Characterization of inverted membrane vesicles from the halophilic archaeon Haloferax volcanii.

Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea.

Making grandma's data secure: a security architecture for home telemedicine.

Home telemedicine presents special challenges for data security and privacy. Experience in the Informatics for Diabetes Education And Telemedicine (IDEATel) project has demonstrated that data security is not a one-size-fits-all problem. The IDEATel users include elderly patients in their homes, nurse case managers, physicians, and researchers. The project supports multiple computer systems that require a variety of user interactions, including: data entry, data review, patient education, videoconferencing, and electronic monitoring. To meet these various needs, a number of different of security solutions were utilized, including: UserID/Password, PKI certificates, time-based tokens, IP filtering, VPNs, symmetric and asymmetric encryption schemes, firewalls and dedicated connections. These were combined in different ways to meet the needs of each user groups.

Alloantigen-driven T cell death mediated by Fas ligand and tumor necrosis factor-alpha is not essential for the induction of allograft acceptance.

BACKGROUND: Fas ligand (FasL)-Fas and tumor necrosis factor alpha (TNFalpha)-tumor necrosis factor receptor (TNFR) interactions regulate immune responses and contribute to self-tolerance by mediating antigen-driven T cell apoptosis. It is not known whether FasL and TNFalpha, expressed by the recipient's lymphoid or nonlymphoid cells, are essential for the apoptosis of alloreactive T lymphocytes and the induction of allograft acceptance. METHODS: We compared the survival of fully allogeneic vascularized cardiac allografts between wild-type (wt) and FasL-mutant (gld) recipient mice. In addition, we studied cardiac allograft survival in gld mice injected with TNFalpha-neutralizing antibody. Allograft acceptance (graft survival >100 days) was induced by treating the recipients with CTLA4Ig, a recombinant fusion protein that blocks B7-CD28 T cell costimulation. In vivo alloantigen-driven apoptosis of mature CD4+ and CD8+ T lymphocytes was analyzed in mice repeatedly stimulated with allogeneic splenocytes. RESULTS: We found that CTLA4Ig induces 100% long-term acceptance of cardiac allografts in wt and gld mice. Similarly, CTLA4Ig induced 100% allograft acceptance in gld recipients injected with TNFalpha-neutralizing antibody. In vivo alloantigen-driven apoptosis of mature CD4+ and CD8+ T cells was significantly reduced in gld mice and in wt mice treated with anti-TNFalpha antibody. However, neutralizing TNFalpha activity in gld mice failed to abrogate alloantigen-driven T cell apoptosis. CONCLUSIONS: These data indicate that: (1) FasL and TNFalpha expression are not obligatory for the induction of long-term allograft acceptance by CTLA4Ig and (2) FasL- and TNFalpha-independent death pathways contribute to alloantigen-driven T cell apoptosis.

Increased susceptibility to immunologically mediated glomerulonephritis in IFN-gamma-deficient mice.

It is postulated that IFN-gamma confers susceptibility to immunologically mediated tissue injury. To test this hypothesis, we compared the intensity of accelerated anti-glomerular basement membrane glomerulonephritis between wild-type (IFN-gamma+/+) and IFN-gamma gene knockout (IFN-gamma-/-) mice. This disease model is initiated by binding of heterologous (sheep) anti-glomerular basement membrane Abs to the glomeruli of mice preimmunized with sheep IgG. The secondary cellular and humoral immune responses to the planted Ag then lead to albuminuria and glomerular pathology. We found that IFN-gamma-/- mice or IFN-gamma+/+ mice injected with IFN-gamma-neutralizing Ab develop worse albuminuria and glomerular pathology than IFN-gamma+/+ mice. The humoral response to sheep IgG (serum mouse anti-sheep IgG titers and intraglomerular mouse IgG deposits) was comparable in the IFN-gamma+/+ and IFN-gamma-/- groups. In contrast, IFN-gamma-/- mice mounted a stronger cellular immune response (cutaneous delayed-type hypersensitivity reaction) to sheep IgG than IFN-gamma+/+ mice. These findings provide evidence that endogenous IFN-gamma has a protective role in immunologically mediated glomerulonephritis initiated by foreign Ags.

