Induction of a differentiated ciliated cell phenotype in primary cultures of Fallopian tube epithelium

Human Fallopian tubal epithelial cells in culture lose morphological features associated with the epithelium in situ and the extent to which they retain their in-vivo phenotype or function is unknown. In order to address this question, immunocytochemical markers were identified which distinguish secretory (HMFG2+, LhS28-) from ciliated (HMFG2-, LhS28+) epithelial cells in tissue sections of Fallopian tube. These markers were used to analyse the phenotype of tubal cells in vitro. Primary cultures of human tubal epithelial cells were seeded onto glass and grown to confluence before addition of oestradiol-17beta. In the absence of hormone, tubal epithelial cells expressed cytokeratins and nuclear receptors for oestrogen and progesterone and adopted a homogeneous (HMFG2+, LhS28-) secretory cell phenotype. Following the addition of oestradiol-17beta, a proportion of cells became positive for LhS28. The induction of a ciliated epithelial cell phenotype was confirmed by scanning electron microscopy, where on permeable collagen membranes, approximately one-third of tubal epithelial cells became ciliated in the presence of oestradiol-17beta. We suggest that in vitro, tubal epithelial cells adopt an immature secretory-like phenotype and that oestrogen can induce differentiation to a ciliated epithelial cell phenotype.      

Stimulation of human monocytes by anti-CD3 monoclonal antibody: Induction of inflammatory mediator release via immobilization of Fc receptor by adsorbed immunoglobulin and T-lymphocytes

Human monocytes released superoxide anion, prostaglandin E₂, leukotriene B₄, IL-1, and TNF when exposed to plastic surfaces coated with murine anti-CD3 monoclonal antibody, OKT 3. Stimulation of mediator release by OKT 3 was dependent on the amount of antibody immobilized onto wells of plastic tissue culture plates. Soluble antibody or antibody adsorbed to monocytes and reacted with an aggregating (cross-linking) second antibody failed to induce mediator release. Monocytes armed with OKT 3 formed rosettes with T cells in a fashion indistinguishable from that seen between monocytes and T cells sensitized with OKT 3. Monocytes with adsorbed OKT 3 antibodies released IL-1 and TNF- when exposed to unsensitized T cells, although increased superoxide release could not be detected. OKT 4a, a murine IgG_2a antibody that reacts with a different T cell epitope (CD4), failed to induce cytokine release from monocytes when cross-linked by T cells or a CD4+ T cell line, even in the presence of IL-2 or IFN-. These data indicate that certain antibodies bound to Fc receptors (FcR) of monocytes may trigger monocyte function when reacting with cells bearing the appropriate target antigens. FcR-mediated signaling resulting in mediator release may be involved in initiating or regulating the immune response. Furthermore, systemically administered monoclonal antibodies may induce inflammatory responses and their attendant symptomatologies via their interaction with FcR-bearing inflammatory cells.     

Cardiac and respiratory patterns in normal infants and victims of the sudden infant death syndrome

Victims of the sudden infant death syndrome (SIDS) have higher overall heart rates prior to death than do control infants (1). The objective of this study was to partition these heart rate differences by state and to identify any state-dependent differences in heart rate variability and respiratory rate and variability. Twenty-two recordings of electrocardiogram (ECG) and respiration from 16 infants who subsequently died of SIDS were compared with 66 recordings of age-matched control infants. Median cardiac and respiratory rate and variability were computed for each sleep state in each recording, and one-way analysis of variance tests were performed for each variable for infants less than 1 month and for infants greater than 1 month of age. Heart rate was higher in SIDS victims less than 1 month of age than in age-matched controls during all sleep-waking states. SIDS victims greater than 1 month showed higher heart rates during rapid eye movement sleep only. Heart rate variability was also diminished during waking in victims less than 1 month, but much of this difference could be attributed to increased heart rate. These results suggest that, as a group, SIDS victims differ physiologically from control infants and that these differences may be especially prominent during particular sleep-waking states.     

Immunofluorescent analysis of blood cells by flow cytometry

Historically, immunofluorescence was one of the first applications of flow cytometry. In the conventional method, forward light scatter and fluorescence are measured for each cell in the flowing sample stream. The size-related forward scatter measurement permits the fluorescence measurement to be made on cells within a particular size range. Fluorescence intensity above a fixed threshold is interpreted to mean a cell is stained or positive. Provided purified cells are used, and that the stained cells are brightly fluorescent, this conventional method provides useful results that are easy to interpret. In this paper we have reported our recent investigations of restrictions, fundamental limitations and basic extensions evidenced in our application of a new method of immunofluorescent analysis of whole blood preparations. These include: limitations due to autofluorescence and nonspecific staining, techniques for optimal staining, and the appropriate evaluation of fluorescent histogram data. Our data indicate that the method reviewed here offers a rapid technique for evaluating T cells and their subclasses with the potential, due to its ease of performance, for application to repeated use in longitudinal studies.     

