Biomedical subjects

Robert Hurwitz

Publications and source records attributed to Robert Hurwitz.

16 recordsLinked to original sources

PubMed2007

Novel cell death program leads to neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are extracellular structures composed of chromatin and granule proteins that bind and kill microorganisms. We show that upon stimulation, the nuclei of neutrophils lose their shape, and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate, allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death process is distinct from apoptosis and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase. Patients with chronic granulomatous disease carry mutations in NADPH oxidase and cannot activate this cell-death pathway or make NETs. This novel ROS-dependent death allows neutrophils to fulfill their antimicrobial function, even beyond their lifespan.

Antibodies↗

PubMed2006

Critical role of methylglyoxal and AGE in mycobacteria-induced macrophage apoptosis and activation.

Apoptosis and activation of macrophages play an important role in the host response to mycobacterial infection involving TNF-alpha as a critical autocrine mediator. The underlying mechanisms are still ill-defined. Here, we demonstrate elevated levels of methylglyoxal (MG), a small and reactive molecule that is usually a physiological product of various metabolic pathways, and advanced glycation end products (AGE) during mycobacterial infection of macrophages, leading to apoptosis and activation of macrophages. Moreover, we demonstrate abundant AGE in pulmonary lesions of tuberculosis (TB) patients. Global gene expression profiling of MG-treated macrophages revealed a diverse spectrum of functions induced by MG, including apoptosis and immune response. Our results not only provide first evidence for the involvement of MG and AGE in TB, but also form a basis for novel intervention strategies against infectious diseases in which MG and AGE play critical roles.

Animals↗

PubMed2006

Plasmodia express two threonine-peptidase complexes during asexual development.

Threonine-peptidases of the T1-family are multi-subunit complexes with broad substrate specificity. In eukaryotes, at least 14 genes encode subunits of the prototypic T1 threonine-peptidase, the proteasome. The proteasome determines the turnover of most proteins and thereby plays a fundamental role in diverse processes such as protein quality control, signal transduction, and cell cycle regulation. While eukaryotes and archaea possess a proteasome, bacteria generally express a second member of the T1-family, the proteasomal predecessor ClpQ/hslV that has a similar structure but is encoded by only one gene. The plasmodial genome is an exception because it encodes proteasomal subunits as well as a ClpQ/hslV-orthologe (Plasmodium falciparum-hslV; PfhslV). Structure, expression, and function of both types of peptidase-complex in P. falciparum are presently unknown. Our aim was to analyze both the coding sequences and derived proteins of both peptidase-complexes because highly specific and potent inhibitors can be designed against this class of enzymes. The proteasome was found expressed throughout the cell cycle, whereas PfhslV was detectable in schizonts and merozoites only. Treatment of P. falciparum with the threonine-peptidase inhibitor epoxomicin blocked two of three catalytically active proteasome subunits. This led to the accumulation of ubiquitinated proteins and, finally, to parasite death. In conclusion, we provide the first functional analysis of plasmodial threonine-peptidase-complexes and identify a lead compound for the development of a novel class of antimalarial drugs.

Amino Acid Sequence↗

PubMed2006

Rapid development of a gamma interferon-secreting glycolipid/CD1d-specific Valpha14+ NK1.1- T-cell subset after bacterial infection.

