Culture of human epidermal Langerhans cells in a skin equivalent

Langerhans cells (LCs) have been cultured in a skin equivalent (SE). Seventy-two SEs were produced by inserting skin biopsies from nine subjects into dermal equivalents consisting of fibroblasts in a collagen matrix. The SEs were cultured in a serum-free medium containing 2-mercaptoethanol with or without 5 ng/mL granulocyte-monocyte colony-stimulating factor (GM-CSF). The SEs were cultured for 12 or 15 days. In the latter case, 0, 1 or 10 microg/mL cyclosporin A (CyA) was added for the last 3 days. The SEs were then snap frozen for immunohistochemistry. The migration of LCs was evaluated by measuring the distances from the inserted skin biopsy in the SEs to the HLA-DR + and CD1a+ dendritic cells localized at the longest distance from the biopsy in the epidermal outgrowth on both sides of the biopsy. The density of these cells was estimated in 15-day-old SEs by counting them on both sides of the inserted skin biopsy and dividing the number of positive cells by the migrated distances. All epidermal outgrowths (range 0.6-3.7 mm) were well differentiated and displayed HLA-DR+, CD1a+ and Lag+ dendritic cells. Only occasionally were CD83+ cells observed. In the 15-day-old SEs cultured with GM-CSF, a few CD86+ cells were seen in the epidermal outgrowths and occasionally CD80+ cells. The median (n = 4) density of CD1a+ and HLA-DR+ cells in the epidermal outgrowths at day 15 was 5.2 and 9.1 cells/mm, respectively. GM-CSF did not influence migration in 12-day-old SEs, but there was a tendency to increased migration of HLA-DR+ dendritic cells in 15-day-old SEs. CyA did not affect migration or density. We conclude that LCs can be cultured with an in vivo-like density in a SE. They express the phenotype of immature antigen-presenting cells efficient in capturing and processing antigen. This model may be suitable for studies of the initial phase of contact allergic reactions.     

Postnatal development of Leydig cells in the opossum (Monodelphis domestica): an immunocytochemical and endocrinological study

Using a mouse early preantral follicle culture system, mature full grown oocytes, arrested in prophase I of meiosis, were produced after 12 days using a recombinant gonadotrophin-supplemented medium. This culture medium does not mimic the normal extracellular environment of the oocyte and might therefore modify meiotic regulation and more particularly progression to metaphase II (MII). The aim of this study was to optimize the treatment using recombinant stimulatory ligands which were known to induce germinal vesicle breakdown (GVBD) and completion of meiosis I, metaphase II (MII), namely recombinant follicle stimulating hormone (r-FSH), chorionic gonadotrophin (r-HCG) and epidermal growth factor (EGF). Full-grown intrafollicular oocytes could not resume meiosis when the 'ovulatory' stimulus was r-FSH, used at a 100 times higher dose than during culture. r-FSH did not increase progesterone production. When 1.5 IU/ml r-HCG was used as meiotic trigger, germinal vesicle breakdown was obtained in 95% of the oocytes 64% of which extruded a first polar body. r-HCG induced a dramatic increase in progesterone production. When EGF was administered as sole stimulus on day 12 to the attached follicle-enclosed oocytes, only doses > or =5 ng/ml could cause GVBD, although less effectively than r-HCG (45 versus 95%; P < 0.0001). Oocytes undergoing GVBD by the EGF pulse reached metaphase II at a rate of 54% (not significant versus r-HCG). EGF did not stimulate progesterone production. Addition of increasing doses of EGF (0.5; 5; 10; 50 ng/ml) to r-HCG did not increase the GVBD-rate, but EGF doses >5 ng/ml improved MI to MII transition (P=0.027), thereby improving the final yield of MII oocytes by 12.5%. These data show that up to a dose of 50 ng/ml, EGF on its own could only override the somatic inhibitory stimuli in less than half of the cultured follicles. However, in addition to HCG, EGF (25 ng/ml) had a stimulatory effect on completing the first meiotic division. It was concluded that, under the present culture conditions, EGF in combination with HCG provided optimal nuclear maturation.     

