Peptide-amidating enzymes are expressed in the stellate epithelial cells of the thymic medulla

C-terminal amidation is a post-translational processing step necessary to convey biological activity to a large number of regulatory peptides. In this study we have demonstrated that the peptidyl-glycine alpha-amidating monooxygenase enzyme complex (PAM) responsible for this activity is located in the medullary stellate epithelial cells of the thymus and in cultured epithelial cells bearing a medullary phenotype, using Northern blot, immunocytochemistry, in situ hybridization, and enzyme assays. Immunocytochemical localization revealed a granular pattern in the cytoplasm of the stellate cells, which were also positive for cytokeratins and a B-lymphocyte-associated antigen. The presence of PAM activity in medium conditioned by thymic epithelial cell lines suggests that PAM is a secreted product of these cells. Among the four epithelial cell lines examined, there was a direct correlation between PAM activity and content of oxytocin, an amidated peptide. Taken together, these data provide convincing evidence that thymic epithelial cells have the capacity to generate amidated peptides that may influence T-cell differentiation and suggest that the amidating enzymes could play an important role in the regulation of thymic physiology.     

Thymic epithelial cells as a diagnostic pitfall in the fine-needle aspiration diagnosis of primary mediastinal lymphoma

This paper reports data on the effect of a new antioxidant, U-83836E, on the lipid peroxidation and antioxidant status of liver, red blood cells (RBCs) and blood serum of rats intoxicated with methanol (3.0 g/kg body weight). Methanol administration slightly increased the levels of peroxidation products in the liver, and markedly increased them in RBCs and serum. In contrast, glutathione-peroxidase, glutathione-reductase activity, reduced glutathione concentration and total antioxidant status were decreased. The use of U-83836E, containing a trolox ring, appeared to be beneficial in reducing lipid peroxidation products and in partially in preventing the decrease in glutathione and antioxidant enzymes induced by methanol in liver and serum. These results show that antioxidant U-83836E may partially prevent methanol toxicity.     

Antigen acquisition by dendritic cells: intestinal dendritic cells acquire antigen administered orally and can prime naive T cells in vivo

In the rat, mesenteric lymphadenectomy allows collection of dendritic cells (DC) derived from the small intestine after cannulation of the thoracic duct. We prepared rats this way and administered antigens by oral feeding or intraintestinal injection. DC enriched from the thoracic duct lymph collected over the first 24 h from these animals are able to stimulate sensitized T cells in vitro and to prime popliteal lymph node CD4+ T cells after footpad injection, while B and T cells from the same thoracic duct lymph are inert in priming. 500 or less DC pulsed in vitro with antigen can prime T cells in vivo, whereas 100 times more B cells or macrophages pulsed in vitro are quite inert. 1 mg of ovalbumin administered orally is sufficient to load DC for in vivo priming of T cells. Antigen could not be detected directly in DC but was present in macrophages in the lamina propria. Direct presentation of antigen by DC to T cells was demonstrated by injecting F1 recipients with parental DC and showing restriction of T cell sensitization to the major histocompatibility complex of the injected DC. Antigen-bearing DC do not induce a detectable primary antibody response but a small secondary antibody response can be detected after a boosting injection. These results show that acquisition of antigens by DC in the intestine is very similar to what occurs in vitro or in other tissues, suggesting that there may be no special difference in antigen handling at mucosal surfaces. One implication of these results is that hypotheses designed to explain oral tolerance must take into account the presence of immunostimulatory, antigen-bearing DC in animals that have received oral antigens.     

Expression of integrin receptors on plasma membranes of primary corneal epithelial cells is matrix specific

The performance evaluation of a semi-supervised fuzzy c-means (SFCM) clustering method for monitoring brain tumor volume changes during the course of routine clinical radiation-therapeutic and chemo-therapeutic regimens is presented. The tumor volume determined using the SFCM method was compared with the volume estimates obtained using three other methods: (a) a k nearest neighbor (kNN) classifier, b) a grey level thresholding and seed growing (ISG-SG) method and c) a manual pixel labeling (GT) method for ground truth estimation. The SFCM and kNN methods are applied to the multispectral, contrast enhanced T1, proton density, and T2 weighted, magnetic resonance images (MRI) whereas the ISG-SG and GT methods are applied only to the contrast enhanced T1 weighted image. Estimations of tumor volume were made on eight patient cases with follow-up MRI scans performed over a 32 week interval during treatment. The tumor cases studied include one meningioma, two brain metastases and five gliomas. Comparisons with manually labeled ground truth estimations showed that there is a limited agreement between the segmentation methods for absolute tumor volume measurements when using images of patients after treatment. The average intraobserver reproducibility for the SFCM, kNN and ISG-SG methods was found to be 5.8%, 6.6% and 8.9%, respectively. The average of the interobserver reproducibility of these methods was found to be 5.5%, 6.5% and 11.4%, respectively. For the measurement of relative change of tumor volume as required for the response assessment, the multi-spectral methods kNN and SFCM are therefore preferred over the seedgrowing method.     