The phenotypic and functional changes of glycolipid presented by CD1d(glycolipid/CD1d) specific Valpha14+ T cells in the liver of mice at early stages of bacterial infection were investigated. After Listeria monocytogenes infection or interleukin-12 (IL-12) treatment, alpha-galactosylceramide/CD1d tetramer-reactive (alpha-GalCer/CD1d+) T cells coexpressing natural killer (NK) 1.1 marker became undetectable and, concomitantly, cells lacking NK1.1 emerged in both euthymic and thymectomized animals. Depletion of the NK1.1+ subpopulation prevented the emergence of alpha-GalCer/CD1d+ NK1.1- T cells. Before infection, NK1.1+, rather than NK1.1-, alpha-GalCer/CD1d+ T cells coexpressing CD4 were responsible for IL-4 production, whereas gamma interferon (IFN-gamma) was produced by cells regardless of NK1.1 or CD4 expression. After infection, IL-4-secreting cells became undetectable among alpha-GalCer/CD1d+ T cells, but considerable numbers of IFN-gamma-secreting cells were found among NK1.1-, but not NK1.1+, cells lacking CD4. Thus, NK1.1 surface expression and functional activities of Valpha14+ T cells underwent dramatic changes at early stages of listeriosis, and these alterations progressed in a thymus-independent manner. In mutant mice lacking all alpha-GalCer/CD1d+ T cells listeriosis was ameliorated, suggesting that the subtle contribution of the NK1.1- T-cell subset to antibacterial protection is covered by more profound detrimental effects of the NK1.1+ T-cell subset.

Animals↗

PubMed2006

IAP-IAP complexes required for apoptosis resistance of C. trachomatis-infected cells.

Host cells infected with obligate intracellular bacteria Chlamydia trachomatis are profoundly resistant to diverse apoptotic stimuli. The molecular mechanisms underlying the block in apoptotic signaling of infected cells is not well understood. Here we investigated the molecular mechanism by which apoptosis induced via the tumor necrosis factor (TNF) receptor is prevented in infected epithelial cells. Infection with C. trachomatis leads to the up-regulation of cellular inhibitor of apoptosis (cIAP)-2, and interfering with cIAP-2 up-regulation sensitized infected cells for TNF-induced apoptosis. Interestingly, besides cIAP-2, cIAP-1 and X-linked IAP, although not differentially regulated by infection, are required to maintain apoptosis resistance in infected cells. We detected that IAPs are constitutively organized in heteromeric complexes and small interfering RNA-mediated silencing of one of these IAPs affects the stability of another IAP. In particular, the stability of cIAP-2 is modulated by the presence of X-linked IAP and their interaction is stabilized in infected cells. Our observations suggest that IAPs are functional and stable as heteromers, a thus far undiscovered mechanism of IAP regulation and its role in modulation of apoptosis.

Apoptosis↗

PubMed2005

Human neutrophils kill Bacillus anthracis.

Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

Animals↗

PubMed2005

Human alpha-defensins neutralize anthrax lethal toxin and protect against its fatal consequences.

Anthrax caused by Bacillus anthracis represents a major bioterroristic threat. B. anthracis produces lethal toxin (LeTx), a combination of lethal factor (LF) and protective antigen that plays a major role in anthrax pathogenesis. We demonstrate that human neutrophil alpha-defensins are potent inhibitors of LF. The inhibition of LF by human neutrophil protein (HNP-1) was noncompetitive. HNP-1 inhibited cleavage of a mitogen-activated protein kinase kinase and restored impaired mitogen-activated protein kinase signaling in LeTx-treated macrophages. HNP-1 rescued murine macrophages from B. anthracis-induced cytotoxicity, and in vivo treatment with HNP-1-3 protected mice against the fatal consequences of LeTx.

Animals↗

PubMed2005

Scant activation of CD8 T cells by antigen loaded on heat shock protein.

Heat shock proteins (HSP) not only function as chaperones for denatured proteins but also for antigenic peptides, thus inducing protective T cell responses. Here we show that vaccination with peptide-loaded HSP70 causes initial interferon-gamma production by murine CD8 T cells but no T cell expansion. These CD8 T cells lacked cytotoxic activity in vitro and in vivo, which was not due to apoptosis. Restimulation with peptide-pulsed dendritic cells both bypassed the proliferative block and suspended the non-protective state of CD8 T lymphocytes in an infection model with the bacterial pathogen, Listeria monocytogenes. Cotransfer of antigen-specific CD4 T cells circumvented the proliferative arrest of CD8 T cells. Our data suggest that HSP vaccines induce CD8 T cell unresponsiveness unless proficient help is provided. Assuming that this model reflects the antigenically experienced human condition where immunological space is restricted and any T cell response possibly leads to suppression of heterologous reactions, our findings bear implications for rational vaccination protocols including those for immunocompromised patients.

