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PubMed2000

Sporadic microsatellite instability is specific to neoplastic and preneoplastic endometrial tissues.

Microsatellite instability is a frequent (13%-24%) finding in sporadic endometrial adenocarcinoma and its precursor lesions, but most studies are limited to patients who already have malignant or premalignant endometrial disease. We performed retrospective testing for microsatellite instability in women in whom cancers showing microsatellite instability developed later and prospective testing in randomly selected normal and anovular endometrial biopsy specimens. Microsatellite instability in cancer-bearing biopsy specimens accurately reflected that seen in matched malignant tissues obtained at hysterectomy. In 1 patient, microsatellite instability developed in a scanty sample of fragmented endometrial tissues 7 years before the onset of endometrial cancer. Prospective testing for microsatellite instability in the endometria of women unselected for subsequent appearance of endometrial cancer showed a very low rate of microsatellite instability. Only 1 endometrial specimen showing microsatellite instability was found among 75 anovulatory endometrial specimens, and none were found in 377 normal endometrial specimens and 46 polyps examined. Microsatellite instability may precede the onset of histologically diagnosed carcinoma but is rare in randomly sampled histologically normal endometrial tissues.

Carcinoma, Endometrioid↗
PubMed2005

Quantitative measurement of fibrillogenesis by mass spectrometry.

In this chapter, a method for the quantitative determination of amyloid conversion by electrospray mass spectrometry is presented. Mass spectrometry is typically used for the purpose of measuring the mass of unknowns. However, the judicious selection of an internal standard permits the quantitative determination of protein concentration. For amyloid formation, this is particularly useful in circumstances where either the protein under study is in limited abundance, or separation of the precursor from other protein factors is impractical or undesirable. For the measurement of amyloid formation, internal standards are typically mass distinct variants of the amyloid precursor. In addition, the extreme stability of amyloid fibers permits assessment of residual precursor concentration with or without separation of fibers from unreacted precursor. Lastly, by using internal standards which are not amyloidogenic and do not interfere with fiber formation, electrospray mass spectrometry permits quantitative measurement of fiber formation in real-time and with small (pmoles) quantities of protein.

Amyloid↗
PubMed1985

Endocrine effects of high-dose ketoconazole therapy in advanced prostatic cancer.

The endocrine effects of ketoconazole (400 mg orally every 8 h) were studied in 9 previously untreated patients with advanced prostatic cancer. Five of these patients were followed for 12 months. A rapid fall in the serum concentration of testosterone was noted in all patients studied. Minimal values were observed on day 4 of treatment but thereafter serum testosterone increased slowly. The effect of the drug on unbound testosterone was relatively more important, since sex hormone binding globulin increased markedly during treatment. An increase in progesterone and LH was observed in all patients. This suggests that ketoconazole limits the conversion of C21-precursors into androgens. This block is compensated in part by activation of the hypothalamo-hypophyseal feedback system. Urinary 17-ketosteroids were decreased but 17-hydroxysteroids were unaffected by the treatment. In 5 patients followed monthly over a period of 12 months the mean testosterone concentration ranged from 69 ng/100 ml in one patient to 428 ng/100 ml in another. An excellent inverse correlation could be demonstrated between the mean serum concentration of testosterone and the mean concentration of ketoconazole. The change of serum dehydroepiandrosterone sulphate also correlated inversely with the mean ketoconazole level. Increased concentrations of oestradiol were noted in 2 patients with slight gynaecomastia. It is concluded that long-term suppression of androgen production can be realized by high-dose ketoconazole treatment and that the degree of suppression is proportional to the serum levels of the drug.

17-Ketosteroids↗
PubMed2000

Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications.

