The influence of amniotic membrane extracts on cell growth depends on the part of membrane and childbirth mode selected: a proof-of-concept study.

OBJECTIVE: The amniotic membrane (AM) is a rich source of biologically active factors, important for wound healing and is widely used in various clinical applications, including tissue engineering, reconstructive surgery and wound management. The aim of the present proof-of-concept study was to assess the influence of amniotic membrane extracts on in vitro proliferation of main cells involved in tissue regeneration. The assessment was done in regards to the content of selected biologically active factors in amniotic membrane extracts. METHOD: The quantitative analysis of EGF, TGF-ß and TIMP-1 in tested samples was assayed by enzyme-linked immunosorbent assay (ELISA) tests. The influence of amniotic membrane extracts on proliferation of keratinocytes (HaCaT), fibroblasts (Wi-38) and endothelial cell lines (HECa-10) was assessed using a colorimetric tetrazolium salt reduction assay. RESULTS: In all of the amnion samples high amounts of EGF, TGF-ß and TIMP-1 were detected. However, the content of these factors varied between placental and cervical portions of the same membrane. Moreover, various concentrations of biologically active factors between physiological at-term delivery and caesarean section-derived membranes were also observed. All of the assessed amnion extracts stimulated proliferation of HaCaT and Wi-38 cells, although samples prepared from caesarean section-derived cervical portion of amniotic membrane stimulated more proliferation of keratinocytes than of fibroblasts. In contrast to HaCaT and Wi-38 cells, proliferation of HECa-10 cell line was inhibited by all tested extracts. CONCLUSION: The results of our proof-of-concept study confirm that biological dressings prepared from amniotic membrane, especially its placental portion, since they stimulated both fibroblasts and keratinocytes, may provide relevant support for wound healing. On the other hand, dressings prepared from caesarean section-derived cervical portion of amniotic membrane, since they stimulate mainly epidermal cells, may be suitable for some specific applications, where more selective action is required, such as in ocular surgery. However, verification of this observation requires further studies.

Morphology and chemical composition of cobalt germanide islands on Ge(001).

The reactive growth of cobalt germanide on Ge(001) was investigated by means of in situ x-ray absorption spectroscopy photoemission electron microscopy (XAS-PEEM), micro-illumination low-energy electron diffraction (µ-LEED), and ex situ atomic force microscopy (AFM). At a Co deposition temperature of 670 °C, a rich morphology with different island shapes and dimensions is observed, and a correlation between island morphology and stoichiometry is found. By combining XAS-PEEM and µ-LEED, we were able to identify a large part of the islands to consist of CoGe2, with many of them having an unusual epitaxial relationship: CoGe2 [Formula: see text] [Formula: see text] Ge [Formula: see text]. Side facets with (112) and (113) orientation have been found for such islands. However, two additional phases were observed, most likely Co5Ge7 and CoGe. Comparing growth on Ge(001) single crystals and on Ge(001)/Si(001) epilayer substrates, the occurrence of these intermediate phases seems to be promoted by defects or residual strain.

Growth and evolution of nickel germanide nanostructures on Ge(001).

Nickel germanide is deemed an excellent material system for low resistance contact formation for future Ge device modules integrated into mainstream, Si-based integrated circuit technologies. In this study, we present a multi-technique experimental study on the formation processes of nickel germanides on Ge(001). We demonstrate that room temperature deposition of ∼1 nm of Ni on Ge(001) is realized in the Volmer-Weber growth mode. Subsequent thermal annealing results first in the formation of a continuous NixGey wetting layer featuring well-defined terrace morphology. Upon increasing the annealing temperature to 300 °C, we observed the onset of a de-wetting process, characterized by the appearance of voids on the NixGey terraces. Annealing above 300 °C enhances this de-wetting process and the surface evolves gradually towards the formation of well-ordered, rectangular NixGey 3D nanostructures. Annealing up to 500 °C induces an Ostwald ripening phenomenon, with smaller nanoislands disappearing and larger ones increasing their size. Subsequent annealing to higher temperatures drives the Ni-germanide diffusion into the bulk and the consequent formation of highly ordered, {111} faceted Ni-Ge nanocrystals featuring an epitaxial relationship with the substrate Ni-Ge (101); (010) || Ge(001); (110).

