CALCIUM HYDROXYLAPATITE MICROSPHERES - BIOCOMPATIBILITY AND CLINICAL EFFECTS.

Soft tissue fillers are used in aesthetic medicine for volumizing and for contouring. Calcium hydroxylapatite (CaHA) is a fully biodegradable biostimulatory filler. The study presents results of in vitro biocompatibility and cytotoxicity investigations performed on CaHA and illustrates its clinical effects. We used a human cell culture model for in vitro studies with HaCaT keratinocytes and human dermal fibroblasts, known to be a sensitive cell type for biocompatibility and cytotoxicity. Cells were exposed to different concentrations of CaHA (Radiesse®). Cell proliferation was calculated by luminometric adenosine triphosphate (ATP) measurement using the ATPLiteTM-M Assay. Possible cytotoxic effects were detected by the calorimetric Cytotoxicity Detection Kit. Clinical data were obtained from our own treatment files. CaHA did neither inhibit cell proliferation nor cause cytotoxicity. Clinical data suggest an excellent tolerability and long-term efficacy superior to hyaluronic acid-based fillers. CaHA is a versatile and well-tolerated biodegradable and biostimulatory filler.

Disturbed glutathione antioxidative defense is associated with structural brain changes in neuroleptic-naïve first-episode psychosis patients.

BACKGROUND: Oxidative stress and impaired antioxidant defense are reported in schizophrenia and are thought to be associated with disturbed neurodevelopment, brain structural alterations, glutamatergic imbalance, negative symptomatology, and cognitive impairment. To test some of these assumptions we investigated the glutathione (GSH) antioxidant defense system (AODS) and brain structural abnormalities in drug-naïve individuals with first acute episode of psychosis (FEP). METHOD: The study involved 27 drug-naïve FEP patients and 31 healthy controls (HC). GSH AODS markers and TBARS (thiobarbituric acid reactive substances) were measured in blood plasma and erythrocytes. High-resolution T1-weighted 3T MRI were acquired from all subjects. To investigate brain structural abnormalities and effects of illness on interactions between GSH metabolites or enzyme activities and local grey matter density, voxel-based morphometry (VBM) with the computational anatomy toolbox (CAT12) was used. Symptomatology was assessed using the Positive and Negative Syndrome Scale (PANSS) and the Symptom Checklist 1990 revised (SCL-90-R). RESULTS: (i) In FEP patients, glutathione reductase activity (GSR) was lower than in the HC group. GSR activity in plasma was inversely correlated with SCL-90-R scores of depression and PANSS scores of the negative symptom subscale. (ii) A reduction of GM was observed in left inferior frontal, bilateral temporal, as well as parietal cortices of FEP patients. (iii) Interaction analyses revealed an influence of illness on GSR/GM associations in the left orbitofrontal cortex (BA 47). CONCLUSION: Our findings support the notion of altered GSH antioxidative defense in untreated acute psychosis as a potential pathomechanism for localized brain structural abnormalities. This pathology relates to a key brain region of social cognition, affective motivation control and decision making, and is clinically accompanied by depressive and negative symptoms.

Epidermal changes during UVB phototherapy assessed by multiphoton laser tomography.

BACKGROUND: Multiphoton laser tomography (MPT) is a non-invasive technique that allows imaging of skin in vivo with very high spatial resolution and contrast. Previous work of our group has demonstrated that known morphological changes due to erythematogenic ultraviolet B (UVB) irradiation may be imaged in vivo by MPT. The present work investigated if morphological skin changes known from experimental erythematogenic UVB irradiation are also demonstrable in the course of a standard phototherapy regime that implies suberythematogenic doses of narrow band UVB. METHODS: Sixteen patients with psoriasis vulgaris receiving a narrow band phototherapy were included. A test field and a light-protected control field were measured with the multiphoton tomograph DermaInspect® at four time points: at baseline, the next day, after 3 days and at the day of the last exposure. RESULTS: In the course of the UVB phototherapy, spongiosis and pleomorphy as parameters of inflammation and cellular damage did not show significant changes. By contrast, an adaptive skin reaction with significant changes of keratosis and pigmentation was observed. CONCLUSION: MPT is a suitable technique for the investigation of qualitative and quantitative skin changes after UVB irradiation. After suberythematogenic UVB irradiation, photoadaptive skin changes, but no cellular damage can be observed with MPT.

