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PubMed2004

Induction of HIV immunity in the genital tract after intranasal delivery of a MVA vector: enhanced immunogenicity after DNA prime-modified vaccinia virus Ankara boost immunization schedule.

Vaccines intended to prevent mucosal transmission of HIV should be able to induce multiple immune effectors in the host including Abs and cell-mediated immune responses at mucosal sites. The aim of this study was to characterize and to enhance the immunogenicity of a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 Env IIIB Ag (MVAenv) inoculated in BALB/c mice by mucosal routes. Intravaginal inoculation of MVAenv was not immunogenic, whereas intranasally it induced a significant immune response to the HIV Ag. Intranasal codelivery of MVAenv plus cholera toxin (CT) significantly enhanced the cellular and humoral immune response against Env in the spleen and genitorectal draining lymph nodes, respectively. Heterologous DNAenv prime-MVAenv boost by intranasal immunization, together with CT, produced a cellular immune response in the spleen 10-fold superior to that in the absence of CT. A key finding of these studies was that both MVAenv/MVAenv and DNAenv/MVAenv schemes, plus CT, induced a specific mucosal CD8(+) T cell response in genital tissue and draining lymph nodes. In addition, both immunizations also generated systemic Abs, and more importantly, mucosal IgA and IgG Abs in vaginal washings. Specific secretion of beta-chemokines was also generated by both immunizations, with a stronger response in mice immunized by the DNA-CT/MVA-CT regimen. Our findings are of relevance in the area of vaccine development and support the optimization of protocols of immunization based on MVA as vaccine vectors to induce mucosal immune responses against HIV.

AIDS Vaccines↗
PubMed2005

Electroporation as a "prime/boost" strategy for naked DNA vaccination against a tumor antigen.

We have developed novel DNA fusion vaccines encoding tumor Ags fused to pathogen-derived sequences. This strategy activates linked T cell help and, using fragment C of tetanus toxin, amplification of anti-tumor Ab, CD4(+), and CD8(+) T cell responses is achievable in mice. However, there is concern that simple DNA vaccine injection may produce inadequate responses in larger humans. To overcome this, we tested electroporation as a method to increase the transfection efficiency and immune responses by these tumor vaccines in vivo in mice. Using a DNA vaccine expressing the CTL epitope AH1 from colon carcinoma CT26, we confirmed that effective priming and tumor protection in mice are highly dependent on vaccine dose and volume. However, suboptimal vaccination was rendered effective by electroporation, priming higher levels of AH1-specific CD8(+) T cells able to protect mice from tumor growth. Electroporation during priming with our optimal vaccination protocol did not improve CD8(+) T cell responses. In contrast, electroporation during boosting strikingly improved vaccine performance. The prime/boost strategy was also effective if electroporation was used at both priming and boosting. For Ab induction, DNA vaccination is generally less effective than protein. However, prime/boost with naked DNA followed by electroporation dramatically increased Ab levels. Thus, the priming qualities of DNA fusion vaccines, integrated with the improved Ag expression offered by electroporation, can be combined in a novel homologous prime/boost approach, to generate superior antitumor immune responses. Therefore, boosting may not require viral vectors, but simply a physical change in delivery, facilitating application to the cancer clinic.

Adjuvants, Immunologic↗
PubMed1999

Chagas' disease diagnosis: comparative analysis of parasitologic, molecular, and serologic methods.

During the course of chronic chagasic infection, low parasitemia levels prevent parasite detection by current techniques such as hemoculture and xenodiagnosis. Since serologic tests have sensitivity but lack specificity, molecular assays based on the polymerase chain reaction (PCR) have been proposed as alternative tools for parasite detection in individuals with chronic Chagas' disease. A variable degree of PCR efficiency has been reported in the literature and illustrates the need for further evaluation of large numbers of chagasic patients. In this study, we compared an optimized PCR technique with hemoculture and complement-mediated lysis (CoML) in 113 individuals from or living in endemic areas of Brazil who had conventional serologic results that were either positive, negative, or inconclusive. The PCR amplification yielded positive results in 83.5% (66 of 79) of individuals with positive serology, 47.6% (10 of 21) with negative serology, and 46.2% (6 of 13) with inconclusive serology. Of 10 patients with negative serology and positive PCR result, eight (80%) had positive CoML, indicating that they could have been chagasic but were not mounting immune responses. The PCR results were also positive for all individuals who had positive hemoculture, for 37 individuals with negative hemoculture and positive serology, and for two of six individuals with inconclusive serology and negative hemoculture. Thirteen individuals living in nonendemic areas who had negative serology were used as a negative control group: 100% had negative PCR results. Our results show that the optimized PCR protocol used here was very sensitive in detecting the presence of Trypanosoma cruzi in chronic chagasic patients. The PCR and CoML results were well correlated in all of the groups studied, which suggests that our PCR protocol may be effective in the evaluation of cure in patients who receive anti-parasite treatment.