Ba termination of Ge(001) studied with STM.

We use controlled annealing to tune the interfacial properties of a sub-monolayer and monolayer coverages of Ba atoms deposited on Ge(001), enabling the generation of either of two fundamentally distinct interfacial phases, as revealed by scanning tunneling microscopy. Firstly we identify the two key structural phases associated with this adsorption system, namely on-top adsorption and surface alloy formation, by performing a deposition and annealing experiment at a coverage low enough (∼0.15 ML) that isolated Ba-related features can be individually resolved. Subsequently we investigate the monolayer coverage case, of interest for passivation schemes of future Ge based devices, for which we find that the thermal evaporation of Ba onto a Ge(001) surface at room temperature results in on-top adsorption. This separation (lack of intermixing) between Ba and Ge layers is retained through successive annealing steps to temperatures of 470, 570, 670 and 770 K although a gradual ordering of the Ba layer is observed at 570 K and above, accompanied by a decrease in Ba layer density. Annealing above 770 K produces the 2D surface alloy phase accompanied by strain relief through monolayer height trench formation. An annealing temperature of 1070 K sees a further change in surface morphology but retention of the 2D surface alloy characteristic. These results are discussed in view of their possible implications for future semiconductor integrated circuit technology.

The direct inhibition of MMP-2 and MMP-9 by an enzyme alginogel: a possible mechanism of healing support for venous leg ulcers.

Flaminal Forte is an enzyme alginogel,whose activity depends on the absorption and binding of matrix metalloproteinases (MMPs), which are known to play a crucial role in delayed wound healing. The aim of the study was to evaluate the influence of Flaminal on MMP-2/-9 activity in ulcer exudate, ex vivo. Eight patients with bilateral venous leg ulcers were treated for 4 weeks with Flaminal Forte covered by hydrocolloid ('F' wounds), or with hydrocolloid alone ('H' wounds) as a reference control. Clinical assessment did not reveal any differences between F and H wounds regarding surface reduction and general wound condition. Nevertheless, although non-significant, there was a visible difference in peri-wound skin appearance in F wounds, as compared to H wounds. The wound exudate contained high MMP-2/-9 levels, which gradually decreased as wounds healed. The attenuation of MMPs was stronger in F than in H exudate, however, in standard zymography this difference appeared non-significant. Real-time zymography revealed that Flaminal mediated a powerful direct inhibition of gelatinolytic activity of wound exudate and of recombinant MMP-2/-9 in vitro.

The effects of sodium diethyldithiocarbamate in fibroblasts V79 cells in relation to cytotoxicity, antioxidative enzymes, glutathione, and apoptosis.

Sodium diethyldithiocarbamate (DETC) is the main metabolite of disulfiram. Recently, we reported that mechanism of disulfiram cytotoxicity in V79 cells might be partially connected with thiol redox-state imbalance. Here, we examined the effect of DETC on the level of intracellular glutathione (GSH), protein oxidation (measured as PC-protein carbonyl content), lipid peroxidation (measured as TBARS-thiobarbituric acid reactive substances), antioxidant enzymatic defense, as well as on apoptosis. We used V79 Chinese hamster fibroblasts cells with and without modulated glutathione (GSH) level by N-acetyl-L-cysteine (NAC). We showed that treatment with DETC at concentrations that cause a moderate increase in thiol-state imbalance but not cell death stimulates oxidative stress measured as increased level of PC and TBARS, adaptive response of GSH-related enzymes and apoptosis. Our results show that cellular effects of DETC are partially attributable to the initial redox cellular state, since the increase of GSH level by NAC pre-treatment prevented the observed changes.

Low molecular weight heparin treatment decreases MMP-9 plasma activity in patients with abdominal aortic aneurysm.