Typha capensis (Rohrb.)N.E.Br. (bulrush) extract scavenges free radicals, inhibits collagenase activity and affects human sperm motility and mitochondrial membrane potential in vitro: a pilot study.

The biodiversity in South Africa provides more than 30,000 higher plants, of which more than 3000 are used by traditional healers to treat diseases. Typha capensis (bulrush) is one of the medicinal plants used in South Africa to treat male fertility problems. Considering that South African traditional healers have been recognised by Law and the health benefits of T. capensis have not been scientifically investigated yet, this study aimed at investigating the in vitro effects of aqueous extracts from this plant on male reproductive functions. Both leaves and rhizomes of T. capensis were dried, infused with distilled water and freeze-dried. Motile sperm from 50 men were isolated by swim-up and incubated with 1 µg ml(-1) aqueous extract of Typha rhizome for 1 h at 37 °C. Vitality, motility, sperm production of reactive oxygen species and mitochondrial membrane potential were analysed in the test sample, a control and in the pellet from the swim-up. Results showed that the rhizome extract had significant (P < 0.0001) negative effects on all parameters. The extracts from the leaves and rhizomes revealed dose-dependent inhibitory activity for collagenase and free radical formation. No inhibitory activity for elastase was found. The inhibitory activity for collagenase might indicate possible anti-cancer effects.

Molecular-weight-dependent toxic effects of chitosans on the human keratinocyte cell line HaCaT.

The cationic polysaccharide chitosan possesses bioactive properties such as antimicrobial activity, antitumor effects, hemostatic assets and positive effects on wound healing. The influence of polycations like chitosan on human cells has been reported to depend on their molecular weight. However, the mechanism of cytotoxicity caused by polycations is not yet fully understood. In the study presented, the influence of two chitosans with a similar degree of deacetylation but different molecular weight, chitosan 1130 (120 kDa) and chitosan oligosaccharide (5 kDa), on the human keratinocyte cell line HaCaT was analyzed. The results obtained indicate that chitosans exhibit a molecular-weight-dependent negative effect on HaCaT cell viability and proliferation in vitro. The chitosans tested also stimulated the release of inflammatory cytokines by HaCaT cells depending on incubation time and concentration. Chitosan 1130 and chitosan oligosaccharide induced apoptotic cell death which was mediated by activation of the effector caspases 3/7. At least for chitosan 1130, the involvement of both, extrinsic and intrinsic signal pathways, was shown by activation of caspases 8 and 9.

Influence of cyclodextrins on the proliferation of HaCaT keratinocytes in vitro.

