Development and evaluation of RADA-PDGF2 self-assembling peptide hydrogel for enhanced skin wound healing.

Background: Wound healing complications affect numerous patients each year, creating significant economic and medical challenges. Currently, available methods are not fully effective in the treatment of chronic or complicated wounds; thus, new methods are constantly sought. Our previous studies showed that a peptide designated as PDGF2 derived from PDGF-BB could be a promising drug candidate for wound treatment and that RADA16-I can serve as a release system for bioactive peptides in wound healing. Based on that, in this work, we designed a new self-assembling hydrogel RADA-PDGF2, connecting both peptides by a sequence specific for neutrophil elastase, and evaluated its activity in wound healing. Methods: The physicochemical properties of the designed scaffold were analyzed using transmission electron microscopy, atomic force microscopy, cryoSEM microscopies, and circular dichroism spectroscopy. The enzymatic cleavage was performed using human neutrophil elastase and monitored using high-performance liquid chromatography and MS spectroscopic techniques. The aforementioned techniques (HPLC and MS) were also used to assess the stability of the peptide in water and human plasma. The biological activity was analyzed on human skin cells using a colorimetric XTT test, collagen synthesis evaluation, and a migration assay. The biocompatibility was analyzed with LDH cytotoxicity assay and flow cytometric analysis of activation of immune cells. Finally, RADA-PDGF2 activity in wound healing was checked in a mouse dorsal skin injury model. Results: The analysis showed that RADA-PDGF2 can self-assemble, form a hydrogel, and release a bioactive sequence when incubated with human elastase. It shows pro-proliferative and pro-migratory properties and accelerates wound closure in the mouse model compared to RADA16-I. In addition, it is not cytotoxic to human cells and does not show immunogenicity. RADA-PDGF2 seems to be a promising drug candidate for wound management.

Novel and recurrent variants of ATP2C1 identified in patients with Hailey-Hailey disease.

Hailey-Hailey disease (HHD) is a rare, late-onset autosomal dominant genodermatosis characterized by blisters, vesicular lesions, crusted erosions, and erythematous scaly plaques predominantly in intertriginous regions. HHD is caused by ATP2C1 mutations. About 180 distinct mutations have been identified so far; however, data of only few cases from Central Europe are available. The aim was to analyze the ATP2C1 gene in a cohort of Polish HHD patients. A group of 18 patients was enrolled in the study based on specific clinical symptoms. Mutations were detected using Sanger or next generation sequencing. In silico analysis was performed by prediction algorisms and dynamic structural modeling. In two cases, mRNA analysis was performed to confirm aberrant splicing. We detected 13 different mutations, including 8 novel, 2 recurrent (p.Gly850Ter and c.325-3 T > G), and 6 sporadic (c.423-1G > T, c.899 + 1G > A, p.Leu539Pro, p.Thr808TyrfsTer16, p.Gln855Arg and a complex allele: c.[1610C > G;1741 + 3A > G]). In silico analysis shows that all novel missense variants are pathogenic or likely pathogenic. We confirmed pathogenic status for two novel variants c.325-3 T > G and c.[1610C > G;1741 + 3A > G] by mRNA analysis. Our results broaden the knowledge about genetic heterogeneity in Central European patients with ATP2C1 mutations and also give further evidence that careful and multifactorial evaluation of variant pathogenicity status is essential.

Matrix metalloproteinase 2 in tumor cell-induced platelet aggregation: regulation by nitric oxide.

