Distribution of cytoskeletal and contractile proteins in normal and tumour bearing salivary and lacrimal glands

Summary We have evaluated by means of immunocytochemistry the distribution of various cytoskeletal and contractile proteins (cytokeratins, vimentin, desmin and -smooth muscle actin) in 23 salivary or lacrimal gland primary tumours (15 pleomorphic adenomas and 8 carcinomas in pleomorphic adenoma), one third of which contained areas of normal gland. Normal epithelial luminal cells were stained by cytokeratin antibodies with a general specificity, while myoepithelial cells were selectively stained by a monoclonal antibody (SK2-27) reacting in immunoblots with cytokeratin polypeptides 14, 16 and 17, according to the classification of Moll et al. (1982) and by an antibody directed against -smooth muscle actin (Skalli et al. 1986). In pleomorphic adenomas, both epithelial and myoepithelial cells displayed typical topographic distributions; moreover, myoepithelial cells showed two distinct cytoskeletal phenotypes. These findings could account in part for the heterogeneity of aspects observed in this tumour. In carcinomas, malignant cells were always positive to cytokeratin antibodies with general specificity and myoepithelial cells were absent as judged by anticytokeratin SK2-27 and anti--smooth muscle actin immunostainings. However, interestingly, there was in all cases a strong positivity for -smooth muscle actin in stromal cells, similarly to what has previously been described for mammary carcinoma (Skalli et al. 1986). Our findings may be useful for the interpretation of the histogenesis of salivary and lacrimal tumour and stromal cells.     

Peripheral nerve grafts lacking viable Schwann cells fail to support central nervous system axonal regeneration

Summary Peripheral nerve grafts were implanted bilaterally into the diencephalon of adult hamsters. One graft segment contained both viable Schwann cells and their basal lamina tubes. The Schwann cell population in the second graft segment was killed by freezing prior to implantation. Seven weeks after graft implantations, the extracranial end of each graft segment was exposed, transected and labelled with a fluorescent tracer substance. One week after the labelling procedure each animal was perfused and the diencephalon and midbrain were examined. Ultrastructural analyses of both types of graft demonstrated the persistence of the Schwann cell-derived basal lamina tubes. Retrogradely labelled neurons were found in all cases in which an intact graft remained in place for two months, but were seen in only one case with a frozen graft. Large numbers of myelinated and unmyelinated axons were seen within the intact grafts, but no axons were found in the previously frozen grafts. These results indicate that lesioned CNS axons are able to regenerate vigorously when provided with an environment which includes viable Schwann cells. But, CNS axons regenerate less well, if at all, when Schwann cells are absent. Further, it appears that Schwann cell-derived basal lamina tubes, when isolated from their parent cells, are insufficient to initiate or sustain CNS axonal regeneration.This material is based upon work supported by the National Science Foundation under grant BNS-8416911     

The relationship between epithelial Ia expression and the inflammatory cell infiltrate during experimental oral carcinogenesis

Summary The development of oral epithelial expression of Ia antigens and its relationship to the presence of IL-2r⁺ (CD25⁺) cells was investigated in rats treated with the water soluble carcinogen 4-nitroquinoline-N-oxide (4NQO). Acetone fixed frozen sections of the palate and tongue were stained using an indirect immunoperoxidase technique and monoclonal antibodies to rat Ia (I-A & I-E) and IL-2 receptor. After 4 weeks 4NQO treatment all rats expressed oral epithelial Ia but thereafter (2–9 months) expression was present in only 20–40% of animals. Epithelial expression of Ia by histologically normal, dysplastic and neoplastic epithelium was always associated with the presence of an underlying inflammatory cell infiltrate containing CD25⁺ cells. Overall there were significantly more CD25⁺ cells in tissue specimens containing Ia⁺ epithelium compared with Ia^– epithelium. Furthermore, during the first 4 weeks of carcinogen treatment, a significant positive correlation was found between the CD25⁺ cell density and occurrence of focal epithelial Ia expression. These results, together with analysis of the T cell, NK cell, macrophage and B cell content of the infiltrates induced by 4NQO, suggest that the CD25⁺ cells represent activated T cells. Thus, our results in this experimental model are consistent with the idea that epithelial expression of Ia is the result of production of IFN- by locally activated T cells.     

