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Biomedical subjects

S H Kaufmann

Publications and source records attributed to S H Kaufmann.

At least 73 records · Page 4Linked to original sources
PubMed2000

Retention of the human Rad9 checkpoint complex in extraction-resistant nuclear complexes after DNA damage.

Studies in yeasts and mammals have identified many genes important for DNA damage-induced checkpoint activation, including Rad9, Hus1, and Rad1; however, the functions of these gene products are unknown. In this study we show by immunolocalization that human Rad9 (hRad9) is localized exclusively in the nucleus. However, hRad9 was readily released from the nucleus into the soluble extract upon biochemical fractionation of un-irradiated cells. In contrast, DNA damage promptly converted hRad9 to an extraction-resistant form that was retained at discrete sites within the nucleus. Conversion of hRad9 to the extraction-resistant nuclear form occurred in response to diverse DNA-damaging agents and the replication inhibitor hydroxyurea but not other cytotoxic stimuli. Additionally, extraction-resistant hRad9 interacted with its binding partners, hHus1 and an inducibly phosphorylated form of hRad1. Thus, these studies demonstrate that hRad9 is a nuclear protein that becomes more firmly anchored to nuclear components after DNA damage, consistent with a proximal function in DNA damage-activated checkpoint signaling pathways.

Animals↗
PubMed2000

Cutting edge: contribution of NK cells to the homing of thymic CD4+NKT cells to the liver.

In contrast to peripheral lymphoid organs, in the liver a high proportion of T cells are CD4+NKT cells. We have previously reported that LFA-1 plays a pivotal role in the homing of thymic CD4+NKT cells to the liver. In the present study, we further assessed which cell type participates in the homing of thymic CD4+NKT cells to the liver. The accumulation of donor thymocyte-derived CD4+NKT cells in the liver of SCID mice that had been reconstituted with thymocytes from C57BL/6 mice was severely impaired by in vivo depletion of NK cells, but not Kupffer cells in recipients. These results suggest that NK cells participate in the homing of thymic CD4+NKT cells to the liver. We assume that LFA-1 expressed on NK cells is involved in this mechanism.

Animals↗
PubMed2000

Isolation of RNA from mycobacteria grown under in vitro and in vivo conditions.

Isolation of RNA from mycobacteria is very difficult to perform, and the yields are generally very low. We describe an approach to isolate RNA from mycobacterial species which combines the disruption of mycobacterial cells by a silica/ceramic matrix in a reciprocal shaker with the ease and efficiency of subsequent RNA purification on spin columns with silica gel-based membranes. This method is rapid, easy to perform and yields high amounts of pure, intact total RNA. Due to its safety, this method is applicable even to group 3 biological hazard organisms like Mycobacterium tuberculosis. By combining a method for the isolation of phagosomal bacteria from infected primary macrophages with the novel RNA isolation technique, we are able to monitor gene expression during infection even in bacteria which are rather resistant to genetic manipulation, like Mycobacterium bovis.

Animals↗
PubMed2000

Intersection of group I CD1 molecules and mycobacteria in different intracellular compartments of dendritic cells.

Human CD1a, CD1b, and CD1c molecules can present mycobacterial glycolipids to T cells. Because phagosomes containing viable mycobacteria represent early endosomal compartments, we studied where mycobacterial glycolipids intersect with CD1 molecules in infected APC. CD1b and CD1c, but not CD1a, localized to late endosomes/lysosomes. CD1a and CD1c were predominantly expressed on the cell surface and in mycobacterial phagosomes of the early endosomal stage. In contrast, CD1b was present in a subset of mycobacterial phagosomes representing mature phagolysosomes. Released mycobacterial glycolipids including lipoarabinomannan and phosphatidylinositol mannosides were transported from the phagosome into late endosomes/lysosomes and to uninfected bystander cells. The macrophage mannose receptor, which has been implicated in glycolipid uptake by APC for CD1b-mediated presentation, was absent from mycobacterial phagosomes and may therefore not be involved in trafficking of glycolipids between phagosomes and late endosomes/lysosomes. In conclusion, all three CD1 molecules have access to mycobacteria and glycolipids thereof, but at different intracellular sites. This allows sampling by CD1a, CD1b, and CD1c of mycobacterial glycolipids from different intracellular sites of the infected cell, which has important implications for processing and presentation of such Ags during mycobacterial infections.

Antigens, CD1↗
PubMed2000

Flavopiridol binds to duplex DNA.

