The bone marrow microenvironment contributes to type I diabetes induced osteoblast death

Type I diabetes increases an individual's risk for bone loss and fracture, predominantly through suppression of osteoblast activity (bone formation). During diabetes onset, levels of blood glucose and pro‐inflammatory cytokines (including tumor necrosis factor α (TNFα)) increased. At the same time, levels of osteoblast markers are rapidly decreased and stay decreased chronically (i.e., 40 days later) at which point bone loss is clearly evident. We hypothesized that early bone marrow inflammation can promote osteoblast death and hence reduced osteoblast markers. Indeed, examination of type I diabetic mouse bones demonstrates a greater than twofold increase in osteoblast TUNEL staining and increased expression of pro‐apoptotic factors. Osteoblast death was amplified in both pharmacologic and spontaneous diabetic mouse models. Given the known signaling and inter‐relationships between marrow cells and osteoblasts, we examined the role of diabetic marrow in causing the osteoblast death. Co‐culture studies demonstrate that compared to control marrow cells, diabetic bone marrow cells increase osteoblast (MC3T3 and bone marrow derived) caspase 3 activity and the ratio of Bax/Bcl‐2 expression. Mouse blood glucose levels positively correlated with bone marrow induced osteoblast death and negatively correlated with osteocalcin expression in bone, suggesting a relationship between type I diabetes, bone marrow and osteoblast death. TNF expression was elevated in diabetic marrow (but not co‐cultured osteoblasts); therefore, we treated co‐cultures with TNFα neutralizing antibodies. The antibody protected osteoblasts from bone marrow induced death. Taken together, our findings implicate the bone marrow microenvironment and TNFα in mediating osteoblast death and contributing to type I diabetic bone loss. J. Cell. Physiol. 226: 477–483, 2011. © 2010 Wiley‐Liss, Inc.     

Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines

Virulence in Toxoplasma gondii is strongly influenced by the genotype of the parasite. Type I strains uniformly cause rapid death in mice regardless of the host genotype or the challenge dose. In contrast, the outcome of infections with type II strains is highly dependent on the challenge dose and the genotype of the host. To understand the basis of acute virulence in toxoplasmosis, we compared low and high doses of the RH strain (type I) and the ME49/PTG strain (type II) of T. gondii in outbred mice. Differences in virulence were reflected in only modestly different growth rates in vivo, and both strains disseminated widely to different tissues. The key difference in the virulent RH strain was the ability to reach high tissue burdens rapidly following a low dose challenge. Lethal infections caused by type I (RH) or type II (PTG) strain infections were accompanied by extremely elevated levels of Th1 cytokines in the serum, including IFN-gamma, TNF-alpha, IL-12, and IL-18. Extensive liver damage and lymphoid degeneration accompanied the elevated levels of cytokines produced during lethal infection. Increased time of survival following lethal infection with the RH strain was provided by neutralization of IL-18, but not TNF-alpha or IFN-gamma. Nonlethal infections with a low dose of type II PTG strain parasites were characterized by a modest induction of Th1 cytokines that led to control of infection and minimal damage to host tissues. Our findings establish that overstimulation of immune responses that are normally necessary for protection is an important feature of acute toxoplasmosis.     

Plasma carbonyls do not correlate with lung function or computed tomography measures of lung density in older smokers.

Oxidative stress and inflammation are hallmarks of chronic obstructive pulmonary disease (COPD). A critical byproduct of oxidative damage is the introduction of carbonyl groups into amino acid residues. We hypothesize that plasma carbonyl content is inversely correlated with lung function and computed tomography (CT) measures of lung density among smokers and is elevated in COPD. Carbonyl was measured in plasma of participants aged 60 years and older by ELISA. Generalized linear and additive models were used to adjust for potential confounders. Among 541 participants (52% male, mean age 67 years, 41% current smokers), mean plasma carbonyl content was 17.9+/-2.9 nmol ml(-1) and mean forced expiratory volume in one second (FEV(1)) was 80.7+/-20.9% of predicted. Plasma carbonyl content was inversely associated with FEV(1), but this relationship was largely explained by age. Multivariate analyses ruled out clinically meaningful associations of plasma carbonyl content with FEV(1), FEV(1)/FVC (forced vital capacity) ratio, severity of airflow obstruction, and CT lung density. Plasma carbonyl content is a poor biomarker of oxidative stress in COPD and emphysema.     