Myoblasts fail to stimulate T cells but induce tolerance

Recent interest in myoblast transfer and in the use of myoblastsas vehicles in gene therapy has made it important to understandthe potential immunogenicity of allogeneic or neoantlgen-expresslngmyoblasts. Given the problems of producing a pure populationof myoblasts, In this study we used a tumour-derived musclecell line (TE671), with phenotyplc features of myoblasts, whichwe transfected to express HLA-DR1. However, this cell line wasunable to stimulate either established HLA-DR1-specific alloreactlveT cell clones or a primary alloresponse. Nor could it presenthaemagglutlnln peptide HA 306–324 to DR1-restricted, HA306–324-speciflc T cell clones or lines. Indeed, prelncubatlonwith DR1-expressing TE671 and HA 306–324 rendered suchT cells tolerant as Judged by their subsequent inability toproliferate in response to a DR1⁺ B cell line plus peptide HA306–324. These results imply that myoblasts do not providecostlmulatory signals, and are therefore unlikely to stimulateallospeclfic T cells following myoblasts transplantation orto initiate neoantlgen-speclfic immune responses following Invivo transfection.     

The functional state of follicular dendritic cells in severe combined immunodeficient (SCID) mice: Role of the lymphocytes

In the present study, we have investigated the capacity of follicular dendritic cells (FDC) to trap immune complexes (IC) in the splenic white pulp of severe combined immunodeficient (SCID) mice and the influence of lymphocyte transfer on FDC function. FDC are absent in the splenic white pulp of naive SCID mice as revealed by in vitro IC trapping assay. One week after transfer of syngeneic lymphocytes, functional FDC with complement receptors appeared in the primary follicles coincident with B cell segregation, and IC were trapped on those FDC in a complement-dependent manner. Next, we immunized the reconstituted SCID mouse to see whether another type of FDC could be induced in the secondary follicle. Antigenic stimulation induced FDC with an additional capacity to capture IC via FcR γ II. As seen in immunocompetent mice, this type of FDC was located only in the light zone of the secondary follicle. The newly generated FDC did not carry H-2 antigen of transferred lymphocytes from F₁ mice. In SCID mice, in which normally no functional FDC are detectable, the microenvironments of the splenic white pulp have a capacity to develop and differentiate normally after transfer of lymphocytes. Apparently, the generation of functional IC-trapping FDC causes the induction of complement receptor(s) and Fc receptor on meshwork cells, which requires the presence of the lymphocytes.     

Ganglion cell density and oil droplet distribution in the retina of brown-eared bulbul (Hysipetes amaurotis)

This study was intended to determine the number and density of both retinal ganglion cells and the oil droplets of cone photoreceptor cells in brown-eared bulbul (Hysipetes amaurotis). For this study birds were killed with proper dose of anesthetic (pentobarbital, 30 mg/kg), and the eyes were removed from the orbital cavity to isolate the retina. For the ganglion cell study retinal whole-mount specimens were prepared and stained with 0.1% cresyl violet. The different types of oil droplets were counted from color microphotographs of freshly prepared retinal samples. The mean total number of ganglion cells was estimated at approximately 2.5×10⁶; with an average density of 16 523 cells/mm². Two high-density areas, namely the central area (CA) and the dorso-temporal area (DTA), are located in the central and dorso-temporal retinas, respectively, in bulbuls (24 032 cells/mm² in the CA; 23 113 cells/mm² in the DTA). Small ganglion cells persisted in the highest density areas, whereas the largest soma sizes were found in the lowest density areas of the retina. Four types of different colored oil droplets — red, orange, green and clear — were identified with an average density of 29 062/mm². Among the different colors, the green oil droplets had a significantly higher population (13 083/mm²) than the others across the retina. The central retina had a significantly higher number of all types of oil droplets, at a density of 60 552/mm². The density and size of the different colored oil droplets were inversely related across the regions of the retina. Taken together, it is concluded that the CA of the retina is an excellent quality area for visual perception due to peak density of ganglion cells and oil droplets. Moreover, each specific oil droplet makes a distinct contribution to visual perception, thereby ensuring that the bird has a retina that best matches its natural environment and feeding behavior.     