A correlation exists between the ability of tumor cells to aggregate platelets and their tendency to metastasize. Tumor cell-induced platelet aggregation (TCIPA) facilitates the embolization of the vasculature with tumor cells and the formation of metastatic foci. It is well documented that matrix metalloproteinases (MMPs) play an integral part in tumor spread and the metastatic cascade. Therefore, we have examined the role of MMPs during TCIPA and its regulation by nitric oxide (NO) in vitro. Human HT-1080 fibrosarcoma and A549 lung epithelial cancer cells induced TCIPA in a concentration-dependent manner that was monitored by aggregometry. This aggregation resulted in the release of MMIP-2 from platelets and cancer cells, as measured by zymography. HT-1080 cells released significantly more MMP-2 than A549 cells and were more efficacious in inducing TCIPA. Inhibition of MMP-2 with phenanthroline (1-1000 microM), a synthetic inhibitor of MMPs, and by neutralizing anti-MMIP-2 antibody (10 microg/ml) reduced TCIPA induced by HT-1080 cells. TCIPA was abolished by simultaneous inhibition of platelet function with acetylsalicylic acid (100 microM; thromboxane pathway inhibitor), apyrase (250 microg/ml; ADP pathway inhibitor), and phenanthroline. NO donors such as S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione (both at 0.01-100 microM) inhibited TCIPA and MMP-2 release from platelets and tumor cells. The inhibitory actions of S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione were reversed by 1H-[1,2,4]oxadiazole[4,3]quinoxalin-1-one (0.01-30 microM), a selective inhibitor of the soluble guanylyl cyclase. We conclude that (a) human fibrosarcoma cells aggregate platelets via mechanism(s) that are mediated, in part, by MMP-2; (b) NO inhibits TCIPA, in part, by attenuating the release of MMP-2; and (c) these effects of NO are cGMP-dependent.

pS2 expression and response to hormonal therapy in patients with advanced breast cancer.

Seventy-two patients with advanced breast carcinoma (42% bone, 25% visceral, 5.5% soft tissue, and 27.5% multiple site metastases) were evaluated to determine the relationship between tumor expression of the estrogen-regulated protein pS2, estrogen receptor (ER) or progesterone receptor (PgR) content, and response to hormonal therapy. Twenty-nine % of tumors were pS2 positive, 64% were ER positive, and 29% were PgR positive. Of the ER-positive patients (n = 43), 15 (35%) had greater than 10% of the invasive carcinoma which immunostained for pS2 (these were considered pS2 positive). Only 3 of 24 ER-negative tumors were pS2 positive. A weak association between pS2 expression and ER content (P = 0.08) but not PgR content was observed. Of pS2-positive patients, 52% had a partial or complete response to hormonal therapy. In 24% of pS2-positive patients the disease stabilized with treatment. In contrast, 27% of pS2-negative patients had a partial or complete response. In 10% of these patients the disease stabilized. Similar associations between therapeutic response and ER or PgR were not observed. The odds of having a clinical response to hormonal therapy was greater for pS2-positive than for ER- or PgR-positive tumors. pS2 expression may define a subset of ER-positive tumors that are more likely to respond to hormonal treatment.

Cytologically negative pericardial effusion complicating combined modality therapy for localized small-cell carcinoma of the lung.

A 17% frequency of cytologically negative pericardial effusion (CNPE), accompanied in some cases by tamponade, occurred a median of 12.6 months from the onset of treatment for localized small-cell carcinoma of the lung. CNPE was apparently caused by toxicity of radiation/chemotherapy treatment rather than recurrent cancer. The occurrence of CNPE does not appear to represent enhanced toxicity of immediate (as opposed to delayed) concurrent chemoradiotherapy, but may be a consequence of the superior survival status of patients treated in this way. The onset of chest pain and/or dyspnea associated with increase in cardiac silhouette and positive echocardiogram allowed accurate diagnosis. Each instance was relatively easily managed by catheter drainage, and, for some patients, with the addition of nonsteroidal antiinflammatory drugs. It is important to recognize the possibility that a radiation/chemotherapy-related syndrome of pericardial effusion/tamponade may occur so that early diagnosis can be made and the risk of fatal tamponade avoided.

Autoantibodies to steroidogenic enzymes in autoimmune polyglandular syndrome, Addison's disease, and premature ovarian failure.