Flavopiridol, the first potent cyclin-dependent kinase inhibitor to enter clinical trials, was recently found to be cytotoxic to noncycling cells. The present studies were performed to examine the hypothesis that flavopiridol, like several other antineoplastic agents that kill noncycling cells, might also interact with DNA. Consistent with this possibility, treatment of A549 human lung cancer cells with clinically achievable concentrations of flavopiridol resulted in rapid elevations of the DNA damage-responsive protein p53. In further studies, the binding of flavopiridol to DNA was examined in vitro by four independent techniques. Absorption spectroscopy revealed that addition of DNA to aqueous flavopiridol solutions resulted in a red shift of the flavopiridol lambda(max) from 311 to 344 nm, demonstrating an isosbestic point typical of changes seen with DNA-binding compounds. Reverse-phase high-performance liquid chromatography demonstrated that flavopiridol binds to genomic DNA to a similar extent as ethidium bromide and Hoechst 33258. Nuclear magnetic resonance spectroscopy revealed that DNA caused extreme broadening of flavopiridol 1H nuclear magnetic resonance signals that could be reversed by addition of ethidium bromide or by DNA melting, suggesting that flavopiridol binds to (and likely intercalates into) duplex DNA. Equilibrium dialysis demonstrated that the equilibrium dissociation constant of the flavopiridol-DNA complex (5.4+/-3.4 x 10(-4) M) was in the same range observed for binding of the intercalators doxorubicin and pyrazoloacridine to DNA. Molecular modeling confirmed the feasibility of flavopiridol intercalation into DNA and analysis of the effects of flavopiridol in the National Cancer Institute tumor cell line panel using the COMPARE algorithm demonstrated that flavopiridol most closely resembles cytotoxic antineoplastic intercalators. Collectively, these data suggest that DNA might be a second target of flavopiridol, providing a potential explanation for the ability of this agent to kill noncycling cancer cells.

Acridines↗
PubMed2000

Induction of apoptosis by cancer chemotherapy.

Studies performed over the past five years have demonstrated that there are two major cell-intrinsic pathways for inducing apoptosis, one that begins with ligation of cell surface death receptors and another that involves mitochondrial release of cytochrome c. Several reports have suggested that anticancer drugs kill susceptible cells by inducing expression of death receptor ligands, especially Fas ligand (FasL). Other reports have indicated that chemotherapeutic agents trigger apoptosis by inducing release of cytochrome c from mitochondria. In this review, we describe the two prototypic death pathways, indicate experimental approaches for distinguishing whether chemotherapeutic agents trigger one pathway or the other, summarize current understanding of the role of the two pathways in chemotherapy-induced apoptosis, and discuss the implications of these studies for mechanisms of resistance to chemotherapeutic agents.

Antineoplastic Agents↗
PubMed2000

A Phase I trial of the farnesyl transferase inhibitor SCH66336: evidence for biological and clinical activity.

Farnesyl protein transferase (FT), an enzyme that catalyzes the first step in the posttranslational modification of ras and a number of other polypeptides, has emerged as an important target for the development of anticancer agents. SCH66336 is one of the first FT inhibitors to undergo clinical testing. We report a Phase I trial to assess the maximum tolerated dose, toxicities, and biological effectiveness of SCH66336 in inhibiting FT in vivo. Twenty patients with solid tumors received 92 courses of escalating SCH66336 doses given orally twice a day (b.i.d.) for 7 days out of every 3 weeks. Gastrointestinal toxicity (nausea, vomiting, and diarrhea) and fatigue were dose-limiting at 400 mg of SCH66336 b.i.d. Moderate reversible renal insufficiency, secondary to dehydration from gastrointestinal toxicity, was also seen. Inhibition of prelamin A farnesylation in buccal mucosa cells of patients treated with SCH66336 was demonstrated, confirming that SCH66336 inhibits protein farnesylation in vivo. One partial response was observed in a patient with previously treated metastatic non-small cell lung cancer, who remained on study for 14 months. This study not only establishes the dose for future testing on this schedule (350 mg b.i.d.) but also provides the first evidence of successful inhibition of FT in the clinical setting and the first hint of clinical activity for this class of agents.

Adult↗
PubMed2000

Cutting edge: role of B lymphocytes in protective immunity against Salmonella typhimurium infection.

Infection of mice with Salmonella typhimurium gives rise to a disease similar to human typhoid fever caused by S. typhi. Since S. typhimurium is a facultative intracellular bacterium, the requirement of B cells in the immune response against S. typhimurium is a longstanding matter of debate. By infecting mice on a susceptible background and deficient in B cells (Igmu-/- mice) with different strains of S. typhimurium, we could for the first time formally clarify the role of B cells in the response against S. typhimurium. Compared with Igmu+/+ mice, LD50 values in Igmu-/- mice were reduced during primary, and particularly secondary, oral infection with virulent S. typhimurium. After systemic infection, Igmu-/- mice cleared attenuated aroA- S. typhimurium, but vaccine-induced protection against systemic infection with virulent S. typhimurium involved both B cell-dependent and -independent effector mechanisms. Thus, B cell-mediated immunity plays a distinct role in control of S. typhimurium in susceptible mice.

