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.

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.

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.

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.

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.

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.

Laparoscopic bilateral hand assisted nephrectomy for autosomal dominant polycystic kidney disease: initial experience.

PURPOSE: The laparoscopic technique for bilateral nephrectomy in patients with autosomal dominant polycystic kidney disease is technically difficult. The procedure may be more acceptable if alterations to the technique made it safer and easier to perform. We describe our initial experience with, and the feasibility and potential benefits of hand assisted laparoscopic nephrectomy for approaching these large kidneys in patients with autosomal dominant polycystic kidney disease. MATERIALS AND METHODS: This approach was successfully applied in 3 patients with end stage renal disease due to autosomal dominant polycystic kidney disease. After obtaining transumbilical pneumoperitoneum ports were placed in the umbilicus (12 mm.), sub-xiphoid in the midline (12 mm.) and subcostal in the midclavicular line on each side (12 mm.). The table was tilted 40 degrees away from the planned side of initial nephrectomy with the patient in the half lateral position. A 7 cm. midline incision was made that incorporated the umbilical port and a commercially available hand assistance device was positioned. One surgeon hand was inserted into the abdomen to serve as a retractor/blunt dissector, while the other operated the electrosurgical instruments. The right hand was inserted for left nephrectomy and the left hand was inserted for right nephrectomy. The laparoscope was passed via the sub-xiphoid port and the instruments were placed through the ipsilateral subcostal laparoscopic port. Nephrectomy was completed and the specimen was removed through the hand port incision by draining the cysts as they were exposed to view via the midline incision. When dissection was difficult, an additional port was placed in the anterior axillary line at the umbilical level. Some cysts were ruptured or aspirated to decrease overall kidney size and make extraction possible via the 6 to 7 cm. midline incision. RESULTS: All procedures were successfully completed. Mean operative time for bilateral hand assisted laparoscopic nephrectomy was 5.5 hours (range 4.5 to 6.6). Estimated blood loss was 200 cc or less. Patients resumed oral intake on postoperative day 1. The mean amount of parenteral analgesics required postoperatively was decreased. Mean hospital stay was 4.3 days but it was 3 days when considering nephrectomy only. Patients returned to normal activity after an average of 2 weeks. There was sustained resolution of preoperative discomfort based on pain analog scales. At 1 month or less all patients recorded absent pain. They uniformly noticed improved preoperative pulmonary and gastrointestinal symptoms CONCLUSIONS: Hand assisted laparoscopic nephrectomy in patients with autosomal dominant polycystic kidney disease makes bilateral nephrectomy a reasonable option. The bilateral procedure may be performed as rapidly as laparoscopic only, unilateral nephrectomy in these cases. The advantages of the hand assisted approach include using tactile sensation to facilitate dissection, rapid blunt finger dissection, hand retraction and the application of immediate tamponade when needed. This procedure provides the benefits of minimal intraoperative blood loss, minimal postoperative pain, brief hospital stay and rapid convalescence in this group of patients at high risk.

Functional and phenotypic characterization of distinct porcine dendritic cells derived from peripheral blood monocytes.

Dendritic cells (DCs) are bone marrow-derived antigen-presenting cells that have an exquisite capacity to interact with T cells and modulate their responses. Little is known about porcine DCs despite the fact that they represent an important target in strategies that are aimed at modulating resistance to infection in pigs and may be of major importance in transplantation biology. We generated immature monocyte-derived porcine dendritic cells (MoDCs) directly from adherent peripheral blood cells treated with porcine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The cells were observed via electron microscopy and their phenotype was characterized using monoclonal antibodies. The functionality of the porcine MoDCs was demonstrated showing that the cells were capable of different specialized functions relevant to antigen capture and were potent stimulators in a primary allo-mixed leucocyte reaction. Treatment of the MoDCs with porcine cell line-derived necrotic factors resulted in the phenotypic and functional maturation of MoDCs. We confirmed also that monocyte-derived DCs were differentially regulated by cytokines, showing that transforming growth factor-beta1 (TGF-beta1) is able to redirect monocytic precursors into the differentiation pathway of Langerhans' cells presenting typical Birbeck granules. Interestingly, and in contrast to the human and murine model, we showed that the monocyte-derived porcine Langerhans'-type cells (MoLCs) were much more potent activators of allogeneic T cells than MoDCs obtained without TGF-beta1.