Reinnervation of the rat olfactory bulb after methyl bromide-induced lesion: timing and extent of reinnervation.

We used the inhalation of methyl bromide gas to produce a near-complete destruction of the rat olfactory epithelium and analyzed the reinnervation of the bulb during reconstitution of the epithelium. The degeneration of olfactory axons elicits a transient up-regulation of glial cell proliferation and glial fibrillary acidic protein expression in the olfactory nerve and olfactory nerve layer of the bulb. Anterograde transport after intranasal infusion of wheat germ agglutinin conjugated horseradish peroxidase demonstrates that the first nascent axons reach the bulb within the first week after lesion. Subsequently, a massive wave of fibers arrives at the bulb between 1 and 2 weeks postlesion, and enters the glomeruli between 2 and 3 weeks postlesion. However, the olfactory projection does not stabilize until 8 weeks after lesion judging from the return in growth associated protein-43 expression to control levels. The extent of reinnervation after lesion is correlated with the completeness with which the epithelium reconstitutes itself. In rats that are lesioned while fed ad libitum, there is near-complete reconstitution of the neuronal population, and the projection onto the bulb fills the glomerular layer in its entirety. However, in rats that are lesioned while food restricted, a significant fraction of olfactory epithelium becomes respiratory during its reconstitution, and the population of reinnervating fibers is less. As a consequence, the posterior half of the bulb remains hypoinnervated overall and denervated at its caudal margin. The preferential reinnervation of the anterior bulb in the food-restricted, methyl bromide gas-lesioned animals indicates that the mechanisms that guide the growth of the olfactory axons and restore receptotopy do not operate with the same precision in this setting as they do during development or during the lower level of turnover associated with the "normal" laboratory existence. Accordingly, we hypothesize that the persistence of a significant population of pre-existing neurons is needed to preserve receptotopy during reinnervation. In addition, the results suggest that in the face of massive turnover and a reduced afferent population, there is a tendency for reinnervating axons to fill available synaptic space.

Regulation of alloantigen-mediated T-cell proliferation by endogenous interferon-gamma: implications for long-term allograft acceptance.

BACKGROUND: Recent data suggest that interferon (IFN)-gamma is not an essential mediator of acute rejection but, instead, is critical for the induction of long-term allograft acceptance. The in vivo mechanisms by which endogenous IFN-gamma regulates the alloimmune response and thus facilitates the induction of long-term allograft survival are not known. METHODS: We examined long-term cardiac and skin allograft survival, alloantigen-induced T-cell proliferation, and alloantigen-induced T-cell apoptosis in wild-type (IFN-gamma+/+) and IFN-gamma gene-knockout (IFN-gamma-/-) mice treated with either B7-CD28 T-cell costimulation blockade alone or B7-CD28 T-cell costimulation blockade combined with donor splenocyte transfusion. RESULTS: We found that IFN-gamma is essential for long-term allograft survival induced by treating mice with either B7-CD28 T-cell costimulation blockade alone or B7-CD28 T-cell costimulation blockade combined with donor splenocyte transfusion. Alloantigen-induced T-cell proliferation in vivo was significantly greater in IFN-gamma-/- mice than in IFN-gamma+/+ mice, and T-cell costimulation blockade abrogated alloantigen-induced T-cell proliferation in wild-type mice but failed to do so in mice that lack IFN-gamma. In contrast, alloantigen-induced T lymphocyte apoptosis in vivo did not differ between IFN-gamma+/+ and IFN-gamma-/- mice, and T-cell costimulation blockade enhanced alloantigen-induced T-cell apoptosis in both mouse strains. CONCLUSIONS: These data suggest that endogenous IFN-gamma facilitates the induction of long-term allograft survival by limiting the proliferation of alloactivated T lymphocytes. The data also suggest that B7-CD28 T-cell costimulation blockade exerts immunosuppressive actions by inhibiting the proliferation of activated T lymphocytes and by promoting their apoptosis.