Animals↗
PubMed2005

Zidovudine: a targeted therapy for endemic Burkitt's lymphoma.

BACKGROUND: Although cyclophosphamide based regimens can produce remission rates approaching 60 to 80% in endemic Burkitts lymphoma, relapses and refractory disease are fairly common in developing countries, due to advanced stage disease and cost-constraints in the implementation of optimal chemotherapeutic protocols. OBJECTIVE: To evaluate an affordable, tolerable and targeted approach to chemotherapy for endemic Burkitt's lymphoma as would be desirable in resource poor settings such as Africa. METHOD: We present data and review pertinent literature that indicates that the antiviral agent Zidovudine specifically targets this tumour through a unique and novel mechanism. DATA SOURCE: Our original studies, publications original and review articles searched in Pubmed indexed for Medline. DATA EXTRACTION: A systematic review to identify studies relating to Zidovudine, EBV+ and Burkitt's lymphoma, indicating antiviral agents zidovudine targeting BL in a unique and novel mechanisms. DATA SYNTHESIS: Our data and a qualitative assessment of the relevant literature was undertaken, given the heterogenicity of the study types making it inappropriate to pool results across studies. CONCLUSION: Our data suggests that the incorporation of Zidovudine into Burkitt's regimens may enhance tumour kill and abbreviate the duration of treatment necessary for this disease. Furthermore, the addition of the widely available and inexpensive agent hydroxyurea, markedly potentiates the tumorcidal activity of Zidovudine in Epstein Barr virus positive Burkitt's lymphomas. We recommend that further clinical studies in patients afflicted with this disease are needed to clearly define this potential use of Zidovudine.

Africa↗
PubMed2006

Application of quantitative real-time PCR in the detection of prion-protein gene species-specific DNA sequences in animal meals and feedstuffs.

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130 degrees C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.

Animal Feed↗
PubMed2006

Genetic control of conventional labeling through the bovine meat production chain by single nucleotide polymorphisms using real-time PCR.

Since January 2002, the European Union has adopted precise guidelines aimed at protecting the safety of meat and controlling the production chain. To this purpose, the conventional traceability of livestock and meat represents the main tool, but verification of traceability requires genetic support. At present, single nucleotide polymorphisms (SNPs) represent the most innovative molecular markers in genotyping studies. The aim of this study was to verify correct labeling in a bovine meat production chain by a real-time PCR protocol based on SNP analysis. Reference hair samples from 5,000 animals were randomly collected from 22 farms. Twelve hundred meat samples were collected at different steps of the bovine meat production chain. In particular, 1,000 meat samples were collected at the slaughterhouse and 200 samples from the same animals directly at the butcher's shop. The protocol was optimized and validated by testing a set of 16 SNP markers on 95 DNA samples from bovine sires of different breeds. Thereafter, the genotyping of 2,200 samples was conducted with a set of 12 selected SNPs to verify traceability of the meat production chain at three different stages: farm, slaughterhouse, and butcher's shop. Irregularities in conventional traceability were evidenced directly in 1.87% of the samples at the slaughterhouse. This percentage increased to 3.25% when sampling was conducted at the butcher's shop. This study demonstrates that despite the precautions adopted over the meat production chain, some critical points still exist that cause the loss of a correct association between registration numbers and samples.

Animal Identification Systems↗
PubMed2004

[Reversible dementias, do they exist?].

Due to the increase of the life expectancy the prevalence of the patients with dementia in the services of Internal Medicine is very high (30% in the patients with more of 80 years old). The incidence of the potentially reversible dementias is very different in the publications consulted (10-30%). The objective of these study is to know the real incidence of these dementias and which is the best diagnostic protocol to optimize the resources. The conclusions obtained in the bibliographical review it is that the effect of this type of dementias is minor that the mentioned (1.5-3%).

Dementia↗
PubMedDate not supplied

Surgical treatment of acetabular fractures in elderly patients with osteoporotic bone.