Safety and efficacy of pharmaceutical agents can be greatly improved by encapsulation within, or covalent attachment to, a biomaterial carrier. Drug delivery systems must deliver the necessary amount of drug to the targeted site for a necessary period of time, both efficiently and precisely. Various kinds of high-performance biomaterials are being constantly developed for this purpose. Cyclodextrins are potential candidates for such a role, because of their ability to alter physical, chemical, and biological properties of guest molecules through the formation of inclusion complexes. The alpha-, beta-, and gamma-cyclodextrins are widely used natural cyclodextrins, consisting of six, seven, and eight D-glucopyranose residues, respectively, linked by -1,4 glycosidic bonds into a macro cycle. Each cyclodextrin has its own ability to form inclusion complexes with specific guests, an ability, which depends on a proper fit of the guest molecule into the hydrophobic cyclodextrin cavity. The most common pharmaceutical application of cyclodextrins is to enhance the solubility, stability, and bioavailability of drug molecules. Such kinds of ligand-receptor complexes can be used for different applications, e.g., for a transdermal therapeutic system (TTS) or in biofunctional textiles. The aim of this study was the investigation of the influence of the different cyclodextrins on the cell proliferation using HaCaT keratinocytes as an in vitro test system. Moreover, the study was performed to find harmless and nontoxic cyclodextrin concentrations for dermal applications. By means of different independent in vitro tests could be confirmed that alpha-, beta-, and gamma-cyclodextrins in concentrations up to 0.1% (w/v) do not show any antiproliferative influence on HaCaT keratinocytes. Sometimes even proliferative effects could be found. However, all used cyclodextrins (besides gamma-cyclodextrin and its derivatives) in concentrations of 0.5 and 1% (w/v), respectively, exert a cytotoxic influence on the proliferation of HaCaT keratinocytes. On the basis of these findings, the following rank order of cyclodextrins regarding their cytotoxicity was proposed: M-beta-CD > beta-CD > HP-beta-CD > alpha-CD > (gamma-CD). It could be confirmed that beta-CD and M-beta-CD trigger the activity of the effectors caspases -3 and -7. A significant increase of LDH release could be found for beta-CD and methyl-beta-CD in concentrations of 0.5 and 1% (w/v). The calculated cytotoxicity amounted 45 and 79%, respectively. The measurements of the interleukins IL-6 and IL-8 confirmed the findings of the proliferation assays as well as the LDH measurements. These findings may provide further rationale to the use of CDs in topical formulations for dermal and transdermal drug delivery or as raw material to functionalize textiles for medical applications.

Effect of caffeine and testosterone on the proliferation of human hair follicles in vitro.

BACKGROUND: Androgenetic alopecia (AGA) is a common problem in men of all ages, affecting approximately 50% at 50 years of age. The underlying cause is an androgen-dependent miniaturization of genetically predetermined hair follicles. Here, the hair organ culture model was used to investigate the effects of testosterone and caffeine; the latter being a promising candidate for hair growth stimulation. METHODS: Hair follicles from 14 biopsies, taken from the vertex areas from male AGA patients, were cultivated for 120-192 h in vitro with normal William's E medium (control) or William's E medium containing different concentrations of testosterone and/or caffeine. Hair shaft elongation was measured daily and at the end of cultivation, cryosections of follicles were stained with Ki-67 to evaluate the degree and localization of keratinocyte proliferation. RESULTS: Significant growth suppression was found in hair follicles treated with 5 microg/ml testosterone. This was counteracted by caffeine in concentrations of 0.001% and 0.005%. Moreover, caffeine alone led to a significant stimulation of hair follicle growth. These results were confirmed immunohistochemically by Ki-67 staining. CONCLUSIONS: Androgen-dependent growth inhibition of ex vivo hair follicles from patients suffering from AGA was present in the human hair organ culture model, a constellation which may serve for future studies to screen new substances against androgen-dependent hair loss. Caffeine was identified as a stimulator of human hair growth in vitro; a fact which may have important clinical impact in the management of AGA.

Induction of apoptosis in HaCaT cells by photodynamic therapy with chlorin e6 or pheophorbide a.

The two photosensitizers, chlorin e6 and pheophorbide a, were tested in an in vitro model of topical photodynamic therapy (PDT). Both dyes accumulate in HaCaT keratinocytes as verified by fluorescence measurement but pheophorbide a is enriched fivefold more strongly than chlorin e6 after 24 h. HaCaT cells are susceptible to PDT with both dyes. The phototoxicity measured by ATP bioluminescence is caused by necrosis and apoptosis depending on the photosensitizer used and the treatment modality. Chlorin e6 shows higher toxic potential because it elicits nearly 90% cell mortality 24 h after PDT comparable to pheophorbide a but with a fivefold lower rate of accumulation. These results implicate caution with topical PDT of oncologic diseases due to the risk of serious side effects on healthy skin in the course of topical photodynamic treatment. But the lack of dark toxicity and the time-dependent enrichment of both dyes in HaCaT cells are arguments for the application of these sensitizers in topical PDT of non-malign skin disorders. Further studies are necessary to discover appropriate lower doses and mechanisms of action of topical PDT with both compounds.

Investigations of interactions of chlormezanone racemate and its enantiomers on human keratinocytes and human leucoytes in vitro.