Administration, Oral↗
PubMed2000

Successful virtual screening of a chemical database for farnesyltransferase inhibitor leads.

Virtual screening of chemical databases is an emerging approach in drug discovery that uses computers to dock chemicals into the active site of a drug target to identify leads through evaluation of binding affinities of the chemicals. However, there are concerns about the validity and scope of the reported virtual screens due to lack of studies to show that randomly selected chemicals are not equally active and due to the fact that metalloproteins were rarely used as drug targets. We have performed a virtual screening of a chemical database to identify prototypic inhibitors of farnesyltransferase (FT) with zinc present in the active site. Among the 21 compounds identified by computers, four inhibited FT in vitro with IC(50) values in the range from 25 to 100 microM. The most potent inhibitor also inhibited FT in human lung cancer cells. In contrast, none of 21 randomly selected compounds have an IC(50) lower than 100 microM. The results demonstrate the validity of virtual screening and the feasibility of applications of this approach to metalloprotein drug targets, such as matrix metalloproteinases, farnesyltransferase, and HIV-1 integrase, for the treatments of cardiovascular diseases, cancers, and AIDS.

Alkyl and Aryl Transferases↗
PubMed2000

Substantial in vivo proliferation of CD4(+) and CD8(+) T lymphocytes during secondary Listeria monocytogenes infection.

In mice Listeria monocytogenes infection induces a strong T cell response. In an attempt to quantitatively analyze the magnitude and kinetics of the CD4(+) and CD8(+) T cell response during L. monocytogenes infection in vivo we used a T cell transfer system that is independent of in vitro cell culture techniques and information about the identity of immunogenic T cell epitopes. Our results demonstrate substantial expansion of the in vivo primed and transferred T cell populations in response to L. monocytogenes. At the peak of response, transferred T cells represented more than one third of the total CD4(+) and CD8(+) T cell populations in blood and spleen of recipient mice. After stimulation in vitro, 40 % of these CD4(+) T cells responded to heat-killed listeriae with the production of IFN-gamma. Thus, our results reveal that in addition to the large CD8(+) T cell population an almost equally large population of Listeria-reactive CD4(+) T cells is generated in response to L. monocytogenes infection.

Adoptive Transfer↗
PubMed2000

Apoptosis in cancer: cause and cure.

The accumulation of neoplastic cells can occur through enhanced proliferation, diminished cell turnover, or a combination of both processes. Although the potential contribution of diminished cell turnover to tumor development has been appreciated for a decade, more recent studies in animal models and clinical cancer specimens have elucidated the mechanisms by which alterations in the apoptotic machinery contribute to the process of carcinogenesis. At the same time, a different group of studies have demonstrated the feasibility of eliminating neoplastic cells by selectively inducing apoptosis. In this essay, we review recent developments in the fields of carcinogenesis and molecular therapeutics in light of new understanding of apoptotic pathways.

Animals↗
PubMed2000

Phenotypic characterization of CD8(+)NKT cells.

We describe a novel CD8(+)NKT cell population expressing TCRalpha /beta or TCRgamma /delta. These CD8(+)NKT cells were prominent in the liver, and except for the thymus, virtually absent in other lymphoid organs. CD8(+)NKT cells expressed activation markers and comprised a high proportion of Ly49(+) cells. The development of the majority of CD8(+)NKT cells expressing TCRalpha /beta, but not TCRgamma /delta, depended on classical MHC class I. No CD8(+)NKT cells were detectable in young athymic mice, whereas the cells expressing TCRgamma /delta, but not TCRalpha /beta, appeared randomly in aged athymic mice. CD8(+)NK1(+) TCRalpha /beta cells showed polyclonal TCRVbeta usage and were virtually devoid of TCRValpha14. CD8(+)NK1(+) TCRgamma /delta cells predominantly expressed TCRVgamma1, 2 and 4, and Vdelta4, 5, 6 and 7. CD8(+)NKT cells, in particular those expressing TCRgamma /delta, were a major population in early life. IFN-gamma, but not IL-4, was induced in CD8(+)NKT cells by in vitro stimulation, independent of the TCRalpha /beta or TCRgamma /delta lineage. Hence, these cells represent a unique, though heterogeneous T cell population that shares markers with, but is distinct from, both conventional NKT cells and conventional CD8(+) T cells, and that may play a role in immune regulation.

