Dysfunction of the non-neuronal cholinergic system in the airways and blood cells of patients with cystic fibrosis

The non-neuronal cholinergic system is widely expressed in human airways, skin and immune cells. Choline acetyltransferase (ChAT), acetylcholine and nicotine/muscarine receptors are demonstrated in epithelial surface cells, submucosal glands, airway smooth muscle fibres and immune cells. Moreover, acetylcholine is involved in the regulation of cell functions like proliferation, differentiation, migration, organization of the cytoskeleton, cell-cell contact, secretion and transport of ions and water. Cystic fibrosis (CF), the most frequent genetic disorder, is known to be caused by a mutation of the CF-gene coding for the cystic fibrosis transmembrane regulator protein (CFTR). CFTR represents a regulating transport protein for ion channels and processes involving endo- and exocytosis. Despite the identification of the genetic mutation knowledge of the underlying cellular pathways is limited. In the present experiments the cholinergic system was investigated in the peripheral blood and in the lung of CF patients undergoing lung transplantation (n=7). Acetylcholine content in bronchi and lung parenchyma of CF was reduced by 70% compared to controls (tumor-free tissue obtained from patients with lung tumor; n=13). In contrast, ChAT activity was elevated to some extent (p>0.05) in CF, and esterase activity did not differ from control. Acetylcholine content extracted from peripheral leucocytes (30 ml) was also reduced by 70% in CF (n=13) compared to healthy volunteers (n=9). Double labelling experiments with anti-CF antibodies and anti-ChAT antibodies showed a co-localization in peripheral lymphocytes, giving first evidence that CFTR may be linked with the intracellular storage/transport of non-neuronal acetylcholine. It is concluded that the non-neuronal cholinergic system is involved in the pathogenesis of CF. A reduced content of non-neuronal acetylcholine could contribute to the deleterious changes of epithelial ion and water movements in CF, because acetylcholine stimulates apical Cl(-) secretion, inhibits apical Na(+) and water absorption and therewith facilitates mucociliary clearance.     

CCL18 is expressed in atopic dermatitis and mediates skin homing of human memory T cells

CCL18 is a human chemokine secreted by monocytes and dendritic cells. The receptor for CCL18 is not yet known and the functions of this chemokine on immune cells are not fully elucidated. In this study, we describe that CCL18 is present in skin biopsies of atopic dermatitis (AD) patients but not in normal or psoriatic skin. CCL18 was specifically expressed by APCs in the dermis and by Langerhans and inflammatory dendritic epidermal cells in the epidermis. In addition, the serum levels of CCL18 and the percentages of CCL18-producing monocyte/macrophages and dendritic cells were significantly increased in AD patients compared with healthy controls. Furthermore, we demonstrate that CCL18 binds to CLA(+) T cells in peripheral blood of AD patients and healthy individuals and induces migration of AD-derived memory T cells in vitro and in human skin-transplanted SCID mice. These findings highlight a unique role of CCL18 in AD and reveal a novel function of this chemokine mediating skin homing of a subpopulation of human memory T cells.     

Increase in NGF content and nerve fiber sprouting in human allergic contact eczema

