PubMed Health⌕ Search

Biomedical subjects

R Hurwitz

Publications and source records attributed to R Hurwitz.

At least 19 recordsLinked to original sources
PubMed2004

Impact of vector-priming on the immunogenicity of a live recombinant Salmonella enterica serovar typhi Ty21a vaccine expressing urease A and B from Helicobacter pylori in human volunteers.

Orally administered recombinant Salmonella vaccines represent an attractive option for mass vaccination programmes against various infectious diseases. Therefore, it is crucial to gather knowledge about the possible impact of preexisiting immunity to carrier antigens on the immunogenicity of recombinant vaccines. Thirteen volunteers were preimmunized with Salmonella typhi Ty21a in order to evaluate the effects of prior immunization with the carrier strain. Then, they received three doses of 1-2 x 10(10) viable organisms of either the vaccine strain S. typhi Ty21a (pDB1) expressing subunits A and B of recombinant Helicobacter pylori urease (n = 9), or placebo strain S. typhi Ty21a (n = 4). Four volunteers were preimmunized and boosted with the vaccine strain S. typhi Ty21a (pDB1). No serious adverse effects were observed in any of the volunteers. Whereas none of the volunteers primed and boosted with the vaccine strain responded to the recombinant antigen, five of the nine volunteers preimmunized with the carrier strain showed cellular immune responses to H. pylori urease (56%). This supports the results of a previous study in non-preimmunized volunteers where 56% (five of nine) of the volunteers showed a cellular immune response to urease after immunisation with S. typhi Ty21a (pDB1).

Adult↗
PubMed2004

Hydrogen peroxide produced by Aplysia ink toxin kills tumor cells independent of apoptosis via peroxiredoxin I sensitive pathways.

Marine snails of the genus Aplysia possess numerous bioactive substances. We have purified a 60 kDa protein, APIT (Aplysia punctata ink toxin), from the defensive ink of A. punctata that triggers cell death with profound tumor specificity. Tumor cell death induced by APIT is independent of apoptosis but is characterized by the rapid loss of metabolic activity, membrane permeabilization, and shrinkage of nuclei. Proteome analysis of APIT-treated tumor cells indicated a modification of peroxiredoxin I, a cytoplasmic peroxidase involved in the detoxification of peroxides. Interestingly, knockdown of peroxiredoxin I expression by RNA interference sensitized cells for APIT-induced cell death. APIT induced the death of tumor cells via the enzymatic production of H2O2 and catalase completely blocked APITs' activity. Our data suggest that H2O2 induced stress and the modulation of peroxiredoxins might be a promising approach for tumor therapy.

Animals↗
PubMed2001

Safety and immunogenicity of live recombinant Salmonella enterica serovar Typhi Ty21a expressing urease A and B from Helicobacter pylori in human volunteers.

Helicobacter pylori urease was expressed in the common live typhoid vaccine Ty21a yielding Ty21a(pDB1). Nine volunteers received Ty21a(pDB1) and three control volunteers received Ty21a. No serious adverse effects were observed in any of the volunteers. Ten out of 12 volunteers developed humoral immune responses to the Salmonella carrier as detected by antigen-specific antibody-secreting cells but only two volunteers seroconverted. A total of five volunteers showed responses in one or two out of three assays for cellular responses to the carrier (proliferation, IFN-gamma-secretion, IFN-gamma-ELISPOT). Three of the volunteers that had received Ty21a(pDB1) showed a weak but significant T-cell response to Helicobacter urease, while no volunteer had detectable humoral responses to urease. Ty21a(pDB1) is a suitable prototype to optimize Salmonella-based vaccination for efficient cellular responses that could mediate protective immunity against Helicobacter.

Adolescent↗
PubMed2001

Immunization with gp96 from Listeria monocytogenes-infected mice is due to N-formylated listerial peptides.

N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice. N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L. monocytogenes were stimulated with these peptides. No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals. Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L. monocytogenes, but not on gp96 from noninfected mice. Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL. We assume a unique role of gp96 in Ag processing through the H2-M3 pathway.

