Georg Nørgaard Hansen

Induction of experimental chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in alginate microspheres

Alginate‐producing, mucoid P. aeruginosa is frequently found in the lungs of patients with cystic fibrosis (CF), where it causes a chronic infection. The importance of alginate in the pathogenesis was demonstrated by the ability to establish chronic P. aeruginosa lung infection in rats if P. aeruginosa entrapped in minute alginate‐beads were inoculated transtracheally. Alginate beads containing P. aeruginosa were formed by nebulizing a suspension of seaweed sodium‐alginate and P. aeruginosa into a calcium solution. The alginate bead method of establishing infection was compared to an agar‐bead method and proved to be quantitatively similar after 4 weeks. The ability of the two methods to induce formation of precipitins, IgA and IgG antibodies against P. aeruginosa antigens, including outer membrane proteins, flagella, exoenzymes and alginate, was assessed by crossed immunoelectrophoresis, enzyme‐linked immunosorbent assay and immunoblotting. The two methods of inducing infection were comparable and infected rats had significantly higher antibody response than rats inoculated with sterile beads. We suggest that the alginate bead model closely resembles the later stages of CF‐lung infection and that it offers the theoretical advantage of using a substance which is chemically similar to the alginate produced in vivo by P. aeruginosa.

Neuropeptides in Cnidarians

Cnidarians are real neuropeptide factories. From a single sea anemone species, 17 different neuropeptides have been isolated, and we believe that this is only the tip of the iceberg. A similar picture is now emerging from Hydra. Cnidarian neuropeptides can be neurotransmitters or neuromodulators involved in signal transduction, but also neurohormones that steer developmental processes such as metamorphosis. Cnidarians synthesize their neuropeptides as preprohormones of varying sizes that may contain up to 38 neuropeptide copies per precursor molecule. The cnidarian prohormones are processed by both known and unknown (novel) processing enzymes. We have cloned the enzymes that are responsible for neuropeptide C-terminal amidation, showing that at least two enzyme genes are involved in this two-step reaction (in contrast to one gene in mammals). By using neuropeptide immunocytochemistry, we found that the cnidarian nervous system is more sophisticated than we believed before, having neuronal concentrations in the form of ganglion-like structures, neuronal plexuses and nerve tracts. By using a whole-mount two-colour in situ hybridization technique and RNA probes coding for various Hydra preprohormones, we found that Hydra has at least six different populations of nerve cells, of which some coexpress two different preprohormone mRNAs. This is the first example of coexpression of two well-characterized preprohormones (yielding two well-characterized neurohormone families) in cnidarians.

Immunoreactive material resembling vertebrate neuropeptides in the corpus cardiacum and corpus allatum of the insect Leucophaea moderae

SummaryThe presence and differential distribution of substances antigenically related to known vertebrate neuropeptides demonstrated within the corpus cardiacum of the insect Leucophaea are as follows: Of ten mammalian antisera tested, six yielded substantial immunoreactive deposits resembling oxytocin, somatostatin, Substance P, met-enkephalin, bombesin, and neurotensin, respectively. In the remaining four, the reaction was moderate (vasopressin, β-endorphin) or marginal (LH-RF, calcitonin). With regard to their regional distribution, these biochemically distinct reaction products seem to fall into two groups: (1) Materials resembling oxytocin, vasopressin, met-enkephalin, β-endorphin (and presumably also neurotensin and LH-RF) predominate in the central release area of the organ and are considered to be of extrinsic (cerebral) origin. (2) Substances localized primarily in areas rich in intrinsic glandular cells of the corpus cardiacum, and revealed by antisera raised against somatostatin, Substance P, and bombesin, are judged to be synthesized and stored within this organ. In peptidergic fibers entering the adjacent corpora allata, thus far Substance P-, β-endorphin-, and LH-RF-like immunoreactivities have been demonstrated. Some of these “new” neuropeptides may be contained in classical neurosecretory neurons, formerly identified by less specific methods, others must be assigned to additional peptidergic neurons heretofore unknown.

Epidermal growth factor receptor expression on oral mucosa dysplastic epithelia and squamous cell carcinomas.

