Highly versatile cancer photoimmunotherapy using photosensitizer-conjugated avidin and biotin-conjugated targeting antibodies.

BACKGROUND: Photoimmunotherapy (PIT) employing antibody-photosensitizer conjugates is a promising treatment for cancer. However, the fixed antigen specificity severely limits the efficacy and the applicability. Here we describe a universal strategy for PIT of cancer by using a near-infrared (NIR) photosensitizer IRDye700DX-conjugated NeutrAvidin, designated as AvIR, together with various biotinylated antibodies (BioAbs) for cellular targeting. METHODS: Cytotoxicity of AvIR-mediated PIT was evaluated by fluorescence imaging and cell viability assay. Phototoxic effect on tumorigenicity was assessed by tumorsphere-formation assay and Matrigel invasion assay. Cancer stem cell-like side-population (SP) cells were identified by flow cytometry. RESULTS: CHO cells stably expressing carcinoembryonic antigen or EpCAM were pre-labeled with each BioAb for the corresponding antigen, followed by AvIR administration. NIR light irradiation specifically killed the targeted cells, but not off-targets, demonstrating that the AvIR-mediated PIT does work as expected. CSC-like subpopulation of MCF-7 cells (CD24low/CD44high) and SP of HuH-7 cells (CD133+/EpCAM+) were effectively targeted and photokilled by AvIR-PIT with anti-CD44 BioAb or anti-CD133/anti-EpCAM BioAbs, respectively. As results, the neoplastic features of the cell lines were sufficiently suppressed. Cancer-associated fibroblast (CAF)-targeted AvIR-PIT by using anti-fibroblast activation protein BioAb showed an abolishment of CAF-enhanced clonogenicity of MCF-7 cells. CONCLUSIONS: Collectively, our results demonstrate that AvIR-mediated PIT can greatly broaden the applicable range of target specificity, with feasibility of efficacious and integrative control of CSC and its microenvironment.

Potent and specific antitumor effect of CEA-targeted photoimmunotherapy.

Conventional photodynamic therapy (PDT) for cancer is limited by the insufficient efficacy and specificity of photosensitizers. We herein describe a highly effective and selective tumor-targeted PDT using a near-infrared (NIR) photosensitizer, IRDye700DX, conjugated to a human monoclonal antibody (Ab) specific for carcinoembryonic antigen (CEA). The antitumor effects of this Ab-assisted PDT, called photoimmunotherapy (PIT), were investigated in vitro and in vivo. The Ab-IRDye conjugate induced potent cytotoxicity against CEA-positive tumor cells after NIR-irradiation, whereas CEA-negative cells were not affected at all, even in the presence of excess photoimmunoconjugate. We found an equivalent phototoxicity and a predominant plasma membrane localization of Ab-IRDye after both one and six hours of incubation. Either no or little caspase activation and membrane peroxidation were observed in PIT-treated cells and a panel of scavengers for reactive oxygen species showed only partial inhibition of the phototoxic effect. Strikingly, Ab-IRDye retained significant phototoxicity even under hypoxia. We established a xenograft model, which allowed us to sensitively investigate the therapeutic efficacy of PIT by non-invasive bioluminescence imaging. Luciferase-expressing MKN-45-luc human gastric carcinoma cells were subcutaneously implanted into both flanks of nude mice. NIR-irradiation was performed for only the tumor on one side. In vivo imaging and measurement of the tumor size revealed that a single PIT treatment, with intraperitoneal administration of Ab-IRDye and subsequent NIR-irradiation, caused rapid cell death and significant inhibition of tumor growth, but only on the irradiated side. Together, these data suggest that Ab-IRDye-mediated PIT has great potential as an anticancer therapeutics targeting CEA-positive tumors.

Cooperative interactions at the SLP-76 complex are critical for actin polymerization.