The incidence of acetabular fractures in the elderly has recently shown a marked increase due to the combination of greater longevity for the population as a whole and a relative decrease in the incidence of alcohol-related trauma in younger adults. The compromised physiologic reserve and the diminished healing capacity of the typical elderly patient have an adverse effect on the potential for a favorable clinical outcome. The presence of osteopenic bone or degenerative arthritis and the effects of previous radiation therapy to the hip and pelvis hamper diagnostic imaging and the utility of some treatment alternatives that were designed primarily for younger patients. The diverse clinical presentations include major polytrauma, minor trauma, and insufficiency fractures. An assessment of the prior health and functional status of the patient is crucial in determining the optimal therapeutic protocol. Treatment options vary according to the clinical presentation and include conservative methods, percutaneous fixation in situ, open reduction, and acute total hip arthroplasty. The feasibility of acute total hip arthroplasty rests on the use of newly developed techniques for minimally invasive stabilization of the acetabular fracture with cables and the application of morselized or structural autograft harvested from the femoral head. Whichever surgical method is chosen, the objective is rapid mobilization of the patient on a walker or crutches. Late complications that may occur after nonoperative or operative treatment include posttraumatic arthritis, nonunion, wound infection, and heterotopic bone formation.

Acetabulum↗
PubMed2006

Computational flow dynamics in abdominal aortic aneurysm using multislice computed tomography.

Following the introduction of a new multislice computed tomography (MSCT) scanner, it has become possible to produce high-speed CT angiography (CTA), the preferred method for imaging in emergent abdominal vascular conditions. Unlike catheter angiography, multislice CTA not only depicts the vessels but also allows perfusion in adjacent organs to be assessed. To make the most effective diagnostic use of multi-detector row CTA and three-dimensional image post-processing, radiologists must be familiar with the optimal CTA protocols and the typical CT findings in various emergent vascular conditions using computational flow dynamics (CFD). This article describes a technical approach to estimating the blood flow state of human abdominal aortic aneurysms (AAA) in more detail by constructing realistic three-dimensional (3D) vessel models using CFD methods, focusing on pre- and postoperative cases.

Aged↗
PubMed2000

Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice.

Mobilized peripheral blood progenitor cells (PBPC) are a potential target for the retrovirus-mediated transfer of cytostatic drug-resistance genes. We analyzed nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating CD34+ PBPC from patients with cancer after retroviral transduction in various cytokine combinations with the hybrid vector SF-MDR, which is based on the Friend mink cell focus-forming/murine embryonic stem-cell virus and carries the human multidrug resistance 1 (MDR1) gene. Five to 13 weeks after transplantation of CD34+ PBPC into NOD/SCID mice (n = 84), a cell dose-dependent multilineage engraftment of human leukocytes up to an average of 33% was observed. The SF-MDR provirus was detected in the bone marrow (BM) and in its granulocyte fractions in 96% and 72%, respectively, of chimeric NOD/SCID mice. SF-MDR provirus integration assessed by quantitative real-time polymerase chain reaction (PCR) was optimal in the presence of Flt-3 ligand/thrombopoietin/stem-cell factor, resulting in a 6-fold (24% +/- 5% [mean +/- SE]) higher average proportion of gene-marked human cells in NOD/SCID mice than that achieved with IL-3 alone (P <.01). A population of clearly rhodamine-123(dull) human myeloid progeny cells could be isolated from BM samples from chimeric NOD/SCID mice. On the basis of PCR and rhodamine-123 efflux data, up to 18% +/- 4% of transduced cells were calculated to express the transgene. Our data suggest that the NOD/SCID model provides a valid assay for estimating the gene-transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation. P-glycoprotein expression sufficient to prevent marrow aplasia in vivo may be obtained with this SF-MDR vector and an optimized transduction protocol. (Blood. 2000;95:1237-1248)

Adipocytes↗
PubMed2000

Three-dimensional clinical PET in lung cancer: validation and practical strategies.

UNLABELLED: The feasibility of 3-dimensional acquisition mode for semiquantitative analysis in thoracic PET studies was compared to the conventional 2-dimensional mode. Several practical considerations were analyzed to propose an optimized scanning protocol for clinical use. METHODS: Twenty-one patients with focal thoracic abnormalities were evaluated with FDG PET. The acquisition consisted of 3 consecutive static scans for a single bed position: 3-dimensional (10 min), 2-dimensional (15 min), and 3-dimensional (5 min). On the basis of the average and maximum activity values per region of interest, standardized uptake value (SUV) normalized for total body weight (TBW), lean body mass (LBM), body surface area (BSA), and blood glucose level (PGL) were evaluated. The effect of the delay between tracer injection and PET scanning on the SUV, as well as on the relative error of the activity distribution, was studied from 40-134 min after tracer injection. RESULTS: A strong positive correlation was observed among SUVs from 2-dimensional and both 3-dimensional acquisitions. The mean SUV percentage differences between both acquisition modes were about 17%, differences that were not statistically significant when time postinjection was addressed in the analysis of covariance. SUVs provided the greatest variability and differences among studies on experimental periods up to 70 min postinjection. Indeed, the variability of 20% observed on the SUVs from 2 PET scans 13 min apart was reduced to 9% when the acquisitions started at least 70 min after tracer injection. In addition, a two-fold reduction in the relative error of the activity distribution was observed over this period of time. The reproducibility coefficient was increased from 0.87 to 0.95 before and after 70 min postinjection, respectively. No correlation was found between different normalization procedures of SUV and LBM, BSA, TBW, or height, whereas a weak correlation was found between SUV and PGL. CONCLUSION: 18F-FDG 3-dimensional PET is a realistic alternative to the gold standard 2-dimensional for clinical nonkinetic studies. A short, 5-min 3-dimensional acquisition at 70 min postinjection is proposed as the best protocol for the clinical evaluation of thoracic pathologies.