Animals↗
PubMed2000

Participation of leukocyte function-associated antigen-1 and NK cells in the homing of thymic CD8+NKT cells to the liver.

A distinct CD8+NKT cell population expressing TCRalpha/beta or TCRgamma/delta has been identified in liver and thymus. We wondered whether cell adhesion molecules play a role in the homing of CD8+NKT cells to the liver. The number of liver CD8+NKT cells was markedly reduced in leukocyte function-associated antigen (LFA)-1-/- mice compared with wild-type (WT) mice. The reduction was restricted to the liver only and no measurable alterations were found in other organs. In the liver of SCID mice reconstituted with thymocytes from LFA-1-/- or WT mice, the number of donor-derived CD8+NKT cells was comparable and the vast majority of these cells expressed TCRalpha/beta. In a reciprocal radiation thymocyte reconstitution system with LFA-1-/- and WT mice, LFA-1 expressed on liver cells other than CD8+NKT cells appeared to be required for the homing of thymic CD8+NKT cells to the liver. The accumulation of donor thymocyte-derived CD8+NKT cells in the liver of SCID mice was severely impaired by in vivo depletion of NK cells, but not of Kupffer cells. These results not only indicate that thymus provides a source for CD8+NKT cells expressing TCRalpha/beta in the liver, but also suggest that LFA-1 expressed on NK cells is involved in the homing of thymic CD8+NKT cells to the liver.

Animals↗
PubMed2000

Immune response to infection with Salmonella typhimurium in mice.

Infection of mice with Salmonella typhimurium results in systemic infection and a disease similar to that seen in humans after infection with S. typhi. The innate immune system can restrict replication of S. typhimurium to a certain degree, but for effective control and eradication of bacteria, acquired immunity is essential. Salmonella infection induces the generation of specific CD4+ and CD8+ T cells, and both T cell populations are important for protection during primary and secondary responses, although the mechanisms underlying T cell-mediated protection are not yet completely understood. Infection with S. typhimurium also results in a strong antibody response to Salmonella antigens and, in contrast to most other intracellular bacteria, this antibody response participates in protection. In summary, the response to S. typhimurium involves both T and B cell-mediated immunity, and mechanisms mediated by both lymphocyte populations are important for control of primary infection and protection against secondary infection.

Animals↗
PubMed2000

CD1 molecules and CD1-dependent T cells in bacterial infections: a link from innate to acquired immunity?

The MHC class I-like, non-polymorphic CD1 molecules represent a novel system for the presentation of glycolipid antigens to T lymphocytes. CD1-mediated T cell responses appear to play distinct roles during bacterial infections such as in tuberculosis. This review deals with two aspects of CD1-mediated immune reactions. First we discuss the role of group II CD1-dependent NK T cells in bacterial infection. Second, we provide an insight into differential intracellular meeting points for antigen processing between group I CD1 molecules, mycobacteria and mycobacterial glycolipid antigens.

Animals↗
PubMed2000

Detection of DNA cleavage in apoptotic cells.

At least two discrete deoxyribonuclease activities can be detected during apoptotic death, one that generates 30- to 500-kilobase pair (kbp) domain-sized fragments and another that mediates internucleosomal DNA degradation. The latter nuclease has been identified as the caspase-activated deoxyribonuclease (CAD)/CPAN, a unique enzyme that is normally inhibited by the regulatory subunit ICAD (inhibitor of CAD)/DFF45 (DNA fragmentation factor). In this chapter, techniques widely used to detect DNA cleavage in apoptotic cells, including pulsed-field gel electrophoresis, conventional agarose gel electrophoresis, and terminal transferase-mediated dUTP nick end-labeling (TUNEL), are briefly reviewed. In addition, the use of ICAD to inhibit apoptosis-associated nuclease activity is illustrated. When properly applied, these techniques are widely applicable to the characterization of apoptotic cells.

Apoptosis↗
PubMed2000

CD1 and CD1-restricted T cells in infections with intracellular bacteria.

Glycolipid-specific, CD1a-, b- and c-dependent cytotoxic T cells have recently been shown to be involved in the host response against tuberculosis. These CD1 molecules 'sample' mycobacterial glycolipids from different intracellular sites in the infected cell. Additionally, upon microbial encounter, CD1d-dependent natural killer T cells promptly produce cytokines and perform regulatory activities. Here, we discuss the intracellular localization of CD1 molecules and mycobacterial lipids and the role of CD1-mediated T-cell responses in mycobacterial infections.

Antigen Presentation↗