T-cell antigen receptor (TCR) engagement induces formation of multi-protein signalling complexes essential for regulating T-cell functions. Generation of a complex of SLP-76, Nck and VAV1 is crucial for regulation of the actin machinery. We define the composition, stoichiometry and specificity of interactions in the SLP-76, Nck and VAV1 complex. Our data reveal that this complex can contain one SLP-76 molecule, two Nck and two VAV1 molecules. A direct interaction between Nck and VAV1 is mediated by binding between the C-terminal SH3 domain of Nck and the VAV1 N-terminal SH3 domain. Disruption of the VAV1:Nck interaction deleteriously affected actin polymerization. These novel findings shed new light on the mechanism of actin polymerization after T-cell activation.

An idea to explore: Determination of single nucleotide polymorphisms in alcohol metabolism-related genes using PCR-based assays to understand the link between an individual's genotype and phenotype.

Here, we propose a laboratory exercise to quickly determine single nucleotide polymorphisms (SNPs) in human alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) genes involved in alcohol metabolism. In this exercise, two different genotyping methods based on polymerase chain reaction (PCR), namely allele-specific (AS) PCR and a PCR-restriction fragment polymorphism (RFLP) analysis, can be performed under the same PCR program (2-step × 35 cycles, 35 min total) in parallel using a hair root lysate as a template. In AS-PCR, the target regions of the G- or A-alleles of both genes are allele-specifically amplified in a single PCR tube. In the PCR-RFLP analysis, the two genes are amplified simultaneously in a single tube, and then a portion of the PCR product is double-digested with restriction enzymes MslI and Eam1104I for 5 min. The resulting reaction products of each method are electrophoresed side by side, and the genotypes are determined from the DNA band patterns. With the optimized protocol, the whole process from template preparation to genotyping can be completed in about 75 min. During PCR, students also perform an ethanol patch test to estimate their ability to metabolize alcohol. This series of experiments can help students learn the principles and applications of PCR/SNP analyses. By comparing the genotypes revealed by PCR and the phenotypes revealed by the patch tests, students can gain a better understanding of the clinical value of genetic testing.

Adipose-derived Mesenchymal Stem Cell Transplantation Improves Ovarian Function and Oocyte Quality in Aged Mice.

BACKGROUND/AIM: Age-related decline in the number of ovulations and ovum quality are major causes of female infertility, and stem cells have been reported to be effective in tissue regeneration. However, current therapeutic modalities are inadequate. This study investigated the effects of adipose-derived mesenchymal stem cells (ASCs) on ovarian functions in aged mice. MATERIALS AND METHODS: Following the characterization of ASCs using flow cytometry, the effects of ASCs on the number of ovulations, fertilization rate, and blastocyst-formation rate were investigated. In addition, the number of ovarian follicles and serum anti-Müllerian hormone (AMH) levels were examined. ASCs marked with Kusabira Orange were used to examine the location after cell administration. The quality of ovulated oocytes was analyzed using next-generation RNA sequencing. RESULTS: ASCs showed characteristics of mesenchymal stem cells and were distributed to various organs, including the ovarian stroma. The transplantation resulted in increased number of oocytes and ovulation in the ovaries and increased AMH values. Genetic analysis revealed improved oocyte quality and increased fertilization and blastocyst-formation rates. CONCLUSION: ASC therapy may be effective in improving fertility in older women.

Molecular characterization of a fully human chimeric T-cell antigen receptor for tumor-associated antigen EpCAM.

The transduction of T cells to express chimeric T-cell antigen receptor (CAR) is an attractive strategy for adaptive immunotherapy for cancer, because the CAR can redirect the recognition specificity of T cells to tumor-associated antigens (TAAs) on the surface of target cells, thereby avoiding the limitations of HLA restriction. However, there are considerable problems with the clinical application of CAR, mostly due to its xenogeneic components, which could be immunogenic in humans. Moreover, while extensive studies on the CARs have been performed, the detailed molecular mechanisms underlying the activation of CAR-grafted T cells remain unclear. In order to eliminate potential immunogenicity and investigate the molecular basis of the CAR-mediated T-cell activation, we constructed a novel CAR (CAR57-28ζ) specific for one of the most important TAAs, epithelial cell adhesion molecule (EpCAM), using only human-derived genes. We revealed that in Jurkat T cells, lentivirally expressed CAR57-28ζ can transmit the T-cell-activating signals sufficient to induce IL-2 production upon EpCAM stimulation. An immunofluorescent analysis clearly showed that the CAR57-28ζ induces the formation of signaling clusters containing endogenous CD3ζ at the CAR/EpCAM interaction interface. These results suggest that this CAR gene may be safely and effectively applied for adaptive T-cell immunotherapy.

