Induction of heme oxygenase-1 is a beneficial response in a murine model of venous thrombosis

The induction of heme oxygenase-1 (HO-1) may protect against tissue injury. The present study examines the induction of HO-1 in a murine model of venous thrombosis and explores the downstream consequences of this induction. In a model of stasis-induced thrombosis created by ligation of the inferior vena cava, HO-1 expression is markedly induced. Such expression occurs primarily in smooth muscle cells in the venous wall and in leukocytes infiltrating the venous wall and clot. To determine the significance of HO-1 induction in venous thrombosis, this model was imposed in HO-1^+/+ and HO-1^−/− mice. The initial clot size did not differ in either group by day 2, but was significantly larger in HO-1^−/− mice by day 10, where an exaggerated inflammatory response in the venous wall was also observed. Following ligation of the inferior vena cava, HO-1^−/− mice exhibited increased nuclear factor κB activation and markedly increased up-regulation of tissue factor, selectins, inflammatory cytokines, and matrix metalloproteinase-9, the latter incriminated in both clot lysis and vascular injury. We conclude that HO-1 deficiency impairs thrombus resolution and exaggerates the inflammatory response to thrombus formation. These findings offer insight into recent observations that polymorphisms in the HO-1 gene may increase the risk for recurrent venous thrombosis and dysfunction of hemodialysis arteriovenous fistulas, the latter caused, in part, by thrombosis.     

Induction in vitro of a primary human antiviral cytotoxic T cell response

We report herein the successful priming of human anti-viral cytotoxic T cells (CTL) in vitro using two induction strategies based on the stimulation of peripheral blood mononuclear cells isolated from uninfected donors with synthetic viral peptides. The peptides used contain HLA-A2 binding motifs and have been identified as HLA-A2-restricted CTL epitopes in patients infected by the hepatitis B and C viruses. One approach uses repetitive long-term stimulation and the other uses bulk cultures containing large numbers of naive peripheral blood mononuclear cells. Both approaches successfully induce HLA-A2-restricted CTL specific for several viral epitopes. Some CTL recognize endogenously synthesized antigen on target cells infected with recombinant vaccinia virus expressing the corresponding viral proteins. This simple technique permits easy analysis of the primary human CTL repertoire, and may be exploitable for production of specific CTL effector cells for adoptive immunotherapy and dissection of the cellular and molecular requirements for priming of naive human CTL.     

Induction of immune response to bovine herpesvirus-1 with anti-idiotypic antibodies.

Previously, we prepared rabbit anti-idiotypic (anti-Id) antibodies against murine monoclonal antibodies (MAbs) specific for the major bovine herpesvirus-1 (BHV-1) envelope glycoproteins. Glycoprotein III (gIII) contains neutralization epitopes and may be the virus attachment protein. Anti-Id antibodies to a neutralizing MAb that reacts with gIII were purified by sequential immunoaffinity chromatography. Immune responses to the purified anti-Id reagent and BHV-1 were compared in mice. Both groups of mice produced BHV-1-specific neutralizing antibodies. However, lymphocyte proliferative responses and interferon and interleukin-2 production were specific for the respective immunizing antigens. These results suggest that the anti-Id reagent may bear an internal image of a B-cell-stimulating epitope of glycoprotein gIII; however, this epitope does not stimulate a virus-specific cellular immune response in mice.     

Knockdown of Akt1 promotes Akt2 upregulation and resistance to oxidative-stress-induced apoptosis through control of multiple signaling pathways

The Akt signaling pathway plays a key role in promoting the survival of various types of cells from stress-induced apoptosis, and different members of the Akt family display distinct physiological roles. Previous studies have shown that in response to UV irradiation, Akt2 is sensitized to counteract the induced apoptosis. However, in response to oxidative stress such as hydrogen peroxide, it remains to be elucidated what member of the Akt family would be activated to initiate the signaling cascades leading to resistance of the induced apoptosis. In the present study, we present the first evidence that knockdown of Akt1 enhances cell survival under exposure to 50 μM H(2)O(2). This survival is derived from selective upregulation and activation of Akt2 but not Akt3, which initiates 3 major signaling cascades. First, murine double minute 2 (MDM2) is hyperphosphorylated, which promotes p53 degradation and attenuates its Ser-15 phosphorylation, significantly attenuating Bcl-2 homologous antagonist killer (Bak) upregulation. Second, Akt2 activation inactivates glycogen synthase kinase 3 beta (GSK-3β) to promote stability of myeloid leukemia cell differentiation protein 1 (MCL-1). Finally, Akt2 activation promotes phosphorylation of FOXO3A toward cytosolic export and thus downregulates Bim expression. Overexpression of Bim enhances H(2)O(2)-induced apoptosis. Together, our results demonstrate that among the Akt family members, Akt2 is an essential kinase in counteracting oxidative-stress-induced apoptosis through multiple signaling pathways.     