There is increasing evidence for an intimate interaction of the skin and the nervous system. As known from animal studies, nerve growth factor (NGF) is essential for the innervation density and functional properties of sensory neurons of the skin during embryogenesis and in adulthood, and possibly during cutaneous inflammation. This study examined NGF content and sprouting of nerves during the elicitation phase of contact allergy in human skin. Skin biopsies from patients (n=14) undergoing patch-testing were taken from positive test sites and control back skin 96 h after antigen application. NGF content was measured by enzyme-linked immunofluorescence assay. Immunohistochemistry was performed for protein gene product 9.5 (PGP9.5), a marker that stains all neuronal elements, and growth-associated protein 43 (GAP43), a marker for axonal growth cones. The NGF content was significantly increased in lesional skin in comparison with normal skin (4.2+/-0.6 pg to 2.9+/-0.5 pg NGF per mg wet weight). The length of epidermal PGP9.5-immunoreactive (ir) fibers in lesional skin significantly increased from 3.4+/-0.9 mm in normal skin to 5.3+/-1.0 mm in contact eczema, whereas dermal fibers were unaltered (11.1+/-2.7 mm vs 9.5+/-2.1 mm, respectively). GAP43-ir nerve endings were significantly increased in both epidermis (1.6+/-0.3 mm to 2.6+/-0.4 mm) and dermis (0.5+/-0.1 mm to 1.8+/-0.2 mm) in contact eczema. Thus, we have provided evidence for an NGF-mediated nerve-fiber sprouting in human contact eczema. This may have a functional impact on skin-associated immune cells, in particular mast cells and Langerhans cells.     

Influence of a genetic variant of CHAT gene over the profile of plasma soluble ChAT in Alzheimer disease

The choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are fundamental to neurophysiological functions of the central cholinergic system. We confirmed and quantified the presence of extracellular ChAT protein in human plasma and also characterized ChAT and VAChT polymorphisms, protein and activity levels in plasma of Alzheimer''s disease patients (AD; N = 112) and in cognitively healthy controls (EC; N = 118). We found no significant differences in plasma levels of ChAT activity and protein between AD and EC groups. Although no differences were observed in plasma ChAT activity and protein concentration among ChEI-treated and untreated AD patients, ChAT activity and protein levels variance in plasma were higher among the rivastigmine-treated group (ChAT protein: p = 0.005; ChAT activity: p = 0.0002). Moreover, AD patients homozygous for SNP rs1880676 A allele exhibited higher levels of ChAT activity. Considering this is the first study to report the influence of genetic variability of CHAT locus over ChAT activity in AD patients plasma, it opens a new set of important questions on peripheral cholinergic signaling in AD.     

The number and distribution of blood dendritic cells in the epidermis and dermis of healthy human subjects

Human blood dendritic cells (BDC) can be divided into three subsets: plasmacytoid DC (PDC) and two myeloid subsets--MDC1 and MDC2. Several studies revealed the presence of both MDC and PDC in blood of healthy subjects, however no precise literature data exist on the number and distribution of BDC in the skin. The aim of our study was to assess the number and distribution of BDC and their subtypes in the healthy skin. The-study included 30 healthy volunteers (age 18-51). Punch biopsies were taken from the buttock skin from each subject, and immunofluorescent staining was performed using monoclonal mouse IgG1 antibodies directed against BDCA-1, BDCA-2, BDCA-3 and BDC-4. The BDC were present both in the epidermis and dermis. PDC were detected mainly in the dermis (mean 1.2 cells per field). Myeloid subtypes were observed mainly in the middle layers of the epidermis and in the upper part of the dermis (mean 1.8 cells per field). The detection of blood dendritic cells in the skin proves their role in immune cutaneous surveillance.     

Developmental differences in L-type calcium current of human atrial myocytes

We investigated differences in L-type Ca2+ current (ICa) between infant (INF, 1-12 mo old), young adult (YAD, 14-18 yr old), and older adult (AD) myocytes from biopsies of right atrial appendages. Basal ICa was smaller in INF myocytes (1.2 +/- 0.1 pA/pF, n = 29, 6 +/- 1 mo old, 11 patients) than in YAD (2.5 +/- 0.2 pA/pF, n = 20, 16 +/- 1 yr old, 5 patients) or AD (2.6 +/- 0.3 pA/pF, n = 19, 66 +/- 3 yr old, 9 patients) myocytes (P < 0.05). Maximal ICa produced by isoproterenol (Iso) was similar in INF, YAD, and AD cells: 8.4 +/- 1.1, 9.6 +/- 1.0, and 9.2 +/- 1.3 pA/pF, respectively. Efficacy (Emax) was larger in INF (607 +/- 50%) than for YAD (371 +/- 29%) or AD (455 +/- 12%) myocytes. Potency (EC50) was 8- to 10-fold higher in AD (0.82 +/- 0.09 nM) or YAD (0.41 +/- 0.14 nM) than in INF (7.6 +/- 3.5 nM) myocytes. Protein levels were similar for Gialpha2 but much greater for Gialpha3 in INF than in AD or YAD atrial tissue. When Gialpha3 activity was inhibited by inclusion of a Gialpha3 COOH-terminal decapeptide in the pipette, basal ICa and the response to 10 nM Iso were increased in INF, but not in YAD, cells. We propose that basal ICa and the response to low-dose beta-adrenergic stimulation are inhibited in INF (but not YAD or AD) cells as a result of constitutive inhibitory effects of Gialpha3.     

