Mouse model for Pneumocystis carinii pneumonia that uses natural transmission to initiate infection.

Animal models for Pneumocystis carinii, for the most part, have been limited to immunosuppressed rats and ferrets, while a dependable mouse model has been more difficult to develop. A P. carinii mouse model has now been established with several strains of mice, including C3Heb/FeJ, C3HeN, BALB/c, DBA/2N, and BALB/c nu/nu (athymic). In lieu of using invasive methods for initiating P. carinii infections, mice harboring P. carinii transmitted the disease to mice without latent infection via short-term cohabitation. After the exposure period, the seed mice were sacrificed to confirm the presence of acute P. carinii pneumonia. Acute infections in recipient mice developed at approximately 7 to 8 weeks, while control unseeded littermates remained uninfected. All recipient mice and their littermates were maintained in isolation hoods to eliminate the possibility of exposure to other sources of P. carinii. This approach allows investigators to consistently transmit P. carinii to mice and to select the strain of mouse desired for use in a particular study. The results presented here suggest that more attention should be given to the potential for patient-to-patient transmission of P. carinii in immunocompromised patients such as those with AIDS.     

Neuroendocrine mechanisms that delay and initiate puberty in higher primates

This paper highlights a series of studies using the male rhesus monkey that has led to a model for the control of the onset of puberty in higher primates. The model proposes that the timing of puberty in these species is governed by the duration of a central brake that, during juvenile development, holds in check the hypothalamic network of gonadotropin-releasing hormone (GnRH) neurons, which, in the adult, drive the pituitary-gonadal axis. The neurobiology of this hypothalamic brake, and the physiological mechanisms that time its application and removal, are incompletely understood. Nevertheless, the pubertal resurgence of pulsatile GnRH release, which terminates the juvenile phase of primate development and triggers the initiation of puberty in man and monkeys, is associated with structural and molecular remodeling of the hypothalamus. A major component of this developmental plasticity appears to involve neuropeptide Y (NPY). NPY inhibits GnRH release, and NPY gene expression in the hypothalamus is elevated during juvenile development when GnRH release is restrained. Since the changes in hypothalamic function and morphology that trigger primate puberty unfold in the absence of gonadal steroid feedback, the possibility is raised that, in addition to activating the pituitary-gonadal axis at this stage of development, they may also contribute directly to the causation of behaviors and affective states that emerge at adolescence.     

Organization of higher-order brain areas that initiate locomotor activity in larval lamprey

In the lamprey, spinal locomotor activity can be initiated by pharmacological microstimulation in several brain areas: rostrolateral rhombencephalon (RLR); dorsolateral mesencephalon (DLM); ventromedial diencephalon (VMD); and reticular nuclei. During DLM- or VMD-initiated locomotor activity in in vitro brain/spinal cord preparations, application of a solution that focally depressed neuronal activity in reticular nuclei often attenuated or abolished the locomotor rhythm. Electrical microstimulation in the DLM or VMD elicited synaptic responses in reticulospinal (RS) neurons, and close temporal stimulation in both areas evoked responses that summated and could elicit action potentials when neither input alone was sufficient. During RLR-initiated locomotor activity, focal application of a solution that depressed neuronal activity in the DLM or VMD abolished or attenuated the rhythm. These new results suggest that neurons in the RLR project rostrally to locomotor areas in the DLM and VMD. These latter areas then appear to project caudally to RS neurons, which probably integrate the synaptic inputs from both areas and activate the spinal locomotor networks. These pathways are likely to be important components of the brain neural networks for the initiation of locomotion and have parallels to locomotor command systems in higher vertebrates.     

Mechanisms that initiate spontaneous network activity in the developing chick spinal cord