Aged↗
PubMed2000

Multifaceted approach to the diagnosis and classification of acute leukemias.

Until recently, the diagnosis and classification of acute myeloid (AML) and acute lymphoblastic (ALL) leukemias was based almost exclusively on well-defined morphologic criteria and cytochemical stains. Although most cases can be diagnosed by these methods, there is only modest correlation between morphologic categories and treatment responsiveness and prognosis. The expansion of therapeutic options and improvement in remission induction and disease-free survival for both AML and ALL have stimulated emphasis on defining good and poor treatment response groups. This is most effectively accomplished by a multifaceted approach to diagnosis and classification using immunophenotyping, cytogenetics, and molecular analysis in addition to the traditional methods. Immunophenotyping is important in characterizing morphologically poorly differentiated acute leukemias and in defining prognostic categories of ALL. Cytogenetic and molecular studies provide important prognostic information and are becoming vitally important in determining the appropriate treatment protocol. With optimal application of these techniques in the diagnosis of acute leukemias, treatment strategies can be more specifically directed and new therapeutic approaches can be evaluated more effectively.

Acute Disease↗
PubMed2001

[Generalized convulsive status epilepticus: analysis apropos of 57 cases].

BACKGROUND: The generalized convulsive status epilepticus (GCSE) is a medical emergency with a high morbimortality rate. We analyzed the casuistry in our hospital and we assessed the causes related to mortality. PATIENTS AND METHODS: This is a retrospective study of 57 cases of GCSE that took place between january 1992 to december 1998. The variables studied were mortality among groups according to age, previous epilepsy and the duration of the convulsive episode. RESULTS: We found male prevalence (63.2%). The 49.2% of patients didn't have previous history of epilepsy. In 38.6% of cases the treatment dose was insufficient and in 61.4% it was delated. The mortality rate was 36.8% with a higher significance in patients older than 65 (p < 0.02), without previous history of epilepsy (p < 0.001) and in episodes lasting more than 4 hours (p < 0.05). Only 12.9% of patients received prehospital treatment. CONCLUSIONS: The mortality of GCSE is very high. The initial treatment is inadequate. We must establish a premature and adequate protocol, with optim pharmacological doses and a good coordination with intensive care units.

Adolescent↗
PubMedDate not supplied

Feasibility of fluorescent detection of pathogens on pork carcasses.

The direct immunofluorescent detection of pathogens on pork skin is evaluated. Calibrate contamination of pork skin with Salmonella Typhimurium (ST) and Listeria monocytogenes (Lm) is developed in 2 h at 4 degrees C. Then a specific indirect immunofluorescent staining protocol is optimized in order to obtain specific and intensive signals able to be detected by electronic cameras (deported microscopy). Despite the individual staining of ST and Lm is possible on pork skin and is specific and bright, the deported microscopy failed to detect these particles. After respectively 3 and 6 h, we obtain micro-colonies of ST and Lm. Due to the limited power of the video camera used, only the microscope permits the detection on the skin. However, our work gives standard conditions to mime the pathogens contamination and staining directly on a biological matrix such as pork skin. This work is a first step in the development of direct and rapid detection of pathogens on biological matrix.

Animals↗
PubMedDate not supplied

[Special aspects of anesthesiology and intensive care in oncological operations with microsurgical autoplasty].

