressive Effects of Two Probiotics, Lactobacillus paracasei DSM 13434 and Lactobacillus plantarum DSM 15312, on CD4+ T Cells of Multiple Sclerosis Patients.

T cells play an important role in the development and progression of multiple sclerosis (MS), an autoimmune disease of the central nervous system. In the present study, the immunomodulatory impacts of two Lactobacillus strains, L paracasei DSM 13434 and L plantarum DSM 15312, on the frequency and cytokine production of CD4+ T cells in MS patients were explored. Thirty MS patients were enrolled in this study. The CD4+ T cells were isolated, cultured, and exposed to the media containing cell-free supernatants of L plantarum (group1), L paracasei (group 2), the mixture group of cell-free supernatants of both probiotics (group 3), and vehicle (control) group (group 4). The frequencies of T helper (Th) 1, Th17, Th2, and T regulatory type 1 (Tr1) cells and mean fluorescent intensity (MFI) of the associated cytokines were assessed using flow cytometry. The levels of interleukin 17 (IL-17), transforming growth factor ß (TGF-ß), and interferon-gamma (IFN-γ) cytokines in supernatants of all groups were measured by enzyme-linked immunosorbent assay. The percentage of Th1 cells and the MFI of IFN-γ in Th1 cells (CD4+ IFN-γ+) in all three probiotic treatment groups were significantly decreased compared to the control group. However, no significant changes were observed in the proportion and MFI of Th2, Th17, and Tr1 cells. A significant decrease was observed in IL-17 secretion in the supernatant of cultured CD4+ T cells in all three treatment groups in comparison with control. The levels of TGF-ß and IFN-γ were not significantly different among any of the study groups.  Collectively, cell-free supernatants of the lactobacilli showed an in vitro anti-inflammatory effect. However, further studies are needed to prove the real effects of probiotics on MS.

Levofloxacin nanoemulsion gel has a powerful healing effect on infected wound in streptozotocin-induced diabetic rats.

The aim of this study was to develop a novel oil-in-water (o/w) nanoemulsion gel containing levofloxacin for enhanced topical efficacy. Average particle size of sesame oil nanoemulsion without (SONE) and containing levofloxacin (SONEL) was found as 25.2 and 26.3 nm, respectively. Results from scratch test showed that SONEL had better proliferation effect in comparison with negative control. Treated animals with SONEL showed significant reduction in period of epithelialization, wound contraction, and number of inflammatory cells among all groups. Also, SONEL-treated group had the greatest collagen synthesis. Immunohistochemical analysis showed high intensity of CD31 and TGF-ß at wound site of treatment groups with SONEL on day 12 post-treatment (P < 0.05). Skin irritation test demonstrated safety of SONEL gel for skin topical application. In conclusion, our studies suggest that SONEL could be an effective formulation for treatment of diabetic wound infection by controlling infection and improving the healing process. Graphical Abstract .

Assessment of structural, biological and drug release properties of electro-sprayed poly lactic acid-dexamethasone coating for biomedical applications.

The efficacy of an implant is highly depends on its coating characteristics mainly determined by polymer properties and coating technique. Electro-spraying is an inexpensive and versatile coating technique with various advantages for biomedical application. In this study, the efficacy of electro-sprayed (ES) poly lactic acid (PLA)-dexamethasone (DEX) coatings for medical implants was evaluated and compared with spin-coated samples as control. Structural properties of coatings were investigated using X-ray diffraction (XRD) and differential scanning calorimetry (DSC). Confocal and scanning electron microscopy (SEM), contact angle measurement and nanoindentation tests were used to study surface properties. Coating degradation rate and drug release profile were studied for 40 days. Cell viability experiments were also performed on human endothelial (HUVEC) and smooth muscle cells (HUASMC) using MTT assay and SEM. XRD and DSC analysis showed electro-spraying significantly reduce PLA and DEX crystallinity. Surface studies showed ES coatings has significantly higher hydrophobicity and roughness with microbead-nanofiber morphology vs. micro-nanoporous structure of spin-coated samples. Initial burst release of DEX was 22% and 10% after 6 h and total release was 71% and 46% after 40 days for ES and spin-coated samples, respectively. HUVEC viability of ES samples was higher than spin-coated ones after 1 and 4 days. However, dexamethasone release profile reduced HUASMC proliferation in ES PLA-DEX samples in comparison to spin-coated after 1 and 3 days. In conclusion, in vitro results showed potential of ES PLA-DEX as a biocompatible and efficient anti-inflammatory coating with suitable drug release profile for future applications such as coronary drug eluting stents.

Synthesis and characterization of magnetic hybrid nanomaterials via RAFT polymerization: A pH sensitive drug delivery system.

