Immune response to vaccination with DNA-hsp65 in a phase I clinical trial with head and neck cancer patients

DNA-hsp65, a DNA vaccine encoding the 65-kDa heat-shock protein of Mycobacterium leprae (Hsp65) is capable of inducing the reduction of established tumors in mouse models. We conducted a phase I clinical trial of DNA-hsp65 in patients with advanced head and neck carcinoma. In this article, we report on the vaccine`s potential to induce immune responses to Hsp65 and to its human homologue, Hsp60, in these patients. Twenty-one patients with unresectable squamous cell carcinoma of the head and neck received three doses of 150, 400 or 600 mu g naked DNA-hsp65 plasmid by ultrasound-guided intratumoral injection. Vaccination did not increase levels of circulating anti-hsp65 IgG or IgM antibody, or lead to detectable Hsp65-specific cell proliferation or interferon-gamma (IFN-gamma) production by blood mononuclear cells. Frequency of antigen-induced IL-10-producing cells increased after vaccination in 4 of 13 patients analyzed. Five patients showed disease stability or regression following immunization; however, we were unable to detect significant differences between these patients and those with disease progression using these parameters. There was also no increase in antibody or IFN-gamma responses to human Hsp60 in these patients. Our results suggest that although DNA-hsp65 was able to induce some degree of immunostimulation with no evidence of pathological autoimmunity, we were unable to differentiate between patients with different clinical outcomes based on the parameters measured. Future studies should focus on characterizing more reliable correlations between immune response parameters and clinical outcome that may be used as predictors of vaccine success in immunosuppressed individuals. Cancer Gene Therapy (2009) 16, 598-608; doi:10.1038/cgt.2009.9; published online 6 February 2009

Phase I trial of DNA-hsp65 immunotherapy for advanced squamous cell carcinoma of the head and neck

Considering that mycobacterial heat-shock protein 65 (hsp65) gene transfer can elicit a profound antitumoral effect, this study aimed to establish the safety, maximum-tolerated dose (MTD) and preliminary efficacy of DNA-hsp65 immunotherapy in patients with advanced head and neck squamous cell carcinoma (HNSCC). For this purpose, 21 patients with unresectable and recurrent HNSCC were studied. Each patient received three ultrasound-guided injections at 21-day intervals of: 150, 600 or 400 mu g of DNA-hsp65. Toxicity was graded according to CTCAE directions. Tumor volume was measured before and after treatment using computed tomography scan. The evaluation included tumor mass variation, delayed-type hypersensitivity response and spontaneous peripheral blood mononuclear cell proliferation before and after treatment. The MTD was 400 mg per dose. DNA-hsp65 immunotherapy was well tolerated with moderate pain, edema and infections as the most frequent adverse effects. None of the patients showed clinical or laboratory alterations compatible with autoimmune reactions. Partial response was observed in 4 out of 14 patients who completed treatment, 2 of which are still alive more than 3 years after the completion of the trial. Therefore, DNA-hsp65 immunotherapy is a feasible and safe approach at the dose of 400 mg per injection in patients with HNSCC refractory to standard treatment. Further studies in a larger number of patients are needed to confirm the efficacy of this novel strategy.

Construction of a 1-Mb restriction-mapped cosmid contig containing the candidate region for the familial mediterranean fever locus (MEFV) on chromosome 16p13.3

In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3. Using a combination of cosmid walking and screening for P1, PAC, BAG, and YAC clones, we have generated a contig of genomic clones spanning similar to 1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAG, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the similar to 700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported. NotI and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene. (C) 1997 Academic Press.

Intramyocardial transplantation of fibroblasts expressing vascular endothelial growth factor attenuates cardiac dysfunction

In this study, we analyzed whether transplantation of cardiac fibroblasts (CFs) expressing vascular endothelial growth factor (VEGF) mitigates cardiac dysfunction after myocardial infarction (MI) in rats. First, we observed that the transgene expression lasts longer (45 vs 7 days) when fibroblasts are used as vectors compared with myoblasts. In a preventive protocol, induction of cardiac neovascularization accompanied by reduction in myocardial scar area was observed when cell transplantation was performed 1 week before ischemia/reperfusion and the animals analyzed 3 weeks later. Finally, the therapeutic efficacy of this approach was tested injecting cells in a fibrin biopolymer, to increase cardiac retention, 24 h post-MI. After 4 weeks, an increase in neovascularization and a decrease in myocardial collagen were observed only in rats that received cells expressing VEGF. Basal indirect or direct hemodynamic measurements showed no differences among the groups whereas under pharmacological stress, only the group that received cells expressing VEGF showed a significant reduction in end-diastolic pressure and improvement in stroke volume and cardiac work. These results indicate that transplantation of CFs expressing VEGF using fibrin biopolymer induces neovascularization and attenuates left ventricle fibrosis and cardiac dysfunction in ischemic heart. Gene Therapy (2010) 17, 305-314; doi:10.1038/gt.2009.146; published online 10 December 2009