Endothelin-induced vasoconstriction and release of atrial natriuretic peptides in the rat

Endothelin-1 (ET-1) was given to male Sprague-Dawley rats in i.v. bolus injections to evaluate its effects on blood pressure and the release of atrial natriuretic peptides (ANP). In awake rats ET-1 (0.3, 1 and 3 nmol kg-1 body wt) transiently reduced mean arterial pressure (MAP) and increased heart rate (HR), followed by a prolonged increase in MAP. The magnitude of these changes and the duration of the increase in MAP were dose-related. The increase in MAP was completely blocked by verapamil, reversed by sodium nitroprusside, slightly reduced by rat atrial natriuretic factor (103-126) and unaffected by saralasin. The initial fall in MAP was also unaltered by these agents. In all groups HR changes were mirror-images of MAP. In anaesthetized rats ET-1 (1 nmol kg-1 body wt) induced a sustained release of ANP. Right atrial pressure increased transiently and then fell below baseline. When the increase in MAP was blocked with sodium nitroprusside, ET-1 still produced an increase in ANP. In conclusion we find that repeated i.v. administration of ET-1 induces immediate vasodilatation, without signs of tachyphylaxis, followed by long-lasting severe vasoconstriction. Baroreceptor function seems to be unchanged. ET-1 appears to induce ANP release by a direct action on atrial myocytes, independent of right atrial and systemic arterial pressure. We hypothesize that endothelin may be a mediator of stretch-induced release of ANP.     

Changes in immunoregulatory lymphocyte populations in patients with histoplasmosis

Circulating T-lymphocyte subpopulations were enumerated in 65 patients with histoplasmosis and correlated with the different clinical manifestations of the disease. Acute pulmonary histoplasmosis, rheumatologic, disseminated, and chronic inflammatory manifestations of histoplasmosis were all associated with a significant elevation above normal of OKT₈+ (suppressor-cytotoxic) lymphocytes and a significantly lower than normal OKT₄+ (helper-inducer)-lymphocyte to OKT₈+-lymphocyte ratio. In contrast, cavitary disease was associated with an increase in OKT₄+ lymphocytes, a decrease in OKT₈+ lymphocytes, and a higher than normal OKT₄/OKT₈ ratio. Clinical recovery was associated with normalization of these values. Functional activity determined by coculture techniques correlated closely with T-lymphocyte subset measurements. These distinct subset abnormalities may help monitor immunological aspects of disease activity.     

Absence of demonstrable phospholipid turnover in B cells stimulated by low mitogenic concentrations of dextran-anti-immunoglobulin conjugates.

Previously we have demonstrated that when anti-immunoglobulin (Ig) is conjugated to high molecular weight dextran (Dex) it stimulates B cell activation at pg/ml concentrations in the absence of detectable phosphoinositide hydrolysis or increases in intracellular ionized calcium. To study carefully whether anti-Ig-Dex recruited a phosphoinositide-dependent pathway of activation, we stimulated B cells that were labeled with 32P and [3H]glycerol with anti-Ig-Dex conjugates at concentrations ranging from 1-1 x 10(-4) micrograms/ml. Thirty seconds to thirty minutes after stimulation lipids were extracted and analyzed by thin layer chromatography and spots correlating with known lipid standards were isolated and counted. There was a four- and tenfold increase in the ratio of 32P/3H incorporated into phosphatidic acid (a metabolite of diacylglycerol) and phosphatidylinositol, respectively, when cells were stimulated with 0.1-1.0 microgram/ml of anti-Ig-Dex for 30 min. Below 1 ng/ml there was no detectable increase in the turnover of these metabolites despite the fact that in parallel cultures B cells were stimulated to proliferate by this concentration of anti-Ig-Dex. To determine whether a cAMP-dependent pathway was recruited by low concentrations of conjugates, we evaluated cAMP levels from B cells that were stimulated with anti-Ig-Dex for 5-60 min using a radioimmunoassay. While cholera toxin stimulated a 50-100-fold increase in the levels of cAMP, we observed no alteration in cAMP in anti-Ig-stimulated cells. These results support and extend our previous findings by demonstrating that B cell activation that is induced by cross-linking of surface Ig may not stimulate phosphoinositide-dependent or cAMP-dependent pathways of activation. Possible alternative mechanisms of activation will be discussed.