Mycoplasma corogypsi-associated polyarthritis and tenosynovitis in black vultures (Coragyps atratus).

Three wild American black vultures (Coragyps atratus) were presented to rehabilitation centers with swelling of multiple joints, including elbows, stifles, hocks, and carpal joints, and of the gastrocnemius tendons. Cytological examination of the joint fluid exudate indicated heterophilic arthritis. Radiographic examination in 2 vultures demonstrated periarticular soft tissue swelling in both birds and irregular articular surfaces with subchondral bone erosion in both elbows in 1 bird. Prolonged antibiotic therapy administered in 2 birds did not improve the clinical signs. Necropsy and histological examination demonstrated a chronic lymphoplasmacytic arthritis involving multiple joints and gastrocnemius tenosynovitis. Articular lesions varied in severity and ranged from moderate synovitis and cartilage erosion and fibrillation to severe synovitis, diffuse cartilage ulceration, subchondral bone loss and/or sclerosis, pannus, synovial cysts, and epiphyseal osteomyelitis. No walled bacteria were observed or isolated from the joints. However, mycoplasmas polymerase chain reactions were positive in at least 1 affected joint from each bird. Mycoplasmas were isolated from joints of 1 vulture that did not receive antibiotic therapy. Sequencing of 16S rRNA gene amplicons from joint samples and the mycoplasma isolate identified Mycoplasma corogypsi in 2 vultures and was suggestive in the third vulture. Mycoplasma corogypsi identification was confirmed by sequencing the 16S-23S intergenic spacer region of mycoplasma isolates. This report provides further evidence that M. corogypsi is a likely cause of arthritis and tenosynovitis in American black vultures. Cases of arthritis and tenosynovitis in New World vultures should be investigated for presence of Mycoplasma spp, especially M. corogypsi.

Cytokine-induced immune deviation as a therapy for inflammatory autoimmune disease.

The properties and outcome of an immune response are best predicted by the lymphokine phenotype of the responding T cells. Cytokines produced by CD4+ T helper type 1 (Th1) T cells mediate delayed type hypersensitivity (DTH) and inflammatory responses, whereas cytokines produced by Th2 T cells mediate helper T cell functions for antibody production. To determine whether induction of Th2-like cells would modulate an inflammatory response, interleukin 4 (IL-4) was administered to animals with experimental allergic encephalomyelitis (EAE), a prototypic autoimmune disease produced by Th1-like T cells specific for myelin basic protein (MBP). IL-4 treatment resulted in amelioration of clinical disease, the induction of MBP-specific Th2 cells, diminished demyelination, and inhibition of the synthesis of inflammatory cytokines in the central nervous system (CNS). Modulation of an immune response from one dominated by excessive activity of Th1-like T cells to one dominated by the protective cytokines produced by Th2-like T cells may have applicability to the therapy of certain human autoimmune diseases.

Declining amphibian populations: the problem of separating human impacts from natural fluctuations.

Reports of declining amphibian populations in many parts of the world are numerous, but supporting long-term census data are generally unavailable. Census data from 1979 to 1990 for three salamander species and one frog species at a breeding pond in South Carolina showed fluctuations of substantial magnitude in both the size of breeding populations and in recruitment of juveniles. Breeding population sizes exhibited no overall trend in three species and increased in the fourth. Recent droughts account satisfactorily for an increase in recruitment failures. These data illustrate that to distinguish between natural population fluctuations and declines with anthropogenic causes may require long-term studies.

Distinct roles for B7-1 (CD-80) and B7-2 (CD-86) in the initiation of experimental allergic encephalomyelitis.

