Differentiated dendritic cells expressing nuclear RelB are predominantly located in rheumatoid synovial tissue perivascular mononuclear cell aggregates

Objective. Differentiated dendritic cells (DC) and other antigen-presenting cells are characterized by the nuclear location of RelB, a member of the nuclear factor kappa B/Rel family. To characterize and enumerate differentiated DC in rheumatoid arthritis (RA) peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST), the expression and location of RelB were examined. Methods. RelB protein expression and cellular location were determined in RA PB, SF, and ST by flow cytometry and immunohistochemical analysis of purified cells or formalin-fixed tissue. DNA-binding activity of RelB was determined by electrophoretic: mobility shift-Western immunoblotting assays. Results. Circulating RA PBDC resembled normal immature PBDC in that they did not express intracellular RelB protein. In RA ST serial sections, cells containing nuclear RelB (nRelB) were enriched in perivascular regions. A mean +/- SD of 84 +/- 10% of these cells were DC. The remaining nRelB+,HLA-DR+ cells comprised B cells and macrophages. Only 3% of sorted SFDC contained nRelB, However, RelB present in the nucleus of these SFDC was capable of binding DNA, and therefore capable of transcriptional activity. Conclusion. Circulating DC precursors differentiate and express RelB after entry into rheumatoid ST. Differentiated DC can thus be identified by immunohistochemistry in formalin-fixed ST. Signals for DC maturation may differ between RA ST and SF, resulting in nuclear location of RelB predominantly in ST. This is likely to have functional consequences for the DC in these sites.

Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a bloom-forming cyanobacterium found in both tropical and temperate climates which produces cylindrospermopsin, a potent hepatotoxic secondary metabolite. This organism is notorious for its association with a significant human poisoning incident on Palm Island, Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYPO2OB were identified by PCR using degenerate primers based on conserved regions within each gene. Examination of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii. (C) 2001 John Wiley & Sons, Inc.

Retrograde delivery of photosensitizer (TPPp-O-beta-GluOH)(3) selectively potentiates its photodynamic activity

Photodynamic therapy involves administration of a photosensitizing drug and its subsequent activation by visible light of the appropriate wavelength. Several approaches to increasing the specificity of photosensitizers for cancerous tissues and, in particular, through their conjugation to ligands that are directed against tumor-associated antigens have been investigated. Here, we have studied the delivery of the photocytotoxic porphyrin compound TPP(p-O-beta-D-GluOH)(3) into tumor cells that overexpress the glycosphingolipid Gb3, using the Gb3-binding nontoxic B-subunit of Shiga toxin (STxB) as a vector. To allow for site-directed chemical coupling, an STxB variant carrying a free sulfhydryl moiety at its C-terminal end has been used. Binding affinity, cellular uptake, singlet oxygen quantum yield, and phototoxicity of the conjugate have been examined. Despite some effect of coupling on both the photophysical properties of TPP(p-O-beta-D-GluOH)(3) and the affinity of STxB for its receptor, the conjugate exhibited a higher photocytotoxic activity than the photosensitizer alone and was exquisitely selective for Gb3-expressing tumor cells. Furthermore, our data strongly suggest that STxB-mediated retrograde delivery of the photosensitizer to the biosynthetic/secretory pathway is critical for optimal cytotoxic activity. In conclusion, a strong rationale for using retrograde delivery tools such as STxB in combination with photosensitizing agents for the photodynamic therapy of tumors is presented.

Local inflammation is crucial for T cell mediated rejection of skin graft expressing foreign antigen