The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: Functional and localization studies

In both animal models and humans, the first and obligatory step in the activation of arylamines is N-hydroxylation. This pathway is primarily mediated by the phase-I enzymes CYP1A1, CYP1A2 and CYP4B1. In the presence of flavonoids such as alpha-naphthoflavone and flavone, both CYP3A4 and CYP3A5 have also been shown to play a minor role in the activation of food-derived heterocyclic amines. The further activation of N-hydroxyarylamines by phase-II metabolism can involve both N,O-acetylation and N,O-sulfonation catalyzed by N-acetyltransferases (NAT1 and NAT2) and sulfotransferases, respectively. Using an array of techniques, we have been unable to detect constitutive CYP1A expression in any segments of the human gastrointestinal tract. This is in contrast to the rabbit where CYP1A1 protein was readily detectable on immunoblots in microsomes prepared from the small intestine. In humans, CYP3A3/3A4 expression was detectable in the esophagus and all segments of the small intestine. Northern blot analysis of eleven human colons showed considerable heterogeneity in CYP3A mRNA between individuals, with the presence of two mRNA species in same subjects. Employing the technique of hybridization histochemistry (also known as in situ hybridization), CYP4B1 expression was observed in some human colons but not in the liver or the small intestine. Hybridization histochemistry studies have also demonstrated variable NAT1 and NAT2 expression in the human gastrointestinal tract. NAT1 and NAT2 mRNA expression was detected in the human liver, small intestine, colon, esophagus, bladder, ureter, stomach and lung. Using a general aryl sulfotransferase riboprobe (HAST1), we have demonstrated marked sulfotransferase expression in the human colon, small intestine, lung, stomach and liver. These studies demonstrate that considerable variability exists in the expression of enzymes involved in the activation of aromatic amines in human tissues. The significance of these results in relation to a role for heterocyclic amines in colon cancer is discussed.

Endostatin- and interleukin-2-expressing retroviral bicistronic vector for gene therapy of metastatic renal cell carcinoma

Background Metastatic renal cell carcinoma (mRCC) is one of the most treatment-resistant malignancies. Despite all new therapeutic advances, almost all patients develop resistance to treatment and cure is rarely seen. In the present study, we evaluated the antitumor effect of a bicistronic retrovirus vector encoding both endostatin (ES) and interleukin (IL)-2 using an orthotopic metastatic RCC mouse model. Methods Balb/C-bearing Renca cells were treated with NIH/3T3-LendIRES-IL-2-SN cells. In the survival studies, mice were monitored daily until they died. At the end of the in vivo experiment, serum levels of IL-2 and ES were measured, the lung was weighed, and the number of metastatic nodules, nodule area, tumor vessels and proliferation of tumor-infiltrating Renca cells were determined. Results Inoculation of NIH/3T3-LendIRES-IL-2-SN cells resulted in an increase in ES and IL-2 levels in the treated group (p < 0.05). There was a significant decrease in lung wet weight, lung nodule area and tumor vessels in the treated group compared to the control group (p < 0.001). The proliferation of Renca cells in the bicistronic-treated group was significantly reduced compared to the control group (p < 0.05). Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice submitted to bicistronic therapy (log-rank test, p = 0.0016). Bicistronic therapy caused an increase in the infiltration of CD4, CD4 interferon (IFN)gamma-producing, CD8, CD8 IFN gamma-producing and natural killer (CD49b) cells. Conclusions Retroviral bicistronic gene transfer led to the secretion of functional ES and IL-2 that was sufficiently active to: (i) inhibit tumor angiogenesis and tumor cell proliferation and (ii) increase the infiltration of immune cells (C) Copyright. 2011 John Wiley & Sons, Ltd.