Interferon-gamma is necessary for initiating the acute rejection of major histocompatibility complex class II-disparate skin allografts.

BACKGROUND: Although interferon (IFN)gamma has immunostimulatory functions, it is not essential for the acute rejection of fully allogeneic grafts in mice. It is not known whether IFNgamma plays a critical role in the acute rejection of MHC class I- or MHC class II-disparate allografts. METHODS: We studied the survival of skin allografts transplanted from fully allogeneic (BALB/c), MHC class I-disparate (bml), or MHC class II-disparate (bm12) donors to C57BL/6 wild-type (IFNgamma+/+) and IFNgamma gene-knockout (IFNgamma-/-) recipients. We also investigated the in vitro responses of IFNgamma+/+ and IFNgamma-/- T cells to MHC class II-disparate splenocytes. RESULTS: We found that IFNgamma-/- recipients reject BALB/c and bml skin grafts at the same rate as IFNgamma+/+ mice but are not capable of rejecting bm12 skin. Despite the inability of IFNgamma-/- mice to reject bm12 skin grafts, IFNgamma-/- T cells displayed vigorous proliferation and cytotoxic responses when stimulated with bm12 splenocytes in vitro. Furthermore, priming IFNgamma-/- recipients with bm12 splenocytes enabled these mice to reject bm12 skin grafts at a normal rate and to mount a cutaneous delayed-type hypersensitivity response to the bm12 antigen. CONCLUSION: The data demonstrate that IFNgamma is not necessary for generating effector mechanisms associated with acute transplant rejection but that it is required for initiating alloimmune responses to MHC class II-disparate skin grafts.

Breakdown of self-tolerance and the pathogenesis of autoimmunity.

Autoimmunity results from a breakdown of physiological mechanisms responsible for maintaining tolerance to self-antigens. These mechanisms are traditionally divided into central and peripheral. T or B lymphocytes that bind to self-antigens with high avidity are deleted or rendered unresponsive during their ontogeny in generative lymphoid organs such as the thymus and the bone marrow (central tolerance). However, this elimination process is incomplete, and regulatory mechanisms that keep mature autoreactive lymphocytes in check are necessary for preventing autoimmunity (peripheral tolerance). Peripheral tolerance mechanisms include passive or activation-induced T and B cell apoptosis, anergy, ignorance, and perhaps suppression of autoreactivity by regulatory lymphocytes. Observations in humans and experimental animals with defined genetic mutations provide examples of autoimmune disorders arising from failure to maintain peripheral tolerance to self. However, multiple factors are necessary for the induction of autoimmunity. For example, bacterial and viral infections may precipitate autoimmune disease in genetically susceptible individuals by exposing autoreactive T cells to cross-reactive peptides (molecular mimicry) or by enhancing lymphocyte stimulation.

Frequent inactivation of CDKN2A and rare mutation of TP53 in PCNSL.

Twenty primary central nervous system lymphomas (PCNSL) from immunocompetent patients (nineteen B-cell lymphomas and one T-cell lymphoma) were investigated for genetic alterations and/or expression of the genes BCL2, CCND1, CDK4, CDKN1A, CDKN2A, MDM2, MYC, RB1, REL, and TP53. The gene found to be altered most frequently was CDKN2A. Eight tumors (40%) showed homozygous and two tumors (10%) hemizygous CDKN2A deletions. Furthermore, methylation analysis of six PCNSL without homozygous CDKN2A loss revealed methylation of the CpG island within exon 1 of CDKN2A in three instances. Reverse transcription PCR analysis of CDKN2A mRNA expression was performed for 11 tumors and showed either no or weak signals. Similarly, immunocytochemistry for the CDKN2A gene product (p16) remained either completely negative or showed expression restricted to single tumor cells. None of the PCNSL showed amplification of CDK4. Similarly, investigation of CCND1 revealed no amplification, rearrangement or overexpression. The retinoblastoma protein was strongly expressed in all tumors. Only one PCNSL showed a mutation of the TP53 gene, i.e., a missense mutation at codon 248 (CGG to TGG:Arg to Trp). No evidence of BCL2 gene rearrangement was found in 11 tumors investigated. The bcl-2 protein, however, was strongly expressed in most tumors. None of the 20 PCNSL demonstrated gene amplification of MDM2, MYC or REL. In summary, inactivation of CDKN2A by either homozygous deletion or DNA methylation represents an important molecular mechanism in PCNSL. Mutation of the TP53 gene and alterations of the other genes investigated appear to be of minor significance in these tumors.

