Search

A prototype ejector chiller with nominal cooling power of 40 kW, designed according to the “CRMC” criterion, has been tested by our research group since 2011. The prototype has undergone several refinements and finally has been upgraded to R1233zd(E), which is non-flammable, has low-GWP and favourable thermodynamic properties, i.e. “dry-expansion” and moderate pressure at generator. In terms of saturation pressure curve, the new fluid is similar to R245fa, which was previously used in the prototype. Hence, R1233zd(E) has been basically used as a “drop-in” replacement. The ejector chiller was tested at different evaporation temperatures (2.5 to 10°C), typical of air conditioning applications. Saturation temperatures at generator from 95 to 105°C have been selected as representative of waste-heat recovery. The experimental findings prove that the CRMC ejector has a satisfactory performance with the new working fluid.

This paper numerically investigates the performance of a high capacity, industrial ejector chiller working with the non-flammable, low GWP refrigerant R1233zd(E). The test procedures adopted to acquire the experimental data are explained in part 1 of this work. Different turbulence models were examined in order to accurately predict the ejector off-design transition and critical pressure. A peculiar difference has been noticed between the numerical results obtained when initializing the CFD simulation from the solution of a case with lower or higher condenser pressure. This difference may partly explain the difficulties in the accurate predictions of the critical conditions reported by this and other works in the literature. A detailed discussion is presented on the nature and possible cause of this phenomenon, which is identified as typical of non-linear fluid dynamics.

Myeloid derived suppressor cells (MDSCs) are an immature heterogeneous population of myeloid lineage that attenuate the anti-tumor immune responses. Depletion of MDSCs has been shown to improve efficacy of cancer immunotherapeutic approaches. Here, we produced and characterized a recombinant peptibody capable of recognizing and depleting murine MDSCs. Using SOE-PCR, the coding sequence of the MDSC binding peptide and linker were synthesized and then ligated into a home-made expression plasmid containing mouse IgG2a Fc. The peptibody construct was transfected into CHO-K1 cells by lipofectamine 3000 reagent and the resulting fusion protein was purified with protein G column and subsequently characterized by ELISA, SDS-PAGE and immunoblotting. The binding profile of the peptibody to splenic MDSCs and its MDSC depletion ability were then tested by flow cytometry. The purified peptibody appeared as a 70 kDa band in Western blot. It could bind to 98.8% of splenic CD11b+/Gr-1+ MDSCs. In addition, the intratumoral MDSCs were significantly depleted after peptibody treatment compared to their PBS-treated negative control counterparts (P

An impulse facility for analysis of water vapour nozzle flows using both shock tunnel and Ludwieg tube operating modes has been developed and tested at the University of Southern Queensland. Unique high-speed flow visualisation of the water vapour condensation shock has been acquired in the throat region of a nominally two-dimensional convergent-divergent nozzle with a 120 mm2 throat area. This paper presents the facility performance and the time-resolved visualisation results for the nozzle flow, and the results are analysed with the aid of image post-processing tools and quasi-one-dimensional (Q1D) thermodynamic calculations. The experiments have produced qualitative and quantitative data on the flow conditions at four different relative humidity values from 25% to 100%, and over a range of nozzle supply temperatures between 293 K and 343 K. The direct measurement of the pressures and wave speeds within the tunnel resulted in a good agreement with the ideal gas Q1D calculations, particularly for the first incident shocks and expansion waves. Differences progressively increased with the number of reflections, due to the effect of viscous dissipation and the non-ideal end-wall interaction caused by the presence of the nozzle inlet. Moreover, flow visualizations of the location and orientation of both the weak disturbance waves and the condensation shock enabled the investigation of the supersonic water vapour condensation within the nozzle of this impulse facility. Based on these measurements, the nozzle flow downstream of the condensation shock was demonstrated to have a lower Mach number relative to the dry nitrogen flow case. This phenomenon is due to the phase change heat release, which appeared to be more significant when the condensation shock intensity was higher. Although further development of the apparatus is possible, the impulse test-rig configuration that we have developed permits a relatively high flow rate of test gas for a short period of time, leading to significantly reduced operating expenses relative to a continuous flow facility with the same mass flow rate. It is therefore suggested that future studies further improve and test the impulse facility techniques for studying condensation phenomena as a potential alternative to high-power, continuous flow facilities.