Chlormezanone is a centrally acting muscle relaxant introduced in human therapy as a racemic substance. The following investigation was performed in order to investigate whether the racemate and both enantiomers differ in their potential cytotoxicty in vitro. We investigated antiproliferative effects and cytotoxicity (PicoGreen and ATP assay) for human HaCaT keratinocytes, production of oxygen radicals (ROS) by human interleukin-3-stimulated leukocytes (Lucigenin assay) and production of sulfoleukotrienes (Cellular Antigen Stimulation Test - CAST) by human leukocytes. In the dosage range of 0.001 to 0.1 mg/ ml chlormezanone, no antiproliferative effects were measured with the racemate and both enantiomers. At 1.0 mg/ml, a decrease of proliferative activity was seen after 48 h incubation time of about 50% for the enantiomers and of about 80% for the racemate (PicoGreen) and 50% (enantiomers) or 21% (racemate) in the ATP assay, respectively. ROS production was significantly inhibited at concentrations < or =0.01 mg/ ml by the racemate and the (+)-enantiomer, whereas the (-)-enantiomer was less effective. There was no stimulation of sulfidoleukotrienes in human leukocytes by chlormezanone. Present data argue for absence of significant cytotoxicity against human HaCaT keratinocytes and a dose-dependent suppression of ROS production by human leukocytes that is not uniform among the racemate and its enantiomers.

HaCaT cell proliferation influenced by melatonin.

The hormone melatonin is characterized by numerous pharmacological effects. The influence of melatonin on the growth of the human hair follicle was shown in previous investigations. In the present study, the effects of melatonin were investigated by means of proliferation tests of HaCaT keratinocytes using the [3H]thymidine incorporation, a fluorescence assay with Hoechst dye 33342 and the ATP bioluminescence assay. The aim of the study was to find melatonin concentrations suitable for treatments of the skin and whether there is a cytotoxic effect on HaCaT cells. The different proliferative activity of melatonin depending on its concentration and the time of incubation could be shown in all investigations.

Inhibition of signal transduction in EGF-dependent epithelial cell lines.

The epidermal growth factor (EGF) receptor plays a pivotal role in growth regulation of epidermal keratinocytes. Its expression and function can be markedly altered during malignant transformation in squamous cell carcinoma. The present study investigated the potential of growth inhibition by signal-transduction inhibitors in EGF-dependent epithelial cell lines in vitro. Benign HaCaT keratinocytes and malignant A431 cells were grown in vitro and exposed to various concentrations of a panel of eleven kinase and phosphodiesterase inhibitors. Cell growth was measured after 24 h and 48 h using fluorescence labeling with Hoechst 33342 and propidium iodide. Significant growth inhibition was achieved with all inhibitors when applied to HaCaT cells. The strongest growth inhibition was achieved with inhibitors H-7, A3 and diacylglycerol kinase inhibitors I and II. A431 cells were inhibited significantly by H-7, A3 and H-9. Selected signal-transduction inhibitors such as A3, H-7 and H-9 acting on intracellular kinases are capable of suppressing growth of EGF-dependent benign and malignant epithelial cell lines in vitro. They might be of future potential in the treatment of epithelial cancer but further studies are necessary.

Serum matrix metalloproteinase-2 in patients with malignant melanoma.

PURPOSE: Matrix metalloproteinases (MMPs) are a family of structurally related zinc-dependent endopeptidases that are able to degrade extracellular matrix components. MMPs play a role in tumor invasion and tumor metastasis. MMP-2 (also known as gelatinase A) is expressed in human melanoma cells. METHODS: In this study, we measured MMP-2 in 337 serum probes of 166 melanoma patients with a recently developed enzyme immunoassay and compared these data with the tumor stage, presence of metastases, and the levels of S100beta and soluble intracellular adhesion molecule-1 (sICAM-1) in serum. RESULTS: The mean levels were (189.2 +/- 50.8) ng/ml for MMP-2, (263.2 +/- 74.1) ng/ml for sICAM-1, and (0.424 +/- 1.568) U/ml for S100beta. There was a statistical significant correlation of MMP-2 with sICAM-1 (P=0.05) and Sl00beta (P=0.01). The mean MMP-2 levels (in ng/ml) in patients with metastatic melanoma were 196.4 +/- 54.0 versus 182.6 +/- 46.9 in non-metastasizing melanoma (P=0.037). However, there was no significant difference in MMP-2 levels between the different tumor stages. CONCLUSION: Determination of MMP-2 serum levels is of limited value as a tumor marker in melanoma, though there are higher levels in the more advanced disease.

