Germline mutation of the RET proto-oncogene in members of Slovak families with multiple endocrine neoplasia 2

Detection of mutations in RET proto-oncogene in Slovak families from different localities and of different ethnic origin with MEN 2 syndrome is reported. Despite the fact that the same mutation of RET oncogene was found in different family members, the latency period of tumor appearance and their pathogenicity differed substantially. In addition, also different phenotypes of the disease were expressed in various family members having the same RET gene mutation. The data indicate that the mechanism of MEN2 syndrome is not only due to the RET gene mutation, and strongly support the conclusion that additional genetic events are involved in the disease formation.     

Human glioma cells expressing herpes simplex virus thymidine kinase gene treated with acyclovir, ganciclovir and bromovinyldeoxyuridine. Evaluation of their activity in vitro and in nude mice

Human glioma cell lines 8-MG-BA and 42-MG-BA were infected with retrovirus vector containing the herpes simplex virus thymidine kinase (HSVtk) gene. The effect of acyclovir (ACV), ganciclovir (GCV), and bromovinyldeoxyuridine (BVDU) on both, parental and HSVtk expressing glioma cells was studied in vitro. BVDU displayed the most potent cytotoxic properties in HSVtk-containing cells, however bystander killing of nontransduced parental cells in a mixture with HSVtk-containing cells was less potent, than observed for ACV or GCV. Taking into account the cytotoxic effect of different prodrugs used, as well as their ability to kill nontransduced bystander cells, ganciclovir was shown to be the most effective. Therefore the effect of GCV treatment on 8-MG-BA xenografts inoculated with PA-317JH5cl13 virus producer cells was further studied on nude mice.     

Construction and testing of gene therapy retroviral vector expressing bacterial cytosine deaminase gene

A retroviral vector containing gene for bacterial enzyme cytosine deaminase (CD) under the control of viral LTR sequences was constructed and transfected into packaging cell line GP+envAm12. High virus titer producing single cell clone (1 x 10(7) cfu/ml, determined on NIH 3T3 cells) was isolated and used to transfer CD gene into human mammary carcinoma cell lines in vitro. Transduced cells exhibited high sensitivity to the antifungal drug 5-fluorocytosine (5-FC), whereas parental cells did not. Cocultivation of CD-positive and CD-negative parental cells showed bystander effect, dependent on the ratio of CD-positive cells. No enhancement of 5-FC cytotoxicity by leucovorin was observed in cells expressing cytosine deaminase.     

Stem cell based glioblastoma gene therapy

There is no curative therapy for glioblastoma multiforme (GBM) thus far. Combined therapies including surgery, followed by concomitant irradiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ), slightly improves patients' survival but the prognosis remains poor. The fatal nature of glioblastoma is caused by tumor-initiating glioblastoma cells. The tumor tropic ability of adult mesenchymal stem cells offers the attractive possibility to use these cells as a?vehicle to deliver therapeutic agents to the site of the tumor. In preclinical studies using animal models, mesenchymal stem cells engineered to express suicide genes were shown to elicit a?significant antitumor response against various tumors including glioblastoma. This review summarizes the current state of knowledge about stem cell directed glioblastoma therapy. Results obtained in a?preclinical study using mesenchymal stem cells engineered to express cytosine deaminase provided evidence that stem cell based gene therapy might also attack glioblastoma stem cells and therefore be curative. In addition to stem cell directed prodrug gene therapies, other immunotherapeutic modalities using mesenchymal stem cells are discussed as well. Encouraging results of preclinical studies of stem cell based gene therapy for glioblastoma support the argument to begin clinical studies. Keywords: glioblastoma, glioblastoma stem cells, chemoresistance, radio-resistance, stem cell driven gene therapy, prodrug gene therapy, preclinical studies.     