Acceptance of Murine Islet Allografts Without Immunosuppression in Inguinal Subcutaneous White Adipose Tissue Pretreated With bFGF.

Prevention of immune rejection without immunosuppression is the ultimate goal of transplant immunobiology. One way to achieve this in cellular transplantation, such as with islet transplantation, is to create a favorable local environment at the transplant site. In the current study, we found that C57BL/6 mice with streptozotocin-induced diabetes remained normoglycemic for >1 year after transplantation of BALB/c islets without immunosuppression when the inguinal subcutaneous white adipose tissue (ISWAT) was the site of transplantation and when the site was pretreated with basic fibroblast growth factor. Mechanistically, mesenchymal stem cells (MSCs) expanded in the ISWAT after the treatment was found to produce transforming growth factor-ß (TGF-ß), and prevention of islet allograft rejection could be achieved by cotransplantation with syngeneic MSCs isolated from the ISWAT after the treatment, which was abolished by anti-TGF-ß antibody treatment. Importantly, TGF-ß-producing cells remained present at the site of cotransplantation up to the end of observation period at 240 days after transplantation. These findings indicate that prevention of islet allograft rejection without immunosuppression is feasible with the use of syngeneic TGF-ß-producing MSCs expanded in the ISWAT after the treatment with bFGF, providing a novel strategy for prevention of islet allograft rejection without immunosuppression.

Trophic and immunomodulatory effects of adipose tissue derived stem cells in a preclinical murine model of endometriosis.

Endometriosis, which exhibits enigmatic pathological features such as stromal fibrosis and proliferation of ectopic epithelial cells, is known as a refractory disease. Mesenchymal stem cells modulate the fibrosis in stromal tissues through their trophic and immunomodulatory properties. To investigate the potential of stem cells in treating endometriosis, we examined the secondary morphology and molecular alterations in endometriosis-like lesions after the administration of adipose tissue-derived stem cells (ASCs) to an experimental murine model of endometriosis. The infused ASCs were found integrated in the endometriosis-like lesions. Accompanied by the suppression of stromal fibrosis and proliferation of endometriotic epithelial cells, the infusion of ASCs with stemness potential (early passage of ASCs) suppressed the growth of endometriosis-like lesions and inhibited the expression of pro-inflammatory and pro-fibrotic cytokines, whereas no significant attenuation of endometriosis-like lesions occurred after the infusion of ASCs without stemness potential (late passage of ASCs). Accordingly, the trophic and immunomodulatory properties of ASCs may regulate fibrosis in endometriosis-like lesions, suggesting that regenerative medicine could be recognized as an innovative treatment for patients with endometriosis through the accumulation of evidence of preclinical efficacy.

MicroRNAs miR-4535 and miR-1915-5p in amniotic fluid as predictive biomarkers for chorioamnionitis.

BACKGROUND: This study was performed to investigate the clinical significance of miR-4535 and miR-1915-5p in severe chorioamnionitis. MATERIALS & METHODS: Amniotic fluid samples from 37 patients with severe chorioamnionitis were subjected to miRNA array analysis and ddPCR™. Diagnostic values were assessed using the receiver operating characteristic curve. The patients were separated into three groups according to Blanc's criteria. RESULTS: The expression of miR-4535 and miR-1915-5p was significantly correlated with the copy number of 16S rDNA, had extremely high diagnostic accuracy for severe chorioamnionitis, and was linked to maternal and fetal inflammation. CONCLUSION: miR-4535 and miR-1915-5p serve as promising biomarkers for the diagnosis of severe chorioamnionitis.

Establishment of human induced pluripotent stem cells derived from skin cells of a patient with Dravet syndrome.