Induction of a type 1 immune response to a recombinant antigen from Mycobacterium tuberculosis expressed in Mycobacterium vaccae.

A 19-kDa lipoprotein from Mycobacterium tuberculosis was expressed as a recombinant antigen in the nonpathogenic mycobacterial host strain M. vaccae. Immunization of mice with the recombinant M. vaccae resulted in induction of a strong type 1 immune response to the 19-kDa antigen, characterized by immunoglobulin G2a (IgG2a) antibodies and gamma interferon (IFN-gamma) production by splenocytes. Immunization with the same antigen in incomplete Freund's adjuvant induced a strong IgG1 response with only low levels of IFN-gamma. Subsequent intravenous and aerosol challenges of immunized mice with virulent M. tuberculosis demonstrated no evidence of protection associated with the response to the 19-kDa antigen; in fact, the presence of the recombinant 19-kDa antigen abrogated the limited protection conferred by M. vaccae (vector control). The recombinant M. vaccae system is a convenient approach to induction of type 1 responses to M. tuberculosis antigens. However, the unexpected reduction in protective efficacy of M. vaccae expressing the 19-kDa antigen highlights the complexity of testing recombinant subunit vaccines and the need for a better understanding of the immune mechanisms required for effective vaccination against tuberculosis.     

THP-1 cell apoptosis in response to Mycobacterial infection

We previously reported that Mycobacterium tuberculosis infection primes human alveolar macrophages (HAM) for tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis and that macrophage apoptosis is associated with killing internalized bacilli. Virulent mycobacterial strains elicit much less apoptosis than attenuated strains, implying that apoptosis is a defense against intracellular infection. The present study evaluated the potential for phorbol myristate acetate-differentiated THP-1 cells to mimic this response of primary macrophages. Consistent with the behavior of alveolar macrophages, attenuated M. tuberculosis H37Ra and Mycobacterium bovis BCG strongly induce THP-1 apoptosis, which requires endogenous TNF. THP-1 apoptosis is associated with reduced viability of infecting BCG. In contrast, virulent wild-type M. tuberculosis H37Rv and M. bovis do not increase THP-1 apoptosis over baseline. BCG induced early activation of caspase 10 and 9, followed by caspase 3. In contrast, wild-type M. bovis infection failed to activate any caspases in THP-1 cells. BCG-induced THP-1 apoptosis is blocked by retroviral transduction with vectors expressing crmA but not bcl-2. We conclude that differentiated THP-1 cells faithfully model the apoptosis response of HAM. Analysis of the THP-1 cell response to infection with virulent mycobacteria suggests that TNF death signals are blocked proximal to initiator caspase activation, at the level of TNF receptor 1 or its associated intracytoplasmic adaptor complex. Interference with TNF death signaling may be a virulence mechanism that allows M. tuberculosis to circumvent innate defenses leading to apoptosis of infected host cells.     

Human primary gastric dendritic cells induce a Th1 response to H. pylori

Adaptive CD4 T-cell responses are important in the pathogenesis of chronic Helicobacter pylori gastritis. However, the gastric antigen-presenting cells that induce these responses have not yet been identified. Here we show that dendritic cells (DCs) are present in the gastric mucosa of healthy subjects and are more prevalent and more activated in the gastric mucosa of H. pylori-infected subjects. H. pylori induced gastric DCs isolated from noninfected subjects to express increased levels of CD11c, CD86 and CD83, and to secrete proinflammatory cytokines, particularly interleukin (IL)-6 and IL-8. Importantly, gastric DCs pulsed with live H. pylori, but not control DCs, mediated T-cell secretion of interferon-γ. The ability of H. pylori to induce gastric DC maturation and stimulate gastric DC activation of Th1 cells implicates gastric DCs as initiators of the immune response to H. pylori.     