Assessment of optimal balloon size for rupture of the ureteropelvic junction and mid-ureter in a porcine model.

PURPOSE: Balloon dilation potentially represents a safer and simpler technique for the treatment of ureteropelvic junction (UPJ) obstruction and ureteral strictures. Using a porcine model, we sought to establish the optimal balloon size for endoballoon rupture of the UPJ and ureter. MATERIALS AND METHODS: The efficacy of endoballoon rupture of the proximal and middle ureter with 24F, 30F, and 36F balloon catheters was compared in 19 female minipigs. At the proximal ureter, the effect of the rate of dilation also was evaluated for each balloon size. Extravasation of methylene blue-stained contrast material was assessed with retrograde pyelograms and direct laparoscopic vision. After acute sacrifice, the dilated segments were evaluated histologically with hematoxylin and eosin and Masson's trichrome staining. RESULTS: At the proximal ureter, free extravasation of contrast was observed in 61% of the rapid inflation and 72% of the slow inflation trials; contained extravasation was noted in 28% of the rapid inflation and 17% of the slow inflation trials. Except for two of the 24F slow inflation trials, all of the proximal ureteral trials produced at least one full-thickness tear into the periureteral fat. Grossly, the tears appeared linear with various lengths and no consistent orientation. Rapid inflation and increasing balloon size tended to produce a ureterotomy with less damage to the ureter surrounding the tear. At the mid-ureter, none of the balloon sizes consistently produced a transmural tear. CONCLUSIONS: Rapid dilation and use of a 36F balloon capable of maintaining a low profile after inflation may result in a cleaner proximal ureterotomy with less distortion of the untorn neighboring proximal ureter. Both 36F and 30F balloons consistently produced a full-thickness proximal ureterotomy in normal porcine tissue. For mid-ureteral strictures, balloon dilation to even 36F may fail to create a suitable ureterotomy. However, it must be noted that dysplastic or scarred tissue may respond differently to dilation than the more elastic normal porcine tissues used in this study.

Laparoscopic renal ablation: an in vitro comparison of currently available electrical tissue morcellators.

OBJECTIVES: Morcellation with the Cook high-speed electrical laparoscopic (HSEL) morcellator in an impermeable nylon/plastic sack (LapSac) has remained unchanged since its inception nearly one decade ago. Sack deployment and specimen entrapment remain relatively difficult, and morcellation with this device is expensive and relatively slow. As such, in an effort to facilitate specimen entrapment and morcellation, we adapted two currently available electrical morcellators (the Steiner gynecologic morcellator and the electrical prostate morcellator [EPM]) for renal morcellation and compared them with the HSEL morcellator. METHODS: All morcellation was performed through a simulated abdominal wall under direct laparoscopic vision. Ten porcine kidneys were ablated with each of the following techniques: HSEL morcellation in a LapSac; HSEL morcellation in a fluid-filled LapSac; Steiner morcellation in an insufflated Endocatch sack; and EPM morcellation in a fluid-filled Endocatch sack. A modified laparoscopic trocar was constructed and used for the Steiner and EPM morcellation. The time to complete morcellation, morcellation product size, and entrapment sack integrity were evaluated for each technique. Cost data for each morcellator are also presented. RESULTS: The mean morcellation time for the Steiner, HSEL dry, HSEL wet, and EPM morcellation was 6.0, 15.9, 14.7, and 26.0 minutes, respectively. The mean fragment size for these morcellators was 2.97, 0.65, 0.62, and 0.013 g, respectively. A single entrapment sack perforation was documented in a LapSac during routine HSEL morcellation. CONCLUSIONS: Renal morcellation with all three morcellators is feasible. The Steiner morcellator combined with an Endocatch resulted in more rapid morcellation and larger morcellation products.

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.

The serum albumin-binding region of streptococcal protein G (BB) potentiates the immunogenicity of the G130-230 RSV-A protein.

BBG2Na is a protein comprising residues 130-230 of the respiratory syncytial virus subgroup A (RSV-A) G protein (G2Na) fused to the albumin-binding domain of streptococcal G protein (BB). BBG2Na was cloned, expressed in Escherichia coli and renaturated. In rodent models, this subunit RSV vaccine adjuvanted in Alhydrogel induced specific antibodies and conferred protection to RSV infection. Comparison of the antibody production in a BALB/c mouse model revealed that BBG2Na induced a stronger and earlier G2Na antibody response than G2Na alone, without altering the IgG subclass distribution. To address the role of the BB part, we explored its carrier properties and showed that it is a Th dependent antigen, generating a more potent G2Na-specific B cell memory response and able to generate Th cells that provide help for G2Na antibody production.