Limited proteolysis of most large protein precursors is carried out in vivo by the subtilisin-like pro-protein convertases. Many important biological processes such as peptide hormone synthesis, viral protein processing and receptor maturation involve proteolytic processing by these enzymes, making them potential targets for the development of novel therapeutic agents. However, the efficient development of such molecules requires a better understanding of the molecular mechanisms of proteolytic protein processing. Herein, we review the most recent findings on the molecular aspects of subtilisin-like convertase activity, such as the structural analysis of the proteases, the mechanisms of enzyme/substrate specificity, their interaction with other proteins such as 7B2, and the comparative tissue and cellular distribution of the enzymes and their substrates. These data are then used as a background for the review of the known biological functions of subtilisin-like pro-protein convertases, the reported clinical cases involving proteolytic processing defects and, finally, the ongoing development of new therapeutic inhibitor molecules based on this knowledge.

Animals↗
PubMed1999

Ethanol induces cell death and cell cycle delay in cultures of pheochromocytoma PC12 cells.

Animal models have clearly established that ethanol exposure can deplete neurons in the developing nervous system. However, the mechanism by which ethanol reduces cell number is unclear. In our study, cultures of pheochromocytoma cells, a neuronal-like cell line, were maintained in media, which supported cell proliferation. Although cell numbers continued to increase in the presence of ethanol, this increase was partially inhibited by ethanol exposure. This inhibitory effect was concentration and duration dependent. Cell proliferation was still partially inhibited after removal of ethanol, but this inhibition was temporary and disappeared after a 24-hr recovery period in ethanol-free conditions. Further study indicated that ethanol partially inhibited the increase in cell numbers by two mechanisms: (1) studies with vital stains indicated that ethanol induced cell death; (2) experiments using synchronized pheochromocytoma cell cultures showed that ethanol can induce cell cycle delay, thereby lengthening the doubling time of the cells. Analysis by flow cytometry indicated that with ethanol exposure, the cells accumulated in the G1 phase of the cell cycle. Our results suggest that in the developing nervous system, ethanol may limit the numbers of proliferating, neuronal precursor cells by two simultaneous mechanisms, cell death and cell cycle delay.

Animals↗
PubMed1999

Generation of IL-2-dependent cytolytic T lymphocytes (CTLs) with altered TCR responses derived from antigen-dependent CTL clones.

Ag-specific CD8+ CTL clones require TCR stimulation to respond to IL-2 for growth. Because IL-2 may be produced in the vicinity of CD8+ CTLs when Ag is limiting at the end of an immune response, we have examined the effect of culturing viral-specific CTL clones in IL-2 in the absence of antigenic stimulation. Limiting dilution analysis revealed a high precursor frequency for CTL clones derived from IL-2 propagation (termed CTL-factor dependent (FD)) that are dependent upon exogenous IL-2 for growth and survival and no longer require TCR stimulation to proliferate. Culturing CTL-FDs with infected splenocytes presenting Ag and IL-2 did not revert the clones but did lead to a TCR-induced inhibition of proliferation. The derived CTL-FDs have lost the ability to kill via the perforin/granule exocytosis mechanism of killing, although they express similar levels of TCR, CD3epsilon, CD8alphabeta, CD45, and LFA-1 compared with the parental clones. The CTL-FDs retain Fas ligand/Fas-mediated cytotoxicity, and IFN-gamma production and regulate the expression of CD69 and IL-2Ralpha when triggered through the TCR. A parental CTL protected BALB/c mice from a lethal challenge of influenza virus, whereas a CTL-FD did not. These findings represent a novel regulatory function of IL-2 in vitro that, if functional in vivo, may serve to down-regulate cellular immune responses.

Administration, Intranasal↗
PubMed2005

Idiopathic pure red cell aplasia: case report with review of literature.

Pure red cell aplasia (PRCA) is characterized by near absence of red blood cell precursors in the bone marrow with associated anemia and reticulocytopenia. Megakaryocytes and white blood cell precursors remain intact. Acute self-limited PRCA is the most common form. Acquired chronic PRCA is often idiopathic but can be associated with underlying disorders such as thymoma or autoimmune diseases. Congenital PRCA is associated with physical abnormalities. Pure red blood cell aplasia should always be included in the differential diagnosis of chronic unexplained anemia. We report a case of idiopathic pure red blood cell aplasia in an elderly male who has had a chronic anemia with recurrent blood transfusions for about 30 years.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1989

A virus-specific CD4+ cell-mediated cytolytic activity revealed by CD8+ cell elimination regularly develops in uncloned human antiviral cell lines.

