Impact of radical and partial nephrectomy on renal function in patients with renal cancer.

OBJECTIVE: To evaluate renal function in renal cancer patients undergoing radical nephrectomy (RN) or partial nephrectomy (PN) (open or laparoscopic - ORN, OPN, LRN or LPN) and to identify risk factors contributing to renal function loss. METHODS: We analysed 228 consecutive renal cancer patients admitted for OPN, LPN, ORN or LRN. The variables analysed were age, gender, weight, type of surgery (radical versus partial), type of surgical access (open versus laparoscopic), preoperative renal function and history of hypertension, diabetes or malignancy. Absolute renal function was calculated as the difference in glomerular filtration rate (ΔGFR) between the renal function before (GFR0) and 12 months after surgery (GFR12). The relative renal function of patients undergoing PN and RN was evaluated by the change in chronic kidney disease stage. RESULTS: LRN caused the greatest loss in absolute renal function, followed by ORN, LPN and OPN. A GFR of ≥60 ml/min was noted for 90 (68.7%) patients before and 65 (49.6%) patients after RN and for 80 (82.5%) patients before and 74 (76.3%) patients after PN. The chronic kidney disease stage dropped to 4 or 5 in the case of 6 (4.6%) patients who underwent RN and 2 (2.1%) patients who underwent PN. Multivariate analysis revealed that only preoperative weight and type of surgery (radical versus partial) had a significant impact on renal function. CONCLUSION: Renal function significantly decreased in patients undergoing RN, irrespective of the access route. Patients with preoperative poor renal function are at risk of postoperative end-stage renal disease.

Unbiased label-free quantitative proteomic profiling and enriched proteomic pathways in seminal plasma of adult men before and after varicocelectomy.

STUDY QUESTION: Does the seminal plasma proteomic profile and functional enrichment of gene ontology terms change after microsurgical varicocelectomy? Are there any potential targets for diagnosis or therapeutic intervention in varicocele? SUMMARY ANSWER: A shift in state from a responsive-to-stress condition before varicocele correction to a responsive-to-environment condition after varicocelectomy was observed in enriched proteomic pathways. WHAT IS KNOWN ALREADY: Varicocele may lead to many adverse effects, including failure of testicular growth and development, and is associated with decreased semen quality and increased semen oxidative stress. Varicocelectomy is the treatment of choice, and is associated with improved semen quality, but little is known regarding the underlying molecular mechanisms and post-genomic pathways following intervention. STUDY DESIGN, SIZE, DURATION: A prospective study was carried out including 18 adult men with varicocele. These patients provided one semen sample before they were submitted for bilateral varicocele repair through microsurgical varicocelectomy, and one other semen sample 90 days after the surgery. PARTICIPANTS/MATERIALS, SETTING, METHODS: An aliquot of each semen sample was used for unbiased proteomics analysis by a label-free quantitative approach (2D nanoUPLC-ESI-MS(E)). Samples were pooled according to group (normalized to protein content) and run in quadruplicate. These quadruplicate runs provided degrees of freedom in order to compare groups using a non-parametric Mann-Whitney test for quantified proteins. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 316 proteins were quantified or identified, of which 91 were exclusively identified or quantified in one of the groups (53 in the pre- and 38 in the post-varicocelectomy group), and 68 were quantified in both groups and submitted to statistical analysis, of which 5 were overrepresented in the pre-varicocelectomy group (P < 0.05). In enriched functional analysis, binding and response to stimulus functions were enriched in a common cluster (present in both groups), nitric oxide metabolism and tetratricopeptide repeat domain-binding functions were enriched in the pre-varicocelectomy group, and response to reactive oxygen species, gluconeogenesis, nicotinamide adenine dinucleotide-binding and protein stabilization were enriched in the post-varicocelectomy. LIMITATIONS, REASONS FOR CAUTION: Because a shotgun proteomics analysis was chosen in order to generate a list of putative biomarkers, a targeted follow-up study should be performed to confirm these biomarkers. WIDER IMPLICATIONS OF THE FINDINGS: The proteins found in both groups possess functions usually found in human semen. The enriched function analysis demonstrated a shift back to homeostasis after varicocelectomy, suggesting that varicocele correction promotes return of semen to a physiological state. STUDY FUNDING/COMPETING INTEREST(S): The funding for this project was received from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) as a scholarship for Ms Camargo. There was no conflict of interest.

