Immunohistochemical detection of the chondroitin sulfate proteoglycan 4 protein in primary and metastatic melanoma.

Treatment of malignant melanoma, the most aggressive form of skin cancer, continues to be a major challenge for clinicians. New targeted therapies with kinase inhibitors or drugs which modify the immune response are often accompanied by the development of resistance or severe side effects. In this context, chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, could be a potential target for alternative therapeutic approaches. The aim of the present study was to identify differences in the levels of CSPG4 protein expression in primary and metastatic melanomas as well as to analyze correlations between CSPG4 expression and histopathological data and patient characteristics. A total of 189 melanoma tissue samples from Lower Austria, including primary melanomas and melanoma metastases, were immunohistochemically stained for the expression of CSPG4 and statistical analyses were performed. A total of 65.6% of melanoma tissue samples stained positive for the expression of CSPG4. Primary nodular and primary superficial spreading melanomas demonstrated a significantly higher number of positively stained tissue samples for CSPG4 compared with primary lentigo maligna melanomas. No significant differences in the expression of CSPG4 were demonstrated between primary melanomas and melanoma metastases. The present study supports the advancement of the understanding of CSPG4 tissue expression patterns in melanoma patients and provides additional information for further investigation of CSPG4 as a potential therapeutic target.

Targeting DNA repair to enhance the efficacy of sorafenib in hepatocellular carcinoma.

The multityrosine kinase inhibitor sorafenib remains an important systemic treatment option for hepatocellular carcinoma (HCC). Signaling pathways, which are targeted by sorafenib, are involved in checkpoint and DNA repair response, RAD51 being a candidate protein. Here, we aim to evaluate the effect of the human RAD51 inhibitor B02 in combination with sorafenib in human HCC cells. Impact of RAD51 expression on HCC patient survival was evaluated by an in silico approach using Human Protein Atlas dataset. Cell viability of HUH7, AKH12, AKH13, and 3P was assessed by neutral red assay. To measure the cytotoxicity, we quantified loss of membrane integrity by lactate dehydrogenase release. We also employed colony formation assay and hanging drop method to assess clonogenic and invasive ability of HCC cell lines upon sorafenib and B02 treatment. Cell cycle distribution and characterization of apoptosis was evaluated by flow cytometry. In silico approach revealed that HCC patients with higher expression of RAD51 messenger RNA had a significantly shorter overall survival. The RAD51 inhibitor B02 alone and in combination with sorafenib significantly reduced viability, colony formation ability, and invasion capacity of HCC cells. Cell cycle analysis revealed that the combination of both agents reduces the proportion of cells in the G2/M phase while leading to an accumulating in the subG1 phase. The RAD51 inhibitor B02 seems to be a promising agent for HCC treatment and enhances the antitumor effects of sorafenib in vitro.

Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs.

(1) Background: Avian influenza viruses (AIVs) promptly evade preexisting immunity by constantly altering the immunodominant neutralizing antibody epitopes (antigenic drift) or by procuring new envelope serotypes (antigenic shift). As a consequence, the majority of antibodies elicited by infection or vaccination protect only against closely related strains. The immunodominance of the globular head of the main glycoprotein has been shown to mask the immunogenicity of the conserved regions located within the hemagglutinin (HA) protein. It has been shown that the broadly neutralizing universal antibodies recognize the HA2 domain in headless hemagglutinin (HA-stalk). Therefore, the HA-stalk is a highly conserved antigen, which makes it a good candidate to be used in universal vaccine development against AIVs. (2) Methods: Sf9 insect cells were used to produce triple H5N1/NA-HA-M1 influenza virus-like particles (VLPs) via co-expression of neuraminidase, hemagglutinin and matrix proteins from a tricistronic expression cassette. Purified influenza VLPs were used to immunize broiler hens. An in-depth characterization of the immune response was performed with an emphasis on the pool of elicited universal antibodies. (3) Results: Our findings suggest, that after vaccination with triple H5N1/NA-HA-M1 VLPs, hens generate a pool of broad-spectrum universal anti-HA-stalk antibodies. Furthermore, these universal antibodies are able to recognize the mammalian-derived HA-stalk recombinant proteins from homologous H5N1 and heterologous H7N9 AIVs as well as from the heterosubtypic human H1N1 influenza strain. (4) Conclusions: Our findings may suggest that highly pathogenic avian influenza H5 HA protein contain functional epitopes that are attractive targets for the generation of broad-spectrum antibodies against AIVs in their native hosts.

A chondroitin sulfate proteoglycan 4­specific monoclonal antibody inhibits melanoma cell invasion in a spheroid model.