The proliferation of mouse mammary epithelial cells in response to specific mitogens is modulated by the mammary fat pad in vitro

A volume-preserving three-dimensional smoothing approach is described that can be directly applied to 3D medical image data consisting of sets of 2D image slices, e.g., segmented intravascular ultrasound image sequences. Two local smoothing filters ℱ and 𝒢 were designed according to different smoothing goals and their performance was compared. Filtering with the ℱ filter of a relatively large frequency window keeps the important local characteristics of the object and results in little shrinkage while removing noise. Filtering with the Gaussian filter G that has an added volume compensation step results in no global shrinkage and may be used for multiscale filtering. The two filters can be easily extended to n-dimensional filtering.     

A comparison of immunohistochemical staining of human cultured mesothelial cells and ovarian tumour cells using epithelial and mesothelial cell markers

Atlantic salmon (Salmo salar) post-smolts were fed diets containing either Fosol (FO), a North Sea fish oil, sunflower oil (SO), linseed oil (LO) or Marinol K (MO), a southern hemisphere fish oil rich in 20:5(n-3) for 12 weeks. A macrophage-enriched leucocyte preparation was obtained from head kidney and the fatty acid compositions of the individual membrane phospholipids measured. In general phospholipids from SO- and LO-fed fish had increased 18:2(n-6), 20:2(n-6) and 20:3(n-6) compared to the fish oil treatments while LO-fed fish had lower 20:4(n-6) than any other dietary treatment. Fish fed LO also had increased 18:3(n-3), 18:4(n-3), 20:3(n-3) and 20:4(n-3). The 20:5(n-3) content of kidney macrophage-enriched leucocyte phospholipids was highest in MO-fed fish followed by FO- and LO-fed fish with the lowest level in fish fed SO. The overall effect on the ratio of eicosanoid precursors, 20:4/20:5, showed the highest value in SO-fed fish and the lowest in fish fed LO. Production of LTB5 by kidney macrophage-enriched leucocytes stimulated with A23187 was highest in MO-fed fish and lowest in those fed SO. Production of LTB4 was greatest in SO-fed fish and lowest in fish fed LO. Serum Ig levels were significantly affected by dietary treatment with highest values in fish fed FO and SO and lowest in fish fed MO and LO.     

The endocytosis of transferrin by rat intestinal epithelial cells

BACKGROUND/AIMS: The transferrin receptor is a prominent protein on the basal and lateral membranes of intestinal epithelial cells, yet little is known of the function of the receptor in the intestine. The aim of the present study was to determine whether intestinal transferrin receptors were capable of facilitating transferrin internalization. METHODS: Using the rat as an experimental model, the uptake of radiolabeled transferrin by cells isolated from different regions along the crypt-villus axis of the proximal small intestine was studied. RESULTS: An intestinal epithelial cell fraction highly enriched in crypt cells bound most radiolabeled transferrin. Cells in this fraction were able to internalize transferrin and recycle it back to the cell surface. A high affinity, saturable pathway of transferrin uptake by these cells predominated at transferrin concentrations below 0.3 mumol/L, whereas at higher concentrations, most uptake was via a nonsaturable process. Intravenously injected radiolabeled transferrin could be detected within intestinal crypt cells, indicating that these cells are able to internalize transferrin in vivo. CONCLUSIONS: These data suggest that intestinal crypt cells have an active transferrin/transferrin receptor system. Transferrin may play an important role in iron delivery to and/or as a growth factor for the rapidly proliferating intestinal epithelium.     

