Distribution of laminin and types IV and III collagen in fetal,infant and adult human spleens

Summary The immunohistochemical distribution of the basement membrane (BM) proteins, laminin and type IV collagen, and interstitial type III collagen was investigated in 12 fetal spleens at the 15th–38th gestational weeks (g.w.) and in spleens of 8 infants from term to 4 years. The results were compared with the distribution of the same proteins in adult human spleen. BM proteins were found to be abundantly present in the red pulp of all spleens during the whole of development. The content of type III collagen gradually decreased with advancing age and, in adult spleen, there were only occasional positively staining fibers in Billroth's cords. This finding indicates that the composition of reticular fibers in the red pulp of spleen is different from the reticular fibers elsewhere in lymphoreticular tissue. Early signs of ring fiber formation in the walls of venous sinuses were detectable at the 15th–19th g.w., although their more complete development occurred relatively late from the 36th g.w. onwards. Ring fibers contained both laminin and type IV collagen in all the investigated spleens. They never stained for type III collagen. The developing white pulp was positive for BM proteins, but showed no staining for type III collagen at the 15th g.w. At later ages, the white pulp stained similarly for both BM proteins and type III collagen.     

Production of colonization factor antigen II of enterotoxigenicEscherichia coli is subject to catabolite repression

Experiments were performed to study the effect of glucose on the production of the fimbrial colonization factor CFA/II of enterotoxigenicEscherichia coli (ETEC). The production of the CFA/II antigen was examined by electron microscopy, quantitative ELISA, and hemagglutination. The results showed that addition of 1% glucose to the growth medium drastically decreased CFA/II production, whereas addition of glycerol or sodium acetate did not have any effect. Bacteria grown in the presence of 1% glucose were essentially devoid of CFA/II fimbriae when examined under the electron microscope. Addition of 1 mM cAMP reversed the repressive effect of glucose, suggesting that the glucose suppression on CFA/II synthesis is via the mechanism of catabolite repression.     

Fractionation, morphological and functional characterization of effector cells responsible for human natural killer activity against cell-line targets

Human natural killer cells cytotoxic against cell-line target cells (NK-CLT) were isolated and characterized by utilizing adsorption-elution of the effector cells from the K-562 target cells. The cell associated with the cytotoxicity was a large lymphocyte with pale and characteristically granular cytoplasm. Thus, its morphology was identical with that of the large granular lymphocyte (LGL) previously shown to be the principal cytotoxic NK cell against fetal fibroblasts (NK-FF). The association of LGL with natural killer activity was verified with contact analysis from mixtures of unfractionated effector cells and target cells, which revealed that the number of contact of LGL with K-562 was correlated to the level of the individually expressed intensity of natural cytotoxicity. The ANAE-staining distribution of LGL was intensively positive with granular or diffuse staining pattern. In direct surface marker analysis LGL were E-rosette forming but, in contrast to NK-FF, heterogenous in regard to the Fc receptors. During in vitro incubation after elution from the target cells, the cytotoxic activity of LGL increased several fold. Also, the presence of K-562 among unfractionated effector cells caused an augmentation of cytotoxicity. This phenomenon was not observed as a result of effector cell-fetal fibroblast coculturing. Evidence from fetal fibroblast adsorption-elution and aggregated IgG blocking experiments suggested that the LGL with strong expression of Fc receptors were initially cytotoxic “mature” NK-cells, whereas the LGL with a weak expression of Fc receptors were initially noncytotoxic, but contact with K-562 “augmented” or “recruited” them to nonselective cytotoxicity.     

Human natural cell-mediated cytotoxicity against fetal fibroblasts. III. Morphological and functional characterization of the effector cells

We have isolated and characterized human natural killer cells cytotoxic to human fetal fibroblasts utilizing adsorption-elution of the effector cells from target cell-coated beads. The cell associated with enriched cytotoxicity was slightly larger than small- to medium-sized lymphocytes, the cytoplasm was pale and characteristically granular. In direct surface marker analysis the cell was Fc-receptor-positive, formed E-rosettes, and displayed strong either diffuse or granular ANAE reactivity in the cytoplasm. The ANAE reactivity could not be inhibited with sodium fluoride and in mitogen and antigen stimulation experiments the cell had T-cell characteristicis. The cell type was termed large granular lymphocyte and we suggest that it is the main direct effector cell for natural killer activity against human fetal fibroblasts.     