Dravet syndrome is known as an intractable infantile epilepsy caused by a heterozygous de novo mutation in SCN1A, with mutations being reported globally. In this study, we established 2 human induced pluripotent stem cell lines by expressing reprogramming factors, OCT3/4, SOX2, KLF4, L-MYC, LIN28 and p53 shRNA in the fibroblast skin cells of a patient with Dravet syndrome harboring the Y1102X pathogenic mutation in SCN1A. These cell lines showed pluripotency, ability for differentiation to the 3 germ layers, and normal karyotype.

Establishment of a human induced stem cell line (FUi002-A) from Dravet syndrome patient carrying heterozygous R1525X mutation in SCN1A gene.

De novo mutations in SCN1A are the most common cause of Dravet syndrome (DS), an infantile-onset epileptic encephalopathy. In this study, human induced pluripotent stem cell (hiPSC) line FUi002-A was generated from skin fibroblasts obtained from a clinically diagnosed 26-year-old male DS patient with the R1525X variant of the SCN1A gene. Skin fibroblasts were reprogrammed using OriP/EBNA-1 based episomal plasmids expressing reprogramming factors expressing OCT4, SOX2, KLF-4, L-MYC, LIN28, and p53 shRNA. The transgene-free FUi002-A showed pluripotency, three germ layer differentiation capacity in vitro, and a normal karyotype. The resulting hiPSCs were heterozygous for the mutation in the SCN1A gene.

Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen.

Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR) is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv) specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy.

Duplex PCR-RFLP for the Simultaneous Genotyping of Single Nucleotide Polymorphisms in ADH1B and ALDH2 Genes.

Single nucleotide polymorphisms (SNPs) in ADH1B and ALDH2, which encode alcohol dehydrogenase 1B and aldehyde dehydrogenase 2, respectively, are responsible for ethanol-metabolizing activity and alcohol-related disease. We developed a simple and rapid polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) method for the simultaneous genotyping of the genes in a single-tube reaction. Using an alkaline lysate of a human hair root as a template, SNP-containing regions of each gene were PCR-amplified simultaneously. The PCR products were directly applied to double restriction digestion, followed by agarose gel band analysis. The duplex PCR-RFLP allows for the simultaneous and accurate determination of the SNP genotypes of ADH1B and ALDH2 within 1.2 h.

Role of Geminin in cell fate determination of hematopoietic stem cells (HSCs).

Geminin exerts two distinct molecular roles. Geminin negatively regulates DNA replication licensing through the direct interaction with Cdt1 to prevent re-replication in proliferating cells. Geminin also regulates chromatin remodeling through the direct interaction with Brahma/Brg1 to maintain undifferentiated states of stem cells. We previously uncovered that Polycomb-group complex 1 and Hoxb4/Hoxa9, well-known intrinsic factors that are essential for maintaining the hematopoietic stem cell (HSC) activity, alternatively act as ubiquitin-proteasome systems for Geminin protein to reduce the protein expression level, and sustain the HSC activity. Thus, Geminin is presumed to play an important role in determining cell fate, i.e., turning on and off cellular quiescence and proliferation/differentiation, in HSCs. We recently generated recombinant cell-penetrating Geminin (CP-Geminin), enabling rapid incorporation and withdraw of Geminin protein in cells. CP-Geminin may be useful in regulating the cell cycle and chromatin configuration. In this article, we summarize current information on the molecular functions of Geminin and the regulatory system for Geminin protein expression, and argue for the molecular role of Geminin in cell fate determination of HSCs, and future perspective of a new technology for manipulating the activities of HSCs and cancer stem cells (CSCs).

Corrigendum to "Molecular Characterization of a Fully Human Chimeric T-Cell Antigen Receptor for Tumor-Associated Antigen EpCAM".

[This corrects the article DOI: 10.1155/2012/853879.].

Molecular cogs of the insect circadian clock.

During the last five years, enormous progress has been made in understanding the molecular basis of circadian systems, mainly by molecular genetic studies using the mouse and fly. Extensive evidence has revealed that the core clock machinery involves "clock genes" and "clock proteins" functioning as molecular cogs. These participate in transcriptional/translational feedback loops and many homologous clock-components in the fruit fly Drosophila are also expressed in mammalian clock tissues with circadian rhythms. Thus, the mechanisms of the central clock seem to be conserved across animal kingdom. However, some recent studies imply that the present widely accepted molecular models of circadian clocks may not always be supported by the experimental evidence.