Animals↗
PubMed2001

Role of CD28 for the generation and expansion of antigen-specific CD8(+) T lymphocytes during infection with Listeria monocytogenes.

Infection of mice with the intracellular bacterium Listeria monocytogenes results in a strong CD8(+) T cell response that is critical for efficient control of infection. We used CD28-deficient mice to characterize the function of CD28 during Listeria infection, with a main emphasis on Listeria-specific CD8(+) T cells. Frequencies and effector functions of these T cells were determined using MHC class I tetramers, single cell IFN-gamma production and Listeria-specific cytotoxicity. During primary Listeria infection of CD28(-/-) mice we observed significantly reduced numbers of Listeria-specific CD8(+) T cells and only marginal levels of specific IFN-gamma production and cytotoxicity. Although frequencies were also reduced in CD28(-/-) mice during secondary response, we detected a considerable population of Listeria-specific CD8(+) T cells in these mice. In parallel, IFN-gamma production and cytotoxicity were observed, revealing that Listeria-specific CD8(+) T cells in CD28(-/-) mice expressed normal effector functions. Consistent with their impaired CD8(+) T cell activation, CD28(-/-) mice suffered from exacerbated listeriosis both after primary and secondary infection. These results demonstrate participation of CD28 signaling in the generation and expansion of Ag-specific CD8(+) T cells in listeriosis. However, Ag-specific CD8(+) T cells generated in the absence of CD28 differentiated into normal effector and memory T cells.

Animals↗
PubMed2001

Autologous antileukemic immune response induced by chronic lymphocytic leukemia B cells expressing the CD40 ligand and interleukin 2 transgenes.

Although the B cells of chronic lymphocytic leukemia (B-CLL cells) express both tumor-specific peptides and major histocompatibility complex (MHC) class I antigens, they lack the capacity for costimulatory signaling, contributing to their protection against host antitumor immunity. To stimulate CLL-specific immune responses, we sought to transfer the human CD40 ligand (hCD40L) gene to B-CLL cells, using an adenoviral vector, in order to upregulate costimulating factors on these cells. Because efficient gene transduction with adenoviral vectors requires the expression of virus receptors on target cells, including the coxsackievirus B-adenovirus receptors (CAR) and alpha(v) integrins, we cocultured B-CLL cells with human embryonic lung fibroblasts (MRC-5 line). This exposure led to increased expression of integrin alpha(v)beta3 on B-CLL cells, which correlated with higher transduction rates. Using this novel prestimulation system, we transduced B-CLL cells with the hCD40L gene. The Ad-hCD40L-infected cells had higher expression of B7 molecules and induced activation of autologous T cells in vitro, but these T cells could not recognize parental leukemic cells. By contrast, an admixture of Ad-hCD40L-positive cells and leukemic cells transduced with the human interleukin 2 (IL-2) gene produced greater T cell activation than did either immunostimulator population alone. Importantly, this combination generated autologous T cells capable of specifically recognizing parental B-CLL cells. These findings suggest that the combined use of genetically modified CD40L-expressing B-CLL cells in combination with IL-2-expressing B-CLL cells may induce therapeutically significant leukemia-specific immune responses.

Adenoviridae↗
PubMed2001

Immunity against Helicobacter pylori: significance of interleukin-4 receptor alpha chain status and gender of infected mice.

Vaccination of interleukin-4 (IL-4) receptor alpha (IL-4Ralpha) chain-deficient BALB/c mice with Helicobacter pylori urease and cholera toxin or with urease-expressing, live attenuated Salmonella enterica serovar Typhimurium cells revealed that protection against H. pylori infection is independent of IL-4- or IL-13-mediated signals. A comparison of male and female mice suggests a sexual dimorphism in the extent of bacterial colonization that is particularly evident in the absence of the IL-4Ralpha chain.

Animals↗
PubMed2000

Targeting of the pro-apoptotic VDAC-like porin (PorB) of Neisseria gonorrhoeae to mitochondria of infected cells.