Antigens↗

PubMed2005

Cloning and biochemical characterization of APIT, a new l-amino acid oxidase from Aplysia punctata.

The purple ink of the sea hare Aplysia punctata contains a 60 kDa protein with tumoricidal activity. This A. punctata ink toxin (APIT) kills tumor cells within 6--8h in an apoptosis independent manner by the production of high amounts of hydrogen peroxide which induce a necrotic form of oxidative stress. Here, we describe the biochemical features of APIT associated with its anti-tumor activity. APIT is a weakly glycosylated FAD-binding L-amino acid oxidase that catalyzes the oxidative deamination of L-lysine and L-arginine and thereby produces hydrogen peroxide (H(2)O(2)), ammonia (NH(4)(+)) and the corresponding alpha-keto acids. The tumoricidal effect is completely abrogated in the absence of the amino acids L-lysine and L-arginine. The enzyme is stable at temperatures from 0 to 50 degrees C. Similar to other FAD-binding enzymes, it is resistant against tryptic digest. Even digest with proteinase K fails to degrade the enzyme. Cloning of the APIT gene and subsequent sequencing revealed a FAD-binding domain followed by a so-called GG-motif, which is typical for L-amino acid oxidases. Strongest homology exists to escapin, aplysianin A precursor, the cyplasins L and S and achacin.

Amino Acid Sequence↗

PubMed2004

Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells.

A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a phosphatidylinositol mannoside (PIM) fulfilled the requirements for CD1d binding and NKT cell stimulation. This PIM activated human and murine NKT cells via CD1d, thereby triggering antigen-specific IFN-gamma production and cell-mediated cytotoxicity, and PIM-loaded CD1d tetramers identified a subpopulation of murine and human NKT cells. This phospholipid, therefore, represents a mycobacterial antigen recognized by T cells in the context of CD1d.

Animals↗

PubMed2004

Saposin C is required for lipid presentation by human CD1b.

Lipids from Mycobacterium tuberculosis are presented through CD1 proteins to T lymphocytes in humans, but the accessory molecules required for antigen loading and presentation remain unidentified. Here we show that fibroblasts deficient in sphingolipid activator proteins (SAPs) transfected with CD1b failed to activate lipid-specific T cells. However, the T cell response was restored when fibroblasts were reconstituted with SAP-C but not other SAPs. Lipid antigen and SAP-C colocalized in lysosomal compartments, and liposome assays showed that SAP-C efficiently extracts antigen from membranes. Coprecipitation demonstrated direct molecular interaction between SAP-C and CD1b. We propose a model in which SAP-C exposes lipid antigens from intralysosomal membranes for loading onto CD1b. Thus, SAP-C represents a missing link in antigen presentation of lipids through CD1b to human T cells.

Antigen Presentation↗

PubMed2003

The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactivation.

The gastric pathogen Helicobacter pylori translocates the CagA protein into epithelial cells by a type IV secretion process. Translocated CagA is tyrosine phosphorylated (CagA(P-Tyr)) on specific EPIYA sequence repeats by Src family tyrosine kinases. Phos phorylation of CagA induces the dephosphorylation of as yet unidentified cellular proteins, rearrangements of the host cell actin cytoskeleton and cell scattering. We show here that CagA(P-Tyr) inhibits the catalytic activity of c-Src in vivo and in vitro. c-Src inactivation leads to tyrosine dephosphorylation of the actin binding protein cortactin. Concomitantly, cortactin is specifically redistributed to actin-rich cellular protrusions. c-Src inactivation and cortactin dephosphorylation are required for rearrangements of the actin cytoskeleton. Moreover, CagA(P-Tyr)-mediated c-Src inhibition downregulates further CagA phosphorylation through a negative feedback loop. This is the first report of a bacterial virulence factor that inhibits signalling of a eukaryotic tyrosine kinase and on a role of c-Src inactivation in host cell cytoskeletal rearrangements.