The efficiency of the most rational antithrombotic, rheological, and antiischemic agents was evaluated: low-molecular heparin fraxiparin, nonsteroid antiinflammatory drug ketoprofene (ketonal), perfluorane, and antikininogen contrycal. Twenty-eight patients divided into 3 groups were examined before, during, and after extensive interventions with simultaneous microsurgical plastic repair by complex autotissue grafts. Results of treatment of 17 patients, in whom therapeutic and prophylactic complex including fraxiparin, perfluorane, and contrycal was used, deserve special attention. Use of this drug complex according to the proposed protocol creates optimal conditions for maintaining blood supply and oxygenation of the transplant at the expense of deceleration of blood clotting, decrease of blood viscosity, and decrease of erythrocyte and platelet aggregation. Complete healing of transplants was attained in all patients, who were discharged with good plastic results.

Adult↗
PubMedDate not supplied

Routine cryopreservation of kiwifruit (Actinidia spp) germplasm by encapsulation-dehydration: importance of plant growth regulators.

The encapsulation-dehydration protocol was optimized for an in vitro cultured hybrid Actinidia arguta x A. deliciosa. Shoot tips from 14-d reactivated mononodal microcuttings were embedded, transferred to liquid culture medium whose sucrose concentration was daily increased (0.3, 0.5, 0.75 M) and then kept at 0.75 M for 2 or 4 d. Dehydration on silica gel was monitored to 20+/-1.5% residual water content (dry weight basis), allowing direct quenching in liquid nitrogen and rewarming at room temperature. Differential scanning calorimetry analysis underlined the importance of reversible glass transition in shoots for survival. Regrowth ranged from 85% to 95%. Growing shoot tips showed no phenotypic abnormalities. Rooting was also achieved. This method was routinely applied to diploid A. chinensis and A. eriantha, and to several diploid hybrids, yielding over 70% regrowth. A slight decrease in sucrose molarity (0.65 M) allowed tetraploid A. chinensis and A. chrysantha x A. arguta to survive dehydration, but not quenching in LN. For A. deliciosa cv Hayward and cv Tomuri, normal regrowth after cryopreservation was achieved only after modification of the pre- and post-culture media, highlighting the importance of monitoring plant growth regulator balance, principally at the post-thaw recovery step.

Actinidia↗
PubMed2001

A rapid polymerase chain reaction based method for identification of the Anopheles dirus sibling species.

A simple polymerase chain reaction (PCR) based method was developed to differentiate the Anopheles dirus, species A, B, C and D in Thailand using specific primers designed from species specific sequences. The PCR protocol was optimized to obtain products of 120 bp, 75 bp, 60 bp and 172 bp for species A, B, C and D, respectively. This method used a cocktail of four primer sets to identify these An. dirus sibling species. The method is very sensitive as only a small portion of mosquito was required allowing the rest of the mosquito to be used for other analyses. Specimens also kept for up to 14 years could be analyzed unambiguously from either larvae or adult. This method is advantageous over other PCR-based methods for identification of malaria vectors because it does not require any specific DNA extraction. A mosquito specimen was homogenized in 1x PCR buffer, then the supernatant directly used for PCR identification, allowing a large number of samples to be processed at the same time. It provides a simple and rapid practical method for screening An. dirus species, which is essential in malaria vector epidemiological studies in Southeast Asia.

Animals↗
PubMed2002

Tissue engineering of cardiac valve prostheses I: development and histological characterization of an acellular porcine scaffold.

BACKGROUND AND AIMS OF THE STUDY: Several deficiencies in current heart valve prostheses make them problematic for use in younger patients. Tissue valve substitutes are non-viable with a life expectancy of only 10-15 years, while mechanical valves require long-term anti-coagulation therapy. A solution to these problems would be to develop a tissue-engineered heart valve containing autologous cells, enabling the valve to maintain its biochemical and mechanical properties, yet grow with the patient. The study aim was to optimize a protocol to produce a porcine acellular matrix scaffold for use in developing a tissue-engineered heart valve. METHODS: Fresh porcine aortic valve leaflets were treated with Triton X-100, sodium dodecyl sulfate (SDS), sodium deoxycholate, MEGA 10, TnBP, CHAPS, and Tween 20, over a range of concentrations, in the presence of protease inhibitors for up to 72 h. Histological analysis was used to detect the major structural proteins of the heart valve, collagen I, elastin and glycosaminoglycans. RESULTS: After 72 h, most protocols resulted in the retention of large numbers of whole cells and cell fragments. Only SDS (0.03-1%) or sodium deoxycholate (0.5-2%) resulted in total decellularization at 24 h. Histological analysis of acellular matrices showed that the major structural proteins had been retained and appeared to be intact. CONCLUSION: Protocols utilizing SDS or sodium deoxycholate were successful for leaflet decellularization, and histological analysis showed that the major structural components of the valve matrix had been maintained. These methods are being developed further with a view to reseeding with autologous cells to produce tissue-engineered solutions for clinical implantation.

Animals↗