Evidence for coexistence of ATP and nitric oxide in non-adrenergic,non-cholinergic (NANC) inhibitory neurones in the rat ileum,colon and anococcygeus muscle

The possible coexistence of the two non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitters, adenosine 5-triphosphate and nitric oxide in the myenteric plexus was investigated using whole-mount preparations of rat ileum, proximal colon and anococcygeus muscle. The presence of adenosine 5-triphosphate in neurones was examined using the quinacrine fluorescence technique. After localizing and taking photographs of quinacrine-fluorescent neurones and nerve fibres, the same tissues were then fixed and processed for NADPH-diaphorase activity, a marker for nitric oxide-containing neurones. We have demonstrated for the first time that almost all quinacrine-fluorescent myenteric neurones in the proximal colon are also NADPH-diaphorase reactive, while only a subpopulation of quinacrine-fluorescent neurones in ileum and anococcygeus muscle were also NADPH-diaphorase reactive.     

A neurochemical characterisation of the golden hamster myenteric plexus

The neurochemical composition of nerve fibres and cell bodies in the myenteric plexus of the proventriculus, stomach and small and large intestines of the golden hamster was investigated by using immunohistochemical and histochemical techniques. In addition, the procedures for localising nitric-oxide-utilising neurones by histochemical (NADPH-diaphorase) and immunohistochemical (nitric oxide synthase) methods were compared. The co-localisation of vasoactive intestinal polypeptide and nitric oxide synthase in the myenteric plexus of all regions of the gut was also assessed. The results demonstrated the presence of nerve fibres and nerve cell bodies immunoreactive to protein gene product, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, tyrosine hydroxylase, 5-hydroxytryptamine and nitric oxide synthase in all regions of the gastrointestinal tract examined. The pattern of distribution of immunoreactive nerve fibres and nerve cell bodies containing the above markers was found to vary in different regions of the gut. Myenteric neurones and nerve fibres containing immunoreactivity to nitric oxide synthase and NADPH-diaphorase reactivity, however, were shown to have an identical distribution throughout the gut. In contrast to some studies on the guinea-pig and rat, the co-existence of vasoactive intestinal polypeptide and nitric oxide synthase was seen in only a small population of myenteric neurones.     

The effects of hibernation on the myenteric plexus of the golden hamster small and large intestine

We have examined the effects of hibernation on the neurochemical composition of myenteric neurones in the small and large intestine of the golden hamster using immunohistochemical and histochemical techniques. Hibernation was induced in golden hamsters by altering the photoperiod and external ambient temperature. Age-matched hamsters kept at room temperature and those kept at 5 degrees C but which failed to hibernate were used as controls. Cell counts were carried out to examine possible changes in the numbers of cell bodies immunoreactive to all of the markers examined. The results demonstrated a significant increase during hibernation in the number of neurones immunoreactive to vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide; cell bodies positive for tyrosine hydroxylase, which were largely absent in the control animals, were prominent in the hibernating animals. There was a significant decrease in the number of neurones immunoreactive to 5-hydroxytryptamine, and no significant changes in the numbers of neurones immunoreactive to protein gene-product and nitric oxide synthase. It is suggested that selective upregulation and downregulation of myenteric neurones containing certain neurotransmitters may occur as a protective mechanism during hibernation to maintain the integrity of the muscular and mucosal layers of the intestine in the absence of luminal contents.     

A developmental study of the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder

A histochemical investigation of age-related changes that occur with respect to the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder was carried out. In all age groups examined (embryonic stages day 34 and 52, 2 to 4-day old, 6-month old and 2-year old), the mean percentage of NADPH-diaphorase-positive neurons per ganglion was obtained by taking the number of neurons that were immunoreactive to protein gene product 9.5 (a general neuronal marker) as 100%. In addition, the possible co-existence of NADPH-diaphorase and nitric oxide synthase in the ganglionated plexus of 2 to 4-day old and 6-month old guinea-pig gallbladder was investigated. NADPH-diaphorase was not present in the ganglionated plexus of the gallbladder at embryonic day 34. At embryonic day 52, all the protein gene product 9.5-immunoreactive neurons showed positive staining to NADPH-diaphorase; this dropped to a minimum at 2–4 days (26.7%), rose slightly at 6 months (33.6%), and finally returned close to the 100% value at 2 years. In the gallbladders of 2-year old guinea-pigs, some (3 out of 10) ganglia were devoid of protein gene product 9.5-immunoreactive neurons, but NADPH-diaphorase-stained granules were found within the ganglia. However, all those neurons that were immunopositive to protein gene product 9.5 also expressed NADPH-diaphorase. Moreover, NADPH-diaphorase-positive neurons in the gallbladder of 2 to 4-day-old and 6-month-old guinea-pigs were found to express nitric oxide synthase.     

Healthy individuals that control a latent infection with Mycobacterium tuberculosis express high levels of Th1 cytokines and the IL-4 antagonist IL-4delta2

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop disease and identifying what constitutes "protective immunity" is one of the holy grails of M. tuberculosis immunology. It is known that IFN-gamma is essential for protection, but it is also apparent that IFN-gamma levels alone do not explain the immunity/susceptibility dichotomy. The controversy regarding correlates of immunity persists because identifying infected but healthy individuals (those who are immune) has been problematic. We have therefore used recognition of the M. tuberculosis virulence factor early secretory antigenic target 6 to identify healthy, but infected individuals from tuberculosis (TB)-endemic and nonendemic regions (Ethiopia and Denmark) and have compared signals for cytokines expressed directly ex vivo with the pattern found in TB patients. We find that TB patients are characterized by decreased levels of Th1 cytokines and increased levels of IL-10 compared with the healthy infected and noninfected community controls. Interestingly, the healthy infected subjects exhibited a selective increase of message for the IL-4 antagonist, IL-4delta2, compared with both TB patients or noninfected individuals. These data suggest that long-term control of M. tuberculosis infection is associated not just with elevated Th1 responses but also with inhibition of the Th2 response.     

