Genesis Biotechnologies (United States)

The synthesis of biobased covalent adaptable networks (CANs) is becoming increasingly popular as a sustainable alternative to thermosetting polymers since they are both recyclable and less dependent on petroleum chemicals. However, biobased CANs centered on the Diels–Alder (DA) reaction typically comprise the aromatic N,N′-(4,4′-methylene diphenyl) bismaleimide (BMI), which is toxic. Here, we synthesize less harmful aliphatic bismaleimides with varying molar mass and biobased multifunctional furan cross-linkers via facile procedures that achieve high conversion. These materials were subsequently copolymerized to prepare a range of thermally reversible cross-linked aliphatic polyester CANs with high gel fractions (88–98%). Structure–property relationships were established, showing that the compositions of the furan cross-linker and bismaleimide controlled the thermal and mechanical properties of the networks, leading to dissociation temperatures up to 136 °C and tensile strengths up to 9.3 MPa. Moreover, network recyclability was demonstrated via compression molding under mild conditions with very good recovery of mechanical properties (>75%), and the materials also displayed rapid healing. This research provides a facile approach to the smart design of recyclable biobased aliphatic polyester Diels–Alder CANs without the use of BMI, while achieving network properties that are tunable via the structures of the aliphatic bismaleimides and biobased furan cross-linkers.

Abstract Background: Tumor endothelial cells (ECs) are a population of stromal cells required for tumor growth that cooperate with tumors to form angiogenic structures. In blood, circulating ECs (CECs) are normal constituents of healthy individuals, although a Cancer Associated Vascular Endothelial cell (CAVE) subtype has been observed in cancer patients. The CAVE population has been isolated and identified using their large size or multicellular clustering and a pooled mixture of classical EC markers (i.e. CD31 and CD146). However, there has been no attempt to differentiate CAVEs from the many EC subtypes. This is not surprising as in-depth phenotyping of ECs requires an array of biomarkers that until recently has not been feasible. A multi-phenotypic screening of EC markers was tested on CAVEs from 116 blood samples in 3 types of solid tumors. This data suggests that CAVEs exist as a common and diverse subtype of tumor derived CECs that may express cytokeratin (CK) and various EC biomarkers, correlating to disease stage. Methods: Peripheral blood samples from 116 cancer patients (stage I-IV) were drawn from 2012-2014 including breast (n=42), lung (n=39) and prostate (n=35), as well as blood from 34 healthy controls. Blood was processed by an established filtration approach, i.e. the CellSieveTM microfiltration technique (Creatv MicroTech), filtering blood by size exclusion and staining cells for CK 8, 18 & 19, EpCAM and CD45. After identification and imaging, the QUAS-R (Quench, Underivatize, Amine-Strip and Restain) technique was used to remove fluorescence signal and restain all cells with CD31, CD146, CD144, & DAPI. After reimaging, QUAS-R was again used to remove fluorescence and restain the cells for CD14, CD105, CD34, & DAPI. Results: Out of 116 patient samples, we identified CAVEs in 63 patients (54%) based on positivity of CD31, CD144 or CD146, but none were found in healthy controls. CAVEs per 7.5mL sample in patients averaged 5.1 (breast), 5.6 (prostate) and 7.9 (lung). Presence of CAVEs appeared related to stage with 26% in stage 1, 61% in stage 2, 68% in stage 3, and 74% in stage 4 patients. No CAVEs were positive for CD14 or CD45. CD31 was the most present marker, found on 93% of CAVEs, followed by CD144 (85%), CD34 (64%), CD146 (45%), & CD105 (4%). In contrast with the previous study on this topic, CK was found in 67% of CAVEs, but was not a universal marker. Conclusions: It has been reported that CK+ and CD45- CECs are isolated from the blood of cancer patients in colon and lung cancers, prompting some to classify them as circulating tumor cells. However, subtyping these CECs is incomplete when characterized with only 3-4 biomarkers. A multi-phenotypic subtyping technique was used to properly identify and subtype these CECs in cancer patients. This data suggest that a subset of CECs, e.g. CAVEs, are found in circulation as CK+/CD45-, but exist as a heterogeneous population of cancer specific circulating cells that require further study. Citation Format: Daniel L. Adams, R. Katherine Alpaugh, Steven H. Lin, Jeffrey R. Marks, Raymond Bergan, Stuart S. Martin, Sarany Chumsri, Massimo Cristofanilli, Cha-Mei Tang, Steingrimur Stefansson. Identifying, subtyping and classifying tumor associated circulating endothelial cells in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 778. doi:10.1158/1538-7445.AM2017-778

HDAC inhibitors are a promising group of epigenetic drugs that show the ability to induce apoptosis in cancer stem cells.