Autoantibodies to steroidogenic enzymes, steroid 17 alpha-hydroxylase (17 alpha-OH), cytochrome P450 side-chain cleavage enzyme (P450scc), and steroid 21-hydroxylase (21-OH), were measured using specific and sensitive immunoprecipitation assays (IPAs) in patients with various forms of autoimmune adrenal disease. Autoantibodies to 17 alpha-OH were detected in 6 of 11 (55%) patients with autoimmune polyglandular syndrome (APS) type I, 8 of 24 (33%) patients with APS type II, 11 of 56 (20%) patients with adrenal cortex antibody (ACA; measured by immunofluorescence)-positive patients without Addison's disease, and only 3 of 64 (5%) patients with Addison's disease. Autoantibodies to P450scc were found at a prevalence similar to those to 17 alpha-OH: in 5 of 11 (45%) APS type I patients, 10 of 24 (42%) APS type II patients, 11 of 56 (20%) ACA-positive patients without Addison's disease, and only 6 of 64 (9%) patients of the Addison disease group. Autoantibodies to 21-OH were found in a majority of patients with APS type I (7 of 11;64%), APS type II (23 of 24; 96%), Addison's disease (41 of 64; 64%), and ACA-positive patients without Addison's disease (48 of 56; 86%). All sera that were positive for 17 alpha-OH or P450scc were also positive for 21-OH autoantibodies, except in 1 case. There was good agreement between the presence of ACA measured by immunofluorescence and 21-OH antibodies measured by IPA in all patient groups studied, and this indicates that 21-OH is a major autoantigen in adrenal autoimmune disease regardless of whether the disease presents as isolated Addison's disease or APS type I or type II. Autoantibodies to 17 alpha-OH and P450scc appeared to be the major components of the steroid-producing cell antibodies measured by immunofluorescence. No autoantibodies to 21-OH, 17 alpha-OH, or P450scc were detected in 17 sera from patients with premature ovarian failure without evidence of adrenal autoimmunity (as judged by immunofluorescence studies), except for 1 serum in which low levels of 17 alpha-OH antibodies were found. Overall, our studies indicate that 35S-labeled 17 alpha-OH, P450scc, and 21-OH can be used successfully in IPAs for their respective autoantibodies. Assays such as these may well be valuable in the immunological assessment of patients at risk for or suspected of adrenal autoimmunity.

Thyrotropin (TSH) receptor autoantibodies do not appear to bind to the TSH receptor produced in an in vitro transcription/translation system.

An in vitro transcription/translation (TnT) system was used to produce 35S-labeled full-length TSH receptor (TSHR) and TSHR extracellular domain (TSHRex). The interaction of the labeled proteins with TSHR autoantibodies in Graves' sera was then studied using an immunoprecipitation assay. In the assay, 35S-labeled TSHR or TSHRex were incubated with test sera, and any immune complexes formed were precipitated with protein A-Sepharose (in the case of mouse monoclonal antibodies, antimouse IgG-agarose was used). Rabbit antibodies to the TSHR and a mouse monoclonal antibody precipitated as much as 50% of the 35S-labeled TSHR preparations compared with about 2% for normal rabbit serum and 4% for a control monoclonal antibody. However, none of 34 Graves' sera (TSHR autoantibody levels ranging from 14-95% inhibition of [125I]TSH binding) were able specifically to immunoprecipitate 35S-labeled TSHR or TSHRex. These negative findings were confirmed by analysis of the immunoprecipitates on SDS-PAGE followed by autoradiography. Our results indicate that the TnT system is not useful for producing labeled TSHR preparations that can bind TSHR autoantibodies well. This is in contrast to TnT produced 35S-labeled glutamic acid decarboxylase, thyroid peroxidase, and 21-hydroxylase, which react well with their respective autoantibodies. One main difference between these 3 autoantigens and the TSHR is that the receptor is highly glycosylated, and this extensive glycosylation may be of critical importance for correct folding of the receptor. Consequently, the inability of the TnT system to glycosylate proteins could explain in part why TnT-produced 35S-labeled TSHR and TSHRex do not bind TSHR autoantibodies.

Analysis of autoantibody epitopes on steroid 21-hydroxylase using a panel of monoclonal antibodies.