In vitro development and preservation of specific features of collecting duct epithelial cells from embryonic rabbit kidney are regulated by the electrolyte environment

During kidney development the embryonic collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into functional principal (P) and intercalated (IC) cells. Aldosterone is able to modulate this differentiation. Consequently we investigated whether increased concentrations of extracellular NaCl or Na gluconate may also have an influence on the development of individual CD cell features. Embryonic CD epithelia were isolated from neonatal rabbit kidneys, placed on tissue carriers and cultured in gradient containers, which were constantly perfused with medium for 13 days. Isotonic culture conditions could be mimicked, when on both the luminal and basal side standard Iscove's Modified Dulbecco's Medium (IMDM) was used. In another set of experiments, gradient culture was performed. Standard IMDM was applied on the basal side and IMDM supplemented with 12 mM NaCl and 17 mM Na gluconate on the luminal side. This adaptation of IMDM led to the same Na concentrations as found in the serum of neonatal rabbits. The development of CD cell features was monitored by cellular markers such as the monoclonal antibodies (Mabs) 703 and 503 recognizing P and IC cell features respectively. Epithelia cultured under isotonic conditions showed less than 5% Mab 703- and 503-immunopositive cells. In contrast, epithelia cultured in a luminal-basal medium gradient revealed more than 80% positive cells. Immunoreactivity started to develop after a long lag period of 4 days, then increased continuously during the following 5 days and reached a maximum at day 14. When the medium gradient was then changed to an isotonic environment for another 5 days immunoreactivity for Mab 703 remained stable, while the number of Mab 503-positive cells was found to be decreased to 10%. Thus, the extra-cellular electrolyte environment not only induces but also preserves individual cell features.     

Uroplakins, specific membrane proteins of urothelial umbrella cells, as histological markers of metastatic transitional cell carcinomas

Uroplakins (UPs) Ia, Ib, II, and III, transmembrane proteins constituting the asymmetrical unit membrane of urothelial umbrella cells, are the first specific urothelial differentiation markers described. We investigated the presence and localization patterns of UPs in various human carcinomas by applying immunohistochemistry (avidin-biotin-peroxidase complex method), using rabbit antibodies against UPs II and III, to paraffin sections. Positive reactions for UP III (sometimes very focal) were noted in 14 of the 16 papillary noninvasive transitional cell carcinomas (TCCs) (88%), 29 of the 55 invasive TCCs (53%), and 23 of the 35 TCC metastases (66%). Different localization patterns of UPs could be distinguished, including superficial membrane staining like that found in normal umbrella cells (in papillary carcinoma), luminal (microluminal) membrane staining (in papillary and invasive carcinoma), and, against expectations, peripheral membrane staining (in invasive carcinoma). Non-TCC carcinomas of various origins (n = 177) were consistently negative for UPs. The presence of UPs in metastatic TCCs represents a prime example of even advanced tumor progression being compatible with the (focal) expression of highly specialized differentiation repertoires. Although of only medium-grade sensitivity, UPs do seem to be highly specific urothelial lineage markers, thus operating up interesting histodiagnostic possibilities in cases of carcinoma metastases of uncertain origin.     

Inhibition of posterior capsule opacification with an immunotoxin specific for lens epithelial cells: 24 month clinical results

PURPOSE: To assess the safety and effectiveness of an immunotoxin, MDX-RA, designed to inhibit posterior capsule opacification (PCO). SETTING: Eleven private practices in the United States. METHODS: This study comprised 63 eyes of 63 patients having extracapsular cataract extraction by phacoemulsification; these patients were enrolled in a Phase I/II clinical investigation of the immunotoxin MDX-RA. At the close of surgery, 21 patients were treated with placebo, 23 patients with 50 units of the immunotoxin, and 19 patients with 175 units of the immunotoxin as an aqueous solution. The patients were monitored for 24 months after primary cataract surgery using external eye and slitlamp examinations, visual acuity assessment, ophthalmoscopy, pachymetry, tonometry, endothelial cell counts, and lens capsule photography. Posterior capsule opacification, recorded on lens capsule photographs, was graded independently by a committee of 3 cataract surgeons. The incidence of neodymium:YAG (Nd:YAG) capsulotomy was projected from the opacification results. RESULTS: The immunotoxin, at the 50 unit dose, was well tolerated and effective in inhibiting PCO. At the 175 unit dose, there was a trend toward increased postoperative inflammation that was transient with no residua. From 6 to 24 months postoperatively, the 50 unit dose significantly inhibited PCO compared with the placebo (P < .05). This significant reduction in PCO translated into a significantly lower projected need for Nd:YAG capsulotomy in the 50 unit than the placebo group (P < .004). About 60% in the placebo group and 4% in the 50 unit group were projected to need an Nd:YAG capsulotomy by 3 years postoperatively. CONCLUSION: The immunotoxin was well tolerated and was effective in reducing PCO for up to 24 months after cataract surgery. Although these preliminary results are encouraging, a larger study is underway to determine whether the reduction in PCO by the immunotoxin decreases the need for Nd:YAG capsulotomy.     