Most of the skin grafts from (K14hGH.FVB C57BL/6) F1 mice, which express foreign antigen (human growth hormone, hGH) in skin keratinocytes driven by keratin 14 promoter, were spontaneously rejected by syngeneic wild type F1 recipients and hGH-specific immune responses such as antibody and hGHspecific T cells were generated in these recipients. Interestingly, a 2nd F1 hGH-expressing skin graft was rejected by graft primed recipients, but was not rejected from such recipients if CD4+ or CD8+ T cells were depleted prior to the placement of the 2nd graft. Surprisingly, this 2nd graft retained healthy even after CD4+ or CD8+ T cells were allowed to recover so that the animal could reject a freshly placed 3rd F1 hGH-expressing graft. Furthermore, inflammatory response induced by topical treatment with imiquimod could lead to the rejection of some well-healed 2nd grafts. This result indicates that both CD4+ and CD8+ T cells are required for the rejection and the ability of effector T cells to reject a graft is determined by local factors in the graft which are presumably determined by inflammation induced by surgery or imiquimod treatment. Taken together, our results suggest that in addition to CD4+ and CD8+ T cells, local environmental factors induced by inflammation are also crucial for effector T cell functions leading to graft destruction. The understanding of these local factors will lead to more effective immunotherapy for established, epithelial cancer in the future.

Clinical changes of cervical dystonia pattern in long-term botulinum toxin treated patients

Cervical dystonia (CD) is a complex disorder but the response to long-term botulinum toxin (BTX) therapy is satisfactory in most cases. Bad results are attributed by some authors to changes in muscle activation. Our purpose is to verify if the change in head deviation affects negatively the response to BTX therapy it) a long-term follow-up, and if there are any differences in clinical parameters of these patients in comparison to those with stable pattern. From a total of 88 patients evaluated at the Movement Disorders Clinics of Hospital das Clinicas - University of Sao Paulo School of Medicine between January 1993 and December 2005, 67 were included. In 24 (35.8%) change in pattern of CID was observed, in a medium follow-up period of 80 months. The time between onset of dystonia and the diagnosis of pattern change was 9.7 years. Comparing with patients with no changes in CD pattern, there were no significant statistical differences. Improvement of symptoms around 60% was reported in both groups. In conclusion, the change in head deviation observed in CD was not responsible for bad response to therapy with BTX and there were no significant differences between both groups. (C) 2009 Elsevier Ltd. All rights reserved.

Trigonal injection of botulinum toxin-A does not cause vesicoureteral reflux in neurogenic patients

Aims: We evaluated the effect of botulinum toxin type A (BTX-A) injections in the trigone on the antireflux mechanism and evaluated its short-term efficacy. Materials and Methods: Between April and December 2006, 21 patients (10 men and 11 women) were prospectively evaluated. All were incontinent due to refractory NDO and underwent detrusor injection of 300 units of BTX-A, including 50 units into the trigone. Baseline and postoperative evaluation after eight weeks included cystogram, urinary tract ultrasound and urodynamics. Results: At baseline, 20 patients had no vesicoureteral (VUR) and one had grade II unilateral VUR. Postoperative evaluation revealed no cases of de novo VUR and the patient with preinjection VUR had complete resolution of the reflux. Ultrasound showed 5 (23.8%) patients with hydronephrosis before BTX-A injection and only one (4.8%) at the followup evaluation (p=0.066). After treatment, 9 (42.8%) patients became dry, 11 (52.4%) were improved and one (4.8%) had no improvement. Improved patients received antimuscarinic treatment and 8 (38.1%) became dry, with a final total continence rate of 80.1%. Cystometric capacity increased from 271 +/- 92 to 390 +/- 1189 ml (p = 0.002), reflex volume varied from 241 +/- 96 to 323 +/- 201 ml (p = 0.020) and maximum detrusor pressure reduced from 66 +/- 39 to 38 +/- 37 cm H2O (p < 0.001). Conclusions: Our results confirm the safety of trigone injections of BTX-A in terms of development of VUR and upper urinary tract damage. Whether they are beneficial for patients with NDO or other causes of voiding dysfunction will need further studies.