Evaluating the clinical performance of the Osseotite implant: defining prosthetic predictability.

A 5-year prospective, multicenter study is in progress at four private dental practices to determine the cumulative implant survival rate and prosthetic outcome when using the Osseotite dental implant in posterior maxillary and mandibular areas. An interim evaluation after 34.4 months of study progress is presented. A total of 219 Osseotite implants were placed in 74 patients (34 women and 40 men with a mean age of 57.8 +/- 15.2 years) using a conventional two-stage surgical protocol and 3- to 6-month healing time. Subsequently, patients were restored with fixed or removable restorations. Nineteen of the 74 patients reported smoking an average of 13.2 cigarettes per day. Restorative treatments included 40 single-unit restorations; 53 splinted 2-, 3-, 4-, and 5-unit implant-supported maxillary and mandibular prostheses; 4 full-arch fixed maxillary prostheses; 1 mandibular fixed/detachable hybrid prosthesis; and 1 mandibular overdenture. The mean time from implant placement to second stage surgery was 6.2 +/- 2.0 months; from restoration and implant loading to the most recent follow-up evaluation was 20.9 +/- 6.8 months. Of the 219 implants placed, three posterior maxillary implants developed infections and were removed prior to second stage surgery. No implant failures occurred at second stage surgery or after implant loading. Using the Kaplan-Meier method, the cumulative implant survival rate was 100% for anterior implants and 98.4% for posterior implants at 28.5 +/- 5.7 months. The cumulative postloading implant survival rate was 100% for both anterior and posterior implants. The results of this study indicate that the Osseotite dental implant achieved a high rate of integration that remained stable during nearly 2 years of implant function. In addition, because no postloading implant failures have occurred, the Osseotite implant has provided a high level of prosthetic predictability.

Challenge of BALB/c mice with respiratory syncytial virus does not enhance the Th2 pathway induced after immunization with a recombinant G fusion protein, BBG2NA, in aluminum hydroxide.

The polypeptide of aa 130-230 of the G protein (G2Na) of respiratory syncytial virus (RSV) was fused to BB, the albumin-binding region of streptococcal G protein, producing BBG2Na, which induced protective immune responses in rodent models. Evaluation of the immune response in mice immunized with BBG2Na in the adjuvant alhydrogel revealed high amounts of interleukin (IL)-5 and some IL-4 in splenocytes restimulated in vitro. This is compatible with a Th2 response. The activation of the Th2 pathway in such mice was further supported by the detection of IL-5 and G2Na-specific IgE in vivo. Of interest, in contrast to immunization with formalin-inactivated RSV, immunization of mice with BBG2Na followed by intranasal RSV challenge did not lead to increased production of IL-5- or G2Na-specific IgE. However, IgG1- and IgG2a-specific antibodies were boosted. These results demonstrate that the Th2 pathway is not enhanced by RSV challenge in BBG2Na-immunized mice.

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.

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.

Modification of human long-term bone marrow cultures: establishment of a functional stromal microenvironment devoid of myeloid progenitors.

Differences in the plastic adhesive properties of bone marrow (BM) cells were used to initiate modified stromal layers (MSL) from long-term cultures by removing non-adherent cells shortly (4 to 18 hours) after initial seeding. Following this early modification, adherent cells generated a confluent layer after 21 days of culture. Cellular characteristics of volume and spontaneous fluorescence determined by flow cytometry showed that the MSL included 82% fibroblastic stromal cells, 8% macrophages and 10% myelomonocytic cells. Furthermore, clonogenic assays revealed that the MSL were devoid of hematopoietic progenitor cells. MSL were found to sustain long-term myelopoiesis for at least 7 weeks from exogenously added hematopoietic progenitors isolated from bone marrow (CD34+ cells), thereby demonstrating their functionality. The present experimental model appears of interest for the study of interactions between defined populations of hematopoietic cells and cells of the adherent layer. Of importance, our present modifications of human long-term bone marrow culture are technically simple and do not involve manipulation of the stromal cells.