High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43^def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43^def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.     

Induction of a Specific N-Methyltransferase Enzyme by Long-Term Heat Stress during Barley Leaf Growth

Previous work showed that the indole alkaloid gramine accumulates in the upper leaves (e.g. the fifth) of barley as a response to high growth temperatures. The biosynthesis of gramine proceeds from tryptophan to 3-aminomethylindole (AMI); sequential N-methylations of AMI then yield N-methyl-3-aminomethylindole (MAMI) and gramine.

To determine whether high-temperature stress increases the activity of gramine pathway enzymes, leaf tissue from plants grown at various temperatures was assayed for N-methyltransferase (NMT) activity using AMI and MAMI as substrates in both in vivo and in vitro assays. NMT activity in expanding fifth leaves was increased 8- to 20-fold by growth at high temperatures (35°C day/30°C night) compared to cool temperatures (15°C/10°C). Several days of high temperature were required for full induction of NMT activity. No induction of NMT activity occurred in leaves which had completed expansion in cool conditions before exposure to high temperature.

To investigate NMT induction at the protein level, NMT activity was purified to homogeneity and used to produce polyclonal antibodies. Throughout enzyme purification, relative NMT activities towards AMI and MAMI remained constant, consistent with a single NMT enzyme. Immunoblot analysis showed that a large increase in NMT polypeptide coincided with induction of NMT activity by heat stress. Our results point to a type of high-temperature regulation of gene expression that is quite distinct from heat shock.

     

Toxoplasma gondii-positive human sera recognise intracellular tachyzoites and bradyzoites with diverse patterns of immunoreactivity

Antibody detection assays have long been the first line test to confirm infection with the zoonotic parasite Toxoplasma gondii. However, challenges exist with serological diagnosis, especially distinguishing between acute, latent and reactivation disease states. The sensitivity and specificity of serological tests might be improved by testing for antibodies against parasite antigens other than those typically found on the parasite surface during the acute stage. To this end, we analysed the reactivity profile of human sera, identified as positive for anti-Toxoplasma gondii IgG in traditional assays, by indirect immunofluorescence reactivity to acute stage intracellular tachyzoites and in vitro-induced latent stage bradyzoites. The majority of anti-Toxoplasma gondii IgG positive sera recognised both intracellularly replicating tachyzoites and in vitro-induced bradyzoites with varying patterns of immune-reactivity. Furthermore, anti-bradyzoite antibodies were not detected in sera that were IgM-positive/IgG-negative. These results demonstrate that anti-Toxoplasma gondii-positive sera may contain antibodies to a variety of antigens in addition to those traditionally used in serological tests, and suggest the need for further investigations into the utility of anti-bradyzoite-specific antibodies to aid in diagnosis of Toxoplasma gondii infection.     

Detection of intracellular signal transduction molecules in PBMC from rhesus macaques and sooty mangabeys

Abstract: One of the manifestations of human HIV-1 and nonhuman primate SIV infection that lead to disease is reasoned to be secondary to generalized T-cell dysfunction. The molecular mechanisms associated with the T-cell dysfunction remain to be elucidated. To address this issue, we sought to utilize the nonhuman primate model to study intracellular signaling events in cells from disease-susceptible rhesus macaques and disease-resistant sooty mangabeys. Because relatively little is known about these events in nonhuman primates, our laboratory defined optimal conditions, reagents, and assays for the study of signal transduction events in cells from nonhuman primates. The protein phosphorylation patterns in the two monkeys exhibited quantitative, qualitative, and kinetic differences. Antibodies to Stat6 detected a unique band in macaque cell lysates. This band is markedly decreased human cell lysates and never seen in mangabey cell lysates. Detection of various other intracellular signaling proteins is also described.     

An exception to Darwin's syndrome: floral position,protogyny, and insect visitation in Besseya bullii (Scrophulariaceae)

Darwin pointed out that plants with vertical inflorescences are likely to be outcrossed if the inflorescence is acropetalous (flowers from the bottom up), the flowers are protandrous (pollen is dispersed before stigmas are receptive), and pollinators move upward on the inflorescence. This syndrome is common in species pollinated by bees and flies, and very few exceptions are known. We investigated flowering phenology and pollinator behavior in Besseya bullii (Scrophulariaceae) and found that it did not fit Darwin's syndrome. The vertical inflorescence was acropetalous but the flowers were distinctly protogynous, so flowers with newly receptive stigmas appeared on the inflorescence above those with dehiscing anthers. A number of small insects visited B. bullii; bees in the family Halictidae (Augochlorella striata and Dialictus spp.) were most common. When insects moved between gender phases within inflorescences, they moved up more often than down (61% versus 39% of observations, respectively) but this difference was only marginally significant. Most visits were to male-phase flowers only, and this preference was more pronounced for pollen-foraging insects than for nectar-foraging insects. B. bullii was self-compatible, so its flowering characteristics potentially could result in considerable self-pollination. However, an average of 38% of the lowermost flowers opened before any pollen was available on the same inflorescence; these solo females had a high probability of outcrossing (though fruit set was relatively low in the bottom portion of the inflorescence). Upper flowers may also be outcrossed because downward insect movement was not uncommon. Therefore protogyny in B. bullii may not necessarily lead to more selfing than would protandry.     