The cutaneous non-neuronal cholinergic system and smoking related dermatoses: studies of the psoriasis variant palmoplantar pustulosis

Palmoplantar pustulosis (PPP) is probably the inflammatory skin disease most strongly associated to smoking. The disease is common in middle-aged, smoking women, and is chronic, sometimes disabling and characterized by pustules, erythema and scaling on the soles and palms. It is often treatment-resistant. PPP patients have a co-morbidity with an increased risk of autoimmune thyroid disease, celiac disease/gluten intolerance, abnormal calcium homeostasis, diabetes type 2, and depression. The sweat gland apparatus is involved in the pathogenesis of PPP since a) the normal structure of the acrosyringium is abolished so the keratin pattern differs to that in normal palmar skin; b) granulocytes migrate outwards in the acrosyringium forming the pustule in the stratum corneum. Acetylcholine (ACh) is the main inducer of sweating. With immunohistochemistry the ACh synthesizing enzyme choline acetyltransferase (ChAT) and the ACh-degrading enzyme acetylcholinesterase (AChE) were found to be strongly expressed in the gland and duct as were the alpha-3 and alpha-7 nicotinic acetylcholine receptors (nAChRs). Smoking influenced the staining intensity of the enzymes and the alpha-3 nAChR in healthy subjects. In involved PPP skin there was a massive infiltration of granulocytes expressing ChAT and alpha-3 nAChR, and mast cells expressing AChE indicating a role for acetylcholine in inflammation. Cessation of smoking resulted in fewer pustules, and less scaling and erythema. The mechanisms for the effect of nicotine/smoking in PPP are still unknown but nicotine may lead to enhanced inflammation in consideration of the properties of the sweat duct and/or nicotine might facilitate autoimmune reactions.     

High-Level Synthesis and Fate of Acetylcholine in Baculovirus-Infected Cells: Characterization and Purification of Recombinant Rat Choline Acetyltransferase

Rat choline acetyltransferase (ChAT) has been expressed at a high level in Spodoptera frugiperda Sf9 cells using a baculovirus expression system. A cDNA containing the coding sequence for ChAT was inserted into the transfer vector pAcYM1 to yield the recombinant vector pAcYM1/ChAT. Sf9 cells were then coinfected with pAcYM1/ChAT and the wild-type Autographa californica virus. One recombinant virus particle, containing the cDNA for ChAT, was selected that expressed a protein of 68.5 kDa. Forty hours after infection of cells with the recombinant virus, the specific activity of ChAT in the cytosol was 190 nmol of acetylcholine/min/mg of protein, accounting for approximately 24% of the cell cytosolic proteins as being ChAT. The apparent Km values of the enzyme for choline and acetyl-CoA were 299 and 221 microM, respectively, whereas the respective Vmax values were 10.6 and 11.4 mumol of acetylcholine/min/mg of protein. In addition, analysis of the protein revealed that ChAT is phosphorylated in Sf9 cells. About 0.5 mg of ChAT was obtained from a one-step purification procedure starting with 10(8) infected Sf9 cells. Addition of choline to the incubation medium led to accumulation of high amounts of acetylcholine in the cytosol of the infected cells. The neurotransmitter was not released by Sf9 cells in response to membrane depolarization or on ionophore-mediated calcium entry. Some acetylcholine, which most likely originated from cell death inherent to viral infection, accumulated in the culture medium. The infected insect cells, which synthesize and store neurotransmitter, provide a new and convenient model for analyzing synaptic transmission at the molecular level.     