The activation and differentiation of T cells require both antigen/MHC recognition and costimulatory signals. The present studies examined the role of B7-1 (CD80) and B7-2 (CD86) costimulation in the prototypic autoimmune disorder, experimental allergic encephalomyelitis (EAE). In adoptively transferred EAE, in vitro activation of myelin basic protein (MBP)-specific lymph node cells was inhibited by the combination of anti-CD80 plus anti-CD86, but not individually. However, in actively induced disease, one injection of anti-CD80 significantly reduced disease, while anti-CD86 exacerbated disease. Interestingly, one injection of CTLA-4Ig suppressed disease, while multiple injections resulted in enhanced disease. Thus, the costimulation provided by B7-1 molecules appears to be important for the development of encephalitogenic T cells. The enhanced disease caused by multiple injections of CTLA-4Ig or a single injection of anti-CD86 suggests an inhibitory function for CD86 interaction with its counterreceptors CD28 and CTLA-4 in EAE. Alternatively, these results are consistent with an essential timing requirement for the coordinated interaction of B7 and CD28 family receptors, and that disruption of this critical timing can have opposing results on the outcome of an immune response.

Age related changes in the endocrine hypothalamus: I. Tanycytes and the blood-brain-cerebrospinal fluid barrier.

The fine structural organization of the floor of the third cerebral ventricle (dorsum of the median eminence of the hypothalamus) of 2 normal adult mice Fisher 344 rats was compared and contrasted with that of 2 aged rats 30 months old. Closely juxtaposed tanycytes (specialized ependymal cells) of normal young adults in the lower walls and floor of the third ventricle. In contrast, tanycytes in aged rats demonstrated significant intracellular separations, with only fine cytoplasmic processes remaining to interlink them. The phenomenon of mechanical separation between tanycytes in aged animals is discussed with respect to a potential impairment in the integrity of the blood-brain-cerebrospinal fluid barrier.

Immunity and protection against Brucella abortus.

Brucella abortus is an intracellular pathogen that causes disease in cattle and in humans. The response against B. abortus involves the whole gamut of the immune system, from innate to adaptive immunity resulting from stimulation of antigen-presenting cells, NK cells, CD4(+) and CD8(+) T cells, and B cells.

Scanning electron microscopy of the third ventricular floor of the rat.

By utilizing a horizontal dissection technique the entire floor of the third ventricle has been examined. When viewed in toto the ventricular floor was seen to have an hourglass shape with the supraoptic and infundibular recesses equalling the widened portions. Consistent regional differnces were also noted. The rostral half of the floor was densely ciliated while the caudal portion, containing ependymal elements of the underlying median emience, possessed few cilia. The ciliated cells ended in an abrupt transition zone located about halfway along the floor. The rostral portion of the infundibular recess had many more apical blebs and microvilli than did the caudal areas. Supraepen dymal cells of both the phagocyte-like and neuron-like variety were observed in all of the animals examined. In some animals, complex, branching, interconnecting networks of fine calibered fibers interconnected neuron-like cells that occurred singly and occasionally in clusters. Female rats examined at all phases of the estrus cycle demonstrated no cyclic alterations of the ependymal surface.

Vasoactive intestinal polypeptide in sacral primary sensory pathways in the cat.

Unmyelinated sensory axons in the sacral spinal cord may play a role in bladder reflexes under certain pathological conditions. Previous data suggested vasoactive intestinal polypeptide (VIP) might be contained exclusively in sensory C-fibers, some of which innervate the bladder. This study was undertaken to describe the morphology of these VIP fibers in the sacral cord of the cat. VIP immunoreactivity was confined to unmyelinated axons observed at several levels of the sensory pathway including the dorsal root ganglia, dorsal roots, Lissauer's tract, and the lateral collateral pathway. A combination of light and electron microscopic observations showed VIP-immunoreactive fibers with labeled varicosities and synaptic terminals in laminae I, IIo, V, VII, and X. VIP-immunolabeled varicosities had a mean diameter of 1.6 microm (range = 0.11-7.4 microm, S.D. = 1.01, n = 311) with a small percentage (8%) being relatively large (3-7.4 microm). VIP varicosities contained a mixture of small clear vesicles (CLV) and large dense core vesicles (LDV). Although most varicosities contained a moderate number of LDVs (14.86 LDVs/microm2), some varicosities contained a large number of LDVs, whereas others contained very few. Varicosities that possessed synaptic specializations were classed as terminals and were divided into three morphological classes. Two of these resembled Gray's Type I terminal, whereas a third was similar to the Gray's Type II terminal. There was no consistent relationship between vesicle content of the terminal and the type of synaptic contact it possessed. This study shows that in the sacral spinal cord of the cat, VIP terminals originate only from C-fibers, terminate primarily in laminae I and V, and exhibit a variety of morphologies consistent with heterogeneous origins and functions of the lower urinary tract.