Antiviral HLA class II-restricted cytotoxic CD4+ clones have been relatively well characterized in vitro but their significance in the immune response remains unknown. Here anti-influenza A and anti-EBV CD4+ CTL have been studied by using permanent cell lines either untreated or depleted of CD8+ cells. In bulk cultures, HLA class I-restricted anti-viral CD8+ CTL account for all of the detectable killer cell activity, whereas after elimination of CD8+ cells an HLA class II-restricted killer activity mediated by CD4+/2H4-/4B4+ cells was consistently observed. The CD4+ CTL were fully differentiated in all of the cultures tested from the third in vitro passage because they could be demonstrated immediately after elimination of CD8+ cells. These CD4+ killer cells were equivalent to the CD8+ cells in terms of their lytic capacity. The absence of any class II-restricted antiviral activity in bulk cultures seems to be related to the very small numbers of CD4+ cells present in these antiviral cell lines. However, CD4+ cytolytic activity could not be detected during the first two in vitro passages, even when limiting dilution analysis of the CTL precursors were performed, showing that the killer function of Th cells differentiate only after several in vitro stimulations.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Antigen-presenting T cells. II. Clonal responses of alloreactive and virus-specific self-restricted human cytotoxic T cell responses stimulated by T lymphoblasts.

The capacity of various stimulator cell types to present alloantigens or viral antigens to resting human CD8+ cytotoxic lymphocyte precursors (CLP) was analyzed in a limiting dilution culture system. Cell sorter-separated T lymphoblasts of both CD4+ and CD8+ phenotypes but not resting T cells were found to efficiently stimulate the clonal development of allogeneic CD8+ CLP. Thus, 5000 CD4+ T lymphoblasts activated as many (one out of 200 to one out of 300) allogeneic CLP as 50,000 peripheral blood mononuclear stimulator cells. This potent stimulator activity was found in CD4+ and CD8+ T lymphoblasts activated by mitogen, anti-T3 monoclonal antibody, or mixed leukocyte reactions. Cytotoxic T cells generated in this system were highly specific for HLA class I antigens. Furthermore, T lymphoblasts infected with mumps virus efficiently induced development of autologous CLP into CTL clones that were virus specific and self-HLA restricted, as shown by split-well analysis. The possible in vivo significance of antigen-presenting T lymphoblasts is discussed.

Antigen-Presenting Cells↗
PubMed1986

Precursor frequency of donor-specific lymphocytes recovered from canine lung transplants.

Dogs receiving left unilateral allo- or auto-grafts were treated with Cyclosporin A (Cy A) for 4 days. Thereafter allografted transplant recipients showed pulmonary pathology consistent with rejection. Blood and bronchoalveolar lavage (BAL)-derived lymphocytes were isolated from the recipient animals before and at various times after operation and the frequency of allospecific precursor cytolytic lymphocytes (pCTL) determined by limiting dilution analysis (LDA). Samples from the autografted control animal did not show any post-operative frequency changes; however, both blood and BAL-derived lymphocytes from allografted recipients showed a significant post-operative increase in the proportion of donor-specific pCTL. This increase was consistently greater in samples from the transplanted than the autochthonous lung. The frequency of pCTL determined using targets from an unrelated dog showed no post-operative increase. It is likely that the increase in frequency of donor-specific pCTL recorded during graft-rejection is a specific consequence of an interaction between the graft and recipient's immune system.

Animals↗
PubMed1980

Frequencies of alloreactive cytotoxic T-lymphocyte precursors responding to H-2 antigens alone.