Large abdominal aortic aneurysms (AAAs) are associated with coagulation abnormalities, which are significantly reduced by low molecular weight heparin (LMWH). Considering anti-inflammatory properties of heparin we verified, whether LMWH influences MMP-2/-9 in AAA patients. The study involved 26 AAA individuals, 10 patients with coagulation abnormalities received LMWH and 16 were a control group. The plasma activity of MMP-2/-9 was measured using zymography. We found that, in addition to the reduction of coagulation abnormalities, LMWH treatment was associated with the decreased MMP-9 but not MMP-2 activity. Therefore, LMWH use could be considered as a valuable pretreatment before an elective aneurysm repair.

Heme oxygenase-1 protects tumor cells against photodynamic therapy-mediated cytotoxicity.

Photodynamic therapy is a promising antitumor treatment modality approved for the management of both early and advanced tumors. The mechanisms of its antitumor action include generation of singlet oxygen and reactive oxygen species that directly damage tumor cells and tumor vasculature. A number of mechanisms seem to be involved in the protective responses to PDT that include activation of transcription factors, heat shock proteins, antioxidant enzymes and antiapoptotic pathways. Elucidation of these mechanisms might result in the design of more effective combination strategies to improve the antitumor efficacy of PDT. Using DNA microarray analysis to identify stress-related genes induced by Photofrin-mediated PDT in colon adenocarcinoma C-26 cells, we observed a marked induction of heme oxygenase-1 (HO-1). Induction of HO-1 with hemin or stable transfection of C-26 with a plasmid vector encoding HO-1 increased resistance of tumor cells to PDT-mediated cytotoxicity. On the other hand, zinc (II) protoporphyrin IX, an HO-1 inhibitor, markedly augmented PDT-mediated cytotoxicity towards C-26 and human ovarian carcinoma MDAH2774 cells. Neither bilirubin, biliverdin nor carbon monoxide, direct products of HO-1 catalysed heme degradation, was responsible for cytoprotection. Importantly, desferrioxamine, a potent iron chelator significantly potentiated cytotoxic effects of PDT. Altogether our results indicate that HO-1 is involved in an important protective mechanism against PDT-mediated phototoxicity and administration of HO-1 inhibitors might be an effective way to potentiate antitumor effectiveness of PDT.

Effect of glutathione depletion on apoptosis induced by thiram in Chinese hamster fibroblasts.

Fungicide thiram, which is also known as an inducer of allergic contact dermatitis (ACD), was used as a model compound of thiuram chemicals, and its cellular effects were investigated in cultured Chinese hamster V79 cells. The level of intracellular reduced glutathione (GSH), protein sulfhydryl (PSH) groups, protein carbonyls (PC), membrane lipid peroxidation reflected by enhanced thiobarbituric acid reactive substrates (TBARS) production, as well as apoptotic effect were determined. The apoptosis induction was determined by assessing DNA fragmentation by TUNEL, annexin V binding, and caspases activation assays, using fluorescent microscope or flow cytometry, respectively. The concentrations of thiram required to induce cellular GSH depletion (by 40-50%), protein, and membrane lipid peroxidation (2-fold, and 1.7-fold, respectively), as well as to induce apoptosis in V79 Chinese hamster fibroblasts without causing necrosis through cytotoxic effects were between 50-100 microM. To investigate the role of decreased GSH content in the toxicity of thiram, GSH level was modified prior to exposure. Pretreatment of V79 cells with N-acetyl-L-cysteine (NAC), a GSH biosynthesis precursor, prevented GSH decrease, PC and TBARS production, as well as caspases activation induced by thiram exposure. On the other hand, thiram effects were enhanced by the previous depletion of cellular GSH by L-buthionine-(S,R)-sulfoximine (BSO).

Pentoxifylline inhibits perforin-dependent natural cytotoxicity in vitro.

Pentoxifylline (PTX) is commonly used in peripheral blood vessel diseases, however it has also been found to decrease the level of proinflammatory cytokines such as IL-12, TNF-alpha and IFN-gamma. Moreover, some authors reported that PTX suppresses spontaneous cytotoxicity of peripheral blood mononuclear cells (PBMC) in vitro. It could influence the mechanism of killing target cells by PBMC. For this reason we evaluated the influence of PTX on spontaneous cytotoxicity of PBMC against K562 and CaSki cell lines. Subsequently, we compared this effect to that evoked by dexamethasone, one of the most effective anti-inflammatory drugs. Our study revealed that PTX inhibits natural cytotoxicity preferentially through inhibition of perforin-mediated cell membrane damage, without a statistically significant influence on apoptosis induction. Furthermore, pentoxifylline inhibits natural cytotoxicity as effectively as dexamethasone. However, the result of PTX inhibitory influence is observed much earlier than that of dexamethasone. Currently PTX is commonly used in diseases that occur more frequently in elderly patients. We suggest that PTX, inhibiting perforin-dependent PBMC cytotoxic activity, could weaken anti-cancer action of immune system thus accelerating the progress of neoplasm formation in these patients.