Hyperosmotic Stress as a Stimulant for Proinflammatory Cytokine Production

The synthesis of proinflammatory cytokines involves members of the mitogen-activated protein (MAP) kinase stress pathway, particularly p38 MAP kinase and c-jun NH₂-terminal kinase. In this report we used hyperosmotic stress to study changes in steady-state mRNA levels and synthesis of proinflammatory cytokines in freshly obtained human peripheral blood mononuclear cells (PBMC)in vitro.There was no evidence of interleukin (IL)-8 gene expression in freshly obtained human blood despite 30 cycles of amplification of reverse-transcribed mRNA using the polymerase chain reaction. In contrast, exposure of PBMC to hyperosmotic conditions (330–410 mOsM) by the addition of NaCl to tissue culture medium induced gene expression for IL-1α, IL-1β, and IL-8. Routine tissue culture medium is hyperosmotic (305 mOsM) compared to human plasma (280–295 mOsM), but decreasing the osmolarity to the physiological range resulted in a 50% reduction in baseline IL-8 synthesis (P< 0.001). Although hyperosmotically induced accumulation of steady-state mRNA levels for IL-1α and IL-1β increased 50- and 7-fold over control, respectively, these were poorly translated into each respective cytokine. However, in PBMC stimulated by hyperosmotic stress, the addition of femtomolar concentrations of bacterial lipopolysaccharide, IL-1, or 1% normal human serum resulted in a synergistic synthesis (at least twice that expected) of IL-1α, IL-1β, TNF-α, and IL-8.     

Immune stimulation may contribute to enhanced progression of SIV induced disease in rhesus macaques

Abstract: A number of rhesus macaques experimentally infected with SIV isolates such as SIVmac251, fail to seroconvert, develop high plasma viremia and die rapidly (within 6–7 months p.i.). We hypothesized that such rapid progression is a result of a state of hyperimmune activation and concomitant immune suppression of these animals at the time of virus challenge. In efforts to test the hypothesis that immune activation leads to rapid progression of lentivirus-induced disease, adult rhesus macaques were infected with SIVmac251 and received an alternate monthly schedule of repeated immunization with allogeneic cells, keyhole limpet hemocyanin and tetanus toxoid (group I). For purposes of controls, a group of monkeys was infected with the same pool and dose of virus but were not immunized (group II) and a group was immunized with the same schedule of multiple antigens as group I but were not infected with SIV (group III). All the animals in group I (n ? 3) either failed to seroconvert or developed very low levels of SIV antibodies, had high plasma p27 defined antigenemia, and died within 8 months (2/3 died within 4 months). Of the animals in group II (n = 8), two patterns emerged as we had noted before. One subgroup (3 animals), displayed the same profile as group I (failure to fully seroconvert, high p27 levels and death by 8 months), whereas the other subgroup (5 animals) seroconverted, had low plasma p27 levels, and survived past 11 months (2/5 still alive past 22 months). All 3 animals in group III remained healthy. The data provided herein suggest that either experimental or natural (due to factors not clear at present) immune stimulation may lead to accelerated lentivirus induced disease progression most likely due to immune suppression and has implications for the understanding of the mechanisms for the rate of disease progression in human HIV-1 infection.     

Establishment of a three-dimensional human prostate organoid coculture under microgravity-simulated conditions: Evaluation of androgen-induced growth and psa expression

Summary A novel in vitro human prostate cancer model was established by using a coculture technique in which isolated human prostate fibroblasts were observed to grow as a mixed culture with isolated human prostate cancer cells (LNCaP) on microcarrier beads under microgravity-simulated conditions. This model appears to be promising and deserves further exploration because: (a) cocultured human prostate fibroblasts and cancer epithelial cells appear to undergo patterns of histogenesis similar to those observed in human prostate tumors and (b) unlike the conventional cell culture on plastic dishes, cocultured human prostate fibroblasts and LNCaP cells in microgravity-simulated conditions responded to the inductive signals of growth and differentiation from dihydrotestosterone in a manner similar to that observed in the in vivo condition. These results offer an opportunity to examine molecular mechanisms of cellular signaling in response to androgen stimulation during normal and aberrant human prostate development. The microgravity-simulated three-dimensional prostate epithelial cell culture with prostate fibroblasts can be further explored as an ideal in vitro model for the study of normal and neoplastic prostate development. This model could also be adopted as a drug screening program for the discovery of novel therapeutic agents in the treatment of human prostate cancer and benign hyperplastic growth.