Cortical dysplasia: an immunocytochemical study of three patients

The role that T and B lymphocytes play in the clearance of Giardia muris in the mouse model is well known, but the cytokines produced by CD4+ T cells in response to Giardia antigenic stimulation are unknown. In this study, we have determined how Giardia trophozoite antigenic crude extract and T cell mitogens can trigger the production of cytokines by Peyer's patch and spleen cells removed from infected animals. When Giardia trophozoite proteins were used to challenge the cells in vitro, IL-4, IL-5 and IFN-gamma were not detected in the culture supernatant. When the cells were challenged with Con-A, all three cytokines were released in vitro. However, the level of each cytokine released by the spleen or Peyer's patch cells varied with the latent, acute and elimination phases of the infection. The high levels of IL-4 and IL-5 released by Peyer's patch cells confirm the importance of IgA in the control of the infection. However, we propose that the relative success of G. muris in completing its life cycle in a primary infection might be due, in part, to the stimulation of a Th2-type response (IL-4, IL-5). A stronger Th1 response (IFN-gamma) may lead to a better control of the primary infection.     

Carnoy's fixative reduces the number of chymase-positive cells in immunocytochemical staining of cord-blood-derived human cultured mast cells

KleinJan et al. (Allergy 1996;51:614-20) reported that Carnoy's fixative reduced the number of chymase-positive mast cells in the nasal mucosa. Therefore, in the present study, we investigated whether Carnoy's fixative reduces the number of chymase-positive cells from cord-blood-derived human cultured mast cells when compared with other types of fixatives. Human mast cells were obtained by culturing cord-blood-derived CD34-positive cells in the presence of stem cell factor and interleukin-6. Staining procedures of the cells in fixation with Carnoy's fixative and with other fixatives gave no differences among the number of tryptase-positive cells, whereas fixation with Carnoy's fixative for 15 min gave a significant decrease in the number of chymase-positive cells compared with acetone for 10 min. The number of chymase-positive cells decreased in a time-dependent manner under fixation with Carnoy's fixative, indicating that Carnoy's fixative had a negative effect on the number of chymase-positive cells from cord-blood-derived human cultured mast cells.     

Langerhans cell histiocytosis of the skin

Cutaneous involvement in Langerhans cell histiocytosis (LCH) occurs in 50% of cases and may be the presenting feature. It is, therefore, important to recognize the wide spectrum of clinical disease that this disorder may adopt in the skin. Cutaneous involvement is not necessarily a benign feature and many patients progress to multi-system disease. There are a number of treatments available for cutaneous LCH. The rationale is to start with the simplest treatment and progress to systemic or interventional therapy as needed.     

Reduction in number and morphologic alterations of Langerhans cells after UVB radiation in vivo are accompanied by an influx of monocytoid cells into the epidermis

Acute, low-dose ultraviolet B (UVB) radiation impairs contact hypersensitivity induction in mice by a mechanism due at least in part to Langerhans cells alterations. To better define the effects of UVB on Langerhans cells, we have compared the action of this agent on the skin of intact mice and in skin explants incubated in vitro up to 24 h. Using immunofluorescence, we detected a reduction in the length of the dendrites of Langerhans cells and a significant reduction in the number of Ia-positive Langerhans cells per unit area within 2 h of UVB; these changes reversed within 24 h in vivo, but not in vitro. By electron microscopy, the number of dendritic cells per 100 basal keratinocytes increased in vivo, but decreased in vitro by 2 h after UVB, a discordance that was significant. On the contrary, the number of dendrite profiles per dendritic cell body decreased significantly 2 h after UVB, both in vivo and in vitro. Many epidermal dendritic cells, 2 h after UVB in vivo, were deficient in cytoplasmic organelles, whereas the few cells that remained after UVB in vitro retained their Birbeck granules, and displayed many, dilated cytoplasmic vesicles. We interpret these data to mean that low doses of UVB radiation destroy the functional and morphologic integrity of epidermal Langerhans cells, and that these cells are rapidly replaced by precursor cells that mature in situ into normal-appearing Langerhans cells.     

Distribution of cholecystokinin immunoreactivity in the BALB/c mouse forebrain: an immunocytochemical study

The present study describes cholecystokinin (CCK) immunoreactivity (CCK-IR) distribution in the brains of control and colchicine-treated mice. In the brains of control mice, the CCK-IR strongly revealed numerous axons and terminals. Perikarya exhibiting a faint to moderate immunoreactivity were also observed in areas such as cortices, hippocampus, amygdala, septum, and thalamus. The colchicine treatment did not seem to notably affect the brain CCK-IR innervation, but resulted in profound changes of the perikaryal staining. Indeed, the regions, which contained numerous moderately stained perikarya in the control animals, exhibited after colchicine treatment immunoreactive perikarya intensely stained but only in moderate number. This feature obviously appeared in the cortex in which, in addition to strongly stained perikarya, colchicine induced the appearance of numerous CCK-IR hillocks. In the lateral amygdala and thalamus of colchicine-treated animals, the somatic immunoreactivity was considerably decreased. The regions, such as paraventricular hypothalamic nucleus and bed nucleus of the stria terminalis, which in the control animals did not exhibit any stained perikaryon, showed a high number of strongly stained cell bodies after colchicine treatment. This study, mapping the mouse forebrain CCK-IR, demonstrated a wide distribution of this peptide. Moreover, CCK-IR is spontaneously visible in neurons of untreated mouse in some brain areas previously shown in the rat to exhibit CCK mRNA, but no clear perikaryal CCK-IR even after colchicine treatment.     

