Image processing,diagnostic information extraction and quantitative assessment in pathology

As we enter the information age we hold strong beliefs in the benefits of digital technology applied to pathology: numerical representation offers objectivity . Digital knowledge may indeed lead to significant information discovery, and, processing systems might be designed to allow a true evolution of capabilities. Questions arise whether the methodology underlying quantitative analysis provides the information that we need and whether it is appropriate for some of the problems encountered in diagnostic and prognostic histopathology. While one certainly would not dispute the value of statistical procedures, the clinical needs call for individual patient targeted prognosis.     

Response to oxidative stress as a welfare parameter in swine

In pigs, the genetic selection for lean, large muscle blocks and fast growth has been linked to an increased prevalence of metabolic diseases such as porcine stress syndrome and mulberry heart disease. These diseases are associated with cardiovascular inadequacy, which may lead to oxidative stress. In the present study, reactive oxygen metabolites (ROMs) and the anti-oxidant power (OXY) in sera of different swine groups were investigated. The following groups were selected (each around 80 kg body weight): wild boars (WB), Cinta Senese (CS), and Landrace x Large White (LxLW), the latter as both specific pathogen-free (SPF) and intensively farmed animals. In addition, a group of LxLW agonic sows (AS) was also investigated; this group is known to be under oxidative stress. Two colorimetric micro-methods were used to measure ROMs and OXY; ROMs were expressed as mM H(2)O(2) and OXY as microM HOCl neutralised. Between groups, average ROM and OXY values were found to be significantly different by one-way ANOVA (P < 0.001). ROM levels were lower in WB (13.41 +/- 1.85) and CS (19.27 +/- 1.68), and highest in LxLW (42.00 +/- 1.36). OXY values ranged from 260.10 +/- 22.13 (WB) to 396.90 +/- 9.83 (LxLW). Only one swine group (the CS group) showed a significant, positive correlation between ROM and OXY values. The AS group even showed a negative correlation between ROM and OXY values. These results imply satisfactory environmental coping occurred only within the CS group. Results are discussed in the light of animal welfare legislation, food safety and consumers' protection.     

Status and metabolism of iron in elite sportsmen during a period of professional competition

The aim of this study was to determine the effect of both acute exercise and maintained training during a period of competition (3 mo, at the start of the season) on iron metabolism in sportsmen on a professional volleyball team. Twelve sportsmen volunteered for this study. The exercise test was performed on a mechanically braked Monark cycle ergometer and consisted of a triangular progressive test. Three blood samples were obtained in each test: at rest, just after exercise, and after recovery. The following hematological parameters were determined: red blood count (RBC), hemoglobin (Hb) and hematocrit (Hto), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total proteins (TP), serum iron (Fe) and total iron-binding capacity (TIBC), ferritin (FER), transferrin (TRF), haptoglobin (HPT), and serum cortisol (COR) concentrations. We have found changes in hematological and biochemical variables related to Fe metabolism during the study. The changes observed could be the result of hemoconcentration processes after exercise and, at least in part, to physical stress and muscular damage. We conclude that athletes, after a period of adaptation, with a good plan of work/recovery series, undergo a biological redistribution on hematological and biochemical parameters concerning Fe metabolism during the training and competition period. Also, daily Fe supplementation could restore and mask the true repercussions of maintained training observed in other sports.     

Apoptotic rate: a new indicator for the quantification of the incidence of apoptosis in cell cultures

BACKGROUND: Late apoptotic cells divide into apoptotic bodies and are missed by current detection methods. This results in an artificially low apoptotic index (AI). METHODS: This study proposes a flow cytometry-based ratiometric method that uses an internal reference standard of microbeads combined with fluorescein-annexin V binding and 7-aminoactinomycin D to enumerate viable, necrotic, and early and late apoptotic cells within specific subsets of a heterogeneous culture. RESULTS: In the absence of cell growth, the number of apoptotic cells that undergo fragmentation into apoptotic bodies in culture can also be determined accurately by this method. This information can then be used to obtain the apoptotic rate (AR), a new indicator of apoptosis that calculates the proportion of cells that have undergone apoptosis with respect to the total number of seeded cells. The main limitation of the method is that the AR is only suitable for the study of apoptosis in noncycling cells. CONCLUSIONS: This study reveals the superiority of the proposed method over the widely used Nicoletti method and current annexin-V binding methods. The AI did not reflect the true incidence of lymphocyte apoptosis, neither in response to lectins or phorbol esters, nor to serum deprivation. AR was more sensitive than AI, detecting apoptosis at lower concentrations of cell death inducers in all the subsets studied.     

Optic nerve degeneration and mitochondrial dysfunction: genetic and acquired optic neuropathies

Selective degeneration of the smallest fibers (papillo-macular bundle) of the human optic nerve occurs in a large number of optic neuropathies characterized primarily by loss of central vision. The pathophysiology that underlies this peculiar pattern of cell involvement probably reflects different forms of genetic and acquired mitochondrial dysfunction.Maternally inherited Leber's hereditary optic neuropathy (LHON), dominant optic atrophy (Kjer disease), the optic atrophy of Leigh's syndrome, Friedreich ataxia and a variety of other conditions are examples of inherited mitochondrial disorders with different etiologies. Tobacco-alcohol amblyopia (TAA), the Cuban epidemic of optic neuropathy (CEON) and other dietary (Vitamins B, folate deficiencies) optic neuropathies, as well as toxic optic neuropathies such as due to chloramphenicol, ethambutol, or more rarely to carbon monoxide, methanol and cyanide are probably all related forms of acquired mitochondrial dysfunction.Biochemical and cellular studies in LHON point to a partial defect of respiratory chain function that may generate either an ATP synthesis defect and/or a chronic increase of oxidative stress. Histopathological studies in LHON cases and a rat model mimicking CEON revealed a selective loss of retinal ganglion cells (RGCs) and the corresponding axons, particularly in the temporal-central part of the optic nerve. Anatomical peculiarities of optic nerve axons, such as the asymmetric pattern of myelination, may have functional implications on energy dependence and distribution of mitochondrial populations in the different sections of the nerve. Histological evidence suggests impaired axonal transport of mitochondria in LHON and in the CEON-like rat model, indicating a possible common pathophysiology for this category of optic neuropathies. Histological evidence of myelin pathology in LHON also suggests a role for oxidative stress, possibly affecting the oligodendrocytes of the optic nerves.     

Involvement of 5-HT(2A/2B/2C) receptors on memory formation: simple agonism,antagonism, or inverse agonism?

1. The 5-HT₂ receptors subdivision into the 5-HT_2A/2B/2C subtypes along with the advent of the selective antagonists has allowed a more detailed investigation on the role and therapeutic significance of these subtypes in cognitive functions. The present study further analyzed the 5-HT₂ receptors role on memory consolidation.2. The SB-200646 (a selective 5-HT_2B/2C receptor antagonist) and LY215840 (a nonselective 5-HT_2/7 receptor antagonist) posttraining administration had no effect on an autoshaped memory consolidation. However, both drugs significantly and differentially antagonized the memory impairments induced by 1-(3-chlorophenyl)piperazine (mCPP), 1-naphtyl-piperazine (1-NP), mesulergine, or N-(3-trifluoromethylphenyl) piperazine (TFMPP).3. In contrast, SB-200646 failed to modify the facilitatory procognitive effect produced by (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) or ketanserin, which were sensitive to MDL100907 (a selective 5-HT_2A receptor antagonist) and to a LY215840 high dose.4. Finally, SB-200646 reversed the learning deficit induced by dizocilpine, but not that by scopolamine; while SB-200646 and MDL100907 coadministration reversed memory deficits induced by both drugs.5. It is suggested that 5-HT_2B/2C receptors might be involved on memory formation probably mediating a suppressive or constraining action. Whether the drug-induced memory impairments in this study are explained by simple agonism, antagonism, or inverse agonism at 5-HT₂ receptors remains unclear at this time.6. Notably, the 5-HT₂ receptor subtypes blockade may provide some benefit to reverse poor memory consolidation conditions associated with decreased cholinergic, glutamatergic, and/or serotonergic neurotransmission.     

Molecular dynamics study of a hyperthermophilic and a mesophilic rubredoxin

In recent years, increased interest in the origin of protein thermal stability has gained attention both for its possible role in understanding the forces governing the folding of a protein and for the design of new highly stable engineered biocatalysts. To study the origin of thermostability, we have performed molecular dynamics simulations of two rubredoxins, from the mesophile Clostridium pasteurianum and from the hyperthermophile Pyrococcus furiosus. The simulations were carried out at two temperatures, 300 and 373 K, for each molecule. The length of the simulations was within the range of 6-7.2 ns. The rubredoxin from the hyperthermophilic organism was more flexible than its mesophilic counterpart at both temperatures; however, the overall flexibility of both molecules at their optimal growth temperature was the same, despite 59% sequence homology. The conformational space sampled by both molecules was larger at 300 K than at 373 K. The essential dynamics analysis showed that the principal overall motions of the two molecules are significantly different. On the contrary, each molecule showed similar directions of motion at both temperatures.     

Highly specific inactivation of triosephosphate isomerase from Trypanosoma cruzi

We searched for molecules that selectively inactivate homodimeric triosephosphate isomerase from Trypanosoma cruzi (TcTIM), the parasite that causes Chagas' disease. We found that some benzothiazoles inactivate the enzyme. The most potent were 3-(2-benzothiazolylthio)-propanesulfonic acid, 2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid, and 2-(2-4(4-aminophenyl)benzothiazole-6-methylbenzothiazole-7-sulfonic acid. Half-maximal inactivation by these compounds was attained with 33, 56, and 8 microM, respectively; in human TIM, half-maximal inactivation required 422 microM, 3.3 mM, and 1.6 mM. In TcTIM, the effect of the benzothiazoles decreased as the concentration of the enzyme was increased. TcTIM has a cysteine (Cys 15) at the dimer interface, whereas human TIM has methionine in that position. In M15C human TIM, the benzothiazole concentrations that caused half-maximal inactivation were much lower than in the wild type. The overall findings suggest that the benzothiazoles perturb the interactions between the two subunits of TcTIM through a process in which the interface cysteine is central in their deleterious action.     

Species of Malassezia associated with various dermatoses and healthy skin in the Mexican population

At the present, eight Malassezia species have been described and their distribution in normal skin and in several skin diseases appears variable. The aim of the present study was to determine the frequency and distribution of Malassezia species in patients with psoriasis, seborrhoeic dermatitis and pityriasis versicolor attended in a Hospital from Mexico City, in addition to a healthy individual group. Scales of abnormal and healthy skin were grown in modified Dixon agar and the species identification was performed by macroscopic and microscopic features; by catalase and urease reaction; growth at 32, 37 and 40 degrees C; and Tween 20, 40, 60 and 80 assimilation. The cultures from 63 persons were included: forty six patients (20 psoriasis, 15 seborrhoeic dermatitis, 11 pityriasis versicolor) and 17 healthy individuals (external auditory canal). A total of 96 isolates were obtained. The more frequently isolated species were: M. sympodialis (38.2%) and M. furfur (26.5%) in psoriasis; M. sympodialis (38.5%) and M. slooffiae (34.6%) in seborrhoeic dermatitis; M. globosa (46.7%) and M. sympodialis (26.7%) in pityriasis versicolor; and M. restricta (47.6%) and M. globosa (23.8%) in normal skin. The number of isolates, the species diversity and association were higher in the patients group than in the healthy individuals group.     

Beauvericin cytotoxicity to the invertebrate cell line SF-9

The cyclic hexadepsipeptide beauvericin, initially known as a secondary metabolite produced by the entomopathogenic fungus Beauveria bassiana and toxic to Artemia salina larvae, has been more recently recognized as an important mycotoxin synthesized by a number of Fusarium strains, which parasite maize, wheat and rice. Therefore, this mycotoxin may enter the food chain, causing yet unknown effects to the health of both domestic animals and humans. The cytotoxic effects of beauvericin on mammalian cells have been studied. We investigated the cytotoxicity of this compound in an in vitro invertebrate model, viz. the insect cell line SF-9 (immortalized pupal ovarian cells of the lepidopter Spodoptera frugiperda). Cultures of SF-9 cells in the stationary phase were exposed to beauvericin at concentrations ranging from 100 nM to 300 microM, for different periods of time (from 30' to 120 h). The effects on cell viability were assessed by the trypan blue exclusion method. After 4 h of incubation no significant decrease in cell viability was recorded in SF-9 cell cultures exposed to low concentrations of beauvericin, i.e. 100 nM and 300 nM. However, a slight decrease in viability (3.9%) was seen already in cells exposed to the mycotoxin at the 1 microM concentration. This effect became gradually more evident at higher concentrations (approximately equal to 28% at 30 microM, approximately equal to 50% at 100 microM, approximately equal to 68% at 300 microM). An even more pronounced reduction in cell viability was observed after a 24 h exposure. Under these conditions, 1 microM beauvericin caused an approx. 10% decrease in the number of viable cells, which became more significant at higher concentrations approximately equal to 23% at 3 microM, approximately equal to 47% at 10 microM, approximately equal to 65% at 30 microM, approximately equal to 90% at 100 microM, approximately equal to 99% at 300 microM). Therefore, the 50% cytotoxic concentrations (CC50) at 4 h and 24 h could be estimated as 85 microM and 10 microM, respectively. In time-course experiments, no effect of beauvericin (30 microM) on cell viability could be seen after exposure for periods of time as long as 30', 1 h and 2 h, respectively. In contrast, when SF-9 cells were exposed to the mycotoxin for longer periods of time, from 8 h to 120 h, we recorded a strong cytotoxic effect already in the low micromolar concentration range. Thus, the CC50 after both 72 h and 120 h exposure times was assessed as 2.5 microM. Higher concentrations caused a virtually 100% cell death. The data collected suggest that beauvericin exerts a substantial dose- and time-dependent cytotoxic effect on invertebrate cells, comparable to the effects described in mammalian cells.