Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy

B cell immunotherapy has emerged as a mainstay in the treatment of lymphomas and autoimmune diseases. Although the microenvironment has recently been demonstrated to play critical roles in B cell homeostasis, its contribution to immunotherapy is unknown. To analyze the in vivo factors that regulate mechanisms involved in B cell immunotherapy, we used a murine model for human CD20 (hCD20) expression in which treatment of hCD20(+) mice with anti-hCD20 mAbs mimics B cell depletion observed in humans. We demonstrate in this study that factors derived from the microenvironment, including signals from the B cell-activating factor belonging to the TNF family/BLyS survival factor, integrin-regulated homeostasis, and circulatory dynamics of B cells define distinct in vivo mechanism(s) and sensitivities of cells in anti-hCD20 mAb-directed therapies. These findings provide new insights into the mechanisms of immunotherapy and define new opportunities in the treatment of cancers and autoimmune diseases.     

Synthesis and activity of 8-substituted benzo[c]quinolizin-3-ones as dual inhibitors of human 5alpha-reductases 1 and 2

Some potent dual inhibitors of 5alpha-reductases 1 and 2, based on the benzo[c]quinolizin-3-one structure and with IC(50) values ranging between 93 and 166nM for both isozymes, were found. The presence of the F atom on the ester moiety at the position 8 was crucial. This result can help in the design of other potent, dual inhibitors to be developed as drugs in the treatment of 5alpha-reductase related diseases.     

Insulin resistance in spontaneously hypertensive rats is associated with endothelial dysfunction characterized by imbalance between NO and ET-1 production

Insulin stimulates production of NO in vascular endothelium via activation of phosphatidylinositol (PI) 3-kinase, Akt, and endothelial NO synthase. We hypothesized that insulin resistance may cause imbalance between endothelial vasodilators and vasoconstrictors (e.g., NO and ET-1), leading to hypertension. Twelve-week-old male spontaneously hypertensive rats (SHR) were hypertensive and insulin resistant compared with control Wistar-Kyoto (WKY) rats (systolic blood pressure 202 +/- 11 vs. 132 +/- 10 mmHg; fasting plasma insulin 5 +/- 1 vs. 0.9 +/- 0.1 ng/ml; P < 0.001). In WKY rats, insulin stimulated dose-dependent relaxation of mesenteric arteries precontracted with norepinephrine (NE) ex vivo. This depended on intact endothelium and was blocked by genistein, wortmannin, or N(omega)-nitro-l-arginine methyl ester (inhibitors of tyrosine kinase, PI3-kinase, and NO synthases, respectively). Vasodilation in response to insulin (but not ACh) was impaired by 20% in SHR (vs. WKY, P < 0.005). Preincubation of arteries with insulin significantly reduced the contractile effect of NE by 20% in WKY but not SHR rats. In SHR, the effect of insulin to reduce NE-mediated vasoconstriction became evident when insulin pretreatment was accompanied by ET-1 receptor blockade (BQ-123, BQ-788). Similar results were observed during treatment with the MEK inhibitor PD-98059. In addition, insulin-stimulated secretion of ET-1 from primary endothelial cells was significantly reduced by pretreatment of cells with PD-98059 (but not wortmannin). We conclude that insulin resistance in SHR is accompanied by endothelial dysfunction in mesenteric vessels with impaired PI3-kinase-dependent NO production and enhanced MAPK-dependent ET-1 secretion. These results may reflect pathophysiology in other vascular beds that directly contribute to elevated peripheral vascular resistance and hypertension.     

ATR/TEM8 is highly expressed in epithelial cells lining Bacillus anthracis' three sites of entry: implications for the pathogenesis of anthrax infection

Anthrax is a disease caused by infection with spores from the bacteria Bacillus anthracis. These spores enter the body, where they germinate into bacteria and secrete a tripartite toxin that causes local edema and, in systemic infections, death. Recent studies identified the cellular receptor for anthrax toxin (ATR), a type I membrane protein. ATR is one of the splice variants of the tumor endothelial marker 8 (TEM8) gene. ATR and TEM8 are identical throughout their extracellular and transmembrane sequence, and both proteins function as receptors for the toxin. ATR/TEM8 function and expression have been associated with development of the vascular system and with tumor angiogenesis. TEM8 is selectively upregulated in endothelial cells during blood vessel formation and tumorigenesis. However, selective expression of TEM8 in endothelial cells contradicts the presumably ubiquitous expression of the receptor. To resolve this controversial issue, we evaluated the distribution of ATR/TEM8 in a variety of tissues. For this purpose, we generated and characterized a novel anti-ATR/TEM8 polyclonal antibody. Here, we show that this novel antibody recognizes all three ATR/TEM8 isoforms, which are widely and differentially expressed in various tissue types. We found that ATR/TEM8 expression is not only associated with tumor endothelial cells, as previously described. Indeed, ATR/TEM8 is highly and selectively expressed in the epithelial cells lining those organs that constitute the anthrax toxin's sites of entry, i.e., the lung, the skin, and the intestine. In fact, we show that ATR/TEM8 is highly expressed in the respiratory epithelium of the bronchi of the lung and is particularly abundant in the ciliated epithelial cells coating the bronchi. Furthermore, immunostaining of skin biopsies revealed that ATR/TEM8 is highly expressed in the keratinocytes of the epidermis. Finally, we show that the epithelial cells lining the small intestine strongly express ATR/TEM8 isoforms. This is the first demonstration that the ATR/TEM8 protein is highly expressed in epithelial cells, which represent the primary location for bacterial invasion. These results suggest that the ATR/TEM8 expression pattern that we describe here is highly relevant for understanding the pathogenesis of anthrax infection. anthrax; epithelia; lung; skin; intestine; toxin entry; receptor; bacterial pathogenesis     