Immunohistochemical identification of discrete subsets of rat olfactory neurons and the glomeruli that they innervate.

Glomeruli at the posterior margin of the main olfactory bulb differ in several respects from those located in the remainder of the bulb; e.g., the olfactory sensory neurons (OSNs) that project here exhibit a distinct biochemical phenotype and signal transduction pathway, the microcircuitry of the glomeruli is substantially altered, and the glomeruli are activated by unconventional odorants. In the present work, we report that the monoclonal antibodies 2C6 and MAb213 label distinct subsets of OSNs in the olfactory epithelium (OE), including their axons to their terminations in the main olfactory bulb (MOB). Neurons immunopositive with 2C6 are concentrated in the cul-de-sacs of ectoturbinates 1 and 2 and of endoturbinate IV. Unlike the vast majority of OSNs, 2C6(+) neurons express olfactory marker protein (OMP) at a low level, but their failure to stain with anti-GAP-43 labeling indicates that the OMP "weak" neurons are nonetheless mature. Glomeruli positive for 2C6 are bilaterally symmetrical and occupy reproducible positions along the posterior margin of the MOB. Three of these are very large, and we refer to them as the lateral, posterior ventral, and anterior ventral 2C6(+) necklace glomeruli. MAb213(+) neurons are concentrated in the posteriormost tips of the cul-de-sacs and recesses at the reflection of the OE at the cribriform plate. Like 2C6(+) neurons, MAb213(+) OSNs are weakly labeled with anti-OMP but are fully mature. MAb213(+) glomeruli are also bilaterally symmetrical; they occupy reproducible positions along the posterior margin of the MOB. The three largest glomeruli occupy lateral, posterior ventral, and posterior positions; the first two are found close to the aforementioned 2C6(+) glomeruli. MAb213 also intensely labels one of the glomeruli of the modified glomerular complex, a string of small glomeruli ventrally, and another string dorsal to the accessory olfactory bulb. Acetylcholinesterase (AChE) histochemical staining of adjacent sections showed that many, but not all, MAb213(+) glomeruli colocalize with dense or moderate AChE staining. Thus, it is likely that the "necklace olfactory glomeruli" (Shinoda et al., 1990, 1993) and the phosphodiesterase (PDE2)(+) glomeruli (Juilfs et al., 1997) are a subset(s) of the MAb213(+) glomeruli. On the other hand, 2C6(+) glomeruli are not associated with AChE staining. These data indicate that the 2C6(+) glomeruli comprise a novel subset in the posterior MOB. In addition to the 2C6(+) and MAb213(+) necklace glomeruli, there is another distinct set of glomeruli at the posterior margin of the bulb that are OMP(-), 2C6(-), and MAb213(-). In summary, the current work indicates that glomeruli at the posterior margin of the bulb, which are necklace glomeruli in terms of location and appearance, are actually heterogeneous and may subserve specialized functions within the olfactory system.

Analysis of p53 mutation and epidermal growth factor receptor amplification in recurrent gliomas with malignant progression.