Large increase of Langerhans cells in human skin lymph derived from irritant contact dermatitis

The pharmacokinetic profile of a sulphamonomethoxine-trimethoprim (SMM-TMP) combination was investigated in five horses. The combination was administered intravenously, intramuscularly and orally at a constant dose of 20 mg SMM plus 4 mg TMP kg-1 bodyweight. Following intravenous administration both drugs dispersed rapidly with distribution half-lives of about 12 minutes for SMM and about 18 minutes for TMP. Elimination half-lives for intravenous, intramuscular and oral administration were closely similar, indicating that elimination was independent of administration route. Bioavailability of the drugs in aqueous solution was good: about 72 per cent and 84 per cent for SMM and about 84 per cent and 98 per cent for TMP following intramuscular and oral administration, respectively. It is concluded that SMM-TMP administered orally once a day at 20 mg and 4 mg kg-1 bodyweight, respectively, maintains therapeutic concentrations, whereas twice daily intramuscular administration would be more effective for treating systemic infections in the horse than the once a day regimen usually adopted in veterinary practice.     

Inhalation of cold air increases the number of inflammatory cells in the lungs in healthy subjects

Prolonged exposure to cold air may induce a chronic asthma-like condition in healthy subjects as has been demonstrated in cross-country skiers. In the present controlled study, our aim was to elucidate further the link between cold air exposure and airway inflammation by assessing the cellular influx and mediator levels within the airways following acute exposure to cold air. Bronchoalveolar (BAL) and nasal lavages were performed after exposure to cold air (-23 degrees C) and normal indoor air (+22 degrees C) during a light, intermittent work for 2 h in a cross-over design in eight healthy, nonsmoking, subjects. Analyses of inflammatory cell number, cell activation markers, pro-inflammatory cytokines, albumin and interleukin (IL)-8 in lavage fluids were performed. The number of granulocytes and of alveolar macrophages in BAL fluid was significantly higher after cold air exposure (p<0.05). No increase in BAL fluid lymphocytes and no signs of lymphocyte activation in BAL fluid were found. The concentration of IL-8 was unchanged. There were no signs of granulocyte activation (myeloperoxidase, eosinphilic cationic protein) in BAL fluid. Cold air did not influence the number of inflammatory cells or the concentration of albumin and IL-8 in nasal lavage fluid. In conclusion, exposure to cold air induces an increased number of granulocytes and macrophages in the lower airways in healthy subjects without influencing other inflammatory indices such as cellular activation, plasma leakage and pro-inflammatory cytokines. These findings support the hypothesis that cold air could be of pathogenetic importance in the asthma-like condition previously found in cross-country skiers.     

Langerhans cells in Langerhans cell granulomatosis are not actively proliferating cells

Pulmonary Langerhans cell granulomatosis (LCG), also called histiocytosis X, is a disorder of unknown etiology characterized by the presence of destructive granulomas containing numerous Langerhans cells (LCs). The process may be localized or multifocal, and it remains unclear whether the same pathogenic mechanism is involved in all forms of the disease. It is often assumed that the massive accumulation of LCs at the sites of the lesions results from the abnormal proliferation of these cells, although it has been suggested that LCG in adults, at least in the lung, could be a reactive disorder initiated by activated LCs. Little is known, however, concerning the mechanisms responsible for the accumulation of large numbers of LCs in the course of the disease, and the relative contribution of recruitment and local proliferation of these cells remains to be established. To investigate this question, the proportion of replicating LCs was evaluated in biopsied granulomas from patients with localized or diffuse form of LCG by means of several histopathological techniques currently used in assessment of cell proliferation. The findings demonstrate that, except for proliferating cell nuclear antigen (PCNA), all parameters measured are low in all forms of the disease. They are similar to those of renewing epithelial cells and clearly less than those of neoplastic cells. These data strongly suggest that LCs in LCG granulomas are not a rapidly dividing cell population and that local LC replication makes only a minimal contribution to granuloma maintenance. Caution appears to be necessary in the use of PCNA as a marker of growth fraction.     