Expression and function of the non-neuronal cholinergic system in endothelial cells

Increasing evidence has shown the expression of the non-neuronal cholinergic system in endothelial cells. In the present experiments the expression of choline acetyltransferase (ChAT) was investigated in human endothelial cells by anti-ChAT immunohistochemistry and anti-ChAT immunofluorescence. Positive ChAT immunoreactivity was found in cultures of human umbilical endothelial cells (HUVEC) and a human angiosarcoma cell line (HAEND). In HUVEC and HAEND choline acetyltransferase activity and small amounts of acetylcholine were also detected. Positive ChAT-immunoreactivity was demonstrated in situ in endothelial cells of the human umbilical cord. In addition, in experiments with confocal laser scanning microscopy positive anti-ChAT immunoreactivity was found in situ in endothelial cells of human skin blood vessels. In the first functional experiments with HUVEC acetylcholine appeared to mediate a small facilitatory effect on the expression of intracellular adhesion molecule-1. The present experiments demonstrate the wide existence of ChAT in human endothelial cells. Further experiments are addressed to elucidate the biological role of acetylcholine in the endothelium and possible differences between the different subtypes of endothelial cells.     

Eosinophil degranulation. An immunologic determinant in the pathogenesis of the Mazzotti reaction in human onchocerciasis

Onchocerciasis patients treated with diethylcarbamazine often undergo a severe inflammatory response, the Mazzotti reaction. To assess the eosinophil's role in the pathogenesis of the Mazzotti reaction, we obtained serial blood, plasma, and skin biopsy specimens from 21 heavily infected patients and 3 endemic controls, both before and during therapy with diethylcarbamazine. Samples were analyzed for blood eosinophils, plasma levels of eosinophil granule major basic protein (MBP) and eosinophil-derived neurotoxin, eosinophil infiltration and eosinophil and mast cell degranulation in the skin. After the first dose of diethylcarbamazine, blood eosinophils fell from a pre-treatment level of 888 +/- 111 to 203 +/- 42 cells/mm3 at 8 h. This decrease was followed by a marked eosinophilia developing over the remaining 7 days of treatment and 14 days of follow-up. Plasma eosinophil-derived neurotoxin levels increased from 56 +/- 4 ng/ml pretreatment to a peak of 82 +/- 9 ng/ml at 8 h and returned to pretreatment levels by 48 h. Beginning at 12 h, plasma MBP levels increased from 730 +/- 74 ng/ml pretreatment to a peak of 1140 +/- 74 ng/ml after 5 days. Pretreatment skin biopsies stained for MBP by immunofluorescence showed a bright fibrillar pattern in the dermis consistent with chronic eosinophil degranulation; the MBP was localized on elastic tissue fibers. After treatment, skin biopsy specimens showed both the pretreatment fibrillar MBP staining pattern as well as focal eosinophil degranulation. Deposition of MBP around microfilariae in the papillary dermis was visible as early as 1.5 h. The lowest blood eosinophil levels and peak plasma eosinophil-derived neurotoxin levels coincided with the infiltration and degranulation of eosinophils in the skin. Mast cell degranulation in the skin was maximal by the first posttreatment biopsy (1.5 h) coincident with the beginning of eosinophil degranulation. Although the pathogenesis of the Mazzotti reaction is clearly complex, our results indicate that eosinophil degranulation is characteristic of the response and that it occurs with a time course suggestive of a role for the eosinophil in determining the clinical and pathologic manifestations of the reaction.     