Herein, a facile and versatile method for the synthesis of a novel magnetic nanocarrier via surface- initiated reversible addition-fragmentation chain transfer (RAFT) polymerization is introduced. At first, RAFT agent was successfully attached to the surface of Fe3O4 nanoparticles and, then, poly (glycidyl methacrylate) (PGMA) chains were grown and anchored onto the surface of Fe3O4 nanoparticles. At the end, hydrazine (Hy) groups were introduced to the PGMA chains via reaction between epoxy rings and hydrazine molecules. Doxorubicin (DOX) was covalently conjugated to the prepared nanocarrier (Fe3O4@PGMA@Hy) through a hydrazone linkage. The in vitro drug release of Fe3O4@PGMA@Hy@DOX examined in buffers with pH 7.4 and pH 5.4 exhibited a strong pH-sensitive behavior. The results showed that Fe3O4@PGMA@Hy@DOX successfully performed the delivery and controlled release of doxorubicin (DOX) anticancer drug.

Anti-elastase and anti-collagenase potential of Lactobacilli exopolysaccharides on human fibroblast.

Polysaccharides could be used as biodegradable and biocompatible polymers for scaffolds and carriers matrix. Numerous algal, fungi and herbal polysaccharides can attenuate degradation of skin matrix by the inhibition of elastase, collagenase and matrix metalloproteinases (MMPs). In this study, we investigate anti-elastase and anti-collagenase potential of Lactobacilli exopolysaccharides (LEPS) on normal human fibroblast. Among 60 Lactobacilli isolated from herbal plants and dairy products, selected LEPS showed high anti-collagenase (up to 100%), anti-elastase (up to 87%) and antioxidant activity (up to 60%). Most of them had no cytotoxicity effect on fibroblast, and some of them promote cell proliferation (up to 10%). In scratch assay, all the investigated EPSs stimulated wound healing process in fibroblast (up to 99%). MMP1, MMP2, MMP3, MMP9 and MMP10 were down-regulated significantly and TIMP1 and TIMP2 were up-regulated slightly in LEPS of B9-1 from L. casei with high anti-collagenase and anti-elastase activity; however, no meaningful alteration was observed in MMPs expression level for LEPS of P35 from L. plantarum with low anti-collagenase and anti-elastase activity. By consideration of high anti-collagenase, anti-elastase, antioxidant activity and wound healing of LEPS, they could be considered as good candidate of skin anti-aging agents for tissue engineering and skin regeneration scaffolds.

Isolation and identification of Komagataeibacter xylinus from Iranian traditional vinegars and molecular analyses.

BACKGROUND AND OBJECTIVES: Acetic acid bacteria (AAB) are one of the major interests of researchers. Traditional vinegars are suitable sources of AAB because they are not undergone industrial process like filtering and adding preservatives. Komagataeibacter xylinus as a member of AAB is known as the main cellulose producer among other bacteria. The purpose of the current study was to isolate the bacteria from traditional vinegars and its molecular analyses. MATERIALS AND METHODS: Vinegar samples were collected. Well-organized bacteriological tests were carried out to differentiate isolated bacteria from other cellulose producers and to identify K. xylinus. NaOH treatment and Calcofluor white staining were used for detecting cellulose. Chromosomal DNA of each strain was extracted via three methods of boiling, phenol-chloroform and sonication. Molecular analyses were performed on the basis of 16S rRNA sequences and cellulose synthase catalytic subunit gene (bcsA) for further confirmation. Phylogenetic tree was constructed for more characterization. Two housekeeping genes were studied including phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA). RESULTS: Of the 97 samples, 43 K. xylinus strains were isolated. They were identified via bacteriological and molecular techniques. 16S rDNA sequence showed 99% similarity with registered sequences of the bacteria. Biodiversity of the genome confirmed by analyzing bcsA, pheS and rpoA genes. CONCLUSION: K. xylinus can be isolated from traditional vinegars. Screening tests ought to include the classical methods and molecular techniques. Different molecular techniques and more genomic research should be developed to expand our knowledge for distinguishing isolated bacteria especially in the fields of AAB.

Lactobacilli Modulate Hypoxia-Inducible Factor (HIF)-1 Regulatory Pathway in Triple Negative Breast Cancer Cell Line.