Wenner, Peter andMichael J. O'Donovan.Mechanisms That Initiate Spontaneous Network Activity in theDeveloping Chick Spinal Cord. J. Neurophysiol. 86: 1481-1498, 2001. Many developing networks exhibit a transient period ofspontaneous activity that is believed to be important developmentally. Here we investigate the initiation of spontaneous episodes of rhythmicactivity in the embryonic chick spinal cord. These episodes recurregularly and are separated by quiescent intervals of many minutes. Weexamined the role of motoneurons and their intraspinal synaptic targets(R-interneurons) in the initiation of these episodes. During the latterpart of the inter-episode interval, we recorded spontaneous,transient ventral root depolarizations that were accompanied by small,spatially diffuse fluorescent signals from interneurons retrogradelylabeled with a calcium-sensitive dye. A transient often could beresolved at episode onset and was accompanied by an intense pre-episode(~500 ms) motoneuronal discharge (particularly in adductor andsartorius) but not by interneuronal discharge monitored from theventrolateral funiculus (VLF). An important role for this pre-episodemotoneuron discharge was suggested by the finding that electricalstimulation of motor axons, sufficient to activate R-interneurons,could trigger episodes prematurely. This effect was mediated throughactivation of R-interneurons because it was prevented bypharmacological blockade of either the cholinergic motoneuronal inputsto R-interneurons or the GABAergic outputs from R-interneurons to otherinterneurons. Whole-cell recording from R-interneurons and imaging ofcalcium dye-labeled interneurons established that R-interneuron cellbodies were located dorsomedial to the lateral motor column(R-interneuron region). This region became active before other labeledinterneurons when an episode was triggered by motor axonstimulation. At the beginning of a spontaneous episode, whole-cellrecordings revealed that R-interneurons fired a high-frequency burstof spikes and optical recordings demonstrated that the R-interneuronregion became active before other labeled interneurons. In the presenceof cholinergic blockade, however, episode initiation slowed and theinter-episode interval lengthened. In addition, optical activityrecorded from the R-interneuron region no longer led that of otherlabeled interneurons. Instead the initial activity occurred bilaterallyin the region medial to the motor column and encompassing thecentral canal. These findings are consistent with the hypothesis thattransient depolarizations and firing in motoneurons, originating fromrandom fluctuations of interneuronal synaptic activity, activateR-interneurons, which then trigger the recruitment of the rest of thespinal interneuronal network. This unusual function for R-interneuronsis likely to arise because the output of these interneurons isfunctionally excitatory during development.

     

Systematic characterization of porcine ileal Peyer's patch, II. A role for CD154 on T cells in the positive selection of immature porcine ileal Peyer's patch B cells

We previously demonstrated that the majority (>/= 90%) of porcine ileal Peyer's patch (IPP) follicular cells are immature B cells destined to die by apoptosis, when incubated at 37 degrees. In this paper we approached the mechanisms responsible for positive selection of porcine IPP follicular immature B-cell selection, by screening for various cell types, cytokines and polyclonal and monoclonal antibodies for promoting the survival of IPP B cells. Of these reagents, only CD3 cross-linked purified T cells from mesenteric lymph nodes were able to rescue IPP follicular B cells from apoptosis, although polyclonal anti-IPP lymphocyte antibodies delayed apoptosis. This survival effect could be reproduced simply by incubating IPP follicular B cells with soluble and cell membrane-expressed CD154, an observation consistent with the demonstrated presence of CD40 and CD154 on porcine IPP follicular B cells and activated T cells, respectively. The IPP follicular B cells rescued in this manner expressed a more mature surface marker phenotype. Immunohistology and fluorescence-activated cell sorter analysis demonstrated that subpopulations of IPP follicular T cells (less than 0.5%) express CD154. Thus, perhaps unexpectedly, CD154 on T cells may play a role in the positive selection of immature B cells in the porcine IPP. The origin and control of the activated T cells identified within the porcine IPP remains to be investigated.     

Presence of K88-specific receptors in porcine ileal mucus is age dependent.

Ileal mucus and epithelial cells were isolated from newborn piglets that had never been fed and 35-day-old unweaned piglets. Both newborn and 35-day-old piglet mucus preparations supported growth of Escherichia coli Bd 1107/75 08, a K88-fimbriated porcine enterotoxigenic strain, equally well (i.e., generation times of 28 min were observed in both cases). Adhesion of E. coli Bd 1107/75 08 to 35-day-old piglet ileal epithelial cells was, at most, 2 times that of the same strain to newborn piglet ileal epithelial cells; however, adhesion of E. coli Bd 1107/75 08 to 35-day-old piglet ileal mucus was 16 times that of the same strain to newborn piglet ileal mucus. The receptor in 35-day-old piglet ileal mucus was K88 specific, since it could be removed by purified K88ab fimbriae. Furthermore, adhesion of E. coli Bd 1107/75 08 to 35-day-old piglet ileal mucus was blocked by PAB10, a K88ab-, K88ac-, K88ad-specific monoclonal antibody. Although E. coli Bd 1107/75 08 traversed both newborn and 35-day-old piglet ileal mucus about equally well in vitro and bound well to underlying ileal epithelial cells after passing through newborn ileal mucus, it did not bind to ileal epithelial cells after passing through 35-day-old piglet ileal mucus. The data are discussed with respect to the role that K88-specific receptors present in newborn and ileal mucus might play in the pathogenesis of porcine enterotoxigenic E. coli strains which bear K88 fimbriae.     