Genomic alterations and expression of the p53 tumor suppressor gene and the epidermal factor receptor gene (EGFR) were investigated in 22 patients with primary World Health Organization (WHO) grade II gliomas that on recurrence had progressed to malignant gliomas of WHO grades III or IV. Mutations of the p53 gene (exons 5 to 8) were found in 12 of 22 primary tumors (10 of 13 astrocytomas, 1 of 7 oligodendrogliomas, 1 of 2 oligoastrocytomas). In each of these cases identical p53 mutations were present in the respective malignant recurrences. In all instances in which the p53 mutation was associated with p53 protein accumulation (10 of 12 cases) the percentage of p53 immunopositive tumor cells had increased from the primary to the recurrent tumor. None of the primary low-grade and none of the recurrent high-grade tumors (7 anaplastic astrocytomas, 10 anaplastic oligodendrogliomas, 4 anaplastic oligoastrocytomas, and 5 glioblastomas) showed evidence of EGFR gene amplification. Our results thus demonstrate p53 is mutated in a high fraction of low-grade astrocytomas with progression to anaplastic astrocytomas and glioblastomas and that progression in such cases is frequently associated with an increase in the fraction of p53 immunopositive tumor cells. The general absence of EGFR amplification in our tumor series supports the hypothesis that the significance of p53 mutation and EGFR amplification may be different in glioblastomas that developed by progression from low-grade astrocytomas (secondary glioblastomas) compared to glioblastomas that developed rapidly in a de novo manner without a history of previous low-grade tumor (primary glioblastomas).

The striatal dopaminergic catalepsy mechanism is not necessary for the expression of pontine catalepsy produced by carbachol injections into the pontine reticular formation.

We have found previously that microinjections of carbachol into the pontine reticular formation (PRF) of rats induce an intense cataleptic state which is similar behaviorally with the catalepsy induced by systemic administration of neuroleptic drugs. In the experiments described in the present article we studied the possibility that the pontine carbachol catalepsy is generated via the intermediary of the dopaminergic cataleptogenic mechanism in the striatum. To this purpose we produced kainic acid lesions in the striatum and in the output stations of the striatal cataleptogenic mechanism-substantia nigra reticulata and the VM thalamic nucleus. Catalepsy was tested after systemic haloperidol (2 mg/kg) and pontine microinjections of carbachol (5 micrograms/1 microliter) before and after the kainic lesions. The cataleptogenic effect of carbachol injected in the pons was not attenuated by any of the three types of lesions. On the contrary, the cataleptogenic effect of haloperidol was greatly attenuated by the same lesions. These results suggest that the pontine catalepsy produced by microinjections of carbachol in PRF is generated independently of the dopaminergic cataleptogenic mechanism in basal ganglia.

[Alleles in chromosome 10p21-26 in malignant gliomas]. / Allelieuntersuchungen auf Chromosom 10q21-26 in malignen Gliomen.

Loss of genetic material on chromosome 10 is regarded as a prominent feature in the genesis of glioblastomas. To use chromosome 10 deletions as diagnostic markers for glioblastomas we investigated, if the loss of chromosome 10 material could be restricted on the region 10q21-26. By PCR microsatellite analysis on frozen tissue and paraffin material from the ZULCH brain tumor collection we found (1) loss of heterozygosity in 10q21-26 in 75% of the investigated DNA from frozen tissue and (2) an interstitial loss in the region of the microsatellite marker D10S186. The combined immunohistochemical analysis of overexpression of EGFR, EGF and TGF alpha with LOH on chromosome 10 showed that chromosome 10 deletions are not exclusively bound to EGFR overexpression.

Troponin T release after heart transplantation.