55 Background: HHV-8 has been implicated in the pathogenesis of Kaposi's sarcoma (KS) as well as body cavity B-cell lymphoma based on PCR analysis of tissues and antibody to viral latent and late proteins. Materials & Methods: We developed an IgG antibody ELISA to HHV-8 using whole viral lysate which contained on average 1.9 × 109 viral particles/ml as well as >1.08 mg/ml of protein. An IFA to HHV-8 lytic antigen was made using the KS-1 cell line. In addition, Western blots to structural virus proteins were developed to elucidate immunodominant proteins that may be detected in sera. Results: Our serological data showed that approximately 11% of the healthy donors in the U.S.A. contained IgG antibody to HHV-8 compared to 12% in Thailand, 4% in India, and 15% in Malaysia (ethnic Chinese, Malays, and Bidayuhians). The antibody titer in healthy donors' sera by IFA ranged 1:20 - 1:320 compared to 1:320 - >1:10,000 in classical KS sera. Similar titers were evident by ELISA, and these assays correlated (p <0.0001, r=0.915). Approximately 50% of HIV-1 (+) homosexuals and >40% HIV-1 (+) IV drug users have HHV-8 serum antibody which suggests being at higher risk for KS. Sera from 93% of KS patients both HIV (+) and HIV (−) contained elevated IgG antibody as detected by IFA and ELISA. Body cavity B-cell lymphoma is the only other disease found associated with increased HHV-8 antibody levels. No evidence of HHV-8 antibody was evident in sera from multiple myeloma, Hodgkin's disease, acute and chronic leukemia, and other B- and T-cell lymphomas from the U.S. population. PCR in situ hybridization for HHV-8 DNA corroborated the disease association results found with the antibody tests. Conclusion: Our data showed variations in infection of HHV-8 in U.S.A. vs. India, Malaysia, and Thailand. Although KS has not been reported in Southeast Asia, HHV-8 infection may be linked to other diseases there. Our ELISA contains a majority of the viral structural proteins and, therefore, detects broad-spectrum antibodies which may be missed by other assays. Our IFA is directed against lytic HHV-8 antigens and correlates well with the ELISA assay. Therefore, these assays may be useful tools in large-scale seroepidemiological studies and clinical follow-up for at-risk individuals. Our findings indicate that the prevalence of HHV-8 is extremely low regardless of the geographic location suggesting that HHV-8 is an important human pathogen associated with KS or other unknown diseases compared to other human gamma, beta, and alpha herpesviruses.

HTLV-I is associated with a neurological syndrome designated Tropical Spastic Paraparesis/HTLV-I associated myelopathy (TSP/HAM). To determine whether HTLV-I can replicate in human primary macrophages and thus contribute to HTLV-I dissemination in the nervous system, elutriated human macrophages were infected cell-free with the HTLV-ICR and HTLV-IBOU isolates from patients with adult T-cell leukemia and TSP/HAM, respectively. Viral production was monitored by measuring the viral p24 gag antigen in the cell culture supernatant, by electron microscopy (EM) and by polymerase chain reaction (PCR) on viral DNA and RNA. The HTLV-I p24 gag antigen was detected 21 days after infection with either isolate, and the presence of mature viral particles was demonstrated by electron microscopy one month after infection. Viral sequences were amplified by PCR analysis of the infected macrophages' DNA. Spliced mRNAs for the p40tax and p27rex proteins, as well as the p12I, and p30II proteins encoded by the pX region were readily identified by reverse transcriptase PCR. Altogether, these data indicate that HTLV-I replication occurs in vitro in primary human macrophages. Whether macrophage infection occurs also in vivo and is a crucial step in the induction of the neurological manifestations observed in TSP/HAM remains an open question.

We investigated the interaction of hydrocortisone (HC) and EBV. The treatment of P3HR-1 cells (propagated at 34°C and 37°C) with various concentrations of HC for 7 and 21 days resulted in enhanced levels of antigen positive cells with a maximum increase at 21 days. Virus harvested from HC-treated P3HR-1 cells grown at 34°C had a 1–2 log higher titer in Raji cells when compared to control virus.