The development of low-cost and sustainable hydrogen production is of primary importance for a future transition to sustainable energy. In this work, the selective and simultaneous production of pure hydrogen and chemicals from renewable alcohols is achieved using an anion exchange membrane electrolysis cell (electrochemical reforming) employing a nanostructured Pd-CeO2/C anode. The catalyst exhibits high activity for alcohol electrooxidation (e.g. 474 mA cm−2 with EtOH at 60 °C) and the electrolysis cell produces high volumes of hydrogen (1.73 l min−1 m−2) at low electrical energy input (Ecost = 6 kWh kgHjavax.xml.bind.JAXBElement@16146147−1 with formate as substrate). A complete analysis of the alcohol oxidation products from several alcohols (methanol, ethanol, 1,2-propandiol, ethylene glycol, glycerol and 1,4-butanediol) shows high selectivity in the formation of valuable chemicals such as acetate from ethanol (100%) and lactate from 1,2-propandiol (84%). Importantly for industrial application, in batch experiments the Pd-CeO2/C catalyst achieves conversion efficiencies above 80% for both formate and methanol, and 95% for ethanol.

Melanoma has increased in incidence worldwide prompting investigators to search for new biomarkers for targeted immunotherapy of this disease. Placenta specific 1 (PLAC1) is a new member of cancer-testis antigens with widespread expression in many types of cancer. Here, we aimed to study for the first time the expression pattern of PLAC1 in skin cancer samples including cutaneous melanoma, basal cell carcinoma (BCC), squamous cell carcinoma (SCC) in comparison to normal skin and nevus tissues and potential therapeutic effect of anti-PLAC1 antibody in melanoma cancer cell linesPolyclonal and monoclonal antibodies were applied for immunohistochemical profiling of PLAC1 expression using tissue microarray. The cytotoxic action of anti-PLAC1 antibody alone or as an antibody drug conjugate (with anti-neoplastic agent SN38) was investigated in melanoma cell lines.We observed that 100% (39 of 39) of melanoma tissues highly expressed PLAC1 with both cytoplasmic and surface expression pattern. Investigation of PLAC1 expression in BCC (Our findings re-inforce the concept of re-expression of embryonic/placental tissue antigens in cancer and highlight the possibility of melanoma targeted therapy by employing anti-PLAC1 antibodies. The data presented here should lead to the future research on targeted immunotherapy of patients with melanoma.

Fluorescent dyes are excited by light and emit light at a longer wavelength. Photobleaching is one the most important obstacles in fluorescent image capturing. Photochemical alteration of a fluorescent dye caused by several excitation/emission cycles results in a fluorophore to be unable to emit light. In this study, R-phycoerythrin (R-PE) and Alexa Fluor 568 were separately conjugated to streptavidin. The efficiency of conjugations, R-PE-streptavidin and streptavidin-Alexa Fluor 568, were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and spectrophotometry, respectively. Herceptin, a humanized therapeutic antibody, was subsequently biotinylated. The reactivity of biotin-labeled Herceptin was examined by enzyme-linked immunosorbent assay. The photobleaching of R-PE-streptavidin and streptavidin-Alexa Fluor 568 were then compared in an immunofluorescent staining on a breast cancer cell line, BT-474. Our data showed that streptavidin-Alexa Fluor 568 was more photostable than R-PE-streptavidin, which provides more time for longer viewing of labeled proteins and image capturing.