Infection of cell cultures with Neisseria gonorrhoeae results in apoptosis that is mediated by the PorB porin. During the infection process porin translocates from the outer bacterial membrane into host cell membranes where its channel activity is regulated by nucleotide binding and voltage-dependent gating, features that are shared by the mitochondrial voltage-dependent anion channel (VDAC). Here we show that porin is selectively and efficiently transported to mitochondria of infected cells. Prevention of porin translocation also blocked the induction of apoptosis. Mitochondria of cells treated with porin both in vitro and in vivo were depleted of cytochrome c and underwent permeability transition. Overexpression of Bcl-2 blocked porin-induced apoptosis. The release of cytochrome c occurred independently of active caspases but was completely prevented by Bcl-2. Our data suggest that the Neisseria porin can, like its eukaryotic homologue, function at the mitochondrial checkpoint to mediate apoptosis.

Apoptosis↗
PubMed1999

Adenosine challenge and boost protocols: new tools for myocardial perfusion imaging.

The validity of pharmacologic stress testing and subsequent myocardial perfusion imaging is uncertain in those patients who may have taken caffeine within the 24 hours before testing. For such patients, two new challenge tests have been developed. An intravenous bolus dose of adenosine is given at a dose of 6 mg in a period of 1 or 2 seconds. A physiologic response qualifies the patient to proceed with the scheduled stress test. In the occasional patient who exhibits no pharmacologic symptoms during an infusion test, a similar bolus dose of 6 mg adenosine can validate perfusion tests. These two applications are successful regardless of whether adenosine or dipyridamole infusions are performed. Based on this multicenter experience over 4 years, the authors estimate that 5% to 10% of patients undergoing pharmacologic testing are appropriate candidates for a challenge test.

Adenosine↗
PubMed1998

Tubularized incised plate hypospadias repair for proximal hypospadias.

PURPOSE: We report a multicenter experience using tubularized incised plate urethroplasty for proximal hypospadias. MATERIALS AND METHODS: From August 1993 to December 1996 tubularized incised plate urethroplasty was performed in 27 boys 6 months to 3 years old with mid shaft and penoscrotal hypospadias. In 7 other boys complex reconstruction combined the tubularized incised plate technique for the glanular urethra with other repairs for the proximal urethra. RESULTS: Tubularized incised plate surgery created a functional neourethra even in penoscrotal hypospadias. Complications of the primary repair developed in only 3 of 27 patients (11%). No complications were attributed to use of the technique for the glanular urethra in complex repairs. CONCLUSIONS: Tubularized incised plate urethroplasty is a versatile operation that corrects proximal hypospadias defects with few complications and superior cosmetic results.

Child, Preschool↗
PubMed1997

Functional characterization of the N-glycosylation sites of human acid sphingomyelinase by site-directed mutagenesis.

Most soluble lysosomal enzymes require a mannose-6-phosphate recognition marker present on asparagine-linked oligosaccharides for proper targeting to lysosomes. We have determined the influence of the six potential N-linked oligosaccharide chains of human acid sphingomyelinase (ASM) on catalytic activity, targeting, and processing of the enzyme. Each N-glycosylation site was modified by site-directed mutagenesis and subsequently expressed in COS-1 cells. Evidence is presented that five of these sites are used. Elimination of the four N-terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity. However, removal of the two C-terminal N-glycosylation sites inhibits the formation of mature enzyme. Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticulum/Golgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticulum/Golgi. Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites.

Animals↗
PubMed1996

Purification of acid sphingomyelinase from human placenta: characterization and N-terminal sequence.

Human placental acid sphingomyelinase (ASM) was purified by sequential chromatography on Con A-Sepharose, octyl-Sepharose and Matrex gel red A. Final purification to apparent homogeneity was achieved by immunoaffinity chromatography employing polyclonal anti-ASM antibodies. The antibodies also allowed specific detection of ASM by Western blotting at various stages of purification. The ASM activity was enriched about 110,000-fold over that of the crude extract, yielding an enzyme preparation with a specific activity of about 1 mmol/h per mg protein in a detergent-containing assay system. Analysis of the final preparation by SDS-PAGE resulted in a single protein band with a molecular mass of approximately 75 kDa, which was reduced to approximately 60 kDa after complete deglycosylation. Microsequencing of the purified ASM revealed the N-terminal amino acid sequence of the mature placental enzyme.