Ultrastructural localization of NADPH-diaphorase and colocalization of nitric oxide synthase in endothelial cells of the rabbit aorta

This is the first report on the ultrastructural pattern of distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in endothelial cells, using the rabbit aorta, and its colocalization with the neuronal isoform (type I) of nitric oxide synthase. About 30% of the endothelial cells showed a positive reaction for NADPH-d compared to about 6% for nitric oxide synthase immunoreactivity. Simultaneous double histochemical-immunocytochemical labelling procedures indicate that all of the cells displaying nitric oxide synthase-positive reactivity also contained NADPH-d; the remainder of NADPH-d-positive endothelial cells were negative for this isoform of nitric oxide synthase. Nitric oxide synthase-immunogold labelling was mostly associated with free ribosomes, while NADPH-d activity was distributed largely in patches in the cytoplasm and in association with the cell membrane.     

Effect of age on NADPH-diaphorase-containing myenteric neurones of rat ileum and proximal colon

The effect of age on the distribution of NADPH-diaphorase-containing neurones was investigated in the myenteric plexus of ileum and proximal colon of embryonic day-19 rats, as well as in rats at postnatal day 4, 6 months and 26 months. The mean percentage of NADPH-diaphorase-stained neurones per ganglion was established using protein gene product 9.5 (protein found in most if not all neurones)-immunostained neurones as 100%. The results revealed that there was a significant relative increase in NADPH-diaphorase-positive neurones with increasing age in the myenteric plexus of proximal colon with nearly all protein gene product 9.5-immunoreactive neurones staining for NADPH-diaphorase in 26-month-old rats. This was in marked contrast with the ileum, where no significant relative increase in NADPH-diaphorase-positive neurones was seen in aged rats. The implications of these findings in relation to programmed cell survival and cell death are discussed.     

Ex vivo cytokine mRNA levels correlate with changing clinical status of ethiopian TB patients and their contacts over time

There is an increasing body of evidence which suggests that IL-4 plays a role in the pathogenesis of TB, but a general consensus on its role remains elusive. We have previously published data from a cohort of Ethiopian TB patients, their contacts, and community controls suggesting that enhanced IL-4 production is associated with infection with M. tuberculosis, rather than overt disease and that long-term protection in infected community controls is associated with co-production of the IL-4 antagonist IL-4d2, alongside elevated IL-4. Here, for the first time, we compare data on expression of IFN-gamma, IL-4 and IL-4delta2 over time in TB patients and their household contacts. During the follow-up period, the TB patients completed therapy and ceased to display TB-like symptoms. This correlated with a decrease in the relative amount of IL-4 expressed. Over the same period, the clinical status of some of their contacts also changed, with a number developing TB-like symptoms or clinically apparent TB. IL-4 expression was disproportionately increased in this group. The findings support the hypothesis that elevated IL-4 production is generally associated with infection, but that TB disease is associated with a relatively increased expression of IL-4 compared to IFN-gamma and IL-4delta2. However, the data also suggest that there are no clear-cut differences between groups: the immune response over time appears to include changes in the expression of IFN-gamma, IL-4 and IL-4delta2, and it is the relative, not absolute levels of cytokine expression that are characteristic of clinical status.     

The role of the thyroid gland hormone in ontogenesis and morphological diversification of Barbus intermedius sensu of Lake Tana in Ethiopia

The role of thyroid gland hormone in the ontogenesis of the Large African Barb Barbus intermedius sensu Banister, 1973 (Teleostei; Cyprinidae)--the supposed ancestral species which produced the species flock of the large African Barbs of the Lake Tana (Ethiopia)--has been studied. It has been shown that the thyroid hormone influences the period of many morphological processes and a change in its level causes heterochrony, reflecting on the definitive morphology of fish. Thus, it can be assumed that even a slight change in activity of the thyroid axis in the process of ontogenesis can be the cause of wide variability of the morphological features of B. intermedius and is at the basis for the fast diversification of the species flock of Big African Barbs of Lake Tana.     

Release of calcitonin gene-related peptide from rat enteric nerves is Ca2+-dependent but is not induced by K+ depolarization

The effect of extracellular calcium on the release of calcitonin gene-related peptide (CGRP) induced by electrical field stimulation from enteric nerves of isolated rat ileum was studied; the effect of high potassium, veratridine and caffeine was also examined. Release of endogenous substance P from enteric nerves was also measured for comparison. Electrical field stimulation (10 Hz, 0.3 ms for 2 min) of the ileum preparation caused a significant (P less than 0.001) increase in the release of CGRP and substance P from enteric nerves. The evoked, but not the basal, release of both CGRP and substance P was inhibited in the presence of tetrodotoxin (TTX). The release of CGRP and substance P induced by electrical stimulation was abolished in Ca2+-free medium containing CDTA and also in normal medium containing the calcium channel blocker cadmium chloride (CdCl2), with no change in the level of the basal release of both peptides. However, potassium depolarization (76 and 110 mM) failed to evoke an increase in the release of endogenous CGRP, although it did cause an increase in the release can be induced by mobilization of calcium from intracellular Ca2+ stores. Veratridine, on the other hand, did not cause an increase in CGRP release, although substance P and VIP release was induced by veratridine from the same preparations. The results of the present study have demonstrated that CGRP release from enteric nerves requires the presence of extracellular calcium but, unlike substance P and most other transmitters reported to show calcium-dependent release, potassium depolarization does not induce CGRP release from enteric nerves of rat ileum.(ABSTRACT TRUNCATED AT 250 WORDS)