Selective recruitment of endothelial progenitor cells to ischemic tissues with increased neovascularization

Tissue ischemia remains a common problem in plastic surgery and one for which proangiogenic approaches have been investigated. Given the recent discovery of circulating endothelial stem or progenitor cells that are able to form new blood vessels, the authors sought to determine whether these cells might selectively traffic to regions of tissue ischemia and induce neovascularization. Endothelial progenitor cells were isolated from the peripheral blood of healthy human volunteers and expanded ex vivo for 7 days. Elevation of a cranially based random-pattern skin flap was performed in nude mice, after which they were injected with fluorescent-labeled endothelial progenitor cells (5 x 10(5); n = 15), fluorescent-labeled human microvascular endothelial cells (5 x 10(5); n = 15), or media alone (n = 15). Histologic examination demonstrated that endothelial progenitor cells were recruited to ischemic tissue and first appeared by postoperative day 3. Subsequently, endothelial progenitor cell numbers increased exponentially over time for the remainder of the study [0 cells/mm2 at day 0 (n = 3), 9.6 +/- 0.9 cells/mm2 at day 3 (n = 3), 24.6 +/- 1.5 cells/mm2 at day 7 (n = 3), and 196.3 +/- 9.6 cells/mm2 at day 14 (n = 9)]. At all time points, endothelial progenitor cells localized preferentially to ischemic tissue and healing wound edges, and were not observed in normal, uninjured tissues. Endothelial progenitor cell transplantation led to a statistically significant increase in vascular density in ischemic tissues by postoperative day 14 [28.7 +/- 1.2 in the endothelial progenitor cell group (n = 9) versus 18 +/- 1.1 in the control media group (n = 9) and 17.7 +/- 1.0 in the human microvascular endothelial cell group (n = 9; p < 0.01)]. Endothelial progenitor cell transplantation also showed trends toward increased flap survival [171.2 +/- 18 mm2 in the endothelial progenitor cell group (n = 12) versus 134.2 +/- 10 mm2 in the media group (n = 12) and 145.0 +/- 13 mm2 in the human microvascular endothelial cell group (n = 12)], but this did not reach statistical significance. These findings indicate that local tissue ischemia is a potent stimulus for the recruitment of circulating endothelial progenitor cells. Systemic delivery of endothelial progenitor cells increased neovascularization and suggests that autologous endothelial progenitor cell transplantation may have a role in the salvage of ischemic tissue.     

A comparison of the proinflammatory effects of 12(R)- and 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid in human skin

Topical application of racemic 12-hydroxy-5,8,10,14-eicosatetraenoic acid [12(R,S)-HETE] produces erythema and leucocyte accumulation in human skin. Since 12(R)-HETE is more potent than its epimer 12(S)-HETE as a neutrophil chemoattractant in vitro, their proinflammatory effects have now been compared in vivo. 12(R)- and 12(S)-HETE (0.5 - 20 ug/site) were applied topically to the forearm skin of 5 healthy volunteers and the sites occluded for 6 h. Five ug each of the two enantiomers were also applied to the opposite forearm. At 6 and 24 h blood flow and the areas of erythematous responses were measured. The 5 ug application sites were biopsied at 24 h. Both enantiomers caused dose related erythema and increased blood flow at 6 and 24 h, which were not significantly different at either of the time points tested. In contrast, pronounced neutrophil infiltrates were seen in the epidermis (25.2 +/- 13 cells/hpf) and dermis (13.2 +/- 5.1 cells/hpf) 24 h after application of 12(R)-, but not 12(S)-HETE (0.02 +/- 0.02 and 1.02 +/- 0.7 cells/hpf in epidermis and dermis respectively). However, the numbers of dermal mononuclear cells accumulating in response to the two enantiomers were similar. 12(R)-HETE thus appears to be a more potent neutrophil chemoattractant than 12(S)-HETE in human skin in vivo and may be of potential importance as a mediator of inflammation in man.     