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO₂, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.     

Mosaic RNA Phage VLPs Carrying Domain III of the West Nile Virus E Protein

The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber- or opal-suppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11–16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIII-induced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine.     

Norepinephrine acts as D1-dopaminergic agonist in the embryonic avian retina: late expression of beta1-adrenergic receptor shifts norepinephrine specificity in the adult tissue

Dopamine is the main catecholamine found in the chick retina whereas norepinephrine is only found in trace amounts. We compared the effectiveness of dopamine and norepinephrine in promoting cyclic AMP accumulation in retinas at embryonic day 13 (E13) and from post-hatched chicken (P15). Dopamine (EC(50)=10microM) and norepinephrine (EC(50)=30microM), but not the beta(1)-adrenergic agonist isoproterenol, stimulated over seven-fold the production of cyclic AMP in E13 retina. The cyclic AMP accumulation induced by both catecholamines in embryonic tissue was entirely blocked by 2microM SCH23390, a D(1) receptor antagonist, but not by alprenolol (beta-adrenoceptor antagonist). In P15 retinas, 100microM isoproterenol stimulated five-fold the accumulation of cAMP. This effect was blocked by propanolol (10microM), but not by 2microM SCH23390. Embryonic and adult retina display beta(1) adrenergic receptor mRNA as detected by RT-PCR, but the beta(1) adrenergic receptor protein was detected only in post-hatched tissue. We conclude that norepinephrine cross-reacts with D(1) dopaminergic receptor with affinity similar to that of dopamine in the embryonic retina. In the mature retina, however, D(1) receptors become restricted to activation by dopamine. Moreover, as opposed to the embryonic tissue, norepinephrine seems to stimulate cAMP accumulation via beta(1)-like adrenergic receptors in the mature tissue.     

Pairing centers recruit a Polo-like kinase to orchestrate meiotic chromosome dynamics in C. elegans

Faithful segregation of homologous chromosomes during meiosis requires pairing, synapsis, and crossing-over. In C.?elegans, homolog pairing and synapsis depend on pairing centers (PCs), special regions near one end of each chromosome that interact with the nuclear envelope (NE) and cytoplasmic microtubules. Here, we report that PCs are required for nuclear reorganization at the onset of meiosis. We demonstrate that PCs recruit the Polo-like kinase PLK-2 to induce NE remodeling, chromosome pairing, and synapsis. Recruitment of PLK-2 is also required to mediate a cell cycle delay and selective apoptosis of nuclei containing unsynapsed chromosomes, establishing a molecular link between these two quality control mechanisms. This work reveals unexpected functions for the conserved family of Polo-like kinases, and advances our understanding of how meiotic processes are properly coordinated to ensure transmission of genetic information from parents to progeny.     

HDL Size is More Accurate than HDL Cholesterol to Predict Carotid Subclinical Atherosclerosis in Individuals Classified as Low Cardiovascular Risk

Background

Misclassification of patients as low cardiovascular risk (LCR) remains a major concern and challenges the efficacy of traditional risk markers. Due to its strong association with cholesterol acceptor capacity, high-density lipoprotein (HDL) size has been appointed as a potential risk marker. Hence, we investigate whether HDL size improves the predictive value of HDL-cholesterol in the identification of carotid atherosclerotic burden in individuals stratified to be at LCR.