Langerhans cells in the neuro-immuno-cutaneous system

Baboon (Papio hamadryas) metaphase chromosomes were analyzed using spectral karyotyping (SKY), a technique combining fluorescence microscopy, CCD-imaging, and Fourier spectroscopy. Results from a comparison of SKY analyses using probes derived from human chromosomes on baboon metaphases were consistent with the majority of comparative gene mapping data between the two species. These data were also compatible with earlier studies comparing macaque and human chromosomes. Human (HSA) chromosome 2 was homologous to baboon (PHA) chromosomes 12 (HSA 2q) and 13 (HSA 2p), whereas three baboon chromosomes corresponded to two different human chromosomes: PHA 3 to HSA 7 and HSA 21, PHA 7 to HSA 14 and HSA 15, and PHA 10 to HSA 20 and HSA 22. These results support the retained synteny between the Hominidae and Cercopithecidae genomes.     

Expression of adhesion molecules and HLA-DR by macrophages and dendritic cells in aphthoid lesions of Crohn's disease: an immunocytochemical study

The phenotypes and ultrastructure of macrophages and dendritic cells in aphthoid lesions of the colon were immunocytochemically observed in patients with Crohn's disease. Biopsy specimens were endoscopically obtained from both aphthoid and advanced lesions in Crohn's disease patients. Biopsy specimens obtained from patients with infectious colitis and from normal individuals served as controls. Aphthoid lesions contained densely aggregated CD68+ macrophages, which were surrounded by numerous ID-1+ dendritic cells. In the normal controls and infectious colitis patients, however, a few scattered CD68+ macrophages and ID-1+ dendritic cells were noted beneath the surface epithelium. CD3+ lymphocytes were significantly increased in both aphthoid and advanced lesions of Crohn's disease, but the CD4/CD8 ratio was similar in all groups studied. The double immunoperoxidase staining method revealed that both CD68+ macrophages and ID-1+ dendritic cells in the aphthoid lesions simultaneously expressed ICAM-1 and HLA-DR antigens. Electronmicroscopic observation revealed that CD68+ macrophages had numerous vesicles and lysosomal granules and few projections, and that ID-1+ dendritic cells had appreciable cytoplasmic protrusions with a few vacuoles. These findings suggested that the colonic mucosa in Crohn's disease contained two types of macrophage/dendritic cells in the same lineage that expressed intercellular adhesion molecules and class-II MHC antigens. It also appeared that the aphthoid lesions of Crohn's disease featured an increase in macrophages and dendritic cells consistent with immunological activation.     

Diversity of pituitary cells in primary cell culture. An immunocytochemical study

A bioassay system for rapid detection of carcinogenic agents has been developed using male Fischer 344 rats to bridge the gap between long-term carcinogenicity tests and short-term screening assays. The system, called the medium-term liver bioassay, is fundamentally based on the 2-stage hypothesis of tumor production, employing initiation by diethylnitrosamine (200 mg/kg, i.p.) in the first stage and test chemical administration during the second, in combination with two-thirds partial hepatectomy. It requires only 8 wk for animal experimentation and a further few weeks for quantitative analysis of immunohistochemically demonstrated glutathione S-transferase placental form positive hepatic foci. A total of 291 chemicals/substances have already been analyzed in our laboratory. Among 63 chemicals that were proved to be carcinogenic in the liver of rat and/or mouse, 57 (90%) gave positive results irrespective of their mutagenicity. Negative compounds include peroxisome proliferators and tamoxifen. Even nonhepatocarcinogens were positive at a rate of 24%. Eighty-six percent (12/14) of mouse liver carcinogens were also positive. On the other hand, only 2 out of 45 noncarcinogens showed very weak positivity. Thus, the efficacy of the system for hepatocarcinogens has been well established. This bioassay is increasingly regarded as an appropriate alternative test for carcinogenicity risk assessment and is practically used for a rapid evaluation of hepatocarcinogenicity of chemicals.     

Anatomical distribution of beta-endorphin (1-27) in the cat brainstem: an immunocytochemical study

IL-4 is a pleiotropic cytokine which exerts its actions on various lineages of hematopoietic and nonhematopoietic cells. This cytokine is one of the central regulators of immunity in health and disease states. An alternative splice variant, in which the second of four exons is omitted, has been recently described and designated as IL-4delta2. The variant has been previously described as a potential naturally occurring antagonist of human IL-4 (hIL-4)-stimulated T cell proliferation. In this study, we investigated the effects of recombinant human (rh) IL-4delta2 on monocytes and B cells. In monocytes, rhIL-4delta2 blocked inhibitory action of hIL-4 on LPS-induced cyclooxygenase-2 expression and subsequent prostaglandin E2 secretion. In B cells, rhIL-4delta2 was an antagonist of the hIL-4-induced synthesis of IgE and expression of CD23. Our results broaden the spectrum of hIL-4-antagonistic activities of rhIL-4delta2, thus creating the background for the potential use of rhIL-4delta2 as a therapeutic anti-hIL-4 agent.     