Therapeutic potential of phosphodiesterase type 4 inhibition in chronic autoimmune demyelinating disease.

It was recently demonstrated that selective phosphodiesterase type 4 (PDE4) inhibition suppresses the clinical manifestations of acute experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), and inhibits the production of tumor necrosis factor-alpha (TNF-alpha), a pathogenetically central cytokine. Since the most common presentation of MS in humans is a relapsing-remitting course, we investigated the therapeutic potential of PDE4 inhibition in the relapsing-remitting EAE model of the SJL mouse. Administration of rolipram, the prototypic PDE4 inhibitor, reduced the clinical signs of EAE during both the initial episode of disease and subsequent relapses. In parallel, there was marked reduction of demyelination and also less inflammation throughout the central nervous system (CNS) of rolipram-treated animals. Gene expression of proinflammatory cytokines in the CNS was reduced in most of the rolipram-treated animals. Additional experiments demonstrated that PDE4 inhibition acted principally by inhibiting the secretion of Th1 cytokines, however, the encephalitogenic potential of myelin basic protein-specific T cells was not impaired. Our findings suggest that PDE4 inhibitors are a promising cytokine-directed therapy in chronic demyelinating disease.

HIV peptide conjugated to heat-killed bacteria promotes antiviral responses in immunodeficient mice.

Enhancement of immunity in the setting of HIV infection is difficult owing to loss of functional CD4+ T cells. The MHC class II-deficient mouse (II-/-) environment simulates that of the immunocompromised HIV-infected individual, since these mice have low CD4+ T cell numbers, defective CD4-dependent responses, and are susceptible to opportunistic infection. This strain was used to test whether heat-killed Brucella abortus (BA), covalently conjugated to the V3 peptide of HIV-1 (MN), could elicit anti-HIV responses. V3-BA, but not the T-dependent antigen V3-KLH, induced high levels of IL-12, IFN-gamma, and IL-10 mRNA in both wild-type (WT) and II-/- mice within 24 hr of injection. V3-BA-treated, but not V3-KLH-treated, II-/- mice developed serum IgG and IgA anti-V3 antibodies, with IgG2b and IgG3 as the predominant isotype. Viral neutralization studies, using a syncytium inhibition assay, demonstrated that the antibodies generated by V3-BA in II-/- mice were capable of neutralizing HIV. These experiments demonstrate that a heat-inactivated bacterium such as BA, when used as a carrier, can generate a cytokine environment that results in the production of neutralizing antiviral antibodies in an immunodeficient host. Such strategies could be important in the development of immunotherapies and vaccines for HIV-1 patients.

Vaccine strategies: targeting helper T cell responses.

Vaccine strategies need to take into account the balance of T helper subsets they induce. TH1 cells, which secrete IFN gamma and IL-2, are associated with CMI, rather than humoral responses, and afford protection against intracellular infections including parasites. In contrast, TH2 cells secrete IL-4, IL-5, and IL-10; elicit high-titer antibody responses and poor CMI; and are associated with susceptibility to infection with intracellular pathogens. Depending on the type of TH cell bias required, it is possible to manipulate the immune response to a protein or peptide by employing (1) different adjuvants, (2) conjugating the protein to various carriers, (3) immunizing in the presence of cytokines, (4) using alternative routes of administration, or (5) using different forms or doses of antigen. To apply these approaches to a particular vaccine, it is necessary to identify which component of the infectious agent (e.g., envelope protein or peptide) or allergen to target. Once the type of TH cell response that is protective is identified, it may be possible to combine a protein with an adjuvant or link it to a carrier that will promote responses towards the most advantageous TH subset.

Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry: I. Localization of catecholamines and gonadotropin-releasing hormone in the rat median eminence.