Characterization of Adherent Umbilical Cord Blood Stromal Cells Regarding Passage, Cell Number, and Nano-biomarking Utilization

Adherent umbilical cord blood stromal cells (AUCBSCs) are multipotent cells with differentiation capacities. Therefore, these cells have been investigated for their potential in cell-based therapies. Quantum Dots (QDs) are an alternative to organic dyes and fluorescent proteins because of their long-term photostability. In this study we determined the effects of the cell passage on AUCBSCs morphology, phenotype, and differentiation potential. QDs labeled AUCBSCs in the fourth cell passage were differentiated in the three mesodermal lineages and were evaluated using cytochemical methods and transmission electron microscopy (TEM). Gene and protein expression of the AUCBSCs immunophenotypic markers were also evaluated in the labeled cells by real-time quantitative PCR and flow cytometry. In this study we were able to define the best cellular passage to work with AUCBSCs and we also demonstrated that the use of fluorescent QDs can be an efficient nano-biotechnological tool in differentiation studies because labeled cells do not have their characteristics compromised.

Application of inter simple sequence repeat (ISSR) markers to plant genetics

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)(n), can be made with a degenerate 3'-anchor, such as (CA)(8)RG or (AGC)(6)TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with P-32 or P-33 via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.

A high-resolution genetic map of the familial Mediterranean fever candidate region allows identification of haplotype-sharing among ethnic groups

Familial Mediterranean fever (FMF) is a recessive disorder of inflammation caused by mutations in a gene (designated MEFV) on chromosome 16p13.3, We have recently constructed a 1-Mb cosmid contig that includes the FMF critical region. Here we show genotype data for 12 markers from our physical map, including 5 newly identified microsatellites, in FMF families. Intrafamilial recombinations placed MEFV in the similar to 285 kb between D16S468/D16S3070 and D16S3376. We observed significant linkage disequilibrium in the North African Jewish population, and historical recombinants in the founder haplotype placed MEFV between D16S3082 and D16S3373 (similar to 200 kb). In smaller panels of Iraqi Jewish, Arab, and Armenian families, there were significant allelic associations only for D16S3370 and D16S2617 among the Armenians. A sizable minority of Iraqi Jewish and Armenian carrier chromosomes appeared to be derived from the North African Jewish ancestral haplotype. We observed a unique FMF haplotype common to Iraqi Jews, Arabs, and Armenians and two other haplotypes restricted to either the Iraqi Jewish or the Armenian population. These data support the view that a few major mutations account for a large percentage of the cases of FMF and suggest that same of these mutations arose before the affected Middle Eastern populations diverged from one another. (C) 1997 Academic Press.

Identification of FAM46D as a novel cancer/testis antigen using EST data and serological analysis

Cancer/testis Antigens (CTAs) are immunogenic proteins with a restricted expression pattern in normal tissues and aberrant expression in different types of tumors being considered promising candidates for immunotherapy. We used the alignment between EST sequences and the human genome sequence to identify novel CT genes. By examining the EST tissue composition of known CT clusters we defined parameters for the selection of 1184 EST clusters corresponding to putative CT genes. The expression pattern of 70 CT gene candidates was evaluated by RT-PCR in 21 normal tissues, 17 tumor cell lines and 160 primary tumors. We were able to identify 4 CT genes expressed in different types of tumors. The presence of antibodies against the protein encoded by 1 of these 4 CT genes (FAM46D) was exclusively detected in plasma samples from cancer patients. Due to its restricted expression pattern and immunogenicity FAM46D represents a novel target for cancer immunotherapy. (c) 2009 Elsevier Inc. All rights reserved.

Primary immunodeficiencies unravel critical aspects of the pathophysiology of autoimmunity and of the genetics of autoimmune disease

Background Primary Immunodeficiencies (PIDs) represent unique opportunities to understand the operation of the human immune system. Accordingly, PIDs associated with autoimmune manifestations provide insights into the pathophysiology of autoimmunity as well as into the genetics of autoimmune diseases (AID). Epidemiological data show that there are PIDs systematically associated with AID, such as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), Omenn syndrome, autoinunune polyendocrinopathy-candidiasis-ectodertnal dystrophy (APECED), autoinumine lymphoproliferative syndrome (ALPS), and C1q deficiency, while strong associations are seen with a handful of other deficits. Conclusion We interpret such stringent disease associations, together with a wealth of observations in experimental systems, as indicating first of all that natural tolerance to body components is an active, dominant process involving many of the components that ensure responsiveness, rather than, as previously believed, the result of the mere purge of autoreactivities. More precisely, it seems that deficits of Treg cell development, functions, numbers, and T cell receptor repertoire are among the main factors for autoimmunity pathogenesis in many (if not all) PIDs most frequently presenting with autoimmune features. Clearly, other pathophysiological mechanisms are also involved in autoimmunity, but these seem less critical in the process of self-tolerance. Comparing the clinical picture of IPEX cases with those, much less severe, of ALPS or APECED, provides some assessment of the relative importance of each set of mechanisms.