Corticosteroid enhances TNF‐α‐mediated leukocyte adhesion to pulmonary microvascular endothelial cells

Background: Some severe asthma patients are characterized by elevated levels of tumor necrosis factor alpha (TNF‐α) and neutrophilic inflammation in the airways. Although such phenotypic changes in asthma might contribute to corticosteroid refractoriness, the role of TNF‐α in the process remains unclear. TNF‐α exerts its biological effects mainly by acting on the vascular endothelium, and thereby upregulates leukocyte recruitment into inflamed tissues. The aim of this study was to investigate the effects of dexamethasone (DEX) on the TNF‐α‐mediated responses of human microvascular endothelial cells from lung blood vessels (HMVEC‐LBl) in vitro. Methods: HMVEC‐LBl were cultured with TNF‐α in the presence and absence of DEX. The effects of DEX on various TNF‐α‐mediated responses, such as the expressions of chemokines and cellular adhesion molecules, leukocyte adhesion were determined. Results: TNF‐α significantly induced growth‐related oncogene alpha (GRO‐α), interleukin 8 (IL‐8), regulated on activation, normal T‐cell expressed and secreted (RANTES) and interferon‐inducible protein 10 (IP‐10) productions and cell surface expressions of intracellular adhesion molecule 1 (ICAM‐1) and vascular cell adhesion molecule 1 (VCAM‐1) on HMVEC‐LBl. TNF‐α‐induced GRO‐α and IL‐8 were slightly attenuated by DEX treatment (reaches to 89% and 79%, respectively), whereas expressions of IP‐10, ICAM‐1 and VCAM‐1 were significantly enhanced by the same treatment (up to 172%, 152% and 139%, respectively). Correspondingly, in vitro adhesion of eosinophils and neutrophils to TNF‐α‐treated HMVEC‐LBl were significantly enhanced by DEX. Conclusions: Some proinflammatory effects of DEX, a corticosteroid, were found in TNF‐α‐mediated in vitro reactions of pulmonary microvascular endothelial cells, i.e. chemokine productions and leukocyte adhesion. These in vitro results may explain, at least in part, the corticosteroid refractoriness accompanied by a marked increase in TNF‐α production that is seen in severe asthmatic patients.     

Dendritic cells and natural killer cells in the pathogenesis of HIV infection

Dendritic cells (DC) and natural killer (NK) cells, the main cellular components of the innate immune system, participate in the most ancient first line of defense against infections. Both types of cells patrol peripheral tissues, whereas their rapid recruitment and activation at mucosal surfaces [the major entry point for the human immunodeficiency virus (HIV)] is a hallmark of acute inflammatory response. The ability of HIV to survive and replicate in the human host relies upon several molecular mechanisms eluding the immune surveillance of both adaptive immunity and of DC and NK cells beginning with the acute phase of primary HIV infection. DC and NK cells, unlike CD4⁺ T cells, are impaired more functionally rather than being depleted by HIV infection. In this article we will review some of the aspects of DC/NK cells interaction with HIV infection both in vitro and in vivo, and we will also speculate on the potential consequence for HIV pathogenesis and for the capacity of the virus to escape the surveillance of the innate immune system.     

Ca2+ permeability of the plasma membrane induced by rotavirus infection in cultured cells is inhibited by tunicamycin and brefeldin A

Rotavirus infection of cultured cells induces a progressive increase in plasma membrane permeability to Ca2+. The viral product responsible for this effect is not known. We have used tunicamycin and brefeldin A to prevent glycosylation and membrane traffic and study the involvement of viral glycoproteins, NSP4 and/or VP7, in rotavirus-infected HT29 and MA104 cells. In infected cells, we observed an increase of plasma membrane Ca2+ permeability and a progressive depletion of agonist-releasable ER pools measured with fura 2 and an enhancement of total Ca2+ content measured as 45Ca2+ uptake. Tunicamycin inhibited the increase in membrane Ca2+ permeability, induced a depletion of agonist-releasable and 45Ca2+-sequestered pools. Brefeldin A inhibited the increase of Ca2+ permeability and the increase in 45Ca2+ uptake induced by infection. We propose that the glycosylated viral product NSP4 (and/or VP7) travels to the plasma membrane to form a Ca2+ channel and hence elevate Ca2+ permeability.     

Cbl-b differentially regulates activation-induced apoptosis in T helper 1 and T helper 2 cells

We previously reported that ligation of CD3 induces antiapoptotic signals in T helper 2 (Th2) cells, and in contrast causes Th1 cells to undergo apoptosis. Here we show that Cbl-b is accountable for the unequal response, revealing a previously unknown cell-specific regulatory function for the molecule. Absence of Cbl-b resulted in resistance to activation-induced apoptosis in murine Th1 cells following CD3 ligation, akin to what is observed in Th2 cells containing Cbl-b. Concurrent with the apoptosis profile, CD3 ligation in the absence of Cbl-b induced raft mobilization and cytoskeletal rearrangement in Th1 cells. Despite their ability to signal from CD3, Th2 cells did not aggregate their rafts, providing an explanation for cell-specific activity of Cbl-b.