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Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

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In cancer, activation of the IRE1/XBP1s axis of the unfolded protein response (UPR) promotes immunosuppression and tumor growth, by acting in cancer cells and tumor infiltrating immune cells. However, the role of IRE1/XBP1s in dendritic cells (DCs) in tumors, particularly in conventional type 1 DCs (cDC1s) which are cellular targets in immunotherapy, has not been fully elucidated. Here, we studied the role of IRE1/XBP1s in subcutaneous B16/B78 melanoma and MC38 tumors by generating loss-of-function models of IRE1 and/or XBP1s in DCs or in cDC1s. Data show that concomitant deletion of the RNase domain of IRE1 and XBP1s in DCs and cDC1s does not influence the kinetics of B16/B78 and MC38 tumor growth or the effector profile of tumor infiltrating T cells. A modest effect is observed in mice bearing single deletion of XBP1s in DCs, which showed slight acceleration of melanoma tumor growth and dysfunctional T cell responses, however, this effect was not recapitulated in animals lacking XBP1 only in cDC1s. Thus, evidence presented here argues against a general pro-tumorigenic role of the IRE1/XBP1s pathway in tumor associated DC subsets.

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We describe the synthesis of polymer monoliths inside polypropylene tubes from ink pens. These tubes are cheap, chemically stable, and resistant to pressure. UV-initiated grafting with 5 wt% benzophenone in methanol for 20 min activated the internal surface, thus enabling the covalent binding of ethylene glycol dimethacrylate, also via photografting. The pendant vinyl groups attached a poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith prepared via photopolymerization. These tubes measured 100-110 mm long, with 2 mm of internal diameter. The parent monoliths were functionalized with Na2 SO3 or iminodiacetate to produce strong and weak cation exchangers, respectively. The columns exhibited permeabilities varying from 2.7 to 3.3 × 10-13  m2 , which enabled the separation of proteins at 500 µL/min and back pressures <2.8 MPa. Neither structure collapse nor monolith detachment occurred at flow rates as high as 2.0 mL/min, which produced back pressures between 6.9 and 9.0 MPa. The retention times of ovalbumin, ribonuclease A, cytochrome C, and lysozyme in salt gradient at pH 7.0 followed the order of increasing isoelectric points, confirming the cation exchange mechanism. Separation and determination of lysozyme in egg white proved the applicability of the columns to the analysis of complex samples.

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This paper describes the preparation of polymer monolithic columns in the confines of fluorinated ethylene propylene (FEP) tubes. These tubes are cheap, chemically stable, and widely used in flow analysis laboratories. UV-initiated grafting with 5 wt% benzophenone in methanol for 1 h activated the internal surface walls, thus enabling the further covalent binding of ethylene glycol dimethacrylate (EDMA) from a 15 wt% solution in methanol, also via photografting. Both steps used 254 nm radiation under a potency of 120 mJ cm2. ATR-FTIR measurements revealed the presence of carbonyl, alkyl and vinyl groups in the functionalized FEP. The density of vinyl groups was high enough to firmly attach a poly(lauryl methacrylate-co-ethylene glycol dimethacrylate) monolith in 120 × 1.57 mm i.d. tubes, prepared via photopolymerization. The total preparation lasts less than 2-h. The columns were permeable, (1.58 ± 0.06) × 10-13 m2, providing reproducible chromatographic parameters of retention times, retention factor, selectivity, and resolution. The monoliths were stable at flow rates of 500 µL min-1, collapsing only at flow rates >700 µL min-1, a condition that increased the backpressure over 1000 psi (experiments at the room temperature). The separation of proteins by reversed-phase liquid chromatography demonstrated the efficiency of the columns. Determination of egg white proteins (ovalbumin and lysozyme) and myoglobin in spiked urine proved the applicability to the analysis of real samples.

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OBJECTIVES: It is to prospectively analyze nailfold capillaroscopy (NC) findings in new-onset dermatomyositis (DM) and to correlate NC findings with serum angiogenic cytokines and DM clinical and laboratory features. MATERIALS AND METHODS: Twenty-three patients with DM who experienced < 12 months of symptoms were included in the study. To assess serum cytokine levels, 23 age-, sex-, and ethnicity-matched healthy volunteers were used. NC characteristics and DM activity parameters were analyzed. RESULTS: Significantly higher serum angiogenin (ANG) and vascular endothelial growth factor-1 (VEGF1) levels were observed in DM patients than in controls. Capillary density and avascular areas correlated positively and negatively, respectively, with serum levels of ANG. Moreover, the capillary density correlated inversely with the number of enlarged and giant capillaries and avascular areas. The number of enlarged capillaries correlated positively with patient and physician visual analogue scales (VAS), the presence of a facial rash, giant capillaries, and microhemorrhages. Giant capillaries had a positive correlation with physician and cutaneous VAS, enlarged capillaries, avascular areas, microhemorrhages and bushy capillaries, and a negative correlation with capillary density. Microhemorrhages correlated positively with the "V-neck" sign and physician VAS. VEGF1 showed no relationship with the NC parameters with DM-related clinical and laboratory features. Additionally, 15 out of 23 patients were assessed prospectively after 3.21 years. All patients had a major clinical response with significant improvement in all NC parameters, except for enlarged and bushy capillaries. CONCLUSIONS: The NC may be a useful tool to assess disease activity in recent-onset DM, and it can also reinforce the role of ANG in the angiogenesis of this myopathy.