The therapeutic potential of recombinant BCG expressing the antigen S1PT in the intravesical treatment of bladder cancer

Purpose: Bacillus Calmette-Guerin (BCG) continues to be employed as the most effective immunotherapy against superficial bladder cancer. We have developed an rBCG-S1PT strain that induces a stronger cellular immune response than BCG. This preclinical study was designed to test the potential of rBCG-S1PT as an immunotherapeutic agent for intravesical bladder cancer therapy. Materials and methods: A tumor was induced in C57BL/6 mice after chemical cauterization of the bladder and inoculation of the tumor cell line MB49. Next, mice were treated by intravesical instillation with BCG, rBCG-S1PT, or PBS once a week for 4 weeks. After 35 days, the bladders were removed and weighed, Th1 (IL-2, IL-12, INOS, INF-gamma, TNF-alpha), and Th2 (IL-5, IL-6, IL-10, TGF-beta) cytokine mRNA responses in individual mice bladders were measured by quantitative real time PCR, and the viability of MB49 cells in 18-hour coculture with splenocytes from treated mice was assessed. In an equivalent experiment, animals were observed for 60 days to quantify their survival. Results: Both BCG and rBCG-S1PT immunotherapy resulted in bladder weight reduction, and rBCG-S1PT increased survival time compared with the control group. There were increases in TNF-alpha in the BCG treated group, as well as increases in TNF-alpha and IL-10 mRNA in the rBCG-S1PT group. The viability of MB49 cells cocultured with splenocytes from rBCG-S1PT-treated mice was lower than in both the BCG and control groups. Conclusions: rBCG-S1PT therapy improved outcomes and lengthened survival times. These results indicate that rBCG could serve as a useful substitute for wild-type BCG. (C) 2010 Elsevier Inc. All rights reserved.

Botulinum Toxin Injection in Long-Standing Facial Paralysis Patients: Improvement of Facial Symmetry Observed up to 6 Months

Despite modern reanimation surgical techniques, facial paralysis presents with functional and aesthetic deficits. We evaluated facial symmetry after treating with botulinum toxin the healthy side of the face of 25 patients with long-standing facial paralysis who had previously been treated by surgical methods, with 6 months follow-up. Evaluation consisted of a clinical score, the two subscales of the Facial Disability Index, and surface electromyography. The mean botulinum toxin dose was 38 +/- A 5 U (range = 15-69 U). The clinical score showed significant reduction of asymmetry of 48.4% at 1 month and 16.8% after 6 months. The initial result was a consequence of reduced motion on the treated side combined with better motion on the paralyzed side. At 6 months, the treated side returned to basal scores. The residual effect seen in symmetry was due to an increase (18%) of motion in the paralyzed side. There was a significant decrease in the action potential of muscles on the nonparalyzed side 1 month post injection but completely reverted after 6 months. The Physical Function Index increased, but not significantly. The Social/Well-Being Function Index showed a significant increase at 6 months compared to pretreatment. The proposed treatment improved facial symmetry for up to 6 months. Even after the end of the clinical effect of the drug, the paralyzed side`s clinical score was 18% higher than pretreatment, with an increased quality of life.

Heterozygosity for the S180L Variant of MAL/TIRAP, a Gene Expressing an Adaptor Protein in the Toll-Like Receptor Pathway, Is Associated with Lower Risk of Developing Chronic Chagas Cardiomyopathy

Background. Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. Among T. cruzi-infected individuals, only a subgroup develops severe chronic Chagas cardiomyopathy (CCC); the majority remain asymptomatic. T. cruzi displays numerous ligands for the Toll-like receptors (TLRs), which are an important component of innate immunity that lead to the transcription of proinflammatory cytokines by nuclear factor-kappa B. Because proinflammatory cytokines play an important role in CCC, we hypothesized that single-nucleotide polymorphisms (SNPs) in the genes that encode proteins in the TLR pathway could explain differential susceptibility to CCC among T. cruzi-infected individuals. Methods. For 169 patients with CCC and 76 T. cruzi-infected, asymptomatic individuals, we analyzed SNPs by use of polymerase chain reaction-restriction fragment length polymorphism analysis for the genes TLR1, TLR2, TLR4, TLR5, TLR9, and MAL/TIRAP, which encodes an adaptor protein. Results. Heterozygous carriers of the MAL/TIRAP variant S180L were more prevalent in the asymptomatic group (24 [32%] of 76 subjects) than in the CCC group (21 [12%] of 169) (chi(2) = 12.6; P = .0004 [adjusted P (P(c)) = .0084]; odds ratio [OR], 0.31 [95% confidence interval {CI}, 0.16-0.60]). Subgroup analysis showed a stronger association when asymptomatic patients were compared with patients who had severe CCC (i.e., patients with left-ventricular ejection fraction <= 40%) (chi(2) = 11.3; P = .0008 [P(c) = .017]; OR, 0.22 [95% CI, 0.09-0.56]) than when asymptomatic patients were compared with patients who had mild CCC (i.e., patients with left-ventricular ejection fraction >40%) (chi(2) = 7.7; P = .005 [P(c) = .11]; OR, 0.33 [95% CI, 0.15-0.73]). Conclusion. T. cruzi-infected individuals who are heterozygous for the MAL/TIRAP S180L variant that leads to a decrease in signal transduction upon ligation of TLR2 or TLR4 to their respective ligand may have a lower risk of developing CCC.