Does infection initiate Graves disease? A population based 10 year study

In order to detect whether micro-organisms could initiate the autoimmune process in Graves' disease we have studied the temporal and spatial distribution of 857 cases of hyperthyroidism occurring in a community over ten years. Cases were identified through biochemistry laboratory records and following the exclusion of patients with toxic nodular goitre or with insufficient clinical data there were 599 with Graves' disease--an average annual incidence of 15.9 per 100,000. There was a tendency for cases to present in the summer months. The reported onset of symptoms, however, peaked in December and June. There was no evidence of clustering of cases in space and time using two different statistical methods. Incidence rates doubled between 1976 and 1980 and then declined--a trend that could neither be explained by changes in laboratory or clinical diagnosis nor did it correlate with any pattern of microbial disease in the area. We conclude that it is unlikely that infections that behave in an epidemic manner have a causative role in triggering Graves' disease.     

Abnormal proinsulin congeners as autoantigens that initiate the pathogenesis of Type 1 diabetes

Exposure of concentrated and purified monomeric insulin solutions to inorganic oxidants as iodine and chlorine lead to the appearance of a minor peak on gel chromatography that was disulfide cross-linked. It exhibited a 20-fold reduction in bioactivity, a markedly decreased immunoreactivity and a different electrophoretic pattern as compared to the starting material. The covalent dimer was formed by way of aggregation. These findings were reproduced by incubating monomeric insulin in normal blood spiked to hyperglycemic levels. In contrast, normoglycemic blood used as incubating medium failed to induce dimerization. Since the conformation of proinsulin is similar to that of insulin, involving the exposure of the anterior A7-B7 disulfide bridge, the authors hypothesize that proinsulin dimers rather than insulin dimers might be formed in Type 1 diabetes (TD1), leading to the autoimmune destruction of pancreatic B-cells. Proinsulin is present in a soluble aggregate state in the coated granule and may further accumulate allowing disulfide exchange due to abnormalities of the processing enzymes. This might be caused by inborn errors in genetically susceptible children or perhaps secondary to viral infection, whereas crystallization of insulin in mature granules is unlikely to be conducive to dimerization. Dimeric proinsulin would then migrate to pancreatic B-cell membranes to be taken up by surface immunoglobulins on B-lymphocytes that would in turn present it to cytotoxic T lymphocytes. The abnormal configuration of this congener would not be recognized as self by the immune system, triggering a selective destruction of pancreatic B-cells in the early pre-clinical development of TD1, resulting eventually in clinical disease. Since proinsulin is continuously converted to insulin in the coated granules of Type 2 diabetics, dimerization is unlikely in this condition. As pointed out by earlier investigators, TD1 is not a homogeneous disease, since several of its clinical features are different in children up to 6 years of age as compared to older patients. It is thus conceivable that there are subsets of TD1 triggered by dissimilar autoantigens and we propose in the present hypothesis that a conformationally altered congener of native proinsulin might play such a role.     

Role of cell-associated pathogen metabolism in infection of tracheal explants byMycoplasma pneumoniae

The effect of environmental conditions on the relative pathogenicity ofMycoplasma pneumoniae for hamster tracheal explants was investigated. Organisms from the early stages of the growth cycle (e.g., day 1 to 2) were more effective in the induction of ciliostasis than were older cultures. Both the degree of ciliostasis and the speed of onset were affected. The type of explant culture medium also affected pathogenic potential.M. pneumoniae infection produced significantly greater ciliostasis and cytonecrosis in a permissive medium, i.e., one capable of supporting mycoplasma metabolism and replication, than in a nonpermissive medium. However,no adenine protection effect could be detected under permissive conditions, though it was quite striking when a nonpermissive medium was used as the post-infection explant medium. This suggests that the cell damage noted under permissive conditions may result from processes distinct from those operative in the actual host-parasite cellular interaction.     

Morphological interactions of human first trimester placental villi co-cultured with decidual explants

Abnormalities of pregnancy such as pre-eclampsia and intrauterinegrowth retardation are characterized by shallow trophoblasticinvasion of the placental bed, the precise molecular pathophysiologyof which remains to be fully elucidated. An in-vitro model involvinga co-culture of first trimester placental villi and deciduaparietalis explants (of 8-12 weeks gestation) was developedand used to characterize the migration and local invasion oftrophoblast cells. Trophoblast proliferation (confirmed by Ki-67immunostaining), differentiation and loose attachment of placentalvilli to the underlying decidual epithelium or stroma occurredwithin the first 24 h of co-culture. This was followed by erosionof the syncytial layer of the placental villi and commencementof a progressive cytotrophoblast invasion after 48 h of co-culture,which continued until 120 h, when the experiments were terminated.E-cadherin was expressed at the interfaces between trophoblastcells within the villi, but expression of this adhesion moleculeseemed to be down-regulated in the invasive trophoblast cells.Our results suggest that the model could be useful in investigatingthe factors that control early human placentation and the feto-maternalinterface.     