Comparability of Mixed IC50 Data – A Statistical Analysis

The biochemical half maximal inhibitory concentration (IC₅₀) is the most commonly used metric for on-target activity in lead optimization. It is used to guide lead optimization, build large-scale chemogenomics analysis, off-target activity and toxicity models based on public data. However, the use of public biochemical IC₅₀ data is problematic, because they are assay specific and comparable only under certain conditions. For large scale analysis it is not feasible to check each data entry manually and it is very tempting to mix all available IC₅₀ values from public database even if assay information is not reported. As previously reported for K_i database analysis, we first analyzed the types of errors, the redundancy and the variability that can be found in ChEMBL IC₅₀ database. For assessing the variability of IC₅₀ data independently measured in two different labs at least ten IC₅₀ data for identical protein-ligand systems against the same target were searched in ChEMBL. As a not sufficient number of cases of this type are available, the variability of IC₅₀ data was assessed by comparing all pairs of independent IC₅₀ measurements on identical protein-ligand systems. The standard deviation of IC₅₀ data is only 25% larger than the standard deviation of K_i data, suggesting that mixing IC₅₀ data from different assays, even not knowing assay conditions details, only adds a moderate amount of noise to the overall data. The standard deviation of public ChEMBL IC₅₀ data, as expected, resulted greater than the standard deviation of in-house intra-laboratory/inter-day IC₅₀ data. Augmenting mixed public IC₅₀ data by public K_i data does not deteriorate the quality of the mixed IC₅₀ data, if the K_i is corrected by an offset. For a broad dataset such as ChEMBL database a K_i- IC₅₀ conversion factor of 2 was found to be the most reasonable.     

A polyamine analogue prevents acute pancreatitis and restores early liver regeneration in transgenic rats with activated polyamine catabolism

We recently generated a transgenic rat model for acute pancreatitis, which was apparently caused by a massive depletion of pancreatic polyamines spermidine and spermine due to inducible activation of their catabolism (Alhonen, L., Parkkinen, J. J., Kein?nen, T., Sinervirta, R., Herzig, K. H., and J?nne, J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8290-8295). When subjected to partial hepatectomy, these animals showed striking activation of polyamine catabolism at 24 h postoperatively with a profound decrease in hepatic spermidine and spermine pools and failure to initiate liver regeneration. Here we show that pancreatitis in this model could be totally prevented, as judged by histopathology and plasma alpha-amylase activity, by administration of 1-methylspermidine, a metabolically stable analogue of spermidine. Similarly, the analogue, given prior to partial hepatectomy, restored early liver regeneration in the transgenic rats, as indicated by a dramatic increase in the number of proliferating cell nuclear antigen-positive hepatocytes from about 1% to more than 40% in response to the drug. The present results suggest that the extremely high concentration of spermidine in the pancreas, in fact the highest in the mammalian body, may have a critical role in maintaining organ integrity. The failure to initiate liver regeneration in the absence of sufficient hepatic polyamine pools similarly indicates that polyamines are required for proper commencement of the regenerative process.     

The alpha 2-adrenergic receptor gene and body fat content and distribution: the HERITAGE Family Study