Treatment of stage II cutaneous T-cell lymphoma with interferon alfa-2a and extracorporeal photochemotherapy: a prospective controlled trial.

BACKGROUND: Both interferon alpha and extracorporeal photochemotherapy have been shown to be effective in primary cutaneous T-cell lymphomas (CTCLs). However, no prospective trial has been published on the combination of both treatments, although retrospective investigations suggested a better efficacy than for either interferon or extracorporeal photochemotherapy. OBJECTIVE: Our purpose was to evaluate the efficacy and toxicity of combined interferon alfa-2a with extracorporeal photochemotherapy in a prospective controlled trial. METHODS: A prospective controlled study was performed. Fourteen patients (all male) aged 38 to 72 years with CTCL of the mycosis fungoides type, stage IIa/IIb, and a 72-year-old male patient with a Ki-1 lymphoma were treated twice a month for 6 months with extracorporeal photochemotherapy using oral 8-methoxypsoralen as photosensitizer in combination with interferon alfa-2a subcutaneously 3 times a week in the maximal tolerable dosage (ie, up to 18 x 10(6) U). The effects were investigated by a skin score, staging, histologic score (density of the T-cell infiltrate; from 0 = absent to 3 = heavy), immunohistology, and laboratory investigations including total peripheral T-cell count, CD4/CD8 ratio, and soluble interleukin 2 receptor (sIL-2R). RESULTS: After 6 months, best response was a complete response (CR) in 4 patients, a partial response (PR) in 3, and a stable disease (SD) in 7 of 14 patients (overall response rate [CR + PR] 50%). In responders the time to best response was 4.3 +/- 1.4 months. The skin score decreased from 22.5 +/- 8.1 to 15.1 +/- 11.0 (P <.001), the histologic score decreased from 2.57 +/- 0.51 to 1.21 +/- 0.80 (P <.001). In the lesional skin the percentage of CD4 cells decreased from 75% to 51% (P =.038) and Ki-67-positive cells decreased from 6.7% to 2.4% (P =.001). The total T-cell count/microL decreased from 1018.9 +/- 557.1 to 667.9 +/- 417.9 (P =.012), and the CD4/CD8 ratio also decreased from 1.88 +/- 0.92 to 1.51 +/- 0.67 (P =.038). The sIL-2R levels did not change significantly during the first 4 months of treatment. Among patients of stage IIa the response rate was 60% in contrast to only 25% of those in stage IIb. Side effects were seen temporarily, ranging from grade 0 to grade 3. There was no need for additional therapy, but interferon dose was decreased because of side effects. After 1 year of follow-up the total response rate was 46.2% (6 of 13 patients): 5 of 9 with stage IIa(55.6%) and 1 of 4 with stage IIb (25.0%). CONCLUSION: These results indicate that patients with CTCL stage IIa can achieve a total response rate of 56% with combined interferon alfa-2a and extracorporeal photochemotherapy. Responders seem to experience their best response within the first 6 months of treatment. The treatment is well tolerated and does not cause severe side effects.

17 Alpha-oestradiol and 17 beta-oestradiol do not affect basal and follicle-stimulating hormone-stimulated inhibin B secretion by highly purified rat Sertoli cells.