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BACKGROUND: Until now, there are few studies evaluating serum levels of angiogenic cytokines in dermatomyositis (DM). Therefore, the aims of the present study were: (a) to analyze systematically and simultaneously serum levels of angiogenin (ANG), angiopoietin (ANGPT)-1, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-1 and - 2, platelet derived growth factor (PDGF)-AA and -BB in DM; (b) to correlate the serum level of these cytokines with the DM clinical and laboratory features. METHODS: This is a cross sectional study, in which 48 patients with DM aged 18 to 45 years were gender-, age- and ethnicity-matched with 48 healthy individuals (control group). The serum levels of cytokines analyses were performed by multiplex immunoassay. The parameters of DM activity were based on the scores established by the International Myositis Assessment & Clinical Studies Group. RESULTS: The mean ages, gender frequencies and ethnicities were comparable between the patients with DM and the control group. A significantly higher serum FGF-1 and FGF-2 levels (P < 0.001 and P < 0.001, respectively), lower VEGF and PDGF-AA levels (P = 0.009 and P = 0.022), and comparable ANG, ANGPT-1 and PDGF-BB levels were observed in DM patients compared to controls. There was a tendency for cytokines (with the exceptions of VEGF and PDGF-BB) to correlate positively with the DM activity parameters, whereas FGF-2 correlated inversely. Moreover, FGF-1 strongly correlated with DM cutaneous manifestations. CONCLUSIONS: The present data provide the relevance of different serum angiogenic cytokines in patients with DM. Additional studies will be needed to validate the data obtained in this work.

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Foot ulceration is one of the most common and complex sequelae of diabetes mellitus, generally posing a therapeutic challenge due to poor healing responses and high rates of complications, including peripheral vascular disease, ischemia and infections. Calcitriol, the most active vitamin D metabolite, induces antimicrobial peptides production in keratinocytes from diabetic foot ulcers (DFU); however, little is known about its effects on angiogenic factors in this pathology. Herein we aimed at studying whether calcitriol induces angiogenic molecules in keratinocytes under normoxic and hypoxic conditions, and if these molecules are able to improve cell migration in vitro. Evaluation of DFU samples by immunohistochemistry showed increased VEGF and decreased angiogenin and HIF-1α expression compared to controls, suggesting an altered pattern of angiogenic factors in DFU. Interestingly, incubation of keratinocytes with calcitriol significantly upregulated VEGFA, HIF-1α and angiogenin gene expression, while the resulting cell culture media stimulated both endothelial cells and keratinocytes migration in an in vitro wound closure assay under a normoxic environment (p<0.05). Moreover, the culture media of calcitriol-treated keratinocytes stimulated cell migration in a similar extent as exogenous VEGF or EGF in endothelial and keratinocytes cells. These results suggest that the altered profile of angiogenic molecules in DFU might be improved by local or systemic treatment with calcitriol under normoxic conditions, which could probably be achieved with hyperbaric oxygen therapy. Given that calcitriol not only augments proangiogenic factors but also induces antimicrobial peptides expression, this hormone should be further investigated in clinical trials of DFU.

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PURPOSE:: To investigate the effect of nicotinamide on the secretion of pro-an giogenic and pro-inflammatory cytokines in uveal melanoma cell lines. METHODS:: Two human uveal melanoma cell lines (92.1 and OCM-1) were treated with nicotinamide (10 mmol/L) or control media for 48 hours in culture. The su perna tant from each culture was used in sandwich enzyme-linked immuno sorbent assay-based angiogenesis and inflammation arrays to evaluate the effects of exogenously administered nicotinamide on the secretion of a total of 20 pro-an gio genic and pro-inflammatory proteins. RESULTS:: Seven pro-angiogenic cytokines were detected under control conditions for both uveal melanoma cell lines. Treatment with nicotinamide resulted in a significant decrease in secretion of the following pro-angiogenic cytokines: angiogenin, angiopoietin-2, epidermal growth factor, and vascular epithelial growth factor-A in the 92.1 cells; basic fibroblast growth factor in the OCM-1 cells; and placenta growth factor in both cell lines. Among the pro-inflammatory proteins, monocyte chemotactic protein-1 and interleukin-8 were expressed in both untreated cell lines and both were significantly reduced when treated with nicotinamide. CONCLUSIONS:: Results from this in vitro model suggest that nicotinamide may have anti-inflammatory and anti-angiogenic properties, which may open the possibility of using it as a chemopreventive agent for uveal melanoma; however, further studies including animal models are warranted.