Amino Acid Sequence↗
PubMed1996

Biosynthesis, processing, and targeting of sphingolipid activator protein (SAP )precursor in cultured human fibroblasts. Mannose 6-phosphate receptor-independent endocytosis of SAP precursor.

Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acidic exohydrolases. SAP-A, -B, -C, and -D derive from proteolysis of a 73-kDa glycoprotein, the SAP precursor. In the present publication, we studied the intracellular transport and the endocytosis of SAP precursor in human skin fibroblasts. Our data indicate that SAP precursor bears phosphate residues on noncomplex carbohydrate chains linked to the SAP-C and the SAP-D domain and sulfate residues on complex carbohydrate chains located within the SAP-A, -C, and possibly the SAP-D domain. Treatment of fibroblasts with either bafilomycin A1 or 3-methyladenine indicates that proteolytic cleavage of SAP precursor begins as early as in the late endosomes. To determine whether targeting of SAP precursor depends on mannose 6-phosphate residues, we analyzed the processing of SAP precursor in I-cell disease fibroblasts. In these cells nearly normal amounts of newly synthesized SAP-C were found, although secretion of SAP precursor was enhanced 2-3-fold. Moreover, SAP-C could be localized to lysosomal structures by indirect immunofluorescence in normal and in I-cell disease fibroblasts. Mannose 6-phosphate was not found to interfere significantly with endocytosis of SAP precursor. Normal fibroblasts internalized SAP precursor secreted from I-cells nearly as efficiently as the protein secreted from normal cells. To our surprise, deglycosylated SAP precursor was taken up by mannose 6-phosphate receptor double knock out mouse fibroblasts more efficiently than the glycosylated protein. We propose that intracellular targeting of SAP precursor to lysosomes is only partially dependent on mannose 6-phosphate residues, whereas its endocytosis occurs in a carbohydrate-independent manner.

Animals↗
PubMed1996

Tubularized incised plate hypospadias repair: results of a multicenter experience.

PURPOSE: We report a multicenter experience using tubularized incised plate urethroplasty to correct distal hypospadias. MATERIALS AND METHODS: A total of 148 patients underwent repair by 6 pediatric urologists at different institutions in the United States and Europe. RESULTS: Tubularized incised plate repair created a functional neourethra with a vertically oriented meatus. Complications, including meatal stenoses and fistulas, occurred in 10 patients (7%). CONCLUSIONS: Tubularized incised plate urethroplasty can be performed in most cases of distal hypospadias. Cosmetic results are superior to those of other popular techniques.

Child↗
PubMed1995

Purification, characterization, and biosynthesis of human acid ceramidase.

Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) is the lysosomal enzyme catalyzing the hydrolysis of ceramide to sphingosine and free fatty acid. Its inherited deficiency causes ceramide accumulation in Farber's disease. The enzyme was purified to apparent homogeneity from human urine by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue-Sepharose, and DEAE-cellulose. The final preparation, which was enriched approximately 4450-fold over the starting material, resulted in a polypeptide of approximately 50 kDa and could be reduced into two subunits of approximately 13 (alpha) and approximately 40 (beta) kDa. Treatment of the purified enzyme with endoglycosidase H or peptido-N-glycanase F reduced the molecular mass of the beta subunit to approximately 30-35 and approximately 27 kDa, respectively. In contrast, the molecular mass of the alpha subunit was unchanged. The purified enzyme had an apparent Km of 149 microM and a Vmax of 136 nmol/mg/h using N-lauroylsphingosine as substrate. Polyclonal antibodies were raised against the purified urinary enzyme and used to investigate the biosynthesis of acid ceramidase. Immunoprecipitation studies on metabolically labeled skin fibroblasts indicated that both subunits arose from a single precursor of approximately 55 kDa. A minor portion of newly synthesized acid ceramidase was secreted into the medium as a monomeric 47-kDa protein, indicating that generation of the mature heterodimeric enzyme occurred in endosomal and/or lysosomal compartments.

Acid Ceramidase↗