Adjacent tissue sections through the rat median eminence were examined for the distribution of gonadotropin-releasing hormone (GnRH) and catecholamines (CA). A simultaneous visualization technique was employed for this correlative neuroanatomical analysis. At rostral and mid-central levels of the median eminence the majority of GnRH terminals do not appear in coexistence with CA terminals; the latter were confined to the outer-most 10 micrometers of the median eminence while the densest concentration of GnRH terminals was located internal to this layer. However, individual GnRH fibers appeared to penetrate the outer CA zone wherein they were found in juxtaposition to portal capillaries. At caudal levels of the median eminence, there was an extensive overlap of CA and GnRH varicosities adjacent to the tubero-infundibular sulcus. In addition, numerous GnRH terminals were seen adjacent to portal vessels. The differences in the positions of CA and GnRH terminals between rostral and caudal median eminence may provide a morphological basis for the hypothesis of separate regulatory mechanisms for CA upon GnRH secretion at these two levels of the median eminence.

The neuroanatomical and neurovascular organization of normal fetal hypothalamic explants in the third cerebral ventricle of Brattleboro rats with homozygous diabetes insipidus.

This investigation has combined microangiography, immunocytochemistry, coupled with transmission and scanning electron microscopy to discuss the neuroanatomical interactions that occur in the brains of Brattleboro rats with diabetes insipidus, following stereotaxic placement of normal fetal hypothalamic fragments into the third cerebral ventricle. Following surgical placement of 17 day post-coitus hypothalamic fragments, host rats with chronic autosomal homozygous diabetes insipidus were killed and their brains were prepared for analysis. A significant degree of explants (68%) flourished and grew in the lumen of the third cerebral ventricle of recipient hosts. Explants were rapidly invaded by host vessels from two routes. Vessels arose from the underlying mantle plexus of portal capillaries which remained fenestrated in the lower one-third of the explants and developed neurovascular (neurohemal) zones. The second source of vessels arose from bed capillaries of the adjacent paraventricular nucleus and adjacent hypothalamus. In contrast to vessels arising from the contact zone, these latter vessels remained unfenestrated. Small clusters of immunocytochemically positive neurons (neurophysin positive) were seen throughout the explants. Numerous healthy magnocellular neurons harboring numerous dense core vesicles and exhibiting multiple axosomatic and axodendritic synapses were seen throughout the neuropil of explants. Axon profiles were noted to terminate upon the abluminal basal lamina of perivascular spaces surrounding fenestrated capillaries in the lower one third of explants. None of the host animals exhibited physiological return to normal parameters of urine output, drinking behavior, and/or urine osmolarity. However the growth and development of explants in the third cerebral ventricle of DI hosts coupled with the emergence of bonafide neurovascular zones supports a potential anatomical substrate for the central delivery of neuropeptide hormones in this experimental model.

Identification of catecholamine and luteinizing hormone-releasing hormone (LHRH)-containing neurons in primary cultures of dispersed cells of the basal hypothalamus.

Primary cultures of dispersed cells were prepared from 3-5 mg pieces of basal hypothalami of 10-12-day-old rats. The tissue included median eminence, arcuate nucleus and variable amounts of adjacent hypothalamus and preoptic area. The dispersion procedure consisted basically of tissue trypsinization and mechanical dissociation of cells. They were cultured in a modified L-15 medium in an air atmosphere. Neurons survived approximately 3 months. On the basis of morphological characteristics, two basic cell types could be distinguished. One was a larger (50 mum diameter) multipolar cell; microspectrofluorometric analysis revealed that a small percentage of these neurons contained a catecholamine. A second type was smaller, fusiform or ovoid and generally bipolar; a significant number of these were immunoreactive for the releasing hormone LHRH.

Fetal hypothalamic transplants: neuronal and neurovascular interrelationships.

This investigation is one of a series aimed at developing an understanding of the neuroanatomical correlates of neuronal and vascular interactions that occur between heterografts of normal fetal hypothalamic tissue transplanted into the third cerebral ventricle of adult Brattleboro rats with homozygous autosomal diabetes insipidus (DI). Rapid vascular invasion of fetal neurografts occurred within 3 days and arose from the mantle plexus of portal capillaries in the underlying host median eminence. Host vessels also invaded from the periventricular stratum and preoptic area. In the ventral regions of heterografts neurites were observed to terminate upon perivascular spaces that surrounded local capillaries. Despite the development of apparently normal neurovascular zones, none of the DI host recipients demonstrated an amelioration of polydipsia or polyuria characteristic of diabeties insipidus.