Chemokine-Like Factor 1 (CLFK1) Is Over-Expressed in Patients with Atopic Dermatitis

Background: Human chemokine-like factor 1 (CKLF1), a recently discovered chemokine, has a broad spectrum of biological functions in immune-mediated diseases. It is highly expressed on Th2 lymphocytes and is a functional ligand for human CCR4. CKLF1 has a major role in the recruitment and activation of leucocytes, which plays an important role in the pathogenesis of allergic diseases. The present study was designed to determine the expression of CKLF1 in skin and serum in patients with atopic dermatitis (AD).Methods: The CKLF1 protein expression in skin lesion was analyzed by immunohistochemistry and ELISA. The mRNA expression of CKLF1 in skin lesion was detected by Real-time PCR. The serum levels of CKLF1, IgE, eotaxin, IL-4, IL-5, and IL-13 were measured by ELISA.Results: Histopathological changes in the skin of AD patients showed local inflammation with epidermal thickening and significant inflammatory cellular infiltration. Immunohistochemistry results demonstrated that CKLF1-staining positive cells were located in the epidermal and dermis, and that the CKLF1 expression in AD patients was significantly higher than that in normal control. The CKLF1 mRNA expression in AD patients was significantly higher than that in healthy controls. Serum CKLF1 and IgE levels were significantly increased in AD patients, as were the serum levels of IL-4, IL-5, IL-13 and eotaxin.Conclusions: Both CKLF1 protien and mRNA levels are overexpressed in the skin lesion of AD patients, along with an increase in serum CKLF1 level, indicating that CKLF1 may play an important role in the development of atopic dermatitis.     

Autoradiographic Imaging of [3H]Phorbol 12,13-Dibutyrate Binding to Protein Kinase C in Alzheimer''s Disease

Quantitative autoradiography was used to examine the distribution of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding to protein kinase C in the middle frontal and temporal cortices and the hippocampal region of nine control and nine elderly subjects with Alzheimer's disease (AD). AD patients had a clinical diagnosis of the disease that was confirmed neuropathologically by the presence of numerous plaques in the hippocampus and cerebral cortex. Choline acetyltransferase (ChAT) activity was significantly reduced in the middle frontal and temporal cortex and in the hippocampus of AD subjects, with the deficit being greater than 60% of control values. Quantitative autoradiographic analysis of [3H]PDBu binding to protein kinase C revealed a heterogeneous pattern in control brain, being particularly high in superficial layers of the cortex and CA1 of the hippocampus. There were no significant differences between control and AD sections in all areas examined within the middle frontal cortex; e.g., layers I-II control, 491 +/- 46 versus AD, 537 +/- 39 pmol/g of tissue; middle temporal cortex, e.g., layers I-II control, 565 +/- 68 versus AD, 465 +/- 72 pmol/g of tissue; and hippocampal formation, e.g., CA1 control, 511 +/- 28 versus AD, 498 +/- 25 pmol/g of tissue. In a parallel study, [3H]PDBu binding to homogenate preparations of control and AD brain confirmed that there was no significant difference in [3H]PDBu binding in either the particulate or the cytosolic fraction. We have demonstrated in a well-defined population of AD patients that [3H]PDBu binding to protein kinase C remains preserved in brain regions that are severely affected by the neuropathological and neurochemical correlates of AD.     

Decreased IL-15 may contribute to elevated IgE and acute inflammation in atopic dermatitis.

