Characterisation of the intracellular protozoan MPX in Scottish mussels, Mytilus edulis Linnaeus, 1758.

Ciliates have been reported as pathogens of many species of economically important bivalves. Mussel protozoan X (MPX), is an uncharacterised intracellular ciliate of mussels and has been widely reported in Mytilus spp. around the world. In order to characterise this ciliate, Mytilus edulis samples were collected from a site on the West coast of Scotland, and four different fixatives for histological examination were tested. Fresh preparations of mussel digestive glands were also examined by laser scanning confocal microscopy. Intracellular ciliates were prepared by laser capture microdissection and partