Morphometric investigations of the colon mucosa in chronic Trypanosoma cruzi infected rats.

The objective of this investigation was to study the morphometry of the epithelial mucosa in the chronic phase of T. cruzi infection. Nine young female Wistar rats were inoculated with T. cruzi. Ten months after inoculation the animals were sacrificed and the proximal colon was collected for morphometric measurements of the thickness of the muscle layers, the number of neurons in the myenteric plexus, the crypt cell population (CCP), crypt cell production per crypt (CCPC) and turnover time (TT) of the epithelium. There was no muscle layer hypertrophy but there was significant denervation in the group inoculated with T. cruzi, which also showed hyperplasia of the epithelium. The data suggest that denervation of the myenteric plexus did not induce hypertrophy of the propria muscle layer itself but altered the morphometry of the colonic epithelium in T. cruzi-infected animals, with increased development of CCP and TT. It is possible that this epithelial hyperplasia, as a consequence of a longer crypt cell TT, increased the absorption and secretion activities of the colon, which in turn may participate in the genesis of the enteromegalies observed in the chronic phase of Chagas' Disease.     

Morphological changes and EGF expression in the granular convoluted tubule cells of submandibular glands of Trypanosoma cruzi infected rats

We have previously demonstrated in rats that Chagas' disease affects the salivary glands, by promoting an enlargement of the submandibular gland. In order to further investigate possible functional alterations on infected submandibular glands, the objective of the present study was to analyze epidermal growth factor (EGF) expression on rat submandibular glands during Trypanosoma cruzi infection. Results demonstrated that infected rats presented lower levels of testosterone, and morphological changes in the granular convoluted tubule (GCT) cells of the submandibular glands, along with acinar enlargement and delayed ductal maturation at the developing granular ducts. Immunohistochemistry analysis additionally showed that only few cells immunolabelled with anti-EGF on infected rats during the acute phase of Chagas' disease, while after 64 and 90 days (chronic phase) of infection, EGF expression was similar to non-infected rats. The present findings suggest that at the acute phase of Chagas' disease, lower levels of testosterone may lead to a delayed maturation of GCT, which positively correlates with decreased EGF production by submandibular glands cells.     

Cloning and characterization of the phosphoglucomutase of Trypanosoma cruzi and functional complementation of a Saccharomyces cerevisiae PGM null mutant

Trypanosoma cruzi is the etiological agent of Chagas' disease, a chronic illness characterized by progressive cardiomyopathy and/or denervation of the digestive tract. The parasite surface is covered with glycoconjugates, such as mucin-type glycoproteins and glycoinositolphospholipids (GIPLs), whose glycans are rich in galactopyranose (Galp) and/or galactofuranose (Galf) residues. These molecules have been implicated in attachment of the parasite to and invasion of mammalian cells and in modulation of the host immune responses during infection. In T. cruzi, galactose (Gal) biosynthesis depends on the conversion of uridine diphosphate (UDP)-glucose (UDP-Glc) into UDP-Gal by an NAD-dependent reduction catalyzed by UDP-Gal 4-epimerase. Phosphoglucomutase (PGM) is a key enzyme in this metabolic pathway catalyzing the interconversion of Glc-6-phosphate (Glc-6-P) and Glc-1-P which is then converted into UDP-Glc. We here report the cloning of T. cruzi PGM, encoding T. cruzi PGM, and the heterologous expression of a functional enzyme in Saccharomyces cerevisiae. T. cruzi PGM is a single copy gene encoding a predicted protein sharing 61% amino acid identity with Leishmania major PGM and 43% with the yeast enzyme. The 59-trans-splicing site of PGM RNA was mapped to a region located at 18 base pairs upstream of the start codon. Expression of T. cruzi PGM in a S. cerevisiae null mutant-lacking genes encoding both isoforms of PGM (pgm1Delta/pgm2Delta) rescued the lethal phenotype induced upon cell growth on Gal as sole carbon source.     

Exacerbation of Experimental Trypanosoma cruzi Infection in Mice by Concomitant Malaria*

SYNOPSIS. The effect of malaria on the chronic phase of Chagas’disease was investigated in mice. The animals were given Plasmodium berghei-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y, CL and Gilmar). In all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas’disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curve of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phase with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Trypanosoma cruzi: preliminary investigation of NADH-positive and somatostatin-immunoreactive neurons in the myenteric plexus of the mouse colon during the infection

In this paper, the distribution of NADH-positive and somatostatin (SOM) immunoreactive neurons in the myenteric plexus of the colon of mice infected with Trypanosoma cruzi was studied. Ten young, male, BALB/c mice were inoculated with the Y strain of T. cruzi, 60 days previously (chronic phase of the infection). Another 10 mice were uninfected controls. Distal and proximal colonic neurons from five chronically infected mice and their controls were stained using the NADH-diaphorase method. Quantitative results showed a significant decrease of 39% in the number of neurons in the proximal colon of infected mice and 58% in the distal colon (p<0.05). SOM was localized in five animals from each group by light microscopy, using an indirect immunofluorescence technique. It was observed that there were far fewer nerve cells and fibres and less intensely stained neuron bodies and varicose SOM-positive nerve fibres in both, control and chronic infected mice. These findings could be related to the disturbances in intestinal motility observed in patients in the chronic phase of Chagas' disease.     

Genesis of Morphometric Invariants of Higher Plants as a Database for the Genesis of Morphometric Invariants of Vertebrates

A hypothesis is proposed on the uniformity of the information encoding the characteristic traits of higher plant macrostructure (phyllotaxis) and vertebrate macrostructure.     

Effects of chronic treatment with atypical neuroleptics on the biosynthesis and release of opioid peptides in guinea-pig ileum

The guinea-pig ileum myenteric plexus is known to contain opioid peptides, which can be released by electric stimulation at high frequency. Haloperidol, a classic neuroleptic drug, increases the biosynthesis and release of endogenous opioid peptides from the myenteric plexus. In the present work we have examined the effects of chronic treatment with sulpiride and clozapine, two atypical neuroleptics, on the release of these peptides in the myenteric plexus of guinea-pig ileum. Both neuroleptics, administered over a period of 7 days, produced an increase of the inhibitory response obtained by electrical stimulation at 10 Hz of the ileum myenteric plexuslongitudinal muscle preparation. The inhibitory response was reversed by the specific opioid antagonist naloxone, which suggests that the increase in the inhibitory response produced by blocking the dopaminergic receptors is mediated by an increase in the release of opioid peptides. When sulpiride- or clozapine-treated guinea-pigs received cycloheximide (an inhibitor of peptide biosynthesis) there was a significant decrease of the inhibitory response, which indicates that neuroleptics produced an increase of the synthesis of opioid peptides in the ileum myenteric plexus. These results reveal a possible influence of the dopaminergic system on the biological turnover of these peptides.     

Trypanosoma cruzi: effect of benznidazole therapy combined with the iron chelator desferrioxamine in infected mice

Iron chelators have been employed in various studies aimed at evaluating the relationship between the iron status of the host and the development of infection. In the present study, the effects of benznidazole (BZ) therapy in combination with the iron chelator desferrioxamine (DFO) on the development of infection in mice inoculated with Trypanosoma cruzi Y strain have been investigated. Infected mice treated with DFO presented lower levels of parasitemia compared with infected untreated animals. Therapy with BZ for 21 days, with or without DFO, led to decreased parasitemia and reduced mortality, but BZ in combination with DFO treatment for 35 days (BZ/DFO-35) gave 0% mortality. All infected groups presented lower levels of iron in the liver, but serum iron concentrations were greater in DFO-35 and BZ/DFO-35, whereas hemoglobin levels were higher in BZ/DFO-35 and lower in DFO-35 compared with other treated groups. The percentage cure, determined from negative hemoculture and PCR results in animals that had survived for 60 days post-infection, was 18% for BZ and BZ/DFO-35, 42% for BZ combined with DFO for 21 days, and 67% for DFO-35. The results demonstrate that modification in iron stores increases BZ efficacy.     

Morphology of the digestive system in carnivorous freshwater dourado Salminus brasiliensis

The digestive system of teleost shows remarkable functional and morphological diversity. In this study, the digestive tract and accessory organs of dourado Salminus brasiliensis are characterized using anatomical, histological, histochemical and immunohistochemical analyses. The existence of taste buds bordered by microridges in the oesophagus of dourado was recorded for the first time, thus showing that the species drives food intake by either swallowing or rejecting the food item. The Y-shaped stomach of dourado consisted of cardiac, cecal and pyloric regions with tubular gastric glands registered solely in the cardiac and cecal segments. The intestine is a short N-shaped tube with two loops, an intestinal coefficient of 0.73. The structure of pyloric caeca is similar to that of the intestine wall, comprising tunica mucosa, tela submucosa, tunica muscularis and tunica serosa layers. Histochemical analyses revealed an increased incidence of goblet cells from the midgut to the hindgut segment. A well-developed enteric plexus of scattered nerve cell and fibres are found along the digestive tract, and the calcitonin gene-related peptide (CGRP) immunoreactive neurons and fibres were identified in the myenteric plexus from the oesophagus to the hindgut. The exocrine pancreas appears diffuse in the mesentery around the stomach, intestine and also reaches the liver, and the endocrine pancreas is organized as a few islets of Langerhans. The liver comprises three distinct, asymmetric lobes, and the portal triad arrangement was registered in this tissue.     

Depressed neuronal growth associated protein (GAP)-43 expression in the small intestines of mice experimentally infected with Neodiplostomum seoulense

Neodiplostomum seoulense (Digenea: Neodiplostomidae) is an intestinal trematode that can cause severe mucosal pathology in the small intestines of mice and even mortality of the infected mice within 28 days after infection. We observed neuronal growth associated protein-43 (GAP-43) expression in the myenteric plexus of the small intestinal wall of N. seoulense-infected mice until day 35 post-infection (PI). BALB/c mice were infected with 200 or 500 N. seoulense metacercariae isolated from naturally infected snakes and were killed every 7 days for immunohistochemical demonstration of GAP-43 in the small intestines. N. seoulense-infected mice showed remarkable dilatation of intestinal loops compared with control mice through days 7-28 PI. Conversely, GAP-43 expression in the mucosal myenteric plexus was markedly (P<0.05) reduced in the small intestines of N. seoulense-infected mice during days 7-28 PI and was slightly normalized at day 35 PI. From this study, it is evident that neuronal damage occurs in the intestinal mucosa of N. seoulense-infected mice. However, the correlation between intestinal pathology, including the loop dilatation, and depressed GAP-43 expression remains to be elucidated.     

Reserpine-induced suppression of cortifugal influences on neostriatal neurons in cats and rats

In acute experiments on cats and rats, we demonstrated that the relative number of neostriatal neurons responding to stimulation of the motor cortex with latencies below 8.0 msec significantly decreased after functional destruction of the nigro-striatal dopaminergic system caused by injections of reserpine. Despite the fact that the level of dopamine (DA) in the neostriatum returned to the initial value 24 h after injection of the above neuroleptic, cortico-striatal impulsation recovered slowly, and the number of short-latency corticofugal reactions attained a near-control value only in one month. The data obtained confirm our earlier hypothesis on the toxic effect of excessive amounts of glutamate (which is observed under conditions of the DA deficiency) on receptors of this neurotransmitter. We conclude that, under normal conditions, DA exerts an inhibitory/protective effect on transmission of impulsation through direct cortico-striatal connections influencing D₂ receptors localized on cortico-striatal glutamatergic efferents. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 47–51, January–February, 2007.     

Increased permeability of gut mucosa in rats infected with Nippostrongylus brasiliensis.

The permeability of the gut mucosa to macromolecules has been examined in 4 different strains of rats infected with Nippostrongylus brasiliensis (N. brasiliensis). There is a marked increase in mucosal permeability during the infection. In addition, a considerable strain difference is observed in both worm burden kinetics and the kinetics of intestinal permeability. The dose-response and drug treatment experiments in outbred Wistar rats suggest that increased mueosal permeability is a function of the worm burden. However, the increased permeability is related neither to the rapid phase of worm expulsion nor to the rise in intestinal mast cell numbers.     

The Effects of Piroxicam in the Attenuation of MPP+/MPTP Toxicity In Vitro and In Vivo

Several lines of evidence support the neuroprotective action of cyclooxygenase-2 (COX-2) inhibitors in various models of Parkinson’s disease (PD). In the current study, we investigated the neuroprotective properties of several COX inhibitors against 1-methyl-4-phenylpyridinium (MPP+) in neuroblastoma Neuro 2A (N-2A) cells in vitro and the protection against degeneration of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons after the administration of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) in C57/BL6 male mice. The data obtained demonstrate a lack of protective effects observed by COX 1-2 inhibitors ibuprofen and acetylsalicylic acid against MPP+ toxicity in N-2A, where piroxicam was protective in a dose dependent manner (MPP+ control: 15 ± 2% MPP+ piroxicam: 5 mM 89 ± 4%). The data also indicate a drop in mitochondrial oxygen (O₂) consumption and ATP during MPP+ toxicity with no restoration of mitochondrial function concurrent to a heightened concentration of somatic ATP during piroxicam rescue. These findings indicate that the neuroprotective effects of COX inhibitors against MPP+ are not consistent, but that piroxicam may work through an unique mechanism to propel anaerobic energy metabolism. On the other hand, using mice, piroxicam (20 mg/kg) was effective against MPTP-induced dopaminergic degeneration in the (SNc) and loss of locomotive function in mice. Administering a 3 day pre-treatment of piroxicam (20 mg/kg) was effective in antagonizing the losses in SNc tyrosine hydroxylase protein expression, SNc DA concentration and associated anomaly in ambulatory locomotor activity. It was concluded from these findings that piroxicam is unique among COX inhibitors in providing very significant neuroprotection against MPP+ in vitro and in vivo.     

The interstitial cells in the colon of the rabbit

Summary The interstitial cells associated with the myenteric plexus of the rabbit colon were studied by scanning and transmission electron microscopy. It was demonstrated that the interstitial cells were stellate or fusiform in shape and located over the ganglia, over nerve bundles and between muscle cells. They were characterized by many slender processes, and resemble fibroblasts. No basal lamina was observed between the interstitial cells and muscle cells. It was concluded that structural features of the interstitial cells are distinctly different from those of neurons, Schwann cells, or of smooth muscle cells, while they show clear similarities to those of fibroblasts. By scanning electron microscopy the shapes and the relations of these cells could be demonstrated in great detail.     

Comparative investigations of growth and solvent formation in ‘Clostridium saccharoperbutylacetonicum’ DSM 2152 and Clostridium acetobutylicum DSM 792

The butanol and acetone-producing strain DSM 2152, invalidly described as ‘Clostridium saccharoperbutylacetonicum’ is compared with the type strain C. acetobutylicum, DSM 792, with respect to solvent and acid formation at varying pH values and growth rates. Batch cultures, product-limited chemostat and pH-auxostat cultures were used for characterization. Under all conditions strain DSM 2152 produced much lower amounts of butyric and acetic acids than the type strain. The pH optimum for solvent formation was higher, ie 5.5 instead of 4.5. Solvent formation occurred at higher dilution rates, but below 0.1 h⁻¹ a lower solvent concentration was obtained, indicating that acid production was too low to provide a sufficient amount for acetone formation. The results are discussed in the light of recent publications on the taxonomy of butanol-acetone producing clostridia using 16S rRNA sequence analysis and other nucleic acid data. The presently suggested ‘phylogenetic’ classification of the collective species, C. acetobutylicum, is also reflected in the fermentation characteristics. Received 21 December 1998/ Accepted in revised form 22 January 1999     

Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites

The investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci – six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.     

The response of the blood-sucking bug Triatoma infestans to carbon dioxide and other host odours

Behavioural responses of Triatoma infestans larvae to carbon dioxide and other odours of vertebrate origin were investigated in a locomotion compensator. T. infestans oriented towards airstreams enriched with carbon dioxide exhibiting a threshold response between 300 and 400 p.p.m. above the ambient CO(2) background. The accuracy of the oriented response to carbon dioxide improved with stimulus intensity. Remarkably, insects did not show any change in their sensitivity threshold to carbon dioxide with the starvation time. The attractiveness to carbon dioxide depended on the time of the day, i.e. these nocturnal bugs only oriented towards carbon dioxide-loaded airstreams during the first hours of the scotophase. L-lactic acid did not evoke oriented responses when it was presented as a single stimulus in a wide range of intensities. However, a marked synergism was evident when L-lactic acid was combined with a sub-threshold concentration of carbon dioxide. Under this condition, the threshold response to carbon dioxide decreased to 75-150 p.p.m. above ambient CO(2) background. The isomer D-lactic acid evoked no response, either alone or in combination with carbon dioxide. When insects were stimulated with 1-octen-3-ol a significant positive orientation was found. This response was not modified by the addition of carbon dioxide.     

Free Copper,Ferroxidase and SOD1 Activities,Lipid Peroxidation and NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> Content in the CSF. A Different Marker Profile in Four Neurodegenerative Diseases

The understanding of oxidative damage in different neurodegenerative diseases could enhance therapeutic strategies. Our objective was to quantify lipoperoxidation and other oxidative products as well as the activity of antioxidant enzymes and cofactors in cerebrospinal fluid (CSF) samples. We recorded data from all new patients with a diagnosis of either one of the four most frequent neurodegenerative diseases: Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD) and lateral amyotrophic sclerosis (ALS). The sum of nitrites and nitrates as end products of nitric oxide (NO) were increased in the four degenerative diseases and fluorescent lipoperoxidation products in three (excepting ALS). A decreased Cu/Zn-dependent superoxide dismutase (SOD) activity characterized the four diseases. A significantly decreased ferroxidase activity was found in PD, HD and AD, agreeing with findings of iron deposition in these entities, while free copper was found to be increased in CSF and appeared to be a good biomarker of PD.     

Impact of the metabolic activity of Streptococcus thermophilus on the colon epithelium of gnotobiotic rats

The thermophilic lactic acid bacterium Streptococcus thermophilus is widely and traditionally used in the dairy industry. Despite the vast level of consumption of S. thermophilus through yogurt or probiotic functional food, very few data are available about its physiology in the gastrointestinal tract (GIT). The objective of the present work was to explore both the metabolic activity and host response of S. thermophilus in vivo. Our study profiles the protein expression of S. thermophilus after its adaptation to the GIT of gnotobiotic rats and describes the impact of S. thermophilus colonization on the colonic epithelium. S. thermophilus colonized progressively the GIT of germ-free rats to reach a stable population in 30 days (10(8) cfu/g of feces). This progressive colonization suggested that S. thermophilus undergoes an adaptation process within GIT. Indeed, we showed that the main response of S. thermophilus in the rat's GIT was the massive induction of the glycolysis pathway, leading to formation of lactate in the cecum. At the level of the colonic epithelium, the abundance of monocarboxylic acid transporter mRNAs (SLC16A1 and SLC5A8) and a protein involved in the cell cycle arrest (p27(kip1)) increased in the presence of S. thermophilus compared with germ-free rats. Based on different mono-associated rats harboring two different strains of S. thermophilus (LMD-9 or LMG18311) or weak lactate-producing commensal bacteria (Bacteroides thetaiotaomicron and Ruminococcus gnavus), we propose that lactate could be a signal produced by S. thermophilus and modulating the colon epithelium.     

Nuclear microscopy in the life sciences at the national university of singapore

The nuclear microscope is now gaining popularity in the field of life sciences. In particular, the combination of proton-induced X-ray emission to measure the elemental concentrations of inorganic elements, Rutherford backscattering spectrometry to characterize the organic matrix, and scanning transmission ion microscopy to provide information on the density and structure of the sample represents a powerful set of techniques that can be applied simultaneously to the specimen under investigation. These techniques are extremely useful for measuring any imbalances in trace elements in localized regions of biological tissue and, as such, can provide unique information on many diseases. In this article, we describe the nuclear microscope and its related ionbeam techniques, and we review the biomedical work carried out using the nuclear microscope in the National University of Singapore.