AIM Placenta-specific 1 (PLAC1) is among recently-discovered placental antigens which exerts fundamental role in placental function and development. Increasing body of literature shows that PLAC1 is frequently activated and expressed in a wide variety of human cancers and promote cancer progression. However, no data is available regarding the expression of mouse orthologue, plac1, in murine cancer cell lines. Materials and Methods: We investigated the expression of plac1 in a series of murine cell lines from different histological origins, mammary carcinoma (4T1), melanoma (B16F10), colorectal carcinoma (CT26), renal carcinoma (Renca), glioma (GL26), B-cell lymphoma (A20 and BCL1) and also two fibroblast cell lines (NIH3T3 and L929), using RT-PCR, Western blotting and flow cytometry. Results: Our data demonstrated that plac1 transcript and plac1 protein were expressed in all examined cell lines, as judged by RT-PCR and Western blot, respectively. The molecular weight of mouse plac1 was experimentally observed to be approximately 24 kD. Flow cytometric analysis showed surface expression of plac1 in aforesaid cell lines ranging from 2% to 42.5%. Conclusion: Based on the ubiquitous expression of plac1, the investigated cancer cell lines or immortalized cell lines can be used to examine the role of plac1 in the process of immortalization.

Research activity on ejectors is ongoing at the University of Florence since the late nineties. The most important achievement is a 40 kW ejector chiller designed according to the “CRMC” criterion. The experimentally validated CFD simulations have given some hints about some possible improvements, i.e. refine the surface finish of the ejector, study the effect of heat transfer and improve the final part of the diffuser, which in its present shape does not produce a measurable compression. The prototype has been recently filled with low-GWP refrigerant R1233zd, as a drop-in replacement of previously used R245fa. Both fluids are “dry-expanding” and hence significantly easier to model in CFD simulations. Synthetic low-GWP refrigerants may be an option for ejector chillers, due to their ability to reach below-zero temperature and high volumetric refrigerant capacity. Some lessons learned with synthetic refrigerants can be transferred to the project of a steam ejector chiller, which remains one of our future targets. Herein we resume the principal findings gathered by means of experimental and numerical activity on our prototype and propose a few ideas for the future research.

The emergence of immunotherapy has revolutionized medical oncology with unprecedented advances in cancer treatment over the past two decades. However, a major obstacle in cancer immunotherapy is identifying appropriate tumor-specific antigens to make targeted therapy achievable with fewer normal cells being impaired. The similarity between placentation and tumor development and growth has inspired many investigators to discover antigens for effective immunotherapy of cancers. Placenta-specific 1 (PLAC1) is one of the recently discovered placental antigens with limited normal tissue expression and fundamental roles in placental function and development. There is a growing body of evidence showing that PLAC1 is frequently activated in a wide variety of cancer types and promotes cancer progression. Based on the restricted expression of PLAC1 in testis, placenta and a wide variety of cancers, we have designated this molecule with new terminology, cancer–testis–placenta (CTP) antigen, a feature that PLAC1 shares with many other cancer testis antigens. Recent reports from our lab provide compelling evidence on the preferential expression of PLAC1 in prostate cancer and its potential utility in prostate cancer immunotherapy. PLAC1 may be regarded as a potential CTP antigen for targeted cancer immunotherapy based on the available data on its promoting function in cancer development and also its expression in cancers of different histological origin. In this review, we will summarize current data on PLAC1 with emphasis on its association with cancer development and immunotherapy.

Actin is one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells with important roles in many cell functions. Antibodies against β-actin and other housekeeping gene-encoded proteins are used as internal loading controls in Western blot analyses. The aim of this study was to produce a monoclonal antibody (mAb) against a synthetic peptide derived from N-terminal region of β-actin and to study its reactivity with different organisms. A synthetic peptide, derived from β-actin, was designed and used to produce a mAb by hybridoma technology. The produced antibody (clone 4E5- A10) was purified by an affinity chromatography column followed by characterization of purified mAb using SDS-PAGE, ELISA and Western blot. Our results showed that 4E5-A10 was an IgM and had desired purity and excellent reactivity with the immunizing peptide with an affinity constant of 2.7x10 8 M -1 >. It could detect a band of about 45 kDa, corresponding to β-actin, in Western blot. Furthermore, it could react in a more sensitive manner and with a wider range of organisms than a known commercial anti β-actin antibody. Our data suggest that 4E5-A10 can act as a sensitive probe for detection of β-actin as an internal loading control, for a wide range of organisms, in Western blot analyses.