SummaryThe expression of the receptor for epidermal growth factor (EGF) has been determined on oral squamous cell carcinomas. Immunoreactive receptor was localized using a monoclonal anti-EGF-receptor antibody which reacts with sequences in the external domain of the receptor. Frozen sections were studied from 40 patients with squamous cell carcinomas. In 16 sections from the patients with the squamous cell carcinomas, normal differentiated oral mucosa was included and in 7 of these the patients had received preoperative radiotherapy. Sections from 6 other patients with squamous cell carcinoma contained dysplastic epithelia. EGF-receptor-positive cells were present in the basal cell layer on normal differentiated oral mucosa. In sections from patients receiving preoperative radiotherapy the EGF-receptor-positive cells were also found in the spinous cells. In dysplastic epithelia nearly all cells stained for the receptor. The distribution and staining intensity of the EGF receptor varied in the oral squamous cell carcinomas, 36 were positive. The staining pattern in the carcinomas obtained from patients receiving preoperative radiotherapy was not altered qualitatively. Nearly all poorly differentiated cells were stained, but when the tumor was moderately to well differentiated a reduction in the extent of staining in certain areas was seen, paralleling the findings observed in the differentiated upper layers of the normal oral mucosa. This was most pronounced for the epithelial pearls, where the EGF-receptor-positive cells were localized to the undifferentiated cells in the periphery. The results of the present investigation confirm the presence of the EGF receptor on undifferentiated cells, with the extent of the staining reaction on oral squamous cell carcinomas varying inversely with cellular differentiation.

Immunoreactive material resembling ovine prolactin in perikarya and nerve terminals of the rat hypothalamus.

SummaryThe presence of prolactin (PRL)-like material is demonstrated in the brain of rats with the aid of anti-ovine PRL (oPRL) IgG as primary antibody in the unlabeled antibody-enzyme method. Immunoreactive deposits are visualized as an intraneuronal constituent with a widespread distribution in the hypothalamus and neural lobe of the pituitary. Dense networks of reactive nerve terminals derived from two prominent fibre tracts, a ventral (VHT) and a dorsal hypothalamo-neurohypophysial tract (DHT) are seen. The VHT is confined to the median eminence and pars oralis tuberis, the DHT to the pars caudalis tuberis. Both fibre tracts pass through the infundibular stalk into the neural lobe. The origin of the immunoreactive nerve terminals can be elucidated only to some extent. The VHT gives off beaded fibres entering the ependymal and glandular layer of the median eminence. Immunoreactive perikarya are observed in the supraoptic nucleus, the paraventricular nucleus, the anterior hypothalamic nucleus, the anterior commissural nucleus, the preoptic nucleus and the interstitial nucleus of the stria terminalis. A few of the immunoreactive perikarya are observed in close connection with brain vessels and the ependymal cells of the third ventricle. The results indicate that the anti-oPRL has a unique region specificity implying that only a segment of the mammalian PRL molecule is present in these nuclei of the brain. Fragments of PRL may function as neuromodulators or neurotransmitters in the rat brain.

Immunocytochemical demonstration of somatotropin-like and prolactin-like activity in the brain of Calamoichthys calabaricus (Actinopterygii)

SummaryCellular binding of anti-bSTH and anti-oPRL IgG is demonstrated in the brain and the pituitary gland of the African freshwater fish Calamoichthys calabaricus by means of the unlabeled antibody enzyme method at the light microscopic level. In the brain, somatotropin and prolactin are demonstrated in separate neurons in the preoptic area. The somatotropinergic and prolactinergic perikarya are distinct from those of the hypothalamic-hypophysial neurosecretory neurons, i.e., those stainable with aldehyde fuchsin presumed to be vasotocinergic and isotocinergic. The somatotropinergic and prolactinergic neuronal perikarya give rise to separate beaded axons which pass either ventroposteriorly into the infundibulum, terminating in the neurohypophysis, or ventro-laterally through the wall of the preoptic recess, terminating near the superficial capillary bed covering this part of the brain surface. Moreover, coarse dendrite-like processes of both kinds of immuno-reactive neurons extend towards, and end in, the third ventricle. Binding sites in the brain to antisera against hLHβ, hFSHβ, hTSHβ and anti-(1–24) ACTH IgG, all reactive in the pituitary, are not observed in the neurons confined to the preoptic area.