Sorbin in the porcine gastrointestinal tract and pancreas: an immunocytochemical analysis

Sorbin is a 153-amino acid peptide that was initially discovered in the porcine duodenum. We have reported previously that this peptide regulates intestinal electrolyte transport and have described accumulation sites in the rat digestive tract. In the present study, we investigated the anatomical distribution and the site(s) of sorbin production in the porcine digestive tract using immunocytochemistry. The use of polyclonal antisera, which by cross-reaction studies were shown to be specific for different regions of the molecule, revealed a diversified distribution. Sorbin predominated in endocrine cells preferentially localized in the pyloric glands, duodenal crypts of Lieberkühn, and pancreatic islets; in the gastrointestinal tract, sorbin coexisted with Met-enkephalin or with substance P in a small fraction of serotonin-storing [enterochromaffin (ED)] cells, i.e. EC2 cells and EC1 cells, respectively; in the pancreas, sorbin coexisted with insulin in the beta-cells, also considered as serotonin-storing cells in the pig, and with EC cells in the exocrine pancreas. An enteric neuronal system containing sorbin was also reported. Our results demonstrate that sorbin is a component of the serotonin-storing cell type in the porcine gastrointestinal tract and pancreas, and suggest potential directions to investigate the functions of this new regulatory peptide.     

Regulation of E-cadherin-mediated adhesion in Langerhans cell-like dendritic cells by inflammatory mediators that mobilize Langerhans cells in vivo

Adhesion of Langerhans cells (LC) to keratinocytes is mediated by E-cadherin. IL-1, TNF-alpha, and LPS mobilize LC from epidermis and presumably attenuate LC-keratinocyte adhesion. To determine whether these mediators modulated LC E-cadherin-dependent adhesion directly, we characterized their effects on LC-like dendritic cells expanded from murine fetal skin (FSDDC). FSDDC were propagated from day 16 C57BL/6 fetal skin and isolated as aggregates (FSDDC-A) in which homophilic adhesion was mediated by E-cadherin. IL-1, TNF-alpha, and LPS induced dissociation of FSDDC-A that began within 4 to 8 h and was complete within 20 h. Anti-IL-1RI mAb inhibited disaggregation caused by IL-1alpha and IL-1beta, but not that induced by TNF-alpha or LPS. Anti-TNF-alpha mAb inhibited the effect of TNF-alpha and LPS, but not that caused by IL-1alpha or IL-1beta. Flow cytometry of FSDDC-A revealed that IL-1, TNF-alpha, and LPS induced increased expression of MHC class II, CD40, and CD86 and decreased E-cadherin expression that was temporally related to dissociation of aggregates. IL-1 and TNF-alpha caused a rapid reduction in FSDDC E-cadherin mRNA levels that preceded the decrease in E-cadherin surface expression. These results demonstrate that cytokines that induce LC emigration in vivo act directly on LC-like cells in vitro, reduce E-cadherin mRNA levels, down-regulate E-cadherin surface expression, and induce a loss of E-cadherin-mediated adhesion.     

Clonidine reduces dopamine and increases GABA in the nucleus accumbens: an in vivo microdialysis study

The spectrum of infectious disease (ID) emergencies in hospitalized patients was assessed in a prospective study of 3,626 inpatient ID consultations in a 1,350-bed teaching hospital. ID emergencies, defined by a need or anticipated need for advanced life support or by irreversible organ damage leading to permanent functional loss, were encountered in 175 patients. Infections of the central nervous system (26.3%), cardiovascular system (14.9%), alimentary system (13.1%), and lower respiratory tract (7.4%) and adverse reactions to antimicrobial agents (7.4%) were most common. In 18.9% of the cases, the referring clinicians were unaware of the emergency at the time of referral. Drug reactions (46.1%), severe alimentary and peritoneal infections (32.0%), upper respiratory tract infections (28.6%), and skin and soft-tissue infections (27.3%) were most frequently missed. The emergency ID conditions were not recognized because they had an atypical presentation (51.5%), were not commonly seen in the referring specialty (24.2%), were due to rare organisms (15.2%), or had unusual anatomical sites of involvement (9.1%). A close liaison between clinicians and the ID team is crucial for recognition of ID emergencies at their early stages so that appropriate investigations and management can be instituted expediently, before the occurrence of irreversible damage.