Our recent findings strongly support the idea of PLAC1 being as a potential immunotherapeutic target in prostate cancer (PCa). Here, we have generated and evaluated an anti-placenta-specific1 (PLAC1)-based antibody drug conjugate (ADC) for targeted immunotherapy of PCa. Prostate cancer cells express considerable levels of PLAC1. The Anti-PLAC1 clone, 2H12C12, showed high reactivity with recombinant PLAC1 and selectivity recognized PLAC1 in prostate cancer cells but not in LS180 cells, the negative control. PLAC1 binding induced rapid internalization of the antibody within a few minutes which reached to about 50% after 15 min and almost completed within an hour. After SN38 conjugation to antibody, a drug-antibody ratio (DAR) of about 5.5 was achieved without apparent negative effect on antibody affinity to cell surface antigen. The ADC retained intrinsic antibody activity and showed enhanced and selective cytotoxicity with an IC50 of 62 nM which was about 15-fold lower compared to free drug. Anti-PLAC1-ADC induced apoptosis in human primary prostate cancer cells and prostate cell lines. No apparent cytotoxic effect was observed in in vivo animal safety experiments. Our newly developed anti-PLAC1-based ADCs might pave the way for a reliable, efficient, and novel immunotherapeutic modality for patients with PCa.

Background:HER2 proto-oncogene is critical in the biology of breast cancer and an important therapeutic target of monoclonal antibodies (mAbs). We have recently established a panel of anti-HER2 mAbs recognizing different epitopes within the extracellular domain of HER2. Materials & methods: In the present study the antiproliferative effect of these mAbs was investigated on HER2-overexpressing human breast cancer cell line BT474, using radioactive thymidine incorporation assay. Results: Our results demonstrated that while two of the mAbs (1T0 and 2A8) inhibited cell proliferation dose dependently, similar to trastuzumab, six mAbs (1F2, 1B5, 1H9, 4C7, 1H6 and 2A9) augmented cell proliferation. Treatment of BT474 cells with different combinations of two mAbs induced either synergistic inhibitory or stimulatory effects. Discussion: Our findings indicate that combination of some stimulatory mAbs could completely abolish the inhibitory effect of other mAbs against HER2. Employment of some combinations of mAbs with significant synergistic inhibitory function may improve the therapeutic efficacy of HER2-specific mAbs.

The coproduction of acrylate and hydrogen from 1,3-propandiol is achieved by electrochemical reforming using a nanoparticle Pd on mixed Vulcan XC-72 carbon and ceria electrocatalyst (Pd/C-CeO2). Electrolysis cell parameters (potential, temperature, and flow conditions) are tuned to favor the formation of acrylate with respect to the other oxidation products (3-hydroxypropanoate and malonate). Using high cell temperature combined with a low cell voltage favors the formation of acrylate, the selectivity for which is further enhanced by flowing the 1,3-propandiol solution in single-pass mode rather than recycling through the electrolyzer. Hence, at a cell temperature of 80 °C and at a fixed cell potential of 400 mV using a one pass continuous flow of 1,3-propandiol, 77% of selectivity for acrylate is obtained.

Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami™ and pCold expression vector to yield substantial amount of soluble truncated PLAC1 without further need for solubilization step. Full length PLAC1, however, expressed mostly as inclusion bodies with higher yield in Origami™ and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production.

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin.

Nowadays, fluorescent dyes have become significant tools in immunostaining experiments. Different fluorescent dyes have various physicochemical traits. Some of these traits, for instance photobleaching and photostability, are applicable calibers in such experiments as immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry (FC). In this study, photobleaching, photostability, and mean fluorescence intensity (MFI) of FITC (fluorescein isothiocyanate)- and Dylight488- conjugated Herceptin, the humanized anti Her2 monoclonal antibody drug, were examined. After conjugation of FITC and Dylight488 to Herceptin, their degree of labeling (DOL) was calculated by a spectrophotometer. Photobleaching, photostability, and MFI of conjugates were subsequently investigated by ICC and FC on BT-474 cells. Dylight488 labeled Herceptin revealed higher fluorescent lifetime and photostability than FITC conjugated Herceptin.