The effects of 17 alpha-oestradiol and 17 beta-oestradiol on basal and follicle-stimulating hormone (FSH)-stimulated inhibin B secretion by rat Sertoli cells were studied. Sertoli cells were isolated and cultivated from testes of 18-day-old Wistar rats in the presence and absence of FSH and different doses of oestrogens. On day 4 of culture, secreted inhibin was measured by enzyme-linked immunosorbent assay. Neither 17 alpha-oestradiol nor 17 beta-oestradiol had any effect on the secreted inhibin level in either the presence or absence of FSH. It is concluded that these oestradiols do not play an essential role in regulatory processes involving inhibin or FSH.

Effects of adjuvant interferon-alpha low-dose therapy in melanoma patients on serum inhibin B.

Because the primary aim of adjuvant therapy for melanoma is not curative, all the possible aspects of quality of life have to be considered. One aspect of increasing importance is fertility. The effect of adjuvant interferon alpha-therapy for malignant melanoma on male fertility has not been systematically investigated. In the present study, twelve male patients with primary cutaneous melanoma (pT3, 4; N0; M0) who were taking adjuvant low-dose interferon alpha2b (3 x 3 mio U/week) for one year were included. Inhibin B--an established marker of male fertility-was measured with an immunosorbent assay before and after one year of interferon alpha-therapy to investigate whether this treatment has any influence on fertility. The results were compared with those from normal controls (n=40). The mean serum inhibin B concentration in melanoma patients before interferon therapy was 225.4 +/- 112.5 pg/mL; after treatment the level was 229.6 +/- 82.0 pg/mL. This difference was not statistically significant (p>0.05). The serum inhibin B concentration in controls was 201.5 +/- 17.1 pg/mL, which was not statistically different from either untreated or interferon-treated melanoma patients (p>0.05). We conclude that low-dose interferon alpha does not have a significant (negative) effect on inhibin B or male fertility.

Stimulation and scavestrogen-induced inhibition of reactive oxygen species generated by rat sertoli cells.

The ability of Sertoli cells harvested from 18-day-old Sprague-Dawley rats to generate reactive oxygen species (ROS) occurs under unstimulated and stimulated conditions. Thus, the generation of ROS and its regulation by stimulating and inhibiting compounds was determined as a lucigenin-dependent chemiluminescence reaction. According to the data, ROS generation was influenced by different cell preparation conditions--stimulating substances such as PMA, FMLP, C5a, A23187, and scavestrogens characterized by antioxidant, radical-scavenging properties. The mechanical homogenization during cell preparation procedures leads to an increase of ROS generation. ROS generation of Sertoli cells was followed by elected substances in the following rank order of efficacy: PMA > FMLP > or = C5a > Ca-ionophore A23187. The registered inhibiting effects of the scavestrogens J811 and J861 were significant. The measured CL counts decreased at 72 and 77%, respectively, of control experiments done without scavestrogens. The generation of reactive oxygen species in Sertoli cells and especially the increase in oxygen free radicals and their effects on cellular structures of spermatids are directly involved in inducing morphological alterations. Sertoli cells play an important role in spermatogenesis. The measurements of ROS may have clinical relevance in the evaluation of infertility.

Serum protein s100beta in patients with malignant melanoma detected by an immunoluminometric assay.

S100 protein is well established as a diagnostic tool in malignant melanoma immunohistology. In this study we measured S100beta in serum with a recently developed luminometric immunoassay with a detection limit of 0.02 microg/l. By measuring S-100beta in a group of apparently healthy individuals a mean value of 0.031 +/- 0.026 microg/l was found. In the reference group, serum S100beta was below 0.12 microg/l in all cases. To assess the sensitivity of the assay we investigated serum S-100beta levels in 371 serum samples of 315 patients with histological proven malignant melanoma at different disease stages. Staging was performed according to the German Society of Dermatology classification. Significant differences were observed between the control group and stages IIb (P = 0.01) and IV (P = 0.001). In tumour-bearing patients of stages IIIb and IV, the difference was highly significant (P < 0.0001). S100beta > 0.20 microg/l helps to distinguish between tumour-free and tumour-bearing patients with a specificity of 97.0% and a sensitivity of 64.6%. Our results demonstrate the serum S100beta is of limited value as a melanoma marker. However, it has clinical significance for identifying tumour-positive patients in advanced malignant melanoma stages III and IV.