Clinical aspects of disrupted Hedgehog signaling (Review).

The Hedgehog (HH) signaling pathway is involved in patterning and development of a variety of organ systems, including the nervous system, the skeletal system, the craniofacial structures, and the gastrointestinal tract. Recent evidence also implicates this signaling pathway in the postembryonic regulation of stem-cell number in epithelia and blood. The family of HH proteins consists of at least three different members, i.e., sonic HH (SHH), Indian HH (IHH), and desert HH (DHH). SHH is the most broadly expressed member of this family and is probably responsible for the major effects of this signaling pathway. The HH signal is received and transduced via a specific receptor complex composed of patched (PTCH) and smoothened (SMOH) transmembrane proteins. Abnormalities in this signaling cascade have been found in various developmental pathologies and neoplasms such as basal cell carcinoma. The abnormalities are associated with congenital or sporadic genetic alteration affecting function of different components of the HH signaling pathway, including SHH, PTCH, SMOH and GLI proteins.

Augmented pro-apoptotic effects of TRAIL and proteasome inhibitor in human promonocytic leukemic U937 cells.

TRAIL, Tumor necrosis factor-related apoptosis-inducing ligand), a member of the TNF family, is known to be cytotoxic for a high proportion of tumor cell lines. However, successful application of TRAIL in tumor therapy may depend on finding other agents that can potentiate its antitumor effects. The present study showed that the cytostatic/cytotoxic TRAIL activity against U937 cells could be significantly augmented by proteasome inhibitor PSI, as revealed by MTT assay. Increased cytostatic/cytotoxic effect on U937 cells by TRAIL/PSI combined treatment was caused by apoptosis, as shown by an increased PARP cleavage rate. TRAIL/PSI did not affect the level of mRNA expression for TRAIL receptors (DR4, DR5, DcR1) and other apoptosis signal transduction molecules (TRADD, caspase-8).

Potential application of cytokine level measurement in corneal epithelium.

The aim of the work was to evaluate whether rat corneal epithelial (RtCE) cell line, spontaneously established from rat corneal epithelium in our laboratory, could be used for the evaluation of cornea inflammatory state. Production of proinflammatory cytokines IL-1beta, IL-6 and TNF-alpha by RtCE line in response to a non-specific irritating agent (Triton) was tested. Supernatants from RtCE cells treated for 1 h with 20 microM, 50 microM and 100 microM Triton, were collected after 1 and 24 h, and tested with enzyme-linked immunosorbent assay (ELISA) for IL-1beta, IL-6 and TNF-alpha. The control groups did not produce significant levels of any of the cytokines. However, after stimulation with Triton, the cells did not produce TNF-alpha, while the concentration of IL-1beta and IL-6 increased over 10 times. These results show that in response to a proinflammatory agent RtCE line produces cytokines that could be used for measuring the effect of irritants on the cornea.

Influence of culture medium pH on cytotoxicity of CD95L in vitro.

CD95L belongs to the tumor necrosis factor-alpha (TNF-alpha) family, the members of which induce apoptosis by activation of their specific receptors. However, there are a few publications suggesting that two of these factors, TNF-alpha and TNF-beta, are able to reveal cytotoxic effect in pH-dependent manner. Therefore we investigated, whether CD95L may also reveal pH-dependent cytotoxicity. We analyzed influence of CD95L on U937 and K562 human cell lines at pH 5.1 and pH 7.4 using radioactive chromium release and tetrazolium salt (MTT) reduction assays. Expression of CD95 in both cell lines was estimated using RNase Protection Assay and FACS analysis. It has been found that short incubation of cells at pH 5.1 did not visibly affect their viability, as measured after 16 or 20 h. Incubation of U937 with CD95L at pH 7.4 resulted in a dose-dependent cell cytotoxicity. The effect was significantly augmented by incubation of cells with CD95L at pH 5.1. K562 cell line was resistant to CD95L at pH 7.4. This result correlated with the lack of CD95 expression in K562 cells. However, incubation at pH 5.1 resulted in a sensitization of K562 cells to CD95L. Our results suggest that CD95L, similarly to TNF-alpha, is able to reveal its cytotoxic activity in a receptor-independent manner and this activity strongly depends on pH of the environment.