HIV coreceptor tropism in paired plasma, peripheral blood mononuclear cell, and cerebrospinal fluid isolates from antiretroviral-naïve subjects.

A survey of HIV coreceptor usage in cerebrospinal fluid (CSF) samples, peripheral blood mononuclear cells (PBMCs), and plasma samples from naïve seropositive patients was conducted. One hundred patients were enrolled in this study. Of the 100 patients, 36 had a primary or recent infection (P-RI), 31 had an early chronic infection (>350 CD4 cells) (ECI), and 33 had a late chronic infection (LCI). All 3 compartments were sampled in a subset of 33 participants, while the remaining 67 patients provided plasma samples and PBMCs only. Seventy-seven patients harbored the R5 virus in plasma samples and had a significantly higher median and percentage of CD4(+) T cells than patients with X4 virus (437 and 281 cells/µl, respectively; P = 0.0086; 20.6% and 18.6%, respectively). The X4 strain was detected more frequently in patients with LCI than in patients with P-RI or ECI (39.3%, 19.4%, and 9.6%, respectively; P = 0.0063). PBMC and plasma tropism was concordant in 90 patients, and 73 had the R5 strain. Among patients with discordant results, 4 had the R5 virus in their plasma and the X4 virus in PBMCs; 6 showed the opposite profile. Plasma, PBMC, and CSF tropism determinations were concordant in 26/33 patients (21 patients had R5, and 5 had X4). The tropism was discordant in 5/33 patients, with the X4 virus in plasma and R5 in CSF; the HIV tropism in PBMCs was X4 in 3 patients. The remaining 2/33 patients had the R5 virus in plasma and PBMCs and the X4 virus in CSF; one of these patients had a P-RI. The discordant tropism in CSF and blood may have implications for chemokine (C-C motif) receptor 5 (CCR5) antagonist use in patients with limited response to antiretroviral therapy (ART) or in responding patients evaluated for simplification of treatment.

Switching of inferred tropism caused by HIV during interruption of antiretroviral therapy.

After interruption of highly active antiretroviral therapy, 15 out of 53 patients with the X4 HIV strain had a significantly larger decrease in CD4(+) T cell count (P = 0.001) and shorter length of treatment interruption (P = 0.02) than patients with the R5 strain. At treatment resumption, HIV inferred tropism switched from the X4 strain to the R5 variant in 9 patients (60%). These patients had a prolonged length of treatment interruption compared to that of those who still carried the X4 strain.

Prognostic factors of long-term CD4+count-guided interruption of antiretroviral treatment.

Aim of the study was to determine predictors of the duration of antiretroviral treatment interruption in patients infected with HIV. This pilot prospective, open-label, multicenter trial comprised 62 HIV-seropositive subjects who decided voluntarily to interrupt therapy after two or more years of successful HAART. The primary end-point was the time to patients being free of therapy before reaching a CD4+ cell count < or =350/microl. Fifteen of 62 patients remained in treatment interruption for more than 180 days. Patients restarting therapy had higher HIV-DNA levels (P = 0.05), were treated more frequently with NNRTI-drugs (P = 0.02), had a shorter period of HAART (P = 0.046), and lower CD4+ cell counts after day 14 of interruption of treatment (P = 0.04). Multivariate regression analysis showed that less than 323 baseline proviral HIV-DNA cp/10(6) PBMCs and more than 564 CD4 cells/microl at day 14 after interruption were associated independently with a reduced risk of restarting treatment (P = 0.041 and P = 0.012, respectively). A score based on CD4+ cell counts at nadir, at baseline, at week 2 of treatment interruption, and on baseline HIV-DNA values can identify patients with a prolonged period free safely of treatment.

A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse.