BACKGROUND: Among adrenergic receptor subtypes that regulate lipid mobilization, the alpha2-adrenergic receptor is involved in the inhibition of fatty acid mobilization from adipose tissue. A C-1291G polymorphism is located in the alpha2-adrenergic receptor gene (ADRA2A) but no association with body fat accumulation has been reported yet. MATERIALS AND METHODS: Body mass index (BMI), fat mass (FAT), percentage body fat (%FAT), trunk-to-extremity skinfold ratio (TER), sum of eight skinfolds (SF8), and abdominal subcutaneous (ASF), visceral (AVF), and total (ATF) fat areas assessed by CT scan have been measured in adult sedentary white (n = 503) and black (n = 276) subjects participating in the HERITAGE Family Study. Association between the C-1291G polymorphism and each phenotype was tested separately in men and women of each race using ANCOVA with the effects of age as covariate in addition to the effects of BMI for TER and of FAT for AVF, ASF, and ATF. RESULTS: The allele frequencies of the ADRA2A C-1291G polymorphism differed between races. No association was observed in white subjects, except for a moderate effect of the polymorphism accounting for less than 1% of the variance in AVF and ATF in women. In black subjects, however, the G-1291 allele was found to be associated with an increase of TER in men (3.8% of variance accounted for by the polymorphism), while in black women it was associated with a decrease in TER (2.9%) and in AVF (2.5%). CONCLUSION: These results suggest a role for the ADRA2A gene in determining the propensity to store fat in the abdominal area, independently of total body fatness.     

Use of spot measurements for assessing residential ELF magnetic field exposure: a validity study

The purpose of this study was to evaluate residential short term "spot" measurements as surrogates for long term personal magnetic field (MF) exposure. In an epidemiological study on birth weight and pregnancy delay, MF exposure was assessed by taking five spot measurements in each room. For a subsample of 30 subjects 24 h personal MF measurements were made, and the following exposure metrics were calculated: 24 h arithmetic mean, 24 h median, percentage of time above 0.15 microT, and percentage of time above 0.29 microT. The 24 h exposure metrics were used as gold standards, when evaluating the validity of various summary measures calculated from spot measurements for assessing personal exposure. Based on Spearman correlation coefficient (r), specificity and sensitivity, the average of the spot measurements of a residence resulted in least exposure measurement error (misclassification). Also the above bed spot value correlated better with the 24 h metrics than any room average. Spot measurements performed about equally well in predicting different types of exposure metrics.     

Comparison of the radiotoxicity of two alpha-particle-emitting immunoconjugates,terbium-149 and bismuth-213, directed against a tumor-specific,exon 9 deleted (d9) E-cadherin adhesion protein

We investigated the effects of the alpha-particle emitters (149)Tb and (213)Bi coupled to a tumor-specific antibody targeting the mutated delta 9 E-cadherin (d9 E-Cad) on single cells and cell pellets. The d9 mutation of the adhesion molecule E-cadherin is found in 10% of diffuse-type gastric cancers and is not expressed in normal tissue. Human breast cancer cells (MDA-MB-435S) transfected with d9 E-Cad or the wild-type E-cadherin gene were used to study the effects of anti-d9 E-Cad MAb coupled to (149)Tb and (213)Bi ((149)Tb-d9 MAb and (213)Bi-d9 MAb). The density of binding sites determined on transfected MDA tumor cells by Scatchard analysis and flow cytometry varied from 4 x 10(4) to 6 x 10(4) antigens per cell. Internalization of radioimmunoconjugates by cells expressing d9 E-Cad was less than 10% of bound antibody within 240 min. The effect of the radioimmunoconjugates on cell suspensions and cell pellets was quantified by [(3)H]thymidine incorporation, and the dose to the cell nuclei was determined using microdosimetric calculations. (149)Tb and (213)Bi immunoconjugates affected cells in suspension similarly. Significant differences in the proliferation capacity of d9 E-cadherin- and wild-type E-cadherin-expressing cells were observed at activity concentrations around 185 kBq/ml, corresponding to antibody concentrations between 200 ng/ml and 1000 ng/ml. Proliferation after incubation with (213)Bi-d9 MAb was 50% greater in pelleted wild-type E-Cad-expressing cells compared to wild-type E-Cad cells in suspension. In contrast, the proliferation of pelleted d9 E-Cad cells was similar to that of d9 E-Cad cells in suspension. For (149)Tb-d9 MAb, no significant difference was found between pelleted cells and cells in suspension for low activity concentrations. However, at high activity concentrations, (149)Tb-d9 MAb had only a small effect on pelleted cells. These in vitro studies demonstrate different effects of (149)Tb and (213)Bi conjugated to a tumor-specific antibody toward single cells and tumor cell pellets. Microdosimetric simulation of single cell survival after alpha-particle irradiation modeled the experimental results with reasonable accuracy.