Actins↗

PubMed2003

The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization.

A 9.5-kb section of DNA called region of deletion 1 (RD1) is present in virulent Mycobacterium tuberculosis strains but is deleted in all attenuated Mycobacterium bovis BCG vaccine strains. This region codes for at least nine genes. Some or all RD1 gene products may be involved in virulence and pathogenesis, and at least two, ESAT-6 and CFP-10, represent potent T- and B-cell antigens. In order to produce the entire set of RD1 proteins with their natural posttranslational modifications, a robust expression system for M. tuberculosis proteins in the fast-growing saprophytic strain Mycobacterium smegmatis was developed. Our system employs the inducible acetamidase promoter and allows translational fusion of recombinant M. tuberculosis proteins with polyhistidine or influenza hemagglutinin epitope tags for affinity purification. Using eGFP as reporter gene, we showed that the acetamidase promoter is tightly regulated in M. smegmatis and that this promoter is much stronger than the widely used constitutive groEL2 promoter. We then cloned 11 open reading frames (ORFs) found within RD1 and successfully expressed and purified the respective proteins. Sera from tuberculosis patients and M. tuberculosis-infected mice reacted with 10 purified RD1 proteins, thus demonstrating that Rv3871, Rv3872, Rv3873, CFP-10, ESAT-6, Rv3876, Rv3878, Rv3879c and ORF-14 are expressed in vivo. Finally, glycosylation of the RD1 proteins was analyzed. We present preliminary evidence that the PPE protein Rv3873 is glycosylated at its C terminus, thus highlighting the ability of M. smegmatis to produce M. tuberculosis proteins bearing posttranslational modifications.

Antigens, Bacterial↗

PubMed2003

F-18 FDG positron emission tomographic imaging in a case of ruptured breast implant: inflammation or recurrent tumor?

F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) has proved clinically useful for both malignant and inflammatory lesions. The author describes a 54-year-old woman with previously treated carcinoma of the breast. There was spontaneous rupture of a breast implant placed previously as part of a reconstruction. While being evaluated clinically, prominent axillary lymph nodes were palpated on the ipsilateral side of the recent implant rupture. An F-18 FDG PET scan demonstrated intense uptake in these nodes. Lymph node biopsy demonstrated benign inflammatory reaction and no recurrence of malignancy.

Breast Implants↗

PubMed2002

Regulatory CD4+CD25+ T cells restrict memory CD8+ T cell responses.

CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.

Adoptive Transfer↗

PubMed2002

Neisserial immunoglobulin A1 protease induces specific T-cell responses in humans.

We have previously shown that immunoglobulin A1 (IgA1) protease, an exoenzyme of pathogenic neisseriae, can trigger the release of proinflammatory cytokines from human monocytic subpopulations. Here, we demonstrate a dose-dependent T-cell response to recombinant gonococcal IgA1 protease (strain MS11) in healthy human blood donors. This response was delayed in comparison to the immune response against tetanus toxoid. Stimulation with IgA1 protease led to the activation of CD4(+) and CD8(+) T cells, as well as CD19(+) B cells and CD56(+) NK cells, indicated by de novo expression of CD69. Only CD4(+) T cells proliferated and stained positive for intracellular gamma interferon (IFN-gamma). Both proliferation and IFN-gamma production were dependent on antigen presentation via major histocompatibility complex class II. Peripheral blood mononuclear cells stimulated with IgA1 protease produce IFN-gamma and tumor necrosis factor alpha but no, or very low amounts of, interleukin-10 (IL-10) or IL-4, indicating a Th1-based proinflammatory immune response. These findings support the significance of IgA1 protease as a virulence determinant of bacterial meningitis and its function as a dominant proinflammatory T-cell antigen.

Adult↗