Successful vaccination against West Nile virus (WNV) requires induction of both neutralizing antibodies and cell-mediated immune responses. In this study, we have assessed the ability of a recombinant ALVAC-WNV vaccine (RECOMBITEK WNV) to elicit neutralizing antibodies and virus-specific cell-mediated immune responses in horses. In addition, we examined whether prior exposure to ALVAC-WNV vaccine would inhibit B and cell-mediated immune responses against the transgene product upon subsequent booster immunizations with the same vaccine. The results demonstrated that the recombinant ALVAC-WNV vaccine induced neutralizing antibodies and prM/E insert-specific IFN-gamma(+) producing cells against WNV in vaccinated horses. Prior exposure to ALVAC-WNV vaccine did not impair the ability of horses to respond to two subsequent booster injections with the same vaccine, although anti-vector-specific antibody and cell-mediated immune responses were induced in vaccinated horses. This report describes, for the first time, the induction of antigen-specific cell-mediated responses following vaccination with an ALVAC virus recombinant vaccine encoding WNV antigens. Moreover, we showed that both WNV-specific IFN-gamma producing cells and anti-WNV neutralizing antibody responses, are not inhibited by subsequent vaccinations with the same vector vaccine.

Porcine reproductive and respiratory syndrome (PRRS) virus-specific interferon-gamma(+) T-cell responses after PRRS virus infection or vaccination with an inactivated PRRS vaccine.

Although field studies have found porcine reproductive and respiratory syndrome (PRRSV) inactivated vaccines to be beneficial in reducing losses linked to PRRSV infection, immune mechanisms induced by these vaccines need better understanding. In the study reported here, we examined the interferon-gamma(+) (IFNgamma(+)) PRRS-specific T cell responses induced after infection and vaccination with an inactivated PRRS vaccine. Autologous monocyte-derived dendritic cells loaded with the PRRSV P120 strain were used to re-stimulate ex vivo T cells that had been primed in vivo by either the virus or the vaccine, or both. Virus-specific IFNgamma(+) T cells were quantified by using a porcine IFNgamma- ELISpot assay. A specific but low live virus-induced response was observed between days 35 and 70 for most of the pigs tested, while a significant inactivated vaccine-induced PRRSV-specific IFNgamma(+) T-cell response was measured soon after vaccination. Moreover, we observed that vaccination of pre-challenged pigs clearly favoured the PRRSV-specific cell-mediated immunity primed by the live virus. To characterize further the nature of the PRRSV-specific T cells, the different T-cell subsets involved in PRRSV immunity were analyzed by flow cytometry. We showed that the inactivated vaccine was able to prime both CD4(+)CD8(int+) and CD8(high) virus-specific T cells and that CD4(+)CD8(int+) were preferentially recalled by the live virus.

Toward a detailed characterization of feline immunodeficiency virus-specific T cell immune responses and mediated immune disorders.

Infection of domestic cats with feline immunodeficiency virus (FIV) is associated with the development of an acquired immunodeficiency syndrome (AIDS). The pathogenesis of FIV is not fully understood but it has been reported that the immune system is progressively impaired during disease progression. As a result, anti-FIV specific immune response will usually not clear the virus and the acute stage is followed by a chronic asymptomatic phase. The overall objective of this study was to characterized FIV-induced immune cellular responses and -mediated immune disorder following the first weeks post-infection. Using both cytokine ELISpot and intracellular staining assays, FIV-specific T cells were monitored at 6, 9 and 12 weeks post-infection. We demonstrated that both IFNgamma(+) and, CD4 and CD8 TNFalpha(+) T cells specifically respond to FIV antigens. These responses were found to reach a peak at 9 weeks post-infection. It was further shown that the TNFalpha(+)CD8(+) responding T cells were contained within a CD8beta(low)CD62L(-) T cell subpopulation, expanded in FIV-infected cats. This T cell subpopulation which present features of activated CD8 T cells was further shown to be susceptible to spontaneous apoptosis following a short-term in vitro culture. Moreover, it was observed that cell death by apoptosis of this T cell subset was increased following FIV antigen-recognition. Therefore, FIV might alter immune homeostasis in inducing chronic activation of TNFalpha(+)CD8(+) T cells which eventually will die following antigen contact while deleting CD4(+) T cells. Interestingly, this study confirmed the strong similarity between FIV and HIV pathogenesis.