Structural analysis and identification of novel isoforms of the circadian clock gene period in the silk moth Bombyx mori.

The molecular basis of the circadian clock is an autoregulatory feedback loop in which the PAS domain-containing protein PERIOD periodically inhibits its own transcription. In the present study on PERIOD of the silk moth Bombyx mori, we have cloned two distinct period mRNA homologues with different PAS domain sequences either with or without the pentapeptide GTQEK. A period cDNA fragment first amplified by PCR exhibited a 15 bp-deleted nucleotide sequence in the PAS domain, compared with the database sequence. A possible alternative splicing mechanism was examined by PCR analyses, and a 15 bp-inserted clone was also amplified. The entire sequences of these period alpha and period beta isoforms were then determined by the 3' and 5' RACE methods. Isoform period alpha consists of a 3,324 bp oligonucleotide encoding 1,108 amino acid residues, whereas isoform period beta comprises 3,309 bp corresponding to 1,103 amino acids. Isoforms period alpha and period beta were found to be exactly identical except for the 15 bp deletion/insertion site. Such a pair of isoforms with a deletion/insertion sequence, namely two splice variants, has previously been reported only for the PERIOD proteins of the two honeybees, Apis mellifera and A. cerana. The occurrence of an alternative splicing mechanism in the B. mori period gene was hypothesized based on the genome structure recently clarified. Bombyx mori PERIOD alpha and beta proteins are the isomers that reveal firstly the different PAS domain sequences.

Simultaneous genotyping of single-nucleotide polymorphisms in alcoholism-related genes using duplex and triplex allele-specific PCR with two-step thermal cycles.

We developed a time- and cost-effective multiplex allele-specific polymerase chain reaction (AS-PCR) method based on the two-step PCR thermal cycles for genotyping single-nucleotide polymorphisms in three alcoholism-related genes: alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and µ-opioid receptor. Applying MightyAmp(®) DNA polymerase with optimized AS-primers and PCR conditions enabled us to achieve effective and selective amplification of the target alleles from alkaline lysates of a human hair root, and simultaneously to determine the genotypes within less than 1.5 h using minimal lab equipment.

Novel treatment strategies for cancer and their tumor-targeting approaches using antibodies against tumor-associated antigens.

Novel treatment strategies for cancer that are based on a more detailed understanding over the tumor biology are based on the latest new technology and are expected to improve the current treatment outcome for patients with cancer. However, many of these strategies still have one common and critical problem, being their limited specificity for tumor cells. In this context, antibodies against tumor-associated antigens (TAAs) are used in several ways to increase the tumor specificity of these novel strategies. Firstly, photodynamic or sonodynamic therapy using anti-TAA antibodies conjugated with new sensitizers offers additional therapeutic approaches. Secondly, re-targeting of T-cell immunotherapy using an anti-TAA antibody fusion protein was shown to be useful for the success of cancer immunotherapy, because the down-regulation of HLA class I molecules in tumor tissues constitutes a major tumor escape mechanism associated with tumor-specific cellular immunity. Thirdly, in oncolytic virotherapy, targeting viral vectors carrying cytolytic activity against tumor tissues by modifying the tropisms with anti-TAA antibodies is also very promising from a practical point of view.

Tumor-targeted photodynamic therapy.

Photodynamic therapy (PDT) is a well-established clinical treatment modality for various diseases, including cancer. It involves the topical or systemic administration of a photosensitizer, followed by selective irradiation of the target lesion with a specific wavelength of non-ionizing light, which triggers oxidative photodamage and subsequent death of the targeted cells. Due to this two-step therapeutic process, PDT is a safe and minimally-invasive therapy. Nevertheless, classical non-targeted photosensitizers lack sufficient tumor selectivity and are taken up in the neighboring normal tissues, resulting in undesirable adverse effects. To overcome this obstacle, diverse tumor-targeting approaches have been developed. In this article, we discuss the current strategies and rationale regarding tumor-targeted PDT.