PBMC and acute skin lesions of patients with atopic dermatitis (AD) are characterized by increased IL-4 and IL-13, but decreased IFN-gamma production. This bias toward an increased Th2 cytokine profile may contribute to the elevated IgE levels and acute skin inflammation seen in AD. In this study, we examined the levels of IL-15, a Th1-like cytokine, in the PBMC and the skin lesions of AD patients. IL-15 secretion by Staphylococcal enterotoxin B-treated PBMC of AD patients was significantly lower than that of normals and psoriasis patients (p < 0.001). Membrane-bound IL-15 expression as measured by mean fluorescence intensity and percentage of IL-15-positive cells in Staphylococcal enterotoxin B-treated monocytes of AD patients (644 +/- 49% and 12.7 +/- 0.6%, respectively) were significantly lower than that of normals (869 +/- 56% and 15.8 +/- 1.2%, respectively) and psoriasis patients (1488 +/- 217% and 22.7 +/- 0.8%, respectively; p < 0.0007 and p < 0.0001, respectively). The membrane-bound IL-15 expression was also significantly lower in the control monocytes of AD patients compared with that in normals and psoriasis patients. There was no significant difference in the absolute number or percentage of monocytes between the study subjects. However, psoriasis skin lesions were found to have significantly more IL-15 mRNA-expressing cells (22.4 +/- 1.7) compared with that in acute AD (7.5 +/- 1.7) and chronic AD (13.7 +/- 1.7) skin lesions (p < 0.05). IL-15 enhanced IFN-gamma production by the PBMC of AD patients (p < 0.01), but not by that of normal individuals or psoriasis patients. In addition, IL-15 was found to suppress IgE synthesis (p < 0.01) by the PBMC of AD patients. These data support the concept that reduced IL-15 expression may contribute to the pathogenesis of AD.     

Properties of multiple kinetic forms of soluble cyclic nucleotide phosphodiesterase activity of rat colonic mucosa

Soluble phosphodiesterase (EC 3.1.4.1) activity is 3-5-fold lower in superficial colonic epithelial cells compared to that in cells isolated from the lower colonic crypt. Higher phosphodiesterase activity in lower crypt cells is correlated with a 5-fold higher rate of incorporation of [3H]thymidine into DNA in these cells. DEAE-cellulose chromatography of the soluble fraction of superficial and proliferative colonic epithelial cells resulted in separation of three enzyme forms: (1) fraction I, an enzyme which hydrolyzes both cAMP and cGMP with high affinity (apparent Km cAMP = 5 +/- 1 microM, Km cGMP = 2.5 +/- 0.5 microM) and is stimulated 3-6-fold by Ca2+ plus calmodulin; (2) fraction II, a form which hydrolyzes both cAMP and cGMP with low affinity (S0.5 cAMP = 52 +/- 7 microM, S0.5 cGMP = 17 +/- 4 microM), exhibits positive copperativity with respect to substrate and shows cGMP stimulation of cAMP hydrolysis and (3) fraction III, a cAMP-specific form which exhibits biphasic kinetics, a low Km for cAMP (Km cAMP = 5 +/- 1 microM) and does not hydrolyze cGMP. The pattern of distribution of phosphodiesterase activities on DEAE-cellulose was similar in superficial and proliferative colonic epithelial cells. The higher specific activity in proliferative cells was reflected in higher activities of each of the three chromatographically distinct forms of the enzyme. In contrast to epithelial cells, the soluble fraction of homogenates of the submucosa and supporting cells exhibited phosphodiesterase forms I and II and was lacking in the form corresponding to fraction III of epithelial cells.     

Increase of Choline Acetyltransferase by Colchicine in Primary Cell Cultures of Spinal Cord

