Logo: to the web site of Uppsala University

Avian antibodies have many advantages to mammalian antibodies due to the phylogenetic differences between birds and mammals, resulting in an increased sensitivity and a decreased background in many immunological assays. Since the avidin-biotin system is an efficient detection system for antibodies with a high sensitivity, we wanted to investigate the activity and unspecific binding of optimally biotin labelled chicken antibodies in ELISA and immunohistochemistry. We report on the conditions for biotinylation of chicken antibodies and that optimally biotinylated antibodies show a high activity and a low background in both ELISA and immunohistochemistry.

A proton beam of about 1 mu m allows the study of inner structures of cells. These studies demand sophisticated preparation methods, not to destroy the morphology or the elemental distribution. Analysing a well-preserved cell may lead to important knowledge about basic regulatory processes at the cellular level. Freezing followed by removal of water by drying or by substitution with an organic solvent will be exemplified. Insulin-producing cells were studied to reach a further understanding of the signal transduction between stimulation to secrete insulin and the secretion.

The impact of the nerve growth factor (NGF) family of neurotrophins and their receptors was examined on the cutaneous innervation in the mystacial pads of mice. Ten sets of unmyelinated and thinly myelinated sensory and autonomic innervation were evaluated that terminated in the epidermis, upper dermis, and upper part of the intervibrissal hair follicles. Mystacial pads were analyzed from newborn to 4-week-old mice that had homozygous functional deletions of the genes for NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), tyrosine kinase (trk) A, trkB, trkC, or p75. Mystacial pads were also analyzed in adult transgenic mice that had overproduction of NGF, BDNF, or NT-3 driven by a keratin promoter gene. The innervation was revealed by using immunofluorescence and immunocytochemistry with antibodies for protein gene product (PGP) 9.5, calcitonin gene-related product (CGRP), substance P (SP), galanin (GAL), neuropeptide Y (NPY), tyrosine hydroxylase (TH), and a neurofilament protein. The cumulative results indicated that NGF/trkA signaling plays a major role in the outgrowth and proliferation of sensory axons, whereas NT-3/ trkA signaling plays a major role in the formation of sensory endings. TrkC is also essential for the development of three sets of trkA-dependent sensory innervation that coexpress CGRP, SP, and GAL. Another set of sensory innervation that only coexpressed CGRP and SP was solely dependent upon NGF and trkA. Surprisingly, most sets of trkA-dependent sensory innervation are suppressed by trkB perhaps interacting with p75. BDNF and NT-4 appear to mediate this suppressing effect in the upper dermis and NT-4 in the epidermis. In contrast to sensory innervation, sympathetic innervation to the necks of intervibrissal hair follicles depends upon NGF/trkA signaling interacting with p75 for both the axon outgrowth and ending formation. Although NT-3/trkA signaling is essential for the full complement of sympathetic neurons, NT-3 is detrimental to the formation of sympathetic terminations to the necks of hair follicles. TrkB signaling mediated by BDNF but not NT-4 also suppresses these sympathetic terminations. One sparse set of innervation, perhaps parasympathetic, terminating at the necks of hair follicles is dependent solely upon NT-3 and trkC. Taken together, our results indicate that the innervation of the epidermis, upper dermis, and the upper portion of hair follicles is regulated by a competitive balance between promoting and suppressing effects of the various neurotrophins.

The neutrophilic granulocyte is a cytotoxic and potentially tissue-injuring cell participating in the destructive processes and symptoms seen in a variety of inflammatory diseases. Sensitive immunoassays have been introduced to measure the levels of specific secretory proteins of various inflammatory cells in blood and other body fluids. The aim has been to develop highly specific markers for each cell type. The results obtained by immunoassay have indicated that human neutrophil lipocalin (HNL) is a protein unique to the neutrophil. The present study investigated the specificity of HNL as a neutrophil marker in peripheral blood and lung tissue by using flow cytometry and immunocytochemistry. Flow cytometry and immunocytochemistry on peripheral blood showed that monoclonal antibodies to HNL only react with neutrophils and not with other types of leukocytes. Immunocytochemistry on plastic-embedded sections and on frozen sections of lung tissue showed that a cocktail of six monoclonal antibodies to HNL specifically reacts with neutrophils and not with epithelial cells or macrophages. By immunoelectron microscopical studies performed on healthy human neutrophils after low temperature embedding in Lowicryl K4M following aldehyde fixation and partial dehydration, it could be shown that HNL colocalized with lactoferrin (a known marker for secondary or specific granules) and that myeloperoxidase was localized in the primary or azurophil granules. The results confirm that HNL is a unique component of the secondary granules of the neutrophil granulocyte.

Several aspects of Cl^- secretion in epithelial cells were investigated. Methods to prepare cultured cellsfor X-ray microanalysis, in order to study volume-regulating chloride transport were evaluated. Inaddition, α2A-adrenergic, muscarinic cholinergic, and purinergic Cl^- efflux as well as Cl^- effluxregulated by G-protein coupled receptors in HT29 cells was investigated. The purpose of the studywas to describe the regulation of Cl^- transport in intestinal epithelial cells.

Exposure of HT29 cells to a hypotonic solution resulted in regulatory volume decrease (RVD)accompanied by loss of K and Cl from the cells, whereas exposure to hypertonic solutions resultedin regulatory volume increase (RVI) coupled to gain of Na and Cl. Chloride channel blockers, suchas NPPB and DPC, blocked RVD, accompanied by a decrease in intracellular Na, Cl and Kconcentration, suggesting that chloride channels are important in volume regulating ion transport.

Stimulation with UTP or ATP mainly induced loss of Cl and Na from the cells, concurrent with an increase in Ca. The ion fluxes induced by UTP could be blocked by NPPB and alloxan (aninhibitor of adenylate cyclase), indicating that UTP-mediated Cl^- secretion may occur through bothcAMP- and Ca²⁺-dependent pathways.

The agonists of muscarinic receptors, carbachol and acetylcholine, caused a decrease inintracellular Cl and K concentration. However, P-F-HHSiD, an antagonist of muscarinic 3 receptors,inhibited secretion induced by carbachol or acetylcholine, suggesting that the ion secretion is mediatedby muscarinic 3 receptors. Moreover, the ion secretion could be inhibited by U-73122, an inhibitorof phospholipase C (PLC), indicating that secretion may be regulated by the PLC/Ca²⁺ dependent pathway.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide(PACAP) elicited Cl^- and K⁺ efflux, whereas UK14,304 and somatostatin-14 were able to reverse VIPor PACAP induced secretion, giving rise to an increase in the content of intracellular Na and Cl. Theantisecretory effect of UK14,304 and somatostatin-14 is probably involved in the inhibition of bothadenylate cyclase and PLC pathways by relevant receptors coupled to G_i Use of U-73122 and alloxantogether inhibited PACAP-induced Cl^- efflux, indicatingthat Cl^- secretion may not only be mediatedby cAMP activated Cl^- channels, but also by Ca²⁺ activated Cl^- channels.