Overexpression of cyclin A and cyclin B1 proteins in astrocytomas

BACKGROUND: Cyclins are proteins that are expressed during the progression of a normal cell through the cell cycle. In a number of cancers, overexpression of cyclin A and cyclin B1 proteins has been reported, and in some instances the levels of expression correlated well with the grades of malignancy. The expression of cyclin A and cyclin B1 proteins in astrocytoma may be linked to the histologic grade or proliferative activities. OBJECTIVE: To study the expression of cyclin A and cyclin B1 proteins in astrocytomas and correlate the labeling indices (LIs) of cyclin A and cyclin B1 with histologic grade and Ki-67 LI. DESIGN: The surgical biopsy specimens from 65 adults with astrocytomas were reviewed and divided into grades based on the World Health Organization system. The paraffin sections were immunostained using primary antibodies against Ki-67, cyclin A, and cyclin B1. The LIs of these astrocytomas for the 3 different antibodies were determined by computerized image analysis. RESULTS: The cyclin A LI showed good correlation with astrocytoma grade and Ki-67 LI. Both the nuclear and cytoplasmic cyclin B LIs correlated well with the tumor grade but showed poor correlation with Ki-67 LI. CONCLUSIONS: This study suggests that although both cyclin A and B protein expression are related to the grade of malignancy in astrocytomas, cyclin A levels more generally reflect the proliferative state of these tumors. We also provide indirect evidence that cyclin B1 is associated with the aberrant progression through the G2-M phase checkpoint in astrocytomas.     

Induction of macrophage membrane interleukin 1 expression by T-cell dependent and T-cell independent pathways is inhibited by cyclosporin A

Here we report that cyclosporin A (CsA) inhibits the induction of membrane interleukin 1 (mIL-1) expression on murine peritoneal macrophages. The inhibition of mIL-1 expression was noted in response to both autoreactive T-cell lines specific for class I or class II MHC determinants as well as bacterial endotoxin. The macrophages were the direct target of this inhibition as shown by pretreating T cells and macrophages separately with CsA. The effective suppression by CsA of endotoxin-induced mIL-1 expression was dependent not only on the concentration of endotoxin employed, but also on the relative time of addition of CsA and endotoxin. Furthermore, CsA pretreatment of macrophages abrogated their ability to stimulate synthesis of IL-4 by a Th2 cell clone. These data suggest that inhibition of induction of accessory molecules such as mIL-1 may be a mechanism by which CsA abrogates the capacity of macrophages to present antigen.     

Chromogranin A activates diverse pathways mediating inducible nitric oxide expression and apoptosis in primary microglia

Chromogranin A (CgA) is associated with microglial activation cascades implicated in neurodegeneration in Alzheimer's, Pick's and Parkinson's diseases. In primary rat microglia, CgA-mediated inducible nitric oxide (iNOS) expression, nitric oxide (NO) production, mitochondrial depolarisation and apoptosis were inhibited by PP2 (Src kinase inhibitor). CgA-mediated iNOS expression and NO production were also inhibited by U0126 (MEK inhibitor), but mitochondrial depolarisation and apoptosis were not. PP2 inhibited ERK phosphorylation; therefore, Src mediates CgA-induced ERK phosphorylation leading to iNOS expression and NO production. Glutamate release induced by CgA was independent of both pathways. These findings provide insights into the way microglia are activated by CgA and the microglial signalling mechanisms associated with neurological disorders such as Alzheimer's disease.     

Infection of neutrophil granulocytes with Leishmania major activates ERK 1/2 and modulates multiple apoptotic pathways to inhibit apoptosis

Neutrophil granulocytes provide the first line of defense against bacterial, fungal, and parasitic infections. They phagocytose and kill many invading pathogens. Certain pathogenic microorganisms such as the intracellular protozoan parasite Leishmania major (L. major) can survive inside neutrophils. Mature neutrophils have a very short life span due to spontaneous apoptosis. Previously, we have reported that infections with L. major are able to delay spontaneous apoptosis. In the present study, we addressed the underlying mechanisms of regulation of both extrinsic and intrinsic apoptosis. We show that interaction with L. major transiently activates ERK1/2 phosphorylation. Pharmacological inhibition of ERK1/2 phosphorylation reversed the apoptosis delay. Moreover, infection leads to the enhanced and sustainable expression of the anti-apoptotic proteins Bcl-2 and Bfl-1, respectively. As downstream events, the release of cytochrome c from mitochondria and processing of caspase-6 were inhibited. We also confirm that infection with L. major results in reduced FAS expression on the surface of neutrophils. The presented data indicate that infection with L. major affects both intrinsic as well as extrinsic pathways of neutrophil apoptosis. Enhanced life span of host neutrophils enables the parasite to survive within neutrophils.     