Inhibition of invasion of epithelial cells by Tiam1-Rac signaling

Tiam1 encodes an exchange factor for the Rho-like guanosine triphosphatase Rac. Both Tiam1 and activated RacV12 promote invasiveness of T lymphoma cells. In epithelial Madin-Darby canine kidney (MDCK) cells, Tiam1 localized to adherens junctions. Ectopic expression of Tiam1 or RacV12 inhibited hepatocyte growth factor-induced scattering by increasing E-cadherin-mediated cell-cell adhesion accompanied by actin polymerization at cell-cell contacts. In Ras-transformed MDCK cells, expression of Tiam1 or RacV12 restored E-cadherin-mediated adhesion, resulting in phenotypic reversion and loss of invasiveness. These data suggest an invasion-suppressor role for Tiam1 and Rac in epithelial cells.     

Bacterial phospholipase C upregulates matrix metalloproteinase expression by cultured epithelial cells

Phospholipase C (PLC) is a putative virulence factor of several pathogenic bacteria. We studied if exogenous PLC would perturb epithelial behavior in infected tissues. Gelatin and casein zymography of cell culture medium indicated that the broad-spectrum PLC of Bacillus cereus induced matrix metalloproteinase (MMP) production in epithelial cells of human skin (NHEK), human gingiva (HGE), and porcine periodontal ligament (PLE). In all three cell types, the strongest increase (ninefold) at 0.1 U/ml was seen in the MMP-9 (92-kDa gelatinase) activity, and the effect was dose dependent in the range of 0.1 to 1.0 U/ml. A relatively weaker increase (twofold) in MMP-2 (72-kDa gelatinase) was also observed in each cell type. PLC induction of MMP-3 (48-kDa stromelysin) was also seen in NHEK and HGE on gelatin and more sensitively for PLE by casein zymography (fivefold). Total gelatinolytic activity as measured by degradation of 14C-labeled denatured type I collagen increased by about 18-fold (NHEK), 12-fold (HGE), and 14-fold (PLE). Northern analysis showed a clear increase in the MMP-9, and a minor increase in MMP-3 mRNA levels but no significant increase in MMP-2 mRNA levels. Further studies with PLE revealed that MMP-9 induction by PLC progressively increased with the length of cell culture time in the absence of serum. PLC induction of MMPs was polar, with MMP-9 and MMP-3 secreted primarily in the apical direction and MMP-2 secreted mainly in the basal direction. The PLC effect was blocked by neomycin, an inhibitor of the phosphoinositol signal pathway. No significant effects were observed in MMP expression with the calcium ionophore A23187 or phospholipase A2. Morphologically, PLC treatment resulted in reduced contacts between the cultured cells and loss of the cell surface microvilli. These results suggest that PLC secreted by bacterial pathogens may disrupt epithelium of infected tissue and increase the subepithelial tissue destruction through induction of MMPs.     

Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells

Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1alpha. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease.     

5'-nucleotidase in rat photoreceptor cells and pigment epithelial cells processed by rapid-freezing enzyme cytochemistry

This report describes the subcellular distribution of 5'-nucleotidase (5'-NT) in rat photoreceptor cells and pigment epithelial cells processed by rapid-freeze enzyme cytochemistry. There was a striking difference in the ultrastructural localization of 5'-NT activity between rod outer segments after freeze-substitution fixation and conventional fixation. By rapid-freezing enzyme cytochemistry, 5'-NT activity was localized in the extradiscal space of intact nonvacuolated discs, whereas by conventional cytochemistry it was shown in the intradiscal space of artifactual vacuolated discs. In the freeze-substituted retinal cells, an appreciable difference in functional 5'-NT molecules was also found. The soluble 5'-NT on the cytoplasmic side of the disc membrane was vital in the rod outer segments, whereas the membrane-bound ecto-5'-NT on the exoplasmic (external) surface of the apical process was active in the pigment epithelial cells. Rapid-freezing enzyme cytochemistry should be worth employing as a method to reveal the fine localization of enzyme activity at the level of cell ultrastructures, which are poorly preserved by conventional fixation, and should provide information approximate to that in living cells.     