A panel of five mouse monoclonal antibodies (MAbs) to human recombinant steroid 21-hydroxylase (21-OH) were produced, characterized, and used to study the interaction of 21-OH autoantibodies (AAbs) with different epitopes on human 21-OH. AAbs in patients with isolated autoimmune Addison's disease, autoimmune polyglandular syndromes types I and II, and 21-OH antibody-positive patients without overt Addison's disease (25 patients in total) were studied. Four MAbs were IgG1 subclass, one was IgG2a, and all had kappa light chains. The affinities of four of the antibodies were in the range 2.0 x 10(8) M(-1) to 7.0 x 10(8) M(-1), and the affinity of the other was 2.3 x 10(7) M(-1) 21-OH MAbs did not cross-react with 17alpha-hydroxylase (17alpha-OH)) or P450 side chain cleavage enzyme. Studies using a series of 21-OH fragments allowed the identification of short stretches of amino acids (AA) that were involved in forming the MAb binding sites. AA 391-405, defined as epitope region (ER) 1, were found to be important for binding of M21-OH1 and M21-OH2, AA 406-411 (ER2) were important for M21-OH3 and M21-OH4 binding, and AA 335-339 (ER3) for M21-OH5 binding. In addition, MAb Fab or F(ab')2 fragments were used to study 21-OH AAb epitopes in competition experiments. These investigations demonstrated that 21-OH AAbs recognize similar epitopes to the MAbs, with ER2 and ER3 being part of two distinct major epitopes, and ER 1 being part of a minor epitope. Mixtures of M21-OH antibody Fab or F(ab')2 fragments caused almost complete inhibition (80%-95%) of AAb binding in 24 out of 25 sera, and in the case of the remaining serum, the effect was marked but incomplete (67% inhibition). There were no major differences between the binding characteristics of AAbs from patients with different forms of autoimmune adrenal disease. All five 21-OH MAbs reacted with human adrenal tissue in an immunofluorescence test, but only M21-OH1 and M21-OH2 reacted with bovine adrenal tissue in these experiments. None of the MAbs reacted with human ovarian tissue in an immunofluorescence test. Overall, these studies indicate that 21-OH AAbs bind to at least three different epitopes in the C-terminal part of 21-OH, and two of these epitopes appear to be human 21-OH specific.

Steroid 21-hydroxylase autoantibodies: measurements with a new immunoprecipitation assay.

Autoantibodies (Abs) to steroid 21-hydroxylase (21-OH) are a major component of adrenal cortex Abs and are characteristic of autoimmune Addison's disease. We have developed a new method for measuring Abs to 21-OH based on 125I-labeled recombinant human 21-OH produced in yeast. With this assay, 21-OH Abs were detected in 43 of 60 (72%) sera from patients with isolated Addison's disease, 11 of 12 (92%) autoimmune polyglandular syndrome type I sera, 27 of 27 (100%) autoimmune polyglandular syndrome type II sera, and 24 of 30 (80%) sera from patients who were positive for adrenal cortex antibodies by immunofluorescence but had no overt Addison's disease. 21-OH Abs were found by 125I assay in 4 of 150 (2.7%) sera from patients with insulin-dependent diabetes mellitus, 1 of 77 (1.3%) Graves' sera, 1 of 67 (1.5%) Hashimoto's sera, and 6 of 243 (2.5%) sera from healthy blood donors. 21-OH Abs were not detected in 9 sera from patients with Addison's disease due to tuberculosis, 32 sera from patients with noninsulin-dependent diabetes mellitus, 35 sera from patients with myasthenia gravis, or 17 sera from patients with premature ovarian failure. There was good agreement between the 125I-labeled 21-OH assay and an assay based on 35S-labeled 21-OH produced in an in vitro transcription/translation system (r = 0.86; n = 129; P < 0.001). In the case of sera from patients with Addison's disease, insulin-dependent diabetes mellitus, Graves' disease, and Hashimoto's disease and from healthy blood donors that were low positive in the 125I assay, neutralization studies with unlabeled 21-OH confirmed the presence of specific 21-OH Abs. Overall, the 21-OH Ab assay based on 125I-labeled 21-OH showed good sensitivity, precision, and disease group specificity. This, combined with a simple assay protocol and the convenience of 125I handling and counting, make it attractive for routine use. Further investigations with the new assay should allow wider assessment of the prevalence and pattern of inheritance of adrenal autoimmunity. In addition, studies of the effect of treatment or possible preventative measures on 21-OH Ab levels in individuals without overt adrenal failure may suggest ways of delaying the onset of autoimmune Addison's disease.

Patient dosimetry quality assurance program with a commercial diode system.