BACKGROUND: For the diagnosis of myocardial cell damage the measurement of the serum concentrations of myofibrillar antigens has several potential advantages over the assessment of traditional serological markers. These include the expression of myofibrillar antigens as cardiospecific isoforms and their high intracellular concentrations. Recently a sensitive and specific enzyme immunoassay for cardiac troponin T has been developed that shows little cross-reactivity with skeletal isoforms. OBJECTIVE: To characterise myocardial cell damage after orthotopic heart transplantation, concentration of circulating troponin T were measured prospectively in serial blood samples from 19 consecutive patients taken during the first three months after transplantation. RESULTS: Mean (SD) serum concentrations of cardiac troponin T reached a maximum of 3.6 (1.8) micrograms/l at 7.1 (4.2) days after transplantation and remained higher than 0.5 micrograms/l (twice the detection limit of the assay) in all patients for at least 43 days (mean (SD) 59 (20) days). There was considerable variation in cumulative troponin T release (area under the concentration curve) between the patients (ranging from 27 to 150 micrograms x days/l) that was not related to the total ischaemic time before transplantation or to the patient's renal or hepatic function, preoperative cardiac diseases, major histocompatibility complex matching or the number of complications related to rejection. CONCLUSIONS: Because the half life of cardiac troponin T serum is 2 h the current data show that antigen continued to be released from implanted hearts during the first postoperative months in quantities similar to minor Q wave myocardial infarction. Troponin T release after transplantation continued for much longer than after myocardial infarction or other cardiac surgery. Processes other than perioperative ischaemic damage must be responsible for the considerable individual differences in the release of cardiac troponin T.

The microscopic structure and function of the vascular retrodiscal pad of the human temporomandibular joint.

The highly vascular retrodiscal pad attaches the articular disc of the temporomandibular joint to osseous structures posterior to it. There is debate as to whether or not the pad includes erectile tissue. Histological examination of 11 retrodiscal pads revealed thick-walled muscular arteries, thin-walled veins and numerous vascular sinuses, which is different from the erectile tissue of the corpora cavernosa. Its histological features and position within the joint suggest that the retrodiscal pad may function differently from other human joints due to: (1) the large amount of distraction it undergoes relative to its size and (2) its location anterior to the ear.

Granzyme B-gene expression: a marker of human lymphocytes "activated" in vitro or in renal allografts.

Human killer cells contain cytoplasmic granules in which serine esterases, or granzymes, and perforin have been identified. We have studied the induction of granzyme B-gene expression in peripheral blood lymphocytes and large granular lymphocytes activated by various stimuli in vitro as well as in cellular infiltrates at the site of renal allografts in patients with and without rejection. Using in situ hybridization, kinetic experiments have shown that granzyme B mRNA is an early marker of in vitro cell activation. Data obtained in vivo also indicate the presence of granzyme B mRNA-bearing cells, which argues in favor of the induction of granzyme B gene in cells activated in vivo.

A C2 muscle cell variant defective in transport of the acetylcholine receptor to the cell surface.

We have previously reported the isolation of variants of the C2 mouse muscle cell line that express reduced amounts of acetylcholine receptors (AChRs) on their surface (Black, R. A., and Hall, Z. W. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 124-128). One of the variants, T-, makes an approximately normal amount of the AChR but accumulates most of it in an intracellular pool. This pool is stable and does not serve as precursor for surface AChR. Surface levels of insulin receptor and transferrin receptor are normal in T- cells, and a normal proportion of total hemagglutinin is expressed on the surface after infection of the T- variant with influenza virus. Pulse-chase experiments and kinetic analysis show: 1) that T- cells synthesize a normal amount of the alpha subunit but degrade it much more slowly than do wild-type cells; and 2) that newly synthesized alpha subunit is assembled into the AChR at a normal rate. A small fraction of the assembled AChR in T- cells is transported to the surface with normal kinetics, but most of it remains in an internal pool. This variant may provide an important tool for investigation of the factors that regulate AChR assembly and transport to the surface membrane.

Regulation of motor axon sprouting.

Our studies on the amphibian and mammalian motor systems suggest that sprouting of intact motoneurons and synapse formation can be regulated by three mechanisms: peripheral, central, and transneuronal. Peripheral mechanisms provide the means of a direct mode of interaction between the periphery of the nerve cell and the target, to determine the extent of target innervation. The central mechanism enables target muscles to signal the cell bodies of their innervating motoneurons to regulate axonal growth and synapse formation, and thus again determine the extent of their innervation. The transneuronal mechanism provides a vehicle by which the pattern of innervation of a muscle can be altered by nerve cells that do not themselves innervate the muscle, but are an integral part of the entire system.