BMP10 as a potent inducer of trophoblast differentiation in human embryonic and induced pluripotent stem cells

Bone morphogenetic proteins (BMPs) are known to induce diverse differentiation fates in human embryonic stem cells (hESCs). In the present study, we compared the potency at which BMP5, BMP10 and BMP13, which are members of distinct BMP subgroups due to differences in sequential and structural homology, induce differentiation in hESCs and human induced pluripotent stem cells (hiPSCs). We observed, in agreement with previous BMP4 model studies, that all ligands induced differentiation to the trophoblast lineage in the absence of bFGF. However, distinct BMPs exerted differences in the kinetics of induced differentiation, with BMP10 being the most potent. hiPSCs and hESCs shared comparable expression patterns of BMP type-I and -II receptor subtypes, which might explain conserved properties with respect to ligand potency and activation of SMAD-dependent (via SMAD1/5/8) and -independent (via MAPK p38) signal transduction pathways. The tested BMPs had distinct and also conserved target genes such as CDX2, DLX3, DLX5, GATA2, GATA3, HAND1, ID2, MSX2 and TFAP2A, known to be associated with the emergence of trophoblast cells. hESCs induced expression of the BMP antagonist NOGGIN as a protection mechanism to constrict extensive BMP action. Unlike BMP4, BMP10 has been shown to be resistant to NOGGIN-induced inhibition which in part might explain its potency. BMPs, in particular BMP4, are commonly used cytokines in differentiation protocols to generate diverse mesoderm- and endoderm-derivates from human pluripotent stem cells. Our study has identified BMP10, a cardiac-specific protein, as a superior alternative to BMP4 for inducing trophoblast differentiation in human pluripotent stem cells.     

An analysis and decision tool to measure cost benefit of newborn screening for severe combined immunodeficiency (SCID) and related T-cell lymphopenia

Severe combined immunodeficiency (SCID) is a group of syndromes resulting from genetic defects causing absence in T-cell and B-cell function, leading to serious and life-threatening infections. SCID is often fatal in the first 2 years of life if not identified and properly treated. While additional laboratory methods are being developed, the current T-cell receptor excision circle assay has proven to have outstanding specificity and sensitivity to accurately identify infants with SCID and other T-cell lymphopenia. The Jeffrey Modell Foundation (JMF) has a long history of advocacy and continues to promote newborn screening for SCID to be implemented in the United States and worldwide. Based on reports provided by California, New York, Texas, and Wisconsin on the results of their population based newborn screening programs, the overall incidence of SCID averaged 1:33,000 and T-cell lymphopenia averaged 1:6,600. JMF has developed a working algorithm or “decision tree”, validated by peer-reviewed scientific literature, to be used by Public Health Departments and Health Ministries in states, countries, and regions throughout the world. This decision tool allows for local or regional data to be applied to measure the threshold and economic impact of implementing newborn screening for SCID and T-cell lymphopenia.     

The influence of tumor stage and metastasis on the biodistribution of gallium-67 citrate in the murine Lewis lung carcinoma model

The biodistribution of i.v. administered [67Ga]citrate was investigated in the Lewis lung carcinoma/male B6D2F1 mouse model. Tumors were implanted intramuscularly (10(5) cells or 10(6) cells in suspension) into the thigh, or subcutaneously (10(7) cells or 2 mm3 fragments) into the tail of recipient mice. Intramuscular tumors were allowed to grow for 16, 24, or 33 days; tail tumors developed for 2 wk (fragment implants) or 3 wk (10(7) cells in suspension) after which the primary tumor was amputated along with adjacent fragments of the tail tissue. Gamma camera scintigraphy and dissection/radiometric biodistribution studies indicated that: (a) tumors and metastases took up 5-6% of the injected dose/g except when large necrotic areas were present in the primary tumor; (b) blood levels of 67Ga increased in all tumor-bearing animals, with up to tenfold increases in the i.m. tumor model at later stages of the growth; (c) hepatic uptake increased as a function of tumor size/age, and (d) all tissue:blood ratios declined as the neoplastic tissues progressed. The results are discussed with respect to tumor progression and metastatic disease.     