Association of the UGT1A1-53(TA)(n) polymorphism with L-thyroxine doses required for thyrotropin suppression in patients with differentiated thyroid cancer

There is a considerable interindividual variation in L-thyroxine [ 3,5,3`,5`-tetraiodo-l-thyronine (T(4))] dose required for thyrotropin (thyroid-stimulating hormone) suppression in patients with differentiated thyroid cancer. To investigate whether uridine diphosphate-glucuronosyl transferase 1A1 (UGT1A1)-mediated T(4) glucuronidation in liver affects T(4) dose, we genotyped 101 patients for the common UGT1A1-53(TA)(n) polymorphism and compared T(4) doses among patients having zero (5/6 and 6/6 genotypes), one (6/7 genotype), or two (7/7 and 7/8 genotypes) copies of the low-expression (TA) 7 and (TA) 8 alleles. A significant trend for decreasing T(4) dose with increasing number of copies of (TA)(7) and (TA)(8) (P = 0.037) and significant difference in T(4) dose across the UGT1A1-53(TA)(n) genotypes (P = 0.048) were observed, despite considerable overlap of T(4) doses among different genotypes. These results are consistent with reduced T(4) glucuronidation in patients with low-expression (TA) 7 and (TA) 8 alleles and provide the first evidence for association between UGT1A1-53(TA)(n) and T(4)-dose requirement for thyroid-stimulating hormone suppression in a natural clinical setting. Pharmacogenetics and Genomics 21: 341-343 (C) 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins. Pharmacogenetics and Genomics 2011, 21: 341-343

Evaluation of the genotoxicity of treated urban sludge in the Tradescantia micronucleus assay

The sludge produced in sewage treatment plants can contain toxic substances. Among these, the genotoxic substances are of great concern. The present paper aimed at evaluating the genotoxicity of treated sludge samples collected at four different sewage treatment plants (STP) located in the State of Sao Paulo Brazil, using the Trad-MN assay. Another objective of the study was to compare the responses of the Clone #4430 with the Tradescantia pallida. Sludge samples mixed with reference soil in concentrations of 10, 25 and 50% (v/v) were tested in experiments with 3 months exposure in the field. Negative and positive controls (arsenic trioxide) were also tested with both plants. In Clone #4430 two sludge samples induced genotoxicity while in T pallida three were positive, although no clear dose-response were observed for both plants. Results with the negative and positive controls suggest that T pallida presented similar results when compared to the Clone #4430. The protocol using plants chronically exposed to sludge mixed with soil seems to be a promising tool to assess the genotoxicity of sludge although time consuming. (C) 2008 Elsevier B.V. All rights reserved.

Deregulated expression of superoxide dismutase-2 correlates with different stages of cervical neoplasia

Objective: Superoxide dismutase-2 (SOD2) is considered one of the most important antioxidant enzymes that regulate cellular redox state in normal and tumorigenic cells. Overexpression of this enzyme may be involved in carcinogenesis, particularly in lung, gastric, colorectal and breast cancer. Methods: In the present study, we have evaluated SOD2 protein levels by immunohistochemistry (IHC) in 331 cervical histological samples including 31 low-grade cervical intraepithelial neoplasia (LSIL), 51 high-grade cervical intraepithelial neoplasia (HSIL), 197 squamous cervical carcinomas (SCC) and 52 cervical adenocarcinomas (ADENO). Results: We observed that SOD2 staining increases with cervical disease severity. Intense SOD2 staining was found in 13% of LSIL, 25.5% of HSIL and 40% of SCC. Moreover, 65.4% of ADENO exhibited intense SOD2 staining. Conclusions: Differences in the expression of SOD2 could potentially be used as a biomarker for the characterization of different stages of cervical disease.