The experiments reported here were designed to investigate the role of major histocompatibility antigens alone in primary allogeneic responses in vitro. Mixed lymphocyte cultures were established using small numbers of responder spleen cells and semiallogeneic or fully allogeneic stimulator spleen cells. The cytotoxicity generated in individual cultures was then detected by a sensitive 51Cr-release assay and the frequencies of responding cytotoxic T lymphocyte precursors (CTL-P) determined by limit dilution analysis. To investigate the responses of spleen cells to major histocompatibility (H-2) antigens alone, strains of BALB/c mice congenic at the H-2 locus were used as responder and stimulator cells. The frequencies of CTL-P responding in such congenic combinations were compared with those obtained using mouse strain combinations differing at minor histocompatibility (minor-H) loci in addition to H-2. A similar fraction of spleen cells responded regardless of whether the stimulator cells differed at the H-2 locus alone or at multiple minor-H loci in addition to H-2. It was concluded that minor-H antigenic differences do not contribute significantly to allogeneic responses.

Animals↗
PubMed1984

Interleukin-2-induced activation of natural killer activity in spleen cells from old and young mice.

Generation of natural killer (NK) activity in response to a partially purified preparation of rat interleukin-2 (IL-2) was compared in spleen cells derived from young (8-10 weeks old) and old (greater than 2 years old) female C57BL/6 mice. Significant NK activation was observed in both young and old mouse spleen cells incubated with 100 U IL-2/ml for 1-4 days, but the levels of cytotoxic activity generated in old mouse spleen cells was always lower than those of similarly treated young mouse spleen cells. Differences in IL-2-induced NK activation in old and young mouse spleen cells was obtained irrespective of the concentration of IL-2 used (25-400 U/ml). Quantitative comparisons indicated that old spleen cells activated by 3 day incubation with IL-2 acquired about two-fold higher NK activity than fresh young mouse spleen cells but still had only one-fourth of the levels of NK activity attained by IL-2-activated young mouse spleen cells. Cytotoxic activity of IL-2-activated young or old mouse spleen cells were totally abrogated by anti-asialo GM-1 antiserum + C but not by anti-Ly-2 + C treatment, indicating that the activated cytotoxic cells fell in the NK cell category. An analysis of NK precursor (NK-p) frequency by limiting dilution assay indicated that the NK-p frequency was about 4-fold higher in young as compared to old mouse spleen cells. The level of cytotoxic activity attained per NK-p cell was not significantly different for NK-p cells of old or young mice.

Aging↗
PubMed1980

Some modern aspects of the mode of action of non-steroidal anti-inflammatory drugs.

The modern concepts of the mode of action of non-steroidal anti-inflammatory drugs with reference to the mediators of inflammatory processes are reviewed. It seems likely that prostaglandins (E1 and E2) and the kinin system are closely associated inflammatory mediators which are responsible for maintenance of the signs and symptoms of chronic inflammatory joint disease. It is suggested that the analgesic action of aspirin-like drugs in rheumatoid arthritis is the result not only of blocking production of prostaglandins but also of limiting the increased availability of kinin precursor (kininogen).

Animals↗
PubMed1995

A metalloprotease inhibitor blocks shedding of the IL-6 receptor and the p60 TNF receptor.

Many cytokines and soluble cytokine receptors are generated by limited proteolysis of membrane-bound precursors. We have examined the ability of the recently described metalloprotease inhibitor, TNF-alpha protease inhibitor (TAPI), and other protease inhibitors to modulate shedding. The membrane-bound forms of the ligands TNF-alpha and CSF-1, the p60 TNFR and the IL-6R, were expressed in COS-7 cells. As expected, TAPI blocked the spontaneous and PMA-induced release of TNF-alpha from transfected cells. Interestingly, TAPI also inhibited the release of soluble forms of p60 TNFR and IL-6R in COS-7 cells. However, the processing of CSF-1, which also requires proteolytic cleavage of a membrane protein, was not affected. The ability of TAPI to inhibit shedding was unique, since several other classes of protease inhibitors, including three other metalloprotease inhibitors, did not inhibit shedding of IL-6R. To determine whether TAPI would prevent shedding under more physiologic conditions, we demonstrated that TAPI was able to prevent unstimulated and PMA-induced release of the soluble forms of TNF-alpha, p60 TNFR, and IL-6R from the monocytic cell line, THP-1, and from human peripheral blood monocytes. In addition, TAPI was able to inhibit LPS-induced shedding of the p60 TNFR and TNF-alpha from monocytes. In summary, our results indicate that a metalloprotease or group of related metalloproteases is responsible for the proteolytic cleavage of several cell surface proteins.