Correlation of cyclin D1 and Rb gene expression with apoptosis in invasive breast cancer.

BACKGROUND: In vitro studies have shown that amplification and overexpression of the cyclin D1 gene can accelerate the progress of cells through the G1 phase. Therefore, cyclin D1 may have an apoptosis inhibiting effect. The retinoblastoma (Rb) gene was shown recently to be an important regulator of apoptosis. AIMS: To evaluate whether expression of the cyclin D1 and Rb genes correlated with apoptotic counts in a group of 97 invasive breast cancers. METHODS: Expression of the cyclin D1 and Rb genes was detected by standard immunnohistochemistry using paraffin wax embedded sections. Apoptotic cells were counted according to a strict protocol, in 10 fields of vision systematically spread over the most poorly differentiated area of the tumour, at a magnification of x630. Apoptotic cells counts were expressed as apoptotic cells/mm2. RESULTS: Cyclin D1 overexpression was found in 49% of cases. Loss of Rb expression was found in 44% of cases, and occurred particularly in poorly differentiated tumours. Cyclin D1 and Rb expression showed a positive correlation (p = 0.003). Apoptotic counts varied from 1 to 62/mm2. There were no significant correlations between cyclin D1 overexpression and apoptotic counts in the total group or in the retinoblastoma protein (pRb) positive tumours. Loss of Rb expression also showed no correlation with apoptotic counts. CONCLUSIONS: Cyclin D1 is frequently overexpressed in pRb positive tumours, but no evidence has been found for an anti-apoptotic effect of cyclin D1 overexpression or Rb expression in invasive breast cancer.     

Craniofrontonasal syndrome in a male due to chromosomal mosaicism involving EFNB1: further insights into a genetic paradox

Craniofrontonasal syndrome (CFNS) is an X‐linked disorder caused by inactivating mutations in the gene for ephrin‐B1 (EFNB1). Paradoxically it shows a more severe phenotype in females than in males. As a result of X inactivation cell populations with and without EFNB1 expression are found in EFNB1+/? females. This is thought to initiate a process termed cellular interference which may be responsible for the phenotype in females. We present a boy with severe clinical features of CFNS. In ～42% of his blood cells we found a supernumerary ring X chromosome containing EFNB1 but lacking XIST. Mosaicism for cell populations with different levels of EFNB1 expression can explain the severe phenotype of this patient. In vitro experiments in Xenopus tissue showed that cells overexpress ephrinB1 cluster and sort out from wild‐type cells. Our report provides further evidence that cellular interference contributes to the paradoxical inheritance pattern of CFNS.     

Human meiosis: the occurrence of interlocked bivalents in a normal male

The mitotic and meiotic chromosomes of a healthy 65-year-old man, ascertained from a survey of surgically removed testes, were investigated. A method for preparation of meiotic chromosomes from testicular material is described. A pair of interlocked bivalents was observed in 9 of the 54 diakinesislearly metaphase I cells examined. This was considered an abnormally high frequency to be explained as the result of a chance event. It is suggested that the chromosomes concerned are derived from those numbered 1–3 and 13–18 in the Denver system.     

MutS homolog 4 localization to meiotic chromosomes is required for chromosome pairing during meiosis in male and female mice

Msh4 (MutS homolog 4) is a member of the mammalian mismatch repair gene family whose members are involved in postreplicative DNA mismatch repair as well as in the control of meiotic recombination. In this report we show that MSH4 has an essential role in the control of male and female meiosis. We demonstrate that MSH4 is present in the nuclei of spermatocytes early in prophase I and that it forms discrete foci along meiotic chromosomes during the zygotene and pachytene stages of meiosis. Disruption of the Msh4 gene in mice results in male and female sterility due to meiotic failure. Although meiosis is initiated in Msh4 mutant male and female mice, as indicated by the chromosomal localization of RAD51 and COR1 during leptonema/zygonema, the chromosomes fail to undergo normal pairing. Our results show that MSH4 localization on chromosomes during the early stages of meiosis is essential for normal chromosome synapsis in prophase I and that it acts in the same pathway as MSH5.     