[3H]HOE166 defines a novel calcium antagonist drug receptor — distinct from the 1,4 dihydropyridine binding domain

Summary Benzothiazinones represent a novel class of drugs which block voltage-dependent L-type calcium channels in different tissues. [³H]HOE166 (R-(±)-3,4-dihydro-2-isopro-opyl-4-methyl-2-[2-[4-[4-[2-(3, 4, 5-trimethoxyphenyl)ethyl]piperazinyl]butoxy]phenyl]-2H -1, 4-benzothiazin-3-on-dihydrochloride; 57 Ci/mmol) a potent optically pure benzothiazinone was employed to characterize receptors associated with skeletal muscle transverse tubule calcium channels. [³H]HOE166 reversibly labels the membrane-bound calcium channels with high affinity (K_d = 0.36 ± 0.05 nM; B_max = 18.2 ± 3.3 pmol/mg of membrane protein; means ± SD, n = 13), HOE166 (K_i = 0.76 nM) is 29-fold more potent than the respective (S)-enantiomer (K_i = 22.1 nM). Binding is inhibited by divalent and trivalent cations (Cd²⁺ and La³⁺ being most potent) and other calcium channel drugs (1,4 dihydropyridines, phenylalkylamines, benzothiazepines). High affinity [³H]HOE166 binding activity is maintained (Kd = 4.5–9.0 nM) after solubilization and purification (554–1350 pmoles/mg of protein) of the calcium channel complex from transverse-tubule membranes. The following data support our recent claim (Striessnig et al. 1985, 1988) that HOE166 labels a domain on L-type calcium channels which is distinct from that defined by 1,4 dihydropyridines, phenylalkylamines or benzothiazepines: (1) All 1,4 dihydropyridine-, phenylalkylamine-and benzothiazepine-receptor-selective drugs tested are only very weak inhibitors of [³H]HOE166 binding. (2) (+)-PN200-110 only partially inhibits [³H]HOE166 binding to the purified calcium. channel complex. (3) The decay of the [³H]HOE166-receptor complex is monoexponential but the dissociation rate constants depend on the ligand concentration; (+)-PN200-100 accelerates the dissociation in the presence of unlabelled HOE166. (4) Nanomolar concentrations of HOE166 and HOE167 completely inhibit (–)-[³H]desmethoxyverapamil binding to a Drosophila phenylalkylamine receptor (which lacks a 1,4 dihydropyridine binding domain). Taken together, these results are incompatible with the view that [³H]HOE166 binds competitively to the calcium channel linked 1,4 dihydropyridine drug receptors.Abbreviations kd dissociation constant - K_i inhibition constant - k_–1,k₊₁ dissociation, association rate constant - SDS sodium dodecyl sulfate - T-tubule transverse tubule - s_20,w sedimentation coefficient Send offprint requests to H. Glossmann at the above address     

Hypothalamic-pituitary-adrenal axis hyporesponsiveness to restraint stress in mice deficient for large-conductance calcium- and voltage-activated potassium (BK) channels

Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, releasing ACTH from the anterior pituitary gland and glucocorticoids from the adrenal cortex. Stress also activates the sympathetic nervous system, evoking adrenaline release from the adrenal medulla. Large-conductance calcium- and voltage-activated potassium (BK) channels have been implicated in regulation of cellular excitability in these systems. Here, we examine the functional role of BK channels in HPA axis regulation in vivo using female mice genetically deficient (BK(-/-)) for the pore-forming subunits of BK channels. BK(-/-) phenotype in the HPA was confirmed by immunohistochemistry, Western blot analysis, and corticotrope patch-clamp recording. Restraint stress-induced plasma concentrations of ACTH and corticosterone were significantly blunted in BK(-/-) mice compared with wild type (WT) controls. This stress hyporesponsiveness was associated with reduced activation of hypothalamic paraventricular nucleus (PVN) neurons. Basal expression of CRH, but not arginine vasopressin mRNA in the PVN was significantly lower in BK(-/-) mice compared with WT controls. Total anterior pituitary ACTH peptide content, but not proopiomelanocortin mRNA expression or corticotrope number, was significantly reduced in BK(-/-) mice compared with WT. However, anterior pituitary corticotropes from BK(-/-) mice fully supported ACTH output, releasing a significantly greater proportion of stored ACTH in response to secretagogue in vitro compared with WT. These results support an important role for BK channels in both the neural circuitry and endocrine output of the HPA axis and indicate that the stress hyporesponsiveness in BK(-/-) mice primarily results from reduced activation of hypothalamic PVN neurosecretory neurons.     