The overexpression of chondroitin sulfate proteoglycan 4 (CSPG4) is associated with several tumor types, including malignant melanoma, squamous cell carcinoma, triple­negative breast carcinoma, oligodendrocytomas or gliomas. Due to its restricted distribution in normal tissues, CSPG4 has been considered a potential target for several antitumor approaches, including monoclonal antibody (mAb) therapies. The aim of the present study was to characterize the impact of the CSPG4­specific mAb clone 9.2.27 on its own or in combination with the commonly used BRAF­selective inhibitor, PLX4032, on different functions of melanoma cells to assess the potential synergistic effects. The BRAF V600­mutant human melanoma cell lines, M14 (CSPG4­negative) and WM164 (CSPG4­positive), were exposed to the CSPG4­specific 9.2.27 mAb and/or PLX4032. Cell viability and colony formation capacity were evaluated. A 3D­cell culture spheroid model was used to assess the invasive properties of the treated cells. In addition, flow cytometric analysis of apoptosis and cell cycle analyses were performed. Incubation of the WM164 cell line with CSPG4­specific 9.2.27 mAb decreased viability, colony formation ability and the invasive capacity of CSPG4­positive tumor cells, which was not the case for the CSPG4­negative M14 cell line. Combined treatment of the WM164 cells with 9.2.27 mAb plus PLX4032 did not exert any significant additional effect in comparison to treatment with PLX4032 alone in the clonogenic and invasion assays. M14 cell cycle distribution was not influenced by the CSPG4­specific 9.2.27 mAb. By contrast, the exposure of WM164 cells to the mAb resulted in an arrest of the cells in the S phase. Moreover, combined treatment of the WM164 cells led to a significantly increased accumulation of cells in the subG1 phase, combined with a decrease of cells in the G2/M phase. On the whole, findings of the present study indicate that the CSPG4­specific 9.2.27 mAb exerts an anti­invasive effect on CSPG4­positive melanoma spheroids, which is not enhanced by BRAF inhibition. These findings provide the basis for further investigations on the effects of anti­CSPG4­based treatments of CSPG4­positive tumors.

Expression of chondroitin sulfate proteoglycan 4 (CSPG4) in melanoma cells is downregulated upon inhibition of BRAF.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a multifunctional transmembrane proteoglycan involved in spreading, migration and invasion of melanoma. In addition to the activating BRAF V600E mutation, CSPG4 was shown to promote MAPK signaling by mediating the growth­factor induced activation of receptor tyrosine kinases. However, it remains elusive which factors regulate CSPG4 expression. Therefore, the aim of the present study was to examine whether BRAF and MEK inhibitors have an effect on the expression of CSPG4. We exposed a panel of BRAF­mutant CSPG4­positive or ­negative melanoma cell lines to BRAF and MEK inhibitors. Protein levels of CSPG4 were analyzed by flow cytometry (FACS), immunofluorescence microscopy (IF), and western blotting. CSPG4 mRNA levels were determined by quantitative PCR (qPCR). The prolonged exposure of cells to BRAF and MEK inhibitors resulted in markedly reduced levels of the CSPG4 protein in permanent resistant melanoma cells as well as decreased levels of its mRNA. We did not observe increasing levels of CSPG4 shedding into the culture supernatants. In addition, patient­derived matched tumor samples following therapy with kinase inhibitors showed decreased numbers of CSPG4­positive cells as compared to pre­therapy tumor samples. Our results indicate that BRAF and MEK inhibition downregulates CSPG4 expression until the cells have developed permanent resistance. Our findings provide the basis for further investigation of the role of CSPG4 in the development of drug­resistance in melanoma cells.

High-Titre Neutralizing Antibodies to H1N1 Influenza Virus after Mouse Immunization with Yeast Expressed H1 Antigen: A Promising Influenza Vaccine Candidate.

H1N1 influenza virus is still regarded as a serious pandemic threat. The most effective method of protection against influenza virus and the way to reduce the risk of epidemic or pandemic spread is vaccination. Influenza vaccine manufactured in a traditional way, though well developed, has some drawbacks and limitations which have stimulated interest in developing alternative approaches. In this study, we demonstrate that the recombinant H1 vaccine based on the hydrophilic haemagglutinin (HA) domain and produced in the yeast system elicited high titres of serum haemagglutination-inhibiting antibodies in mice. Transmission electron microscopy showed that H1 antigen oligomerizes into functional higher molecular forms similar to rosette-like structures. Analysis of the N-linked glycans using mass spectrometry revealed that the H1 protein is glycosylated at the same sites as the native HA. The recombinant antigen was secreted into a culture medium reaching approximately 10 mg/l. These results suggest that H1 produced in Pichia pastoris can be considered as the vaccine candidate against H1N1 virus.

Hemagglutinin stalk domain from H5N1 strain as a potentially universal antigen.

Influenza A virus infections are the major public health concern and cause significant morbidity and mortality each year worldwide. Vaccination is the main strategy of influenza epidemic prevention. However, seasonal vaccines induce strain-specific immunity and must be reformulated annually based on prediction of the strains that will circulate in the next season. Thus, it is essential to develop vaccines that would induce broad and persistent immunity to influenza viruses. Hemagglutinin is the major surface antigen of the influenza virus. Recent studies revealed the importance of HA stalk-specific antibodies in neutralization of different influenza virus strains. Therefore, it is important to design an immunogen that would focus the immune response on the HA stalk domain in order to elicit neutralizing antibodies. In the present study, we report characterization of a conserved truncated protein, potentially a universal influenza virus antigen from the H5N1 Highly Pathogenic Avian Influenza A virus strain. Our results indicate that exposure of the HA stalk domain containing conserved epitopes results in cross reactivity with different antibodies (against group 1 and 2 HAs). Additionally, we conclude that HA stalk domain contains not only conformational epitopes recognized by universal FI6 antibody, but also linear epitopes recognized by other antibodies.