PURPOSE: To evaluate a commercial silicone diode dosimeter for a patient dosimetry quality assurance program. METHODS AND MATERIALS: The diode dosimeter was calibrated against an ion chamber and percentage depth dose, linearity, anisotropy, virtual source position, and field size factor studies were performed. Correction factors for lack of full scatter medium in the diode entrance and exit dose measurements were acquired. Dosimetry equations were proposed for calculation of dose delivered at isocenter. Diode dose accuracy and reproducibility were tested on phantom and on four patients. A patient dosimetry quality assurance program based on diode measured dose was instituted and patient dose data were collected. RESULTS: Diode measured percentage depth dose and field factors agreed to within 3% with those measured with an ion chamber. The diode exhibited less than 1.7% angular dose anisotropy and less than 0.5% nonlinearity up to 4 Gy. Diode dose measurements in phantom showed that the calculated doses differed from the prescribed dose by less than 1.5%; the diode exhibited a daily dose reproducibility of better than 0.2%. On four selected patients, the measured dose reproducibility was 1.5%; the average calculated doses were all within +/- 7% of the prescribed doses. For 33 of 40 patients treated with a 6 MV beam, measured doses were within +/- 7% of the prescribed doses. For 58 of 63 patients treated with an 18 MV beam, measured doses were within +/- 7% of the prescribed doses. For 11 out of 12 patients, a second repeat measurements yielded doses within +/- 7% of the prescribed doses. CONCLUSIONS: The proposed diode-based patient dosimetry quality assurance program with dose tolerance at +/- 7% is simple and feasible. It is capable of detecting certain serious treatment errors such as incorrect daily dose greater than 7%, incorrect wedge use, incorrect photon energy and patient setup errors involving some incorrect source-to-surface-distance vs. source-to-axis-distance treatments.

Management of locally advanced carcinoma of the breast by primary radiation therapy.

We performed a retrospective review of 137 patients with locally advanced breast cancer, but without distant metastases, who were treated with radical radiation therapy. Ninety percent of patients had an initial complete response to their radiation therapy. The 5 year rates of local tumor control, survival free of distant failure, and overall survival were 54%, 28% and 30%, respectively. Multivariate analysis revealed that the following features were associated with improved local tumor control: clinically negative axillary nodes, excisional biopsy, radiation dose greater than 6000 rad, and the use of adjuvant systemic therapy. Improved freedom from distant relapse was seen in patients with small primaries and non-inflammatory carcinoma, as well as clinically negative axillary nodes, excisional biopsy, radiation dose greater than 6000 rad, and the use of adjuvant systemic therapy. The results suggest that adequate levels of radiation therapy can provide local tumor control in a significant proportion of patients with locally advanced-breast cancer and that adjuvant systemic therapy is useful in improving both local tumor control and freedom from distant relapse in these patients.

Glutamic acid decarboxylase autoantibody assay using 125I-labelled recombinant GAD65 produced in yeast.

We describe a new method for measuring autoantibodies (Ab) to the 65 kDa isoform of glutamic acid carboxylase (GAD65). In particular, GAD65 without the hydrophobic N-terminal region has been produced in yeast, purified, labelled with 125I and reacted with GAD65 Ab. Antibody bound 125I-GAD65 is then precipitated by the addition of solid phase protein A. With the assay, GAD65 Ab were detected in 59 of 71 (83%) islet cell antibody (ICA) positive IDDM patients and in 8 of 23 (35%) ICA negative IDDM patients (overall 67 of 94 (71%) of IDDM patients). Low concentrations of GAD65 Ab were also detected in 2/98 (2%) healthy blood donors and 1/27 (4%) Graves' disease patients had a high level of antibody. GAD65 Ab were not detected in any of 10 Hashimoto's thyroiditis, 20 Addison's disease or 19 myasthenia gravis sera. There was good agreement between the 125I assay and the current reference method based on 35S-labelled full-length GAD65 (produced by in vitro transcription/translation reaction) and solid phase protein A (r = 0.91, n = 108). Overall, our 125I assay showed sensitivity, precision and disease group specificity at least as good as any assay so far described. These features, combined with a simple assay protocol and the convenience of 125I counting and handling indicate that the method is suitable for routine GAD65 Ab measurements.

Fatigue syndrome due to localized radiation.

