CYTODYNAMICS IN THE THYMUS OF YOUNG ADULT MICE:

Cell proliferation and cell loss in the thymic blast cell population were studied in young adult mice by (1) stathmokinetic methods combined with an analysis of the PLMe-curve after a pulse ³H-TdR, and (2) nigrosin-dye exclusion combined with ³H-TdR-autoradiography. It was calculated that about 17% of the blast cells do not progress into mitosis within the period of an average cell cycle. The dye exclusion studies indicated a rate of blast cell death of about 2–5 %/hr. The two methods of assessing blast cell loss (death) support each other very well. In spite of these findings scintillation countings on thymuses removed from 1 to 17 hr after ³H-TdR injection showed fairly constant levels of thymic radioactivity. This suggests a very extensive reutilization of ³H-labelled break-down products from dying blast cells. The very sparse labelling of pyknotic thymocytes strongly suggests that thymic blast cells do not become pyknotic. The rate of small thymocyte production and disappearance was studied by pulse and repeated ³H-TdR labelling techniques combined with dye exclusion studies and pyknotic counts. The data from the repeated labelling experiment were analysed by use of a model based on the assumption of first order kinetics of small viable, dead, and pyknotic thymocytes. The rate of cell production was estimated to 1–6 %/hr whereas the rates of cell loss due to disintegration, i.e. supravital stainability and nuclear pyknosis, were calculated to 0–02 %/hr and 0–0006 %/hr respectively. Cell loss due to disintegration was less than 2 % of the total loss of small thymocytes. It was concluded that pyknotic counts are a useless method of assessing the cell death in the population of thymic blast cells and small thymocytes. On the basis of a model for thymocyte proliferation, production and loss it is suggested that about 45 % of the small viable thymocytes re-enter the generative cell pool, whereas about 55% disappear by emigration.     

Relative position of trypsin banded homologous chromosomes in human (female) metaphase figures.

"Generalized distances" between centromeres were statistically analyzed (chi2 test) on 50 normal female trypsin-banded metaphase figures. This study revealed that the homologous chromosomes of the pairs 13, 17, 14, and 21 lie closer together than would be expected by a reference distribution, and this in a statistically significant way. The same relative position was demonstrated for the chromosome groups 13-14, 13-21, 14-21, 15-22, and 14-22. Evidences were collected that also showed that homologous chromosomes of the pairs 1, 19, and 20 and the chromosome groups 15-21, 13-15, and 18-20 tend to lie closer together. Giving a functional interpretation to the phenomenon of non-random distribution of chromosomes in metaphase figures, it may be suggested that the chromosomes 13, 14, and 21 are involved in the organization of the human nucleolar organizers, more frequently than the other D- and G-group chromosomes.     

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.     

No evidence of mineralization abnormalities in iliac bone of premenopausal women with type 2 diabetes mellitus

Objectives:Patients with type-2 diabetes mellitus (T2DM) have increased risk for bone fractures which points towards impaired bone quality.Methods:We measured bone mineralization density distribution (BMDD) and osteocyte lacunae section (OLS) characteristics based on quantitative backscattered electron images of transiliac biopsy samples from n=26 premenopausal women with T2DM. Outcomes were compared to those from reference cohorts as well as between T2DM subgroups defined by clinical characteristics.Results:Comparison to references did not reveal any differences in BMDD (all p>0.05) but a lowered OLS-density in cancellous bone in T2DM (-14.9%, p<0.001). Neither BMDD nor OLS-characteristics differed in T2DM subgroups defined by HbA1c (<7% versus >7%). The average degree of bone mineralization (CaMean) was higher (0.44 wt%Ca in T2DM, 0.30 wt%Ca in reference) and consistently the calcium concentration between the tetracycline double labels (CaYoung) was higher (0.76 wt%Ca, all p<0.001) in cancellous versus cortical bone.Conclusions:Our findings suggest that bone matrix mineralization was neither affected by the presence nor by the glycemic control of T2DM in our study cohort. The intra-individual differences between cancellous and cortical bone mineralization gave evidence for differences in the time course of the early mineralization process in these compartments in general.     

The role of adhesion molecules and chemokine receptor CXCR4 (CD184) in small cell lung cancer

Small-cell lung cancer (SCLC) is a particularly aggressive form of lung cancer. Responsible for this highly malignant phenotype is an early and widespread metastasis with a high propensity of SCLC cells for bone marrow involvement and the ability to develop resistance against chemotherapeutic agents. Tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and adhesion molecules. There is growing evidence that the chemokine stromal derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 (CD184) regulate migration and metastasis of a variety of cancers including SCLC. SCLC cells express high levels of functional CXCR4 receptors. Engagement of CXCR4 by CXCL12 leads to an upregulation of integrin-mediated adhesion in SCLC and other tumor cells. Activation of CXCR4 chemokine receptors and integrins on SCLC cells promotes adhesion to accessory cells (such as stromal cells) and extracellular matrix molecules within the tumor microenvironment. These adhesive interactions result in an increased resistance of SCLC cells to chemotherapy. As such, inhibitors of the CXCR4/CXCL12 axis and/or integrin activation may increase the chemosensitivity of SCLC cells and lead to new therapeutic avenues for patients with SCLC.