Loss of functional cell surface transforming growth factor β (TGF-β) type 1 receptor correlates with insensitivity to TGF-β in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries, and there is significant variability in survival within CLL clinical stages. Earlier studies showed that CLL cells produce and are usually growth inhibited by transforming growth factor β type 1 (TGF-β1), suggesting a mechanism for the clinically indolent course of most CLL. Here we studied the mechanism by which CLL cells from about one-third of the patients are insensitive to TGF-β1. Of the 13 patients studied, CLL cells isolated from the peripheral blood of 8 patients were sensitive to growth inhibition by TGF-β1, as determined by incorporation of tritiated thymidine, whereas those from 5 patients were completely resistant to TGF-β1. As judged by binding of radiolabeled TGF-β1 followed by cross-linking and immunoprecipitation with anti-receptor antisera, CLL cells sensitive to TGF-β1 exhibited normal cell surface expression of both types 1 and 2 TGF-β receptors. In contrast, all CLL cells resistant to TGF-β1 exhibited no detectable surface type I receptors able to bind TGF-β1, but normal expression of type II receptors. Both TGF-β1-sensitive and TGF-β1-resistant CLL cells contained normal amounts of both type 1 and type 2 receptor mRNAs. Specific loss of type 1 receptor expression represents a new mechanism by which cells acquire resistance to TGF-β1-mediated growth inhibition in the development and progression of human lymphoproliferative malignancies.     

Rickettsia felis and Bartonella spp. in fleas from cats in Albania

Fleas can serve as vectors for bacterial pathogens like Bartonella and Rickettsia species, which have been isolated worldwide. However, the knowledge of the epidemiology of vector-borne diseases in general and thus on flea-borne diseases in Albania is limited. Therefore, from 78 free-roaming cats in Tirana, Albania, fleas (371 Ctenocephalides felis and 5 Ctenocephalides canis) were collected to examine them for the presence of Rickettsia and Bartonella species. Ten of the 371 C. felis (2.7%) were positive for Rickettsia felis, and 24 (6.5%) for Bartonella spp. (B. henselae and B. clarridgeiae). In total, fleas from 15 cats (19.2%) were positive for either one or the other of the pathogens. The results of this study provided evidence for the presence of R. felis (causing flea-borne spotted fever) and Bartonella spp. (causing cat scratch disease) in Albania. Thus, these infectious diseases should be considered as differential diagnoses when febrile symptoms are presented, especially after contact with cats or their fleas.     

The AIDS Memorial Quilt as preventative education: a developmental analysis of the Quilt.

This study consisted of a survey given to college students (N = 560) at a rural university in the Pacific Northwest. The sample was randomly assigned into four groups, following the Solomon four-group study design. The two levels of treatment included interventions consisting of a visit to the AIDS Memorial Quilt for the experimental groups and attendance at an unrelated event for the control groups. Pretests were completed 4 weeks prior to interventions; posttests were completed by the entire sample 4 weeks after the interventions. Results confirmed expected differences among the four groups in terms of social distance, perceptions of people with AIDS, self-efficacy, and discussion of risky behavior. The results suggest that the AIDS Memorial Quilt addresses issues centrally related to behavior change and indicates support for the message interpretation process and stages of change models.