A general approach to site-specific antibody drug conjugates

Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low–antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.Antibody drug conjugates (ADCs) are emerging as a new class of anticancer therapeutics that combine the efficacy of small-molecule therapeutics with the targeting ability of an antibody (Ab) (1, 2). By combining these two components into a single molecular entity, highly cytotoxic small-molecule drugs (SMDs) can be delivered to cancerous target tissues, thereby enhancing efficacy while reducing the potential systemic toxic side effects of the SMD. Conventional ADCs are typically produced by conjugating the SMD to the Ab through the side chains of either surface-exposed lysines or free cysteines generated through reduction of interchain disulfide bonds (3, 4). Because antibodies contain many lysine and cysteine residues, conventional conjugation typically produces heterogeneous mixtures that present challenges with respect to analytical characterization and manufacturing. Furthermore, the individual constituents of these mixtures exhibit different pharmacology with respect to their pharmacokinetic, efficacy, and safety profiles, hindering a rational approach to optimizing this modality (5).Recently, it was reported that the pharmacological profile of ADCs may be improved by applying site-specific conjugation technologies that make use of surface-exposed cysteine residues engineered into antibodies (THIOMABS) that are then conjugated to the SMD, resulting in site-specifically conjugated ADCs (TDCs) with defined Ab–drug ratios. Relative to the heterogeneous mixtures created using conventional conjugation methodologies, site-specifically conjugated TDCs demonstrated equivalent in vivo potency, improved PK, and an expanded therapeutic window (6, 7). Although this approach may be useful for generating site-specifically conjugated ADCs, THIOMABS produced using this process are not directly amenable to conjugation, but instead, require a multistep process that includes decapping of the engineered cysteine residues, which inevitably results in the partial breaking and reformation of structurally important internal disulfide bonds. Site-specific ADCs generated by enzymatic modification also have demonstrated improved stability and pharmacokinetics; however, a surface-exposed transglutamase tag (LLQG) needs to be engineered into antibodies at a permissive site (8).To provide a more facile and generally applicable approach for synthesizing site-specifically conjugated ADCs, we developed a recombinant DNA-based eukaryotic protein expression system using Chinese hamster ovary (CHO) cells to biosynthetically incorporate nonnative amino acids into a given Ab scaffold (9). Nonnative amino acids, such as para-acetylphenylalanine (pAF) and para-azidophenylalanine (pAZ), can provide orthogonal conjugation chemistries that otherwise are not available from functional groups present in the 20 canonical amino acids. Nonnative amino acid incorporation technology using Escherichia coli expression systems can provide large quantities (>5 g/L) of proteins for clinical use (10). However, E. coli expression is limited to relatively simple, nonglycosylated proteins. The production of more complex glycosylated proteins, such as full-length antibodies, requires a eukaryotic expression system such as CHO cells. Previous attempts to incorporate nonnative amino acids in eukaryotic organisms have met with limited success as the product titers achieved were not high enough for product development and commercialization (11, 12).We report here, the development of a stable expression system using CHO cells (EuCODE) that produces antibodies incorporating nonnative amino acids with titers over 1 g/L. We have applied this technology to the generation of site-specific Ab drug conjugates (NDCs) synthesized by site-specific coupling of the potent tubulin inhibitor, monomethyl auristatin D (MMAD) (13), to genetically encoded pAF residues in anti-5T4 (A1) and anti-Her2 (Her) antibodies. 5T4, also known as trophoblast glycoprotein, is a cell surface antigen that internalizes rapidly, and is highly expressed on colorectal and gastric cancers (14). Human epidermal growth factor receptor 2 (Her2) is the target of the therapeutic agents Trastuzumab and Trastuzumab-mcc-DM1 (T-DM1), and is highly expressed in breast cancers (15). The resulting NDCs were compared in rodent efficacy studies to both conventional ADCs (generated from reduced interchain cysteines) and engineered cysteine TDCs. The NDCs demonstrated superior efficacy and pharmacokinetic properties, and in a single-dose rat toxicology study, the anti-Her2 NDC was well tolerated at dose up to 90 mg/kg. Finally, in vitro plasma stability studies showed that the site of conjugation had a significant impact on the stability of rationally designed cleavable linkers.     

Is it a sarcoidal foreign-body granuloma or a cutaneous sarcoidosis on a permanent eyebrow make-up?

Permanent make-up is a kind of cosmetic tattoo in which the colorants (pigments) are deposited in dermis after piercing the skin by tiny solid needles. It may cause some adverse effects such as local inflammation, infection, and allergic reactions on the skin and even systemic adverse effects such as sarcoidal reactions. Here the case of a 34-year-old woman who has some yellowish hard shiny papules on her eyebrows after having a permanent make-up is described. The histopathological examinations of the papules are diagnosed as sarcoidal foreign-body reactions. All the laboratory investigations were in normal limits except a mild elevation in angiotensin converting enzyme or ACE level. The lesions mostly improved after topical corticosteroid treatment. Sarcoidal foreign-body reaction due to permanent make-up is discussed with this presentation.     

The relationship among PDX1, CDX2, and mucin profiles in gastric carcinomas; correlations with clinicopathologic parameters

Purpose  

Several studies performed on pancreatic-duodenal homeobox 1 (PDX1) have demonstrated a loss of expression and negative tumor modulator effect in gastric carcinoma. Relations between PDX1 and gastric metaplasia, differentiated type of gastric carcinoma, and the early stage of the disease have been exhibited in previous reports. The aim of this study was to examine expressions of PDX1, caudal type homeobox 2 (CDX2) and mucin (MUC) profiles to address the role of PDX1 in gastric carcinogenesis and its relationship with CDX2.