OBJECTIVE: Hypoxia-Inducible Factor (HIF)-1 plays an essential role in the body's response to low oxygen concentrations and regulates expression of several genes implicated in homeostasis, vascularization, anaerobic metabolism as well as immunological responses. Increased levels of HIF-1α are associated with increased proliferation and more aggressive breast tumor development. Lactobacilli have been shown to exert anti-cancer effects on several malignancies including breast cancer. However, the exact mechanism of such effect is not clear yet. The aim of this study was to analyze the expression of selected genes from HIF pathway in a triple negative breast cancer cell line (expressing no estrogen and progesterone receptors as well as HER-2/Neu), MDA-MB-231, following treatment with two lactobacilli culture supernatants. MATERIALS AND METHODS: In this experimental study, we analyzed the expression of HIF-1α, SLC2A1, VHL, HSP90, XBP1 and SHARP1 genes from HIF pathway in MDA-MB-231 cells, before and after treatment with Lactobacillus crispatus and Lactobacillus rhamnosus culture supernatants (LCS and LRS, respectively) by means of quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: Both LRS and LCS had cytotoxic effects on MDA-MB-231 cells, while the former type was more cytotoxic. LRS dramatically down-regulated expression levels of the HIF-1α, HSP90 and SLC2A1 in the MDA-MB-231 cells. LCS had similar effect on the expression of HSP90, to what was observed in the LRS treatment. The expression level of tumor suppressor genes VHL and SHARP1 were also decreased in LCS treated cells. CONCLUSION: Although both LCS and LRS had cytotoxic effects on the MDA-MB-231 cells, it is proposed that LRS could be more appropriate for pathway directed treatment modalities, as it did not decrease expression of tumor suppressor genes involved in HIF pathway. Down-regulation of HIF pathway mediated oncogenes by LRS suggests that the cytotoxic effects of this Lactobacillus may at least be partly caused by this mechanism. As previous studies have shown that inhibition of HIF-1α and HSP90 expressions have therapeutic impact on cancer treatment, the inhibitory effect of LRS on expression of these genes implies that this Lactobacillus can be used in treatment strategies.

Normal and tumour cervical cells respond differently to vaginal lactobacilli, independent of pH and lactate.

Cervical cancer is a human papilloma virus (HPV)-related cancer, but most HPV infections are transient or intermittent and resolve spontaneously. Thus, other factors, such as cervical microflora, which are dominated by lactobacilli, must be involved in invasive cervical carcinoma development after HPV infection. Previous studies have demonstrated that lactobacilli have antitumour effects, and it is possible that vaginal lactobacilli prevent cervical cancer. Here we examined the proliferative and apoptotic responses of normal and tumour cervical cells to common vaginal lactobacilli components by investigating human normal fibroblast-like cervical (normal cervical) and HeLa (cervical tumour) cell responses to Lactobacillus gasseri and Lactobacillus crispatus. The effects of different lactobacilli components, such as culture supernatants, cytoplasmic extracts, cell-wall extracts and live cells, were determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, trypan blue staining, lactate dehydrogenase assay and colorimetric caspase-3 activity assay. Changes in caspase-3 and human chorionic gonadotropin ß (hCGß) expression were analysed by quantitative RT-PCR. Tumour cell growth inhibition by culture supernatants was higher than that by pH- and lactate-adjusted controls. However, the effects of the supernatants on normal cells were similar to those of lactate-adjusted controls. Apoptosis was inhibited by supernatants, which was consistent with higher hCGß expression since hCG inhibits apoptosis. Our study demonstrated that common vaginal lactobacilli exert cytotoxic effects on cervical tumour cells, but not on normal cells, and that this cytotoxicity is independent of pH and lactate. Our results encourage further studies on the interaction between lactobacilli and cervical cells, and administration of common vaginal lactobacilli as probiotics.

Differences in vaginal lactobacilli composition of Iranian healthy and bacterial vaginosis infected women: a comparative analysis of their cytotoxic effects with commercial vaginal probiotics.

BACKGROUND: Vaginal flora of healthy women is dominated by Lactobacillus species which can prevent bacterial vaginosis. OBJECTIVES: The current study aimed to determine the differences in vaginal lactobacilli composition of Iranian healthy and bacterial vaginosis (BV) infected women and compared their cytotoxic effects with commercial vaginal probiotics. PATIENTS AND METHODS: One hundred and seventy eight vaginal specimens were collected from healthy and BV infected women. Lactobacillus colonies were obtained by culturing on laked blood BHI and MRS medias and genetically defined by 16s rRNA sequencing. Differentiating the specimens to normal, intermediate and BV infected were carried out by Ison and Hey grading protocol. Identification of Lactobacillus strains in vaginal specimens were performed by Multiplex PCR. The inhibitory effects of lactobacilli on Hela (tumoral cervical cells) and HNCF-pi52 (normal cervical cells) were conducted by MTT and trypan blue assays. RESULTS: L. crispatus, L. gasseri, L. iners, L. jensenii, L. acidophilus and L. rhamnosus were the most frequently occurring species in vagina of healthy Iranian women. L. crispatus and L. jensenni were significantly higher in the normal than in the BV infected groups. Also the cytotoxic effect of L. crispatus on tumoral cervical cells was higher than other lactobacilli including commercial probiotics. CONCLUSIONS: As L. crispatus and L. jensenni were significantly higher in BV infected women and the cytotoxic effect of L. crispatus on tumoral cervical cells was high, introduction of new probiotics seems necessary.