In vitro culture of porcine respiratory nasal mucosa explants for studying the interaction of porcine viruses with the respiratory tract

The mucosal surface of the respiratory tract is a common site of entry of many viruses. Molecular and cellular aspects of the interactions of respiratory viruses with the respiratory nasal mucosa are largely unknown. In order to be able to study those interactions in depth, an in vitro model was set up. This model consists of porcine respiratory nasal mucosa explants, cultured at an air-liquid interface. Light microscopy, scanning electron microscopy and transmission electron microscopy, combined with morphometric analysis and a fluorescent Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labelling (TUNEL) staining were used to evaluate the effects of in vitro culture on the integrity and viability of the explants. The explants were maintained in culture for up to 60 h post-sampling without significant morphometric (epithelial thickness, epithelial morphology, thickness of the lamina reticularis, continuity of the lamina densa, relative amounts of collagen and nuclei) changes and changes in viability. The potential to infect the explants was demonstrated for two porcine respiratory viruses of major importance: suid herpesvirus 1 and swine influenza virus H1N1. In conclusion, this in vitro model represents an ideal tool to study interactions between infectious agents and porcine respiratory nasal mucosa.     

Reproduction of porcine proliferative enteropathy with pure cultures of ileal symbiont intracellularis.

Porcine proliferative enteropathy is consistently associated with the presence of intracellular curved bacteria in epithelial cells in affected portions of intestine. Two strains of these intracellular bacteria were cultured in a cell culture system with rat enterocytes (IEC-18) and passaged several times and used as oral inocula for 14 gnotobiotic and 8 conventional pigs. DNA and immunological studies had identified these bacteria as belonging to a new taxon, Ileal symbiont (IS) intracellularis. Conventional pigs dosed with approximately 3.7 x 10(6) of these organisms passaged six times in cell culture developed severe lesions of proliferative enteropathy in the ileum. Other conventional pigs dosed with a lower titer or with organisms passaged 13 times developed moderate and minor lesions, respectively. All gnotobiotic pigs dosed with organisms failed to develop lesions. Control pigs, eight conventional and two gnotobiotic, dosed with diluent, uninfected cell material or left undosed failed to develop lesions also. Reisolation of IS intracellularis and demonstration of the organism in mucosal and fecal samples only occurred in conventional pigs dosed with organisms. Gnotobiotic pigs lacking a normal intestinal flora have not been shown to be colonized by the organism. Seroconversion to IS intracellularis or mucosal infiltration by inflammatory cells was not observed in experimentally affected pigs, confirming the weak immune response characteristic of the natural disease. These results support the identification of IS intracellularis as an etiological agent of proliferative enteropathy in pigs.     

Isolation from cultured porcine gingival explants of a neutral proteinase with collagen telopeptidase activity

A neutral proteinase, distinct from collagenase, was isolated by gel-filtration from medium that had been conditioned by the culture of explanted porcine gingiva. This enzyme was shown to cleave the alpha 1 (I) chain carboxyterminal telopeptide in native collagen proximal to (i.e. nearer to the helix than) the lysyl residue at position 17 which is known to be important in the formation of covalent intermolecular cross-links. We refer to this activity as 'telopeptidase'. The enzyme had an apparent Mr of 70,000, as determined by gel-filtration. It was inhibited by ethylene diamine tetraacetic acid but not by N-ethyl-maleimide nor by phenylmethylsulphonyl fluoride. It is therefore probably a metalloproteinase. The pH optimum for this activity was 7.0-7.5. Incubation of the enzyme with fibrillar (acid-insoluble) calf-skin collagen resulted in solubilization of collagen in which shortening of the carboxy-terminal telopeptides could be demonstrated. It is suggested that the telopeptidase, acting within a region of the Type I collagen molecule which is known to be essential for the stability of collagen fibrils, could potentially play an important role in collagen degradation in vivo.     

Experimental infection of rabbit ligated ileal loops with Treponema hyodysenteriae

An in vivo animal model was used to assess the enteropathogenicity of the etiological agent (Treponema hyodysenteriae) of swine dysentery. Multiple ligated ileal loops, prepared in New Zealand white rabbits, were challenged with either pathogenic (B78 and B204) or nonpathogenic (Pu) isolates of the organism. The pathogenic isolates induced the onset of intestinal fluid accumulation as early as 4 h, with maximal fluid induction at 18 h postchallenge. Gross lesions of the intestinal mucosa, observed in ileal loops of rabbits sacrificed 24 h postchallenge, were characteristic of swine dysentery. Both pathogenic isolates colonized the epithelial surface and eroded the mucosal barrier, as determined by histological and scanning electron microscopic observations. Intestinal fluid accumulation and erosion of the mucosal barrier were not observed in ileal loops exposed to the nonpathogenic isolate (Pu) or to either of the nonviable pathogenic (B78 and B204) isolates. The ability of pathogenic isolates to initiate and produce infection in rabbit ligated ileal loops, which closely resembles the disease in swine, provides a system with which to study experimental swine dysentery.