For cancer patients, fatigue is a disturbing symptom caused by many factors. Since fatigue is the most common side effect of localized radiation to the breast, this treatment provides a unique opportunity to follow patients prospectively as they develop one type of fatigue. We evaluated the effect of radiation treatment in 15 women with Stage I or II node-negative breast cancer who were otherwise healthy. Fatigue, contrary to our hypothesis, did not increase linearly with cumulative radiation dose over time. It dropped from the first to second week and rose in the third week. The cumulative effects reached a plateau in the fourth week (after an average of 17 fractions), which was maintained during the remaining weeks of treatment. Within 3 wk after treatment, fatigue had diminished. No patient had sustained depressive symptoms. Cardiopulmonary exercise capacity in 5 patients at 6 and 12 wk did not change from just before radiation. Other markers, including reverse triiodothyronine and pulse change with orthostatic stress, did not correlate with subjective fatigue nor cumulative radiation in 15 patients. The curve of the fatigue syndrome during treatment conforms to the adaptation of the organism to a continuing stress and begins to describe a mild fatigue syndrome associated with radiation.

Evaluation of the mechanisms of todralazine effect on mutagenicity of benzo(a)pyrene.

We have previously described that todralazine markedly decreased mutagenicity of several indirect- and direct-acting mutagens. In this paper we report the results of experiments conducted in order to evaluate the involvement of desmutagenic and bio-antimutagenic activities in the observed antimutagenic effect of todralazine. The results of the Ames test suggest a bio-antimutagenic, and not desmutagenic effect of todralazine. The separation of B(a)P and their derivatives by thin layer chromatography, performed after in vitro incubation of this promutagen with S9 fraction and todralazine revealed almost complete decline of B(a)P derived products in the presence of todralazine. The results indicate that the observed antimutagenic effect of todralazine on B(a)P mutagenicity is bio-antimutagenic rather than desmutagenic in their nature.

The influence of excipients and technological process on cholecalciferol stability and its liberation from tablets.

The influence of excipients, used in tablet technology, and technological processes on cholecalciferol (1) stability was investigated. It was observed that 1 degrades in the presence of some excipients. The stability of 1 may be increased by antioxidant addition. The best stabilizing effect was achieved by a alpha-tocopherol and ascorbic acid mixture. On the basis of the achieved results, tablets with 1 were prepared. Their physical and chemical properties, the content of 1, its stability and its pharmaceutical availability were determined.

[Microencapsulization of cholecalciferol by coacervation]. / Mikroverkapselung von Cholecalciferol mittels Koazervation.

Microcapsules with crystalline cholecalciferol were obtained by the use of gelatin (type A and B) as a main covering component by both simple and complex coacervation methods. The properties of the obtained microcapsules were determined by their size, release in gastric and intestinal juices, coat/core ratio, the content of cholecalciferol and its liberation. It was observed that both in the simple and complex coacervation, regardless of the type of gelatin used in the procedure, the effectiveness of the process and the properties of the microcapsules depend on coat/core ratio. In simple coacervation method optimal results were achieved when coat/core ratio was 0.25:1. In complex coacervation method optimal ratio was 0.5:1.

[Effect of lanreotide on prolactin level in patients with pituitary mixed tumors]. / Influence du lanréotide sur la concentration sérique de prolactine chez les patients atteints de tumeurs somato-lactotropes.

Acromegaly is a disease caused by a pituitary tumor (somatotropinoma) or by ectopic secretion of GH or IGF-1. About 15% of tumors secrete not only GH but PRL as well. Last time a lanreotide and an octreotide (the somatostatine analogues) are useful in the therapy of acromegaly. We observed the influence of the lanreotide on GH and prolactin. We noticed that the lanreotide caused not only serum level reduction of a growth hormone but also prolactine in patients with mixed pituitary tumors.

[Isolation and purification of transcortin (cortisol binding globulin--CBG) using two-step chromatography]. / Izolacja i oczyszczanie transcortyny globuliny wiazacej kortyzol--(CBG)--za pomoca dwustopniowej chromatografii.

The isolation of cortisol and progesterone binding globulin (CBG) from pregnant women serum was performed using affinity and hydrophobic chromatography. The purity and specificity of isolated transcortin was tested by agarose gel electrophoresis using racket and cross immunoelectrophoresis and specific CBG antibodies. High purity and immunoreactivity of the isolated globulin destitute of other proteins contamination, were obtained.