Neural pathways involved in the endocrine response of anestrous ewes to the male or its odor

During the non-breeding season, anestrous ewes do not experience ovarian cycles but exposure to a ram or its odor results in the activation of the luteinizing hormone secretion leading to ovulation. The aim of our work was to identify the neural pathways involved in this phenomenon. Using Fos immunocytochemistry, we examined the brain areas activated by the male or its fleece, in comparison with ewes exposed to the female fleece or the testing room (control group). In comparison with the control group, the male or its odor significantly increases Fos neuronal expression in the main and accessory olfactory bulbs, anterior olfactory nucleus, cortical and basal amygdala, dentate gyrus, ventromedial nucleus of the hypothalamus, piriform and orbitofrontal cortices. The main olfactory bulb, the cortical amygdala and the dentate gyrus are specifically more activated by the male odor than the female odor. Using a procedure of double labeling for Fos and gonadotropin-releasing hormone, we also compared the number of gonadotropin-releasing hormone neurons activated in the four groups of females. The male or its odor significantly increases the number and the proportion of gonadotropin-releasing hormone cells expressing Fos-immunoreactivity in the preoptic area and the organum vasculosum of the lamina terminalis, whereas no such induction of Fos-immunoreactivity was found in gonadotropin-releasing hormone neurons of ewes exposed to the female odor or the testing room. These findings emphasize the role of the main olfactory system in the detection and the integration of the ram odor, and also suggest the participation of the accessory olfactory system. Numerous structures widely distributed seem involved in the processing of the male olfactory cue to reach the gonadotropin-releasing hormone neurons.     

Induction of apoptosis by protease-defective particle preparations of human immunodeficiency virus type 1 is specific to a subset of U937-derived subclones

Several recent reports support the hypothesis that apoptosisoccurring in leukocytes of human immunodeficiency virus type1 (HIV–1)-infected individuals is important in progressionto AIDS. Specifically, apoptosis of uninfected bystander cellsappears critical in the pathogenesis of disease. Here, we presentevidence that protease-defective, gp120-containing HIV–1(L-2) particle preparations specifically induce apoptosis incells obtained from a subset of promonocytic U937- derived subclones.The rate of apoptosis induction was inversely correlated withthe susceptibility of the U937 subclones to wild-type HIV–1infection. Three types of apoptosis experiments were performed:DNA content analysis by flow cytometry, apoptotic nuclear degradationby fluorescent microscopy and DNA fragmentation analysis byagarose gel electrophoresis. Kinetic analysis revealed thatthere was a slower induction of apoptosis by L-2 particle preparationsthan with tumor necrosis factor (TNF)-a or anti-Fas antibody.However, there were no significant differences in the initialbinding rates of L-2 particles as well as the binding of TNF-aor anti-Fas antibody to the U937 subclones. The basal levelof protein kinase C activity was higher in high-type subclonescompared with low-type subclones. These results suggest thatU937 cells can be divided into at least two subpopulations,one that permits a productive HIV–1 infection but is notsubjected to L-2 particle preparation-induced apoptosis, whilethe other poorly replicates HIV–1 and is subjected to, L-2 mediated apoptosis, although at a slower rate than foundwith TNF–a or anti-Fas antibody. Received 7 May 1996, accepted 25 July 1996.     

CX3CR1-deficiency is associated with increased severity of disease in experimental autoimmune uveitis

The role of CX3CR1 in regulating the function of monocytes and microglia was examined in mice in which CX3CR1 had been replaced by green fluorescent protein (GFP). Induction of experimental autoimmune uveitis (EAU) in these mice resulted in increased disease severity at day 23 postimmunization with uveitogenic peptide when compared with CX3CR1-positive mice and increased apoptosis of neuronal cells in the inner nuclear layer. Resident microglia within the retina were activated equally as EAU developed in mice with or without CX3CR1, as determined by changes in morphology, suggesting that the microglial cell response did not account for the differences. Although the inflammatory infiltrate had increased in mice without CX3CR1 at day 23 postimmunization, the percentage of natural killer cells in the infiltrate was not changed in these mice. Similarly, increased disease severity at this stage was not associated with an overall increased percentage of macrophages in the retinal inflammatory infiltrate or in increased activation of these cells. The increased recruitment of monocytes to the retina in response to EAU induction in CX3CR1^GFP/GFP mice compared with CX3CR1^GFP/+ mice was not reflected in increased migration away from vessels, leading to marked clustering of GFP⁺ cells around veins and venules in these mice. It is possible that this monocyte/macrophage clustering leads to the increased severity of disease seen in the mice by focusing and so intensifying the inflammatory response.