Fetal hypothalamic transplants in diabetic rats analysed by scanning electron microscopy.

Adult male Brattleboro rats with chronic diabetes insipidus underwent stereotaxic surgery wherein minced fragments of anterior hypothalamus from fetal rats, 17 days post-coitus, were stereotaxically positioned into the lumen of the host third cerebral ventricle. Host rats with fetal donor tissue were killed at various times following surgery and were prepared for correlative scanning-transmission electron microscopy. Examination with this technique revealed the presence of large neurografts which grew to occupy the entire lumen of the host third ventricle. Grafts were well vascularized and in addition exhibited remarkable numbers of supraependymal, cerebrospinal fluid-contacting neurons. The physical emergence of this cell line in proximity to viable grafts is discussed with respect to the biochemical influences that a neuropeptide producing fetal transplant has upon a peptide-deficient host.

The fetal baboon median eminence as a circumventricular organ: I. Transmission electron microscopy.

This investigation has focused upon a set of neuroanatomical correlates that underscore functional changes in the median eminence of the fetal baboon Papio anubis. Eight fetal primate brains were harvested at mid-gestation (100 days post-coitus) and prepared for routine light and transmission election microscopic examination following ventriculo-cisternal perfusion with high osmolarity aldehyde fixatives. The median eminence and other adjacent circumventricular organs (CVO) were blocked and embedded in epon. Routine transmission electron microscopic examination revealed discreet regional differentiation and zonal maturation of the fetal baboon median eminence at 100 days post-coitus. The ependymal and hypendymal zones were anatomically separate from the underlying fibrous and palisade zones of the median eminence. The dominant cell type of the ependymal and hypendymal zones were tanycytes whose apical surfaces constituted the floor of the third cerebral ventricle. The distal processes of tanycytes terminated upon the abluminal basal lamina of well developed perivascular spaces in the contact zone of the fetal median eminence. Numerous axon profiles that harbored both dense core and microvesicles were also observed to terminate upon the system of perivascular spaces that contained numerous fenestrated capillaries. Sharing this common perivascular system were cells of the pars tuberalis that contained numerous secretory inclusions. These data strongly suggest that by mid-gestation the fetal primate median eminence is highly differentiated and may be capable of a wide range of functional activities in response to changes in the maternal-placental environment. The ultrastructural correlates of an active neuroendocrine axis are apparent at this period of development.

Neuronal and neurovascular integration following transplantation of the fetal hypothalamus into the third cerebral ventricle of adult Brattleboro rats. Neurological transplants: I.

This investigation has confronted some very basic questions of neurobiology and specifically deals with the neurovascular and neuroanatomical interactions that occur between graft and host following neural transplantation. Host Long-Evans rats with chronic autosomal diabetes insipidus (DI) received stereotaxic implants of normal 17 day post-coitus fetal hypothalamic fragments from the rostral (anterior) hypothalamus of normal Long-Evans pups. Following stereotaxic surgery DI hosts were killed 60 or 90 days later and their brain prepared for correlative microangiography-immunocytochemistry coupled with transmission electron microscopy. Explants were rapidly invaded by host vessels from several routes. (1) Vessels appeared to arise from portal capillaries in the underlying median eminence and (2) from adjacent vessels from the paraventricular nucleus and surrounding endocrine hypothalamus and (3) possibly from intrinsic vessels of the graft. The former remained fenestrated and established bonafide neurovascular zones in the ventral regions and in actively growing explants. Small clusters of arginine vasopressin-positive fibers and neurophysin positive neurons were noted throughout the parenchyma of explants. Despite the presence of neurosecretory neurons and neurovascular (neurohemal) zones, none of the host rats exhibited a physiological return to normal parameters of water balance. However the active growth and development of explants in the third cerebral ventricle of DI host rats coupled with emergence of neurovascular zones lends support to a potential model for analyzing the development of anatomical substrates for the central delivery of neuropeptide hormones.