Two-color double-labeling in situ hybridization of whole-mount Hydra using RNA probes for five different Hydra neuropeptide preprohormones: evidence for colocalization

Abstract. The freshwater polyp Hydra magnipapillata has a primitive nervous system that produces at least three distinct classes of neuropeptides: various peptides having the C-terminal sequence Arg-Phe-NH2 (the Hydra-RFamide family), Leu-Trp-NH2 (the Hydra-LWamide family), and a single peptide having the C-terminal sequence Lys-Val-NH2 (Hydra-KVamide). The various Hydra-RFamides are synthesized by three different preprohormones: preprohormone-A, -B, and -C. The various Hydra-LWamides are synthesized by a single preprohormone (prepro-Hydra-LWamide), as is Hydra-KVamide (prepro-Hydra-KVamide). Using a whole-mount double-labeling two-color in situ hybridization technique and RNA probes specific for each of these five Hydra preprohormone mRNAs, we found that specific sets of neurons express each of the five preprohormones, except for the peduncle region of Hydra (an area just above the basal disk), where a population of neurons exists that expresses both preprohormones-A and prepro-Hydra-KVamide mRNAs. The functional significance of this coexpression is unclear. This is the first report on the coexpression of two well-characterized preprohormones (yielding two well-characterized neurohormone families) in cnidarians. This report also shows that there are at least six neurochemically different populations of neurons in Hydra.

Vitellogenin and vitellin from the blowfly Calliphora vicina: Occurrence, purification and antigenic characterization

Abstract The occurrence and purification of vitellogenin and vitellin from Calliphora vicina Rob.-Dev. (= C. erythrocephala (Meig.)) are described together with the preparation of specific anti-vitellogenin antibodies. C. vicina vitellogenin and vitellin were purified from ovaries and eggs respectively; both proteins contain two polypeptide subunits identical to the dominating polypeptides in the growing oocytes. The polypeptides show molecular weights of 52,000 and 48,500 respectively, and are associated with carbohydrate and lipid. Polypeptides of similar size could be identified in haemolymph from yolk-depositing females, but were absent in ovariectomized females. The anti-bodies specifically precipitated the vitellogenin polypeptides from fat body homogenates of females depositing yolk or from the purified vitellogenin. Therefore, these antibodies were judged suitable for use in a study on the ultrastructural localization of vitellogenin in fat body cells ( Thomsen et al. , 1980).

Ultrastructural immunocytochemical localization of vitellogenin in the fat body of the blowfly, Calliphora vicina Rob.-Desv. (erythrocephala Meig.) by use of the unlabeled antibody-enzyme method

SummaryThe unlabeled antibody-enzyme method was used to demonstrate ultrastructurally the specific localization of vitellogenin in the fat body of Calliphora. In control flies the binding sites to vitellogenin were localized in secretory granules situated in the Golgi complex, and in larger bodies named composite secretory granules. These composite granules appear to be formed when a part of a Golgi complex containing secretory granules and a number of small vesicles become surrounded by a common membrane. Ovariectomized flies, which apparently do not produce secretory granules, exhibited no immunocytochemical staining. Ovariectomized flies in which the administration of ecdysterone induced formation of secretory granules, also revealed specific staining on these granules. This is the first ultrastructural evidence of: (a) the specific localization of vitellogenin in secretory granules of the fat body of an insect; (b) the relationship between the presence of the ovary, and of ecdysterone, and the synthesis of vitellogenin by the fat body.

Immunohistochemical localization and identification of the prolactin and growth hormone producing cells in the pituitary gland of the toad Bufo bufo bufo (L)

The two acidophilic cell types in the pars distalis of the pituitary gland of Bufo bufo were tinctorially differentiated and immunohistochemically identified with rabbit antiserum to ovine prolactin and to bovine growth hormone. By means of immunoglobulin-enzyme bridge and indirect immunofluorescence technique, the antio vine prolactin was localized in only the carminophils and antibovine growth hormone was localized in only the orangeophils.