CD40 cross-linking inhibits specific antibody production by human B cells

Ligation of CD40 on B cells is a co-stimulatory signal for proliferation, antibody secretion, heavy chain switching and rescue from apoptosis after somatic mutation in the germinal centre. The importance of these manifold responses to CD40 activation for humoral immunity is exemplified by the inability of boys with X-linked hyper IgM syndrome to make IgG, IgE or IgA due to a mutation in in the gene coding for CD40 ligand (CD40L). In the present study, we have investigated the effect of CD40 ligation on specific antibody production by human B cells to influenza virus. The antibody response was T cell dependent and specific for the strain of influenza virus used as antigen. Addition of either CD40 mAb or recombinant trimeric CD40L profoundly inhibited specific antibody production. Antibody production by unseparated tonsillar mononuclear cells and by T-depleted B cells stimulated with antigen in the presence of T cell replacing factor were equally inhibited with CD40 antibody showing that the effect was due to ligation of CD40 on B cells rather than blocking of T cell help. The specific antibody detected in these experiments was mostly IgG with little or no IgM and was obtained from surface IgM B cells consistent with activation of a secondary (memory) response. Co-stimulation of tonsillar B cells with CD40 antibody and anti-IgG induced proliferation of IgG+ B cells. These results suggest that CD40 ligation can inhibit specific antibody responses and stimulate proliferation in the same IgG+ (memory) B cell subpopulation. Addition of CD40 antibody during the first 24-48 h of the response was required for inhibition, suggesting that the effect was on early B cell activation and/or proliferation required for antibody production. There was no correlation, however, between the ability of CD40 mAb to stimulate proliferation and inhibit antibody production. We suggest that early activation of CD40 in the specific antibody response inhibits the formation of plasma cells and promotes instead the generation of memory cells.     

Induction of specific T cell tolerance by Fas ligand-expressing antigen-presenting cells

Autocrine interaction of Fas and Fas ligand leads to apoptosis of activated T cells, a process that is critical for the maintenance of peripheral T cell tolerance. Paracrine interactions of Fas ligand with T cells also may play an important role in the maintenance of tolerance, as Fas ligand can create immune-privileged sites and prevent graft rejection by inducing apoptosis in T cells. We surmised that APCs that express Fas ligand might directly induce apoptosis of T cells during presentation of Ag to the T cells, thus inducing Ag-specific, systemic T cell tolerance. Here, we show that profound, specific T cell unresponsiveness to alloantigen was induced by treatment of H-2k mice with H-2b APCs that expressed Fas ligand and that profound T cell unresponsiveness specific for the H-Y Ag was induced by treatment of H-2Db/H-Y TCR transgenic female mice with H-2Db/H-Y APCs that expressed Fas ligand. The induction of this systemic T cell tolerance required the expression of Fas ligand on the APCs as well as the expression of Fas on the T cells. The tolerance was restricted to the Ag presented by the APCs. The rapid and profound clonal deletion of the Ag-specific, peripheral T cells mediated by the Fas ligand-expressing APCs contributed to the induction of tolerance. These findings demonstrate that Ag-specific T cell tolerance can be induced by APCs that express Fas ligand and suggest a novel function for APCs in the induction of T cell apoptosis. Furthermore, they indicate a novel immunointervention strategy for treatment of graft rejection and autoantigen-specific autoimmune diseases.     

Functional maturation of neuroendocrine gonadal axis is altered by specific phase relations of circadian neurotransmitter activity in Japanese quail

The present study was designed to ascertain the effects of temporal relationship of circadian neural oscillations on puberty attainment and reproductive growth of Japanese Quail, Coturnix coturnix japonica. Serotonin and dopamine precursors (5-hydroxytryptophan, 5-HTP and L-dihydroxyphenylalanine, L-DOPA; 5 mg/100 g body weight) were injected daily, 8 and 12 h apart in two groups of one-day old chicks, while controls received two daily injections of normal saline. Weekly/biweekly observations (body weight, cloacal gland size, testicular volume and activity, ovarian follicular diameter and rate of egg production) were made until 9 weeks of age, when the experiment was terminated. Results indicate that 8 h relationship completely suppressed gonadal growth even under long photoperiod (LD 16:8), while a 12 h relationship induced precocious sexual maturity and increased the rate of reproduction (spermatogenesis and egg production). It is concluded that circadian phase relationship of serotonergic and dopaminergic activity may not only determine the onset of reproduction in this poultry species, but may also alter the rate of reproduction possibly by affecting photoperiodic mechanism of reproductive regulation.