Correlative scanning-immunoelectromicroscopic analysis of neuropeptide localization and neuronal plasticity in the endocrine hypothalamus.

Thirty-two Sprague-Dawley rats were divided into four groups, eight rats per group. Animals were hypophysectomized with removal of both the pars distalis and the neural lobe of the neurohypophysis. Groups of eight rats were euthanized 1, 2, 4 and 8 weeks following hypophysectomy and prepared for routine scanning electron microscopy (SEM) and correlative immunoelectron microscopy employing antisera against arginine vasopressin (AVP). Eight normal rats served as controls. In experimental rats that survived one to eight weeks posthypophysectomy, remarkable neuroanatomical alterations were notable in the median eminence and adjacent third cerebral ventricular lumen. In contrast to normal control rats, large numbers of neurites were observed with SEM to insinuate from the lateral recess into the cerebral ventricular lumen and as early as one week following hypophysectomy they overgrew the apical surfaces of ependymal cells that constitute the lining of the cerebral ventricle. Immunoelectron microscopy revealed that a significant proportion of these neurites were magnocellular in origin in that they harbored AVP-positive neurosecretory vesicles. In addition to large numbers of invading magnocellular neurites, neuronal perikayria with apparent axosomatic synapses were observed to emerge upon the thick feltwork of invading axons, the latter of which appeared to freely terminate within the ventricular lumen. AVP-positive axon profiles were, in addition, seen to terminate upon the basal lamina of portal perivascular spaces in the zona externa of the median eminence. These data are consistent with the idea that following hypophysectomy (to include high stalk section of the neurohypophyseal system), that there is rapid, and dynamic sprouting and regrowth of AVP-positive axons into the adjacent third cerebral ventricular lumen and to the contact zone of the median eminence as well. This phenomenon may represent a compensatory physiological response to injury of the neurohypophyseal system characterized by a highly plastic neuroanatomical reorganization of magnocellular elements which appear to utilize the CSF of the third cerebral ventricle as a functional terminus for the neurocisternal secretion of AVP which ultimately enters the systemic circulation.

Hypothalamic neuroendocrine correlates of cutaneous burn injury in the rat: I. Scanning electron microscopy.

Rats were given a standard scald burn on 60% of the body surface or only a sham burn and were sacrificed at intervals from 6 hr to 14 days later. Serum thyroxine (T4), free thyroxine index (FT4I) and triiodothyronine (T3) were depressed compared to values in respective shams as early as 6 hr post-burn. T4 and FT4I were less depressed on post-burn days (PBD) 2-3 than on PBD 1 and then exhibited a further fall. T3 remained depressed through PBD 14. Pineal melatonin content was elevated at 6 hr and fell to the normal daytime range in subsequent samples. The ventral portion of the diencephalon was prepared for scanning electron microscopy. Only in the burned rats and beginning on PBD 2, large numbers of supraependymal neurons (SEN) appeared in the ventricular space attached to the inferior walls and floor of the third cerebral ventricle. Transmission electron microscopy was used to confirm the neuronal nature of the SEN. Viewed by scanning electron microscopy, these persisted through PBD 14. SEN were interconnected by cables of their neurites exhibiting varicosities on individual neurites as they passed over perikarya of other SEN. Some SEN were seen to be only partially emerged from the underlying tissue and others were seen to send a thick process into the hypothalamic tissue. These observations indicate that after peripheral injury there is marked plasticity of the brain in an area thought to control the endocrine systems that show abnormalities after such a peripheral injury. The timing, location and nature of these anatomic changes indicate the possibility that at least some aspects of central nervous orchestration of the endocrine metabolic response to injury may be related to the emergence of a neuronal system receiving or sending messages through the cerebrospinal fluid and/or through new neurite circuits along the surface of the third ventricular wall. These structures may appear in response to initial primary hormonal changes and/or may play a role in maintaining the post-injury hormonal milieu manifested in part by a subsequent second fall in serum T4.