Porphyrin substituted phosphoramidites: new building blocks for porphyrin-oligonucleotide syntheses

Thymidine phosphoramidites containing trispyridylphenyl and tetraphenylporphyrin chromophores attached via a short amide linker in the 3'-position have been synthesized and used as building blocks in solid-phase synthesis of self-complementary 8-mer oligonucleotides 3'-T-5'-GCGCGCA-3' and 5'-ACGCGCGT-3'. To our knowledge, these are the first porphyrin-oligonucleotide conjugates carrying the porphyrin chromophores in the 3'-position. Chain assembly was achieved by automated solid-phase synthesis and by inexpensive straightforward 'in flask' modification of commercially available solid supported oligonucleotides. This approach allows the synthesis of modified oligonucleotides without using costly instrumentation for automated DNA synthesis. Porphyrin-containing self-complementary oligonucleotides are expected to be a valuable model for drug binding studies and determination of conformational changes in DNA sequences using circular dichroism.     

Postnatal development of rat pups after maternal exposure to diethanolamine

BACKGROUND: Diethanolamine (DEA), a widely used surfactant, was administered to pregnant mice at the oral LD10 resulting in failure of pups to grow and thrive through postnatal day (PND) 3 [National Toxicology Program, 1987; York et al., Teratology 37:503-504, 1988]. The toxicity profile for DEA differs among rodent species. This study investigated DEA-induced postnatal toxicity in a second species. METHODS: Timed-mated Sprague-Dawley rats were dosed (0, 50, 125, 200, 250, or 300 mg DEA/kg/day, p.o.) on gestational days (GD) 6-19. Dams and pups were monitored for body weight, feed/water intake, clinical signs, litter size, and sex ratio. At necropsy (PND 21), maternal liver and kidney weights and number of uterine implantation sites were recorded. RESULTS: The high-dose group was terminated early due to excessive toxicity. The estimated maternal LD10 was 218 mg/kg/day. Maternal effects included decreased body weight and relative feed intake (>or=200 mg/kg/day), transiently reduced relative water intake (125 and 250 mg/kg/day), and increased absolute kidney weight (>or=125 mg/kg/day). Postimplantation loss (PND 0) and pup mortality (PND 0-4) were increased (>or=200 and >or=125 mg/kg/day, respectively). Pup body weight was reduced (>or=200 mg/kg/day) as late as PND 21. CONCLUSIONS: This study demonstrates reduced postnatal growth and survival in a second species after gestational exposure to DEA, persistence of toxic effects through the end of lactation, possibly due to long elimination half-life, and maternal and developmental toxicity no-observed-adverse-effect level (NOAELs) (50 mg/kg/day) and lowest-observed-adverse-effect level (LOAELs) (125 mg/kg/day) for oral DEA exposure during embryo/fetal development in the rat.     

Lack of effect of maternal body mass index on anthropometric characteristics of newborns in twin gestations

We examined the correlation between maternal prepregnancy body mass index (BMI) and newborn weight, length, BMI, and gestational order, in singleton and twin births. The sample comprised 381 mothers of multiple babies (562 twins), and 7979 singleton pregnancies, used as controls. The Mann-Whitney non-parametric test was used to compare the values between the two groups, and the Spearman's correlation test (rS) was applied to the quantitative variables. A significant positive correlation was found with singleton baby variables: the higher the maternal BMI, the higher the newborn's BMI, weight, length, and gestational order. However, no significant correlation was found between maternal BMI and any of these variables in twins. Maternal weight gain, in the twin group, showed a significant positive correlation with the newborn gestational order (rS = 0.154; P = 0.002), weight (rS = 0.493; P < 0.001), length (rS = 0.469; P < 0.001), and BMI (rS = 0.418; P < 0.001). In singletons, the correlation was positive with all the variables, except for the gestational order. The newborn BMI was significantly higher in twins born by C-section than those born by vaginal birth (Z = -4.974; P < 0.001). Mothers of singletons delivered by C-section had a significantly higher BMI than those of singletons born by vaginal birth (Z = -1.642; P < 0.001); however, no significant differences were observed in mothers of twins. Prepregnancy maternal BMI in twin births would not be predictive of newborns weight, length and BMI in this population. Maternal weight gain during pregnancy proved to be the most adequate for predicting the weight, length and BMI of twins delivered by C-section.     

Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei

The predominant secretory cargo of bloodstream form Trypanosoma brucei is variant surface glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre‐cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post‐mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of endoplasmic reticulum exit sites (ERES) in post‐mitotic cells dropped from 3.9 ± 0.6 to 2.7 ± 0.1 eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and glycosylphosphatidylinositol‐anchor biosynthesis remained relatively unaffected, except for the level of sphingomyelin which increased. However, both endoplasmic reticulum and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans‐face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, that is, VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei, including the ERES and Golgi.