Butyric acid enhances in vivo expression of hTNF-alpha in transduced melanoma cell line.

Butyric acid (NaBut) and its derivatives are well-known agents eliciting tumor cell differentiation and apoptosis. In experimental models, NaBut is also used to enhance the efficacy of viral vectors. With the use of B78 murine melanoma cells transduced with the retroviral vector containing human tumor necrosis factor alpha (hTNF-alpha) gene, we investigated the ability of NaBut to increase the cytokine expression. We observed an increase in hTNF-alpha expression in vitro after incubation with NaBut. We also describe that the NaBut pro-drug tributyrin is able to increase hTNF-alpha expression in transduced B78 cells in a tumor vaccination model in mice. This observation strongly suggests a novel potential role for NaBut and its derivatives in tumor therapy. It could be used not only as a therapeutic directly acting on tumor cells but, in parallel, as a genetic vaccine "enhancer".

[Induction of apoptosis by receptors for factors from the TNF family]. / Indukcja apoptozy przez receptory dla czynników z rodziny TNF.

Mechanisms leading to induction of apoptosis by TNF family receptors involve intracellular activation of cysteinyl-aspartate-specific proteases (caspases). Caspase activation requires engagement of adaptor proteins. It is plausible, that caspase activation is sufficient for cell death in course of receptor-dependent induction of apoptosis. However, there are some data that programmed cell death involves also generation of ceramides, arachidonic acid metabolism, or MAP kinase (SAPK/JNK) activation. On the other hand, TNF receptor family triggers some protective, anti-apoptotic mechanisms, i.e. protein kinase C (PKC) and NF-kappa B. The outcome of induction of apoptosis by TNF receptor family depends on the cell type, its physiological condition and influence of environmental factors.

Microwave-assisted staining of mucosal mast cells and granulated intra-epithelial lymphocytes after formalin fixation.

Rat jejunum was fixed with either formalin or methanol-formalin acetic acid (MFAA) and stained with Astra Blue or Alcian Blue with or without microwave irradiation. Staining of both mucosal mast cells and granulated intra-epithelial lymphocytes after formalin fixation was considerably improved by microwave irradiation. On the other hand, microwave irradiation slightly impaired staining of mucosal mast cells (MMC) and even more strongly granulated intra-epithelial lymphocytes (GIEL) after MFAA fixation.

Differences in cartilage formed intramuscularly or in joint surface defects by syngeneic rat chondrocytes isolated from the articular-epiphyseal cartilage complex.

Syngeneic rat chondrocytes isolated from the articular-epiphyseal cartilage complex were suspended in hyaluronic acid and transplanted intramuscularly or into joint surface defects. Transplants were fixed in ruthenium hexammonium trichloride and embedded in glycol methacrylate. In cartilage nodules produced intramuscularly, chondrocyte hypertrophy and matrix calcification were observed after 2 wk. Partial ossification occurred after 4 wk and the cartilage was almost completely replaced by an ossicle after 8 wk. Only small, dispersed groups of chondrocytes remained within the ossicle. In cartilage formed in joint surface defects a superficial and a deep zone were distinguished. Chondrocytes in the superficial zone did not hypertrophy and cartilage remained unossified. In the deep zone matrix calcification and bone formation occurred. These processes were, however, retarded in comparison with intramuscular transplants. Thus, either intraarticular environment exerted an inhibitory effect on chondrocyte hypertrophy and matrix calcification or articular chondrocytes present among transplanted cells accumulated close to the joint lumen and reconstructed normal articular cartilage.