Abstract: Colchicine (5–10 μ M ) increased choline ace-tyltransferase (ChAT) activity 5–10-fold and suppressed acetylcholinesterase (AChE) and glutamate decarboxylase (GAD) activities to 30% and 50%, respectively, of the levels of control cells in mouse spinal cord cells cultured for several days. The synthesis of radiolaheled acetylcholine (ACh) from [¹⁴C]choline was also enhanced 4.6-fold, although the uptake of [¹⁴C]choline into cells was decreased to 80% of control level. Neither the incorporation of [³H]Ieucine into protein nor the total amount of protein was increased by colchicine. Vinblastine also increased ChAT activity while cytochalasin B was not effective. Immunochemical titration study revealed that the increase of ChAT activity by colchicine was due to the accumulation of ChAT molecules. Co-culture of spinalcord cells with skeletal muscle markedly stimulated ChAT activity, and the addition of colchicine to the co- cultures showed greater than additive effect. These observations indicate that colchicine increases ChAT molecules in a specific manner, that the stimulatory effect of colchicine on ChAT activity is possibly mediated via the interaction with microtubules, and that the increase of ChAT activity is based on a mechanism different from that of co-cultures with skeletal muscle cells.     

Mental Stress in Atopic Dermatitis – Neuronal Plasticity and the Cholinergic System Are Affected in Atopic Dermatitis and in Response to Acute Experimental Mental Stress in a Randomized Controlled Pilot Study

Rationale

In mouse models for atopic dermatitis (AD) hypothalamus pituitary adrenal axis (HPA) dysfunction and neuropeptide-dependent neurogenic inflammation explain stress-aggravated flares to some extent. Lately, cholinergic signaling has emerged as a link between innate and adaptive immunity as well as stress responses in chronic inflammatory diseases. Here we aim to determine in humans the impact of acute stress on neuro-immune interaction as well as on the non-neuronal cholinergic system (NNCS).

Methods

Skin biopsies were obtained from 22 individuals (AD patients and matched healthy control subjects) before and after the Trier social stress test (TSST). To assess neuro-immune interaction, nerve fiber (NF)-density, NF-mast cell contacts and mast cell activation were determined by immunohistomorphometry. To evaluate NNCS effects, expression of secreted mammal Ly-6/urokinase-type plasminogen activator receptor-related protein (SLURP) 1 and 2 (endogenous nicotinic acetylcholine receptor ligands) and their main corresponding receptors were assessed by quantitative RT-PCR.

Results

With respect to neuro-immune interaction we found higher numbers of NGF+ dermal NF in lesional compared to non-lesional AD but lower numbers of Gap43+ growing NF at baseline. Mast cell-NF contacts correlated with SCORAD and itch in lesional skin. With respect to the NNCS, nicotinic acetylcholine receptor α7 (α7nAChR) mRNA was significantly lower in lesional AD skin at baseline. After TSST, PGP 9.5+ NF numbers dropped in lesional AD as did their contacts with mast cells. NGF+ NF now correlated with SCORAD and mast cell-NF contacts with itch in non-lesional skin. At the same time, SLURP-2 levels increased in lesional AD skin.

Conclusions

In humans chronic inflammatory and highly acute psycho-emotional stress interact to modulate cutaneous neuro-immune communication and NNCS marker expression. These findings may have consequences for understanding and treatment of chronic inflammatory diseases in the future.     

Setup and dosimetry for exposure of human skin in vivo to RF-EMF at 900 MHz

The aim of this study was a dosimetrical analysis of an experimental setup used in the exposure of 10 female volunteers to GSM 900 radiation. The exposure was carried out by irradiating a small region of the right forearms of the volunteers for 1 h, after which biopsies were taken from the exposed skin for protein analysis. The source of irradiation was a half-wave dipole fed with a computer controlled GSM phone. The specific absorption rate (SAR) induced in the skin biopsy was assessed by computer simulations. The numerical model of the arm consisted of a muscle tissue simulating cylinder covered with thin skin (1 mm) and fat (3 mm) layers. The simulation models were validated by measurements with a homogeneous cylindrical liquid phantom. The average SAR value in the biopsy was 1.3 W/kg and the estimated uncertainty +/-20% (K = 2). The main source of error was found to be variations in the distance of the forearm from the dipole (10 +/- 1 mm). Other significant sources of uncertainty are individual variations of the fat layer and arm thicknesses, and the uncertainty of radio frequency (RF) power measurement.