Citation Jeddi-Tehrani M, Torabi R, Mohammadzadeh A, Arefi S, Keramatipour M, Zeraati H, Zarnani AH, Akhondi MM, Mahmoudian J, Mahmoudi AR, Zarei S. Investigating association of three polymorphisms of coagulation factor XIII and recurrent pregnancy loss. Am J Reprod Immunol 2010; 64: 212–217 Problem Among important suspected causes of thrombophilia in women with recurrent pregnancy loss (RPL) are the polymorphisms of coagulation factor XIII (FXIII) gene. We performed a case–control study on the association between three polymorphisms of factor XIII (FXIII G103T, FXIII A614T and FXIII C1694T) and RPL in Iranian women. Method of study DNA samples from peripheral blood of 100 female patients with at least two recurrent abortions, as case group, and 100 healthy women with history of at least two successful deliveries were subjected to PCR-RFLP, and the frequencies of the polymorphisms were calculated and compared between the two groups. Results The prevalence of FXIII G103T polymorphism was 29% in the case group and 17% in the control group (P = 0.158). The frequencies of FXIII A614T and FXIII C1694T were 84% and 66% in the case group and 48% and 31% in the control group (P

Actin is one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells with important roles in many cell functions. Antibodies against β-actin and other housekeeping gene-encoded proteins are used as internal loading controls in Western blot analyses. The aim of this study was to produce a monoclonal antibody (mAb) against a synthetic peptide derived from N-terminal region of β-actin and to study its reactivity with different organisms. A synthetic peptide, derived from β-actin, was designed and used to produce a mAb by hybridoma technology. The produced antibody (clone 4E5- A10) was purified by an affinity chromatography column followed by characterization of purified mAb using SDS-PAGE, ELISA and Western blot. Our results showed that 4E5-A10 was an IgM and had desired purity and excellent reactivity with the immunizing peptide with an affinity constant of 2.7x10(8) M(-1). It could detect a band of about 45 kDa, corresponding to β-actin, in Western blot. Furthermore, it could react in a more sensitive manner and with a wider range of organisms than a known commercial anti β-actin antibody. Our data suggest that 4E5-A10 can act as a sensitive probe for detection of β-actin as an internal loading control, for a wide range of organisms, in Western blot analyses.

‎Human CD34 is a transmembrane glycoprotein which is expressed in human hematopoietic stem ‎cells (HSCs) and the small-‎vessel endothelial cells of a variety of tissues. CD34 plays a critical role as a ‎marker for diagnosis ‎and classification of leukemia. Anti CD34 antibodies are used for isolation and ‎purification ‎of HSCs from bone marrow, peripheral blood and cord blood.To characterize a newly produced monoclonal antibody against a human CD34 peptide.Anti CD34 monoclonal antibody (Clone 2C10-D3) was purified from mouse ascitic fluid and hybridoma cell culture supernatants by affinity chromatography and its immune reactivity was examined by ELISA. The purified antibody was further characterized using Western blot and flow cytometry on TF1 (Human Erythroblast) cell line.‎ELISA experiment revealed that the antibody recognized CD34 peptide. Western ‎blot analysis on TF1 ‎cell lysate confirmed the reactivity of the antibody with a 42 KDa protein. Blocking the antibody with a saturating concentration of specific CD34 peptide resulted in loss of its activity with TF1 lysate in Western blot. The 2C10-D3 antibody reacted with TF1 cells in flow cytometry in a similar manner to a commercial anti CD34 monoclonal antibody.‎‎Our data suggest that the anti CD34 monoclonal antibody (Clone 2C10-D3) is an appropriate antibody to study the CD34+ cells by flow cytometry and Western blot.