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pH is a key parameter for technological and biological processes, intimately related to biomolecular charge. As such, it controls biomolecular conformation and intermolecular interactions, for example, protein/RNA stability and folding, enzyme activity, regulation through conformational switches, protein-polyelectrolyte association, and protein-RNA interactions. pH also plays an important role in technological systems in food, brewing, pharma, bioseparations, and biomaterials in general. Predicting the structure of large proteins and complexes remains a great challenge experimentally, industrially, and theoretically, despite the variety of numerical schemes available ranging from Poisson-Boltzmann approaches to explicit solvent based methods. In this work we benchmark a fast proton titration scheme against experiment and several theoretical methods on the following set of representative proteins: [HP36, BBL, HEWL (triclinic and orthorhombic), RNase, SNASE (V66K/WT, V66K/PHS, V66K/Δ+PHS, L38D/Δ+PHS, L38E/Δ+PHS, L38K/Δ+PHS), ALAC, and OMTKY3]; routinely used in similar tests due to the diversity of their structural features. Our scheme is rooted in the classical Tanford-Kirkwood model of impenetrable spheres, where salt is treated at the Debye-Hückel level. Treating salt implicitly dramatically reduces the computation time, thereby circumventing sampling difficulties faced by other numerical schemes. In comparison with experimental measurements, our calculated pKa values have the average, maximum absolute, and root-mean-square deviations of 0.4-0.9, 1.0-5.2, and 0.5-1.2 pH units, respectively. These values are within the ranges commonly observed in theoretical models. They are also in the large majority of the cases studied here more accurate than the NULL model. For BBL, ALAC, and OMTKY3, the predicted pKa are closer to experimental results than other analyzed theoretical data. Despite the intrinsic approximations of the fast titration scheme, its robustness and ability to properly describe the main system physics is confirmed.

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ABSTRACT Purpose: To investigate the effect of nicotinamide on the secretion of pro-an giogenic and pro-inflammatory cytokines in uveal melanoma cell lines. Methods: Two human uveal melanoma cell lines (92.1 and OCM-1) were treated with nicotinamide (10 mmol/L) or control media for 48 hours in culture. The su perna tant from each culture was used in sandwich enzyme-linked immuno sorbent assay-based angiogenesis and inflammation arrays to evaluate the effects of exogenously administered nicotinamide on the secretion of a total of 20 pro-an gio genic and pro-inflammatory proteins. Results: Seven pro-angiogenic cytokines were detected under control conditions for both uveal melanoma cell lines. Treatment with nicotinamide resulted in a significant decrease in secretion of the following pro-angiogenic cytokines: angiogenin, angiopoietin-2, epidermal growth factor, and vascular epithelial growth factor-A in the 92.1 cells; basic fibroblast growth factor in the OCM-1 cells; and placenta growth factor in both cell lines. Among the pro-inflammatory proteins, monocyte chemotactic protein-1 and interleukin-8 were expressed in both untreated cell lines and both were significantly reduced when treated with nicotinamide. Conclusions: Results from this in vitro model suggest that nicotinamide may have anti-inflammatory and anti-angiogenic properties, which may open the possibility of using it as a chemopreventive agent for uveal melanoma; however, further studies including animal models are warranted.

RESUMO Objetivo: Acredita-se que a nicotinamida (NIC) seja capaz de diminuir a angiogênese induzida pelo fator de crescimento endotelial vascular (VEGF). Investigar os efeitos da nicotinamida sobre a secreção de citocinas pró-angiogênicas e pró-inflamatórias em linhagens de células de melanoma uveal humano (UM). Métodos: Duas linhagens de células humanas de UM (92,1 e OCM-1) foram tratadas com NIC (10 mmol/L) ou apenas com meio de cultura por 48 horas. O sobrenadante das culturas obtido após a administração de nicotinamida foi comparado com o sobrenadante das culturas controle quanto à expressão de 20 fatores pró-angiogênicos e pró-inflamatórios, pela técnica de enzyme-linked immunosorbent assay (ELISA). Resultados: Sete citocinas pró-angiogênicas foram detectadas nas condições de controle em ambas as linhagens de células de UM. O tratamento com nicotinamida promoveu uma redução significativa da secreção das seguintes citocinas angiogênicas: Angiogenina, ANG2, EGF e VEGF-A em células 92.1; bFGF em células OCM-1; PIGF em ambas as linhagens celulares. Quanto às proteínas pró-inflamatórias, a expressão de MCP-1 e IL-8 foi significativamente reduzida com a administração de nicotinamida em relação às culturas de células que não receberam o tratamento. Conclusões: Nicotinamida apresenta propriedades anti-inflamatórias e anti-angiogênicas em modelo experimental in vitro. Tais efeitos sugerem a possibilidade de utilizar esta substância na quimioprevenção do UM. Entretanto, estudos com modelos experimentais in vivo são necessários para melhor avaliar o benefício do tratamento do UM com nicotinamida.

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