Long-term culture of human bone marrow. I. characterization of adherent cells in flow cytometry.

Using long-term culture techniques, we were able to characterize the different adherent cell elements of the in vitro bone marrow microenvironment by flow cytometry. After 2 weeks of culture, four adherent cell subsets, L, MM, F, and M, were distinguished according to their intrinsic cellular characteristics of volume and spontaneous fluorescence. Using four immunological markers, we identified each population as a hematopoietic lymphocytic group (L), CD45+, VIM2-, CD14-; a hematopoietic myelomonocytic group (MM), CD45+, VIM2+, CD14-; a hematopoietic macrophage group (M), brightly autofluorescent, CD45+, VIM2-, CD14+; and a group of fibroblastoid cells (F) with a very high volume, CD45-, VIM2-, CD14-, and collagen III+. Isolation of the different hematopoietic and nonhematopoietic components of long-term bone marrow culture is thus possible using flow cytometry analysis according to the cell characteristic of volume and spontaneous fluorescence alone.

Severe Respiratory Distress in a Child with Pulmonary Idiopathic Hemosiderosis Initially Presenting with Iron-Deficiency Anemia.

Idiopathic pulmonary hemosiderosis (IPH) is a rare cause of alveolar hemorrhage in children but should be considered in children with anemia of unknown origin who develop respiratory complications. It is commonly characterized by the triad of recurrent hemoptysis, diffuse parenchymal infiltrates, and iron-deficiency anemia. Pathogenesis is unclear and diagnosis may be difficult along with a variable clinical course. A 6-year-old boy was admitted to the hospital with a severe iron-deficiency anemia, but he later developed severe acute respiratory failure and hemoptysis requiring intubation and mechanical ventilation. The suspicion of IPH led to the use of immunosuppressive therapy with high dose of corticosteroids with rapid improvement in clinical condition and discharge from hospital.

Surface display on staphylococci: a comparative study.

Two different host-vector expression systems, designed for cell surface display of heterologous receptors on Staphylococcus xylosus and Staphylococcus carnosus, respectively, were compared for the surface display of four variants of a 101 amino acid region derived from the G glycoprotein of human respiratory syncytial virus (RSV). Surface localization of the different chimeric receptors was evaluated by a colorimetric assay and by fluorescence-activated cell sorting. It was concluded that the S. carnosus system was better both in the ability to translocate inefficiently secreted peptides and in the number of exposed hybrid receptors. The potential use of the described staphylococci as live bacterial vaccine vehicles or alternatives to filamentous phages for surface display of protein libraries is discussed.

Avidin-binding to peripheral blood B lymphocytes and monocytes.

Avidin-coated magnetic beads bind peripheral blood B lymphocytes and monocytes. This unwanted reactivity is not due to the membrane expression of avidin target molecules since beads coated with a biotin-binding analogue are non-reactive and binding occurs even when cellular carbohydrate-binding sites are not active, in the absence of Mg2+ and Ca2+ cations, or when they are blocked by a alpha-D-glucose or alpha-D-mannose in presence of Ca2+ and Mg2+. The non-polar residues of avidin appear not to be engaged in a hydrophobic bond with the membrane molecule since suroptimal quantities of serum albumin do not prevent the avidin binding. It is suggested that ionic interactions explain the binding of avidin-coated beads to B lymphocytes and monocytes and that these can be inhibited with high molecular weight serum molecules or with 0.4 M NaCl.

Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding.