Methods and Findings

284 individuals (40–75 years) classified as LCR by the current US guidelines were selected in a three-step procedure from primary care centers of the cities of Campinas and Americana, SP, Brazil. Apolipoprotein B-containing lipoproteins were precipitated by polyethylene glycol and HDL size was measured by dynamic light scattering (DLS) technique. Participants were classified in tertiles of HDL size (<7.57; 7.57–8.22; >8.22 nm). Carotid intima-media thickness (cIMT) <0.90 mm (80^th percentile) was determined by high resolution ultrasonography and multivariate ordinal regression models were used to assess the association between cIMT across HDL size and levels of lipid parameters. HDL-cholesterol was not associated with cIMT. In contrast, HDL size >8.22 nm was independently associated with low cIMT in either unadjusted and adjusted models for age, gender and Homeostasis Model Assessment 2 index for insulin sensitivity, ethnicity and body mass index (Odds ratio 0.23; 95% confidence interval 0.07–0.74, p = 0.013).

Conclusion

The mean HDL size estimated with DLS constitutes a better predictor for subclinical carotid atherosclerosis than the conventional measurements of plasma HDL-cholesterol in individuals classified as LCR.     

Genome-Wide and Candidate Gene Association Study of Cigarette Smoking Behaviors

The contribution of common genetic variation to one or more established smoking behaviors was investigated in a joint analysis of two genome wide association studies (GWAS) performed as part of the Cancer Genetic Markers of Susceptibility (CGEMS) project in 2,329 men from the Prostate, Lung, Colon and Ovarian (PLCO) Trial, and 2,282 women from the Nurses'' Health Study (NHS). We analyzed seven measures of smoking behavior, four continuous (cigarettes per day [CPD], age at initiation of smoking, duration of smoking, and pack years), and three binary (ever versus never smoking, ≤10 versus >10 cigarettes per day [CPDBI], and current versus former smoking). Association testing for each single nucleotide polymorphism (SNP) was conducted by study and adjusted for age, cohabitation/marital status, education, site, and principal components of population substructure. None of the SNPs achieved genome-wide significance (p<10⁻⁷) in any combined analysis pooling evidence for association across the two studies; we observed between two and seven SNPs with p<10⁻⁵ for each of the seven measures. In the chr15q25.1 region spanning the nicotinic receptors CHRNA3 and CHRNA5, we identified multiple SNPs associated with CPD (p<10⁻³), including rs1051730, which has been associated with nicotine dependence, smoking intensity and lung cancer risk. In parallel, we selected 11,199 SNPs drawn from 359 a priori candidate genes and performed individual-gene and gene-group analyses. After adjusting for multiple tests conducted within each gene, we identified between two and five genes associated with each measure of smoking behavior. Besides CHRNA3 and CHRNA5, MAOA was associated with CPDBI (gene-level p<5.4×10⁻⁵), our analysis provides independent replication of the association between the chr15q25.1 region and smoking intensity and data for multiple other loci associated with smoking behavior that merit further follow-up.     

TGF-beta-associated enhanced susceptibility to leishmaniasis following intramuscular vaccination of mice with Leishmania amazonensis antigens

Leishmania amazonensis and Leishmania braziliensis are the main causal agents of anergic diffuse cutaneous leishmaniasis and hyperergic mucosal leishmaniasis in man, respectively. In this work we demonstrate that intramuscular vaccination of BALB/c mice with whole antigens of L. amazonensis (LaAg) but not L. braziliensis (LbAg) results in increased susceptibility to cutaneous leishmaniasis. LaAg vaccination resulted in an increased capacity of the draining lymph nodes to produce IL-10 and TGF-beta during antigen recall responses. In vitro cultivation with LaAg but not LbAg induced increased apoptosis of CD8+ T cells. Following infection with L. amazonensis, LaAg-vaccinated mice produced significantly more TGF-beta and a higher serum IgG1/IgG2a antibody ratio compared with LbAg-vaccinated and non-vaccinated animals. The association of TGF-beta with enhanced susceptibility to infection was confirmed in mice co-vaccinated with LaAg and neutralizing anti-TGF-beta antibodies. Upon parasite challenge, these animals developed much smaller lesion sizes and parasite burdens, comparable with non-vaccinated controls. The disease-promoting effect of LaAg vaccination is not a general event, as in contrast to BALB/c, the disease outcome in C57Bl/6 mice was unaltered. Together, these findings indicate that species-specific components of L. amazonensis activate overt TGF-beta production that predisposes more susceptible individuals to aggravated disease following vaccination.