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

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.

Transcriptome changes induced by docetaxel in human mammary cell lines expressing different levels of ERBB2

The taxane docetaxel is currently the most effective chemotherapeutic drug for the treatment of advanced breast cancer. However, a considerable proportion of breast cancer patients do not respond positively to docetaxel. The mechanisms of docetaxel resistance are poorly understood. Overexpression of ERBB2 occurs in 15-30% of breast tumors and is associated with chemoresistance to a variety of anticancer drugs. In the present study, we sought to identify genes involved in ERBB2-mediated chemoresistance to docetaxel. We generated SAGE libraries from two human mammary cell lines expressing basal (HB4a) and high (C5.2) levels of ERBB2 before and after intensive exposure to docetaxel and identified potential ERBB2 target genes implicated in a variety of cellular processes including cell proliferation, cell adhesion, apoptosis and cytoskeleton organization. Comparison of the transcriptome of the cell lines before and after docetaxel exposure revealed substantially different expression patterns. Twenty-one differentially expressed genes between HB4a and C5.2 cell lines, before and after docetaxel treatment, were further analyzed by qPCR. The alterations in the expression patterns in HB4a and C5.2 cell lines in response to docetaxel treatment observed by SAGE analysis were confirmed by qPCR for the majority of the genes analyzed. Our study provides a comprehensive view of the expression changes induced in two human mammary cells expressing different levels of ERBB2 in response to docetaxel that could contribute to the elucidation of the mechanisms involved in ERBB2-mediated chemoresistance in breast cancer.

Passive Acquisition of Protective Antibodies Reactive with Bordetella pertussis in Newborns via Placental Transfer and Breast-feeding

Although acquisition of anti-pertussis antibodies by the newborn via placental transfer has been demonstrated, a subsequent recrudescence of pertussis infection is often observed, particularly in infants. The present study investigated the passive transfer of anti-pertussis IgG and IgA antibodies to term newborns and their ability to neutralize bacterial pathogenicity in an in vivo experimental model using mice intracerebrally challenged with viable Bordetella pertussis. Forty paired samples of maternal/umbilical cord sera and colostrum were obtained. Anti-pertussis antibodies were analysed by immunoenzymatic assay and by Immunoblotting. Antibody neutralizing ability was assessed through intracerebral B. pertussis challenges in mice. Anti-pertussis IgG titres were equivalent in both maternal and newborn sera (medians = 1:225 and 1:265), with a transfer rate of 118%. The colostrum samples had variable specific IgA titres (median = 1:74). The immunoblotting assays demonstrated identical recognition profiles of paired maternal and newborn serum pools but different bacterial recognition intensities by colostrum pools. In the animal model, significant differences were always observed when the serum and colostrum samples and pools were compared with the positive control (P < 0.05). Unlike samples with lower anti-pertussis titres, samples with high titres showed protective capacities above 50%. Pertussis-absorbed serum and colostrum pools protected 30% of mice and purified IgG antibodies protected 65%. Both pooled and single-sample protective abilities were correlated with antibody titres (P < 0.01). Our data demonstrated the effectiveness of anti-pertussis antibodies in bacterial pathogenesis neutralization, emphasizing the importance of placental transfer and breast-feeding in protecting infants against respiratory infections caused by Bordetella pertussis.