Antigens, CD↗
PubMed1996

TGF-beta-induced apoptosis of cerebellar granule neurons is prevented by depolarization.

The regulation of programmed cell death in the developing nervous system involves target-derived survival factors, afferent synaptic activity, and hormone- and cytokine-dependent signaling. Cultured immature cerebellar granule neurons die by apoptosis within several days in vitro unless maintained in depolarizing (high) concentrations of potassium (25 mM K+). Here we report that transforming growth factors (TGF)-beta1, -beta2, and -beta3 accelerate apoptosis of these neurons when maintained in physiological (low) K+ medium (5mM K+) as assessed by measures of viability, quantitative DNA fragmentation, and nuclear morphology. TGF-beta-induced apoptosis of these neurons is not blocked by CNTF and LIF, cytokines that enhance neuronal survival when applied alone, or by IGF-I, which prevents apoptosis upon potassium withdrawal. In contrast, neurons that differentiate in high K+ medium for several days in vitro acquire resistance to TGF-beta-mediated cell death. Granule neurons maintained in either low or high K+ medium produce latent, but not bioactive, TGF-beta1 and -beta2. Because neutralizing TGF-beta antibodies fail to augment survival of low K+ neurons, the cerebellar neurons are apparently unable to activate latent TGF-beta. Thus, apoptosis of low K+ neurons is not attributable to endogenous production of TGF-beta. Taken together, our data suggest that TGF-beta may limit the expansion of postmitotic neuronal precursor populations by promoting their apoptosis but may support survival of those neurons that have maturated, differentiated, and established supportive synaptic connectivity.

Animals↗
PubMed1998

Reconstitution of EBV-specific T cell immunity in solid organ transplant recipients.

EBV-specific autologous CTL were grown in vitro from three adults (two liver transplant recipients and one patient on hemodialysis awaiting kidney retransplant). All CTL lines were TCR alphabeta, CD8 positive cells, EBV specific, and MHC class I restricted. The CTL lines were expanded in vitro and infused in three escalating doses (5 x 10(7), 1 x 10(8), and 2 x 10(8) at monthly intervals. Weekly blood samples were collected following each infusion. EBV-specific CTL precursor cells in peripheral blood were quantitated by limiting dilution analysis, and their effect on EBV load in vivo was assessed by semiquantitative PCR. In all three patients, the numbers of CTL precursor cells increased during the weeks following the infusions and were highest after the third infusion. This level gradually declined but remained above the preinfusion levels for up to 3 mo. EBV genome copy number, on the other hand, dropped following the first infusion and became undetectable thereafter. The EBV DNA level remained lower than the pretransplant level in all patients for up to 3 mo after the last infusion. Our study shows that it is feasible to generate and expand EBV-specific CTL from pretransplant blood samples of solid organ transplant recipients, that these CTL can be stored and infused posttransplant, and that they remain cytotoxic and EBV specific in vivo. The aim of this study is to use these CTL for prevention and treatment of lymphoproliferative disease in solid organ transplant recipients.

Adult↗
PubMed2001

Lack of replicative senescence in cultured rat oligodendrocyte precursor cells.

Most mammalian somatic cells are thought to have a limited proliferative capacity because they permanently stop dividing after a finite number of divisions in culture, a state termed replicative cell senescence. Here we show that most oligodendrocyte precursor cells purified from postnatal rat optic nerve can proliferate indefinitely in serum-free culture if prevented from differentiating; various cell cycle-inhibitory proteins increase, but the cells do not stop dividing. The cells maintain high telomerase activity and p53- and Rb-dependent cell cycle checkpoint responses, and serum or genotoxic drugs induce them to acquire a senescence-like phenotype. Our findings suggest that some normal rodent precursor cells have an unlimited proliferative capacity if cultured in conditions that avoid both differentiation and the activation of checkpoint responses that arrest the cell cycle.

Animals↗