The receptor tyrosine kinase (RTK) ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL). There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs) and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8) induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375) including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy.

Objective: Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. Based on dye chemical structures, their photobleaching and photostability indices are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate (FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleahing profiles than FITC. Materials and Methods: In this experimental study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleaching profiles and photostability indices. Then, the fluorophore/antibody ratios were calculated using a spectrophotometer. The photobleaching profiles and photostability indices of conjugated antibodies were subsequently studied by immunocytochemistry (ICC). Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software. Results: Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC. Conclusion: Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC.

We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140–250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays.

e22198 Background: The receptor tyrosine kinase ROR1 has important functions during normal embryogenesis. It is overexpressed and of importance for the survival of different malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL). ROR1 inhibition in CLL cells and cell lines with high expression of ROR1 induced specific apoptosis of the cells. There is however little information on the expression and function of ROR1 in melanoma. Here, we investigated ROR1 expression, phosphorylation and the effects of anti-ROR1 monoclonal antibodies (mAbs) and ROR1 suppressing siRNA on seven melanoma cell lines. Methods: ROR1 expression and phosphorylation was analysed in a series of malignant melanoma cell lines using RT-PCR, immunocytochemistry, flow cytometry and western blot. Annexin V/PI staining, MTT assay, PARP and caspase 8 and 9 cleavage as well as MCL-1 protein were used for detection of apoptosis induced by anti-ROR1 mAbs. The cytotoxic effects of anti-ROR1 mAbs were evaluated in the absence or presence of complement (CDC) or immune effector cells (ADCC). Transfection of melanoma cell lines using ROR1 siRNA was used for gene silencing. Results: ROR1 was shown to be overexpressed to a varying degree at protein level. ROR1 was phosphorylated at tyrosine and serine residues. Three out of four anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8) induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in the presence of complement or immune effector cells. ESTDAB081 and 094 cell lines were resistant to direct apoptosis of the four anti-ROR1 mAbs alone compared to the other melanoma cell lines, but not to complement dependent cytotoxicity (CDC) and antibody dependent cell mediated cytotoxicity (ADCC). ROR1 siRNA transfection induced down-regulation of the ROR1 expression both at the mRNA and protein levels proceeded by a significant apoptosis of the melanoma cells. Conclusions: The results indicate that ROR1 may play an important role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable targeted for mAb therapy.

Background Opticin (OPTC) is a member of the small leucine-rich proteoglycan (SLRP) family and is localized particularly in certain extracellular matrices. We have previously reported the unique expression of another SLRP, fibromodulin (FMOD) in the leukemic cells of patients with chronic lymphocytic leukemia (CLL). OPTC is located in the same region as FMOD on chromosome 1 (1q32.1). Cluster up-regulation of genes may be observed in malignancies and the aim of the present study was to analyze the expression of OPTC in CLL cells. Methods The expression of OPTC was tested by RT-PCR and realtime qPCR in PBMC from CLL patients, other hematological malignancies and healthy controls. The presence of OPTC protein, and its subcellular localization, was investigated using fractionation methods where the obtained lysate fractions were analyzed by Western blotting. Deglycosylation experiments were performed to investigate the glycosylation status of the CLL OPTC. Results OPTC was expressed at the gene level in all patients with CLL (n = 90) and in 2/8 patients with mantle cell lymphoma (MCL) but not in blood mononuclear cells of healthy control donors (n = 20) or in tumor samples from nine other types of hematological malignancies. OPTC was detected by Western blot in all CLL samples analyzed (n = 30) but not in normal leukocytes (n = 10). Further analysis revealed a CLL-unique unglycosylated 37 kDa core protein that was found to be located preferentially in the cell nucleus and endoplasmic reticulum (ER) of the CLL cells. Conclusions A 37 kDa unglycosylated OPTC protein was detected in ER and in the nucleus of CLL cells and not in healthy control donors. The function of this OPTC core protein remains unclear but its CLL-specific expression and subcellular localization warrants further investigations in the pathobiology of CLL.