The cholera toxin B (CTB) subunit has been used extensively in vaccine research as a carrier for peptide immunogens due to its immunopotentiating properties, where coupling has been obtained either by genetic fusion or chemical conjugation. For genetically fused immunogens both N- and C-terminal fusions have been used. Only shorter extensions have previously been evaluated and in some reports these fusions have impaired the biological functions of CTB, such as the ability to form pentamers and to adhere to its cell receptor, the GM1 ganglioside. Here we report the first systematic study where the same fusion partner has been used for either C-terminal, N-terminal or dual fusions to CTB. The serum albumin binding region (BB, approximately 25 kDa) from streptococcal protein G, which is known to fold independently of N- or C-terminal fusions, was selected as fusion partner. The three fusion proteins CTB-BB, BB-CTB and BB-CTB-BB were expressed in Escherichia coli, where they were efficiently secreted to the periplasmic space, and could be purified by affinity chromatography on human serum albumin (HSA) columns. The CTB fusion proteins were compared for their ability to form pentamers, by gel electrophoresis and size-exclusion chromatography, and it was concluded that all three fusion proteins were able to pentamerize. Interestingly, the C-terminal fusion to CTB showed most efficient pentamerization, while the dual fusion was much less efficient. Purified pentamer fractions from all three fusions where found to react to a monoclonal antibody described to react only to pentameric forms of CTB. In addition, the purified pentamer fractions were analyzed in an enzyme-linked immunosorbent assay (ELISA) for their ability to bind GM1, and it was found that the C-terminal fusion (CTB-BB) showed significant GM1-binding, but that also the N-terminal and dual CTB fusion proteins bound GM1, although less efficiently. The implications of the results for the design and use of CTB fusion proteins as subunit vaccines are discussed.

Flow cytometric quantification of surface-displayed recombinant receptors on staphylococci.

Surface display of recombinant proteins on bacteria and phages has become an important tool in bioscience. To evaluate the various host systems, a great need exists for quantitative methods to determine the densities of displayed proteins and peptides on the bacteria and phage surfaces. Here we describe how a method previously applied for quantification of surface proteins on mammalian cells has been adapted for quantification of chimeric receptors surface-displayed on bacteria; in this study, the bacteria being recombinant staphylococci. The presented method takes advantage of fluorescence-activated cell sorting (FACS) technology and a new type of nonfluorescent plastic beads, similar in size (2 microns in diameter) to bacterial cells, and thus suitable for generation of calibration curves from which the number of chimeric receptors can be obtained. The method was used to estimate the number of antigenic sites on two types of recombinant staphylococci, both carrying heterologous chimeric receptors, and it was found that the recombinant Staphylococcus carnosus cells carried approximately 10(4) surface-displayed antigenic sites, while recombinant Staphylococcus xylosus exposed approximately 3 x 10(3) sites per cell. The use of the deviced method for different applications is discussed.

Differential expression of the LFA-1 molecule on the human peripheral blood mononuclear cell subpopulations.

The expression of leukocyte function associated antigen 1 (LFA-1) was studied on human peripheral blood mononuclear cells (PBMNC) by a double immunofluorescence method. Monoclonal antibodies IOT18 (recognizing the beta chain, 95 kDa, CD18) and SPVL7 (recognizing the alpha chain, 180 kDa, CD11a) were used. These antibodies revealed the same heterogeneous distribution of the LFA-1 molecule among peripheral blood mononuclear cells. A bimodal profile characterized the lymphocytes, whereas the monocyte subpopulation presented a unimodal profile. NK cells, including CD16, CD8 dim, expressed the LFA-1 with a high density. B cells expressing the CD19 phenotype showed surface LFA-1 at a low level, whereas it appeared with an intermediate intensity on the T helpers/inducers (CD4 cells). Among the cytotoxic cells (CD8) and the immunoregulatory subpopulations associated with an immunosuppressive response (CD8+ CD11b+, CD8+ CD11b- and CD8+ leu8+) or with a contrasuppressive response (Vicia villosa cells), an heterogeneous LFA-1 expression was observed, discriminating the LFA-1 dim and the LFA-1 bright cells. Anti LFA-1 antibodies also allowed us to define three novel subsets among the CD8 cells: the CD8 dim LFA-1 bright, the CD8 bright LFA-1 bright, and the CD8 bright LFA-1 dim cell subpopulations. This heterogeneous distribution of LFA-1 among PBMNC may be associated with different functions of the different cell subsets analyzed in this study.

Comparison of renal ablation with cryotherapy, dry radiofrequency, and saline augmented radiofrequency in a porcine model.

BACKGROUND: Needle ablative therapy has recently generated a lot of interest in the urologic community. We compare renal lesions produced in a porcine model using three forms of needle ablative energy: cryoablation (CR), dry radiofrequency (RF), and saline augmented radiofrequency (SARF). STUDY DESIGN: In 10 farm pigs, under ultrasonographic guidance, 40 laparoscopic renal lesions were produced: 825-mm CR lesions were produced with 2.4-mm cryoprobes (Endocare Inc, Irvine, CA), after 1-mL preinfusions of 14.6% saline, 12 SARF lesions were created with 22-gauge needles (2 mL/minute 14.6% saline, 50 W 510 kHz RF for 60 seconds), 12 RF lesions were created with a 2-cm array LeVeen electrode and an RF2000 generator using impedance limited 30 to 60 W double activations (Radiotherapeutics Corp, Mountain View, CA), and 8 RF lesions were produced using 22-gauge needles and double 10 W activations with the RF2000 generator. Eight animals were sacrificed after 1 week for acute pathology. An additional two animals were sacrificed at 8 weeks to provide chronic pathology results for the LeVeen dry RF and SARF modalities. RESULTS: CR produced a regular 18- to 22-mm zone of complete necrosis bordered by a 1.5- to 2.5-mm zone of partial necrosis. Acutely, LeVeen RF and single-needle RF produced lesions 25 to 45 mm and 6 to 10 mm wide, respectively. Acutely, SARF produced irregular cone-shaped lesions 15 to 31 mm wide. Only one of eight acute LeVeen RF lesions showed complete necrosis; none of the four 8-week LeVeen RF lesions displayed complete necrosis. Two of the four 8-week SARF lesions displayed complete necrosis. The remainder of the LeVeen RF, single-needle RF, and SARF lesions showed early, indeterminate tubular damage with relative glomerular sparing and bands of complete necrosis (0.5 to 1.5 mm) and inflammation (0.5 to 2 mm) at the periphery. Only CR could be consistently monitored with laparoscopic ultrasonography. CONCLUSIONS: Renal cryoablation produces well-defined, completely necrotic lesions that can be monitored reliably with ultrasonography. Longer followup may be required to characterize the full extent of renal necrosis produced by RF, but in the short run, none of the RF modalities reliably produced 100% necrosis in all cases.

Hydrophobicity engineering to facilitate surface display of heterologous gene products on Staphylococcus xylosus.

Protein engineering has been employed to investigate the effect of specific amino acid changes on the targeting of heterologous proteins to the outer cell surface of the Gram-positive bacterium Staphylococcus xylosus. Three different variants, corresponding to a 101 amino acid region of the major glycoprotein (G protein) of human respiratory syncytial virus (RSV), were generated in which multiple hydrophobic phenylalanine residues were either substituted or deleted. The different G protein fragments were expressed as one part of recombinant receptors designed for surface display on S. xylosus cells. The engineered variants of the RSV G protein hybrid receptors were, in contrast to a non-engineered fragment, efficiently targeted to the outer cell surface of recombinant S. xylosus cells as determined by different methods, including fluorescence-activated cell sorting. In addition, immunization of mice with live recombinant S. xylosus demonstrated that surface exposure was required to generate receptor-specific antibodies. The present strategy of hydrophobic engineering should be of general interest in surface-display applications and for secretion of proteins otherwise difficult to translocate through host cell membranes.

Enhanced protective response and immuno-adjuvant effects of porcine GM-CSF on DNA vaccination of pigs against Aujeszky's disease virus.

This study was conducted to investigate whether the co-delivery of DNA encoding porcine cytokines would enhance a protective immune response in pigs to a Pseudorabies virus (PRV; or Aujeszky's disease virus) DNA vaccine. Aujeszky's disease in pigs results in respiratory and nervous symptoms with important economic losses. To evaluate cytokine effects, eukaryotic expression vectors were constructed for porcine GM-CSF, IL-2 and IFN-gamma. cDNA for each of these cytokines was inserted under the control of a CMV promoter in the pcDNA3 plasmid and cytokine expression was confirmed after DNA transfection in various mammalian cell cultures by bioassays (GM-CSF and IL2) and ELISA (IFN-gamma). Pigs were vaccinated by single intramuscular injection with plasmid DNA encoding PRV gB and gD along with various combinations of cytokine plasmid constructs. Pig serum was tested for the production of antibody by isotype specific anti-PRV ELISA. Pigs were then challenged with the highly virulent PRV strain NIA3 on day 21 after vaccination. The survival and growth rate of pigs were monitored for seven days after the viral challenge. The co-administration of GM-CSF plasmid increased the immune response induced by gB and gD PRV DNA vaccine. This immune response was characterized by an earlier appearance of anti-PRV IgG2, a significantly enhanced anti-PRV IgG1 and IgG2 antibody response, a significantly decreased and shortened viral excretion in nasal swabs and an improved protection to the viral challenge. In contrast, the co-administration of porcine IL-2 or IFN-gamma had no adjuvant effects. Our results thus demonstrate for the first time that the application of porcine GM-CSF gene in a DNA vaccine formulation can exert immuno-adjuvant and protective effects with single vaccination in the natural host pig against Aujeszky's disease.

Quantitative analysis of the antigen-specific IFNgamma+ T cell-mediated immune response in conventional outbred pigs: kinetics and duration of the DNA-induced IFNgamma+ CD8+ T cell response.

It is now well established that antigen-specific CD8(+) T cells play a major role in vaccine-induced immunity against intracellular pathogens and tumor cells. The detection of these immune cells in outbred animals has been hampered mainly by the need to generate individual autologous antigen-presenting cells (APCs) due to the high degree of polymorphism of the major histocompatibility complex (MHC) Class I loci. We used individually derived immature porcine dendritic cells infected with a pox-based recombinant viral vector to ex vivo stimulate PBMCs from vaccinated conventional pigs. The frequencies of antigen-specific T cells was determined by the number of IFNgamma-secreting cells in a quantitative enzyme-linked immune spot (ELISPOT) assay. Using this approach we were able to rank different pseudorabies virus (PRV) vaccines strategies for their ability to prime viral-specific IFNgamma(+) T cells. Plasmid DNA has recently emerged as a promising tool with multiple applications in the field of infectious diseases, allergy and cancer. We showed for the first time in this study that DNA immunization induced a long-lived antigen-specific IFNgamma(+) T cells response in conventional pigs. Additional studies allowed us to show that these virus-specific IFNgamma(+) responding cells detected in this ELISPOT assay were MHC-restricted and comprised in the CD8alpha(bright) pig T cell subset. These new data confirm the usefulness of DNA vaccines to control diseases requiring cellular immunity in pigs.

Flexible ureteroscopic lithotripsy: first-line therapy for proximal ureteral and renal calculi in the morbidly obese and superobese patient.

BACKGROUND AND PURPOSE: The surgical treatment of kidney and proximal ureteral stones in morbidly obese patients (>14 kg/m2) remains difficult because shockwave lithotripsy is precluded by weight limitations and percutaneous nephrolithotomy is associated with difficult access and a high (9%) rate of transfusion. We review our experience with retrograde ureteroscopic lithotripsy in morbidly obese patients with renal and proximal ureteral stones. PATIENTS AND METHODS: Between December 1992 and April 2000, five women and three men with a mean age of 46.5 years (range 33-68 years) and a mean body mass index of 54 (range 45-65.2) underwent 10 independent ureteroscopic procedures for urolithiasis. The average stone size was 11.1 mm (range 5-25 mm). Lithotripsy was performed with the holmium laser in eight patients (60%) the electrohydraulic lithotripter in four (30%), and the tunable-dye laser in the remaining patient. Stone-free status was defined as no stones visible on a plain film with nephrotomograms or CT scan at 3 months. RESULTS: The mean operation time was 101 minutes (range 45-160 minutes), and 60% of the procedures were done on an outpatient basis. After the initial procedure, the stone-free rate was 70%. Two patients had fragments <4 mm, and no further therapy was undertaken. There was one complication: transient renal insufficiency (serum creatinine concentration 3.7 mg/dL) secondary to aminoglycoside toxicity. No transfusions were needed. CONCLUSION: In the morbidly obese patient with symptomatic stones <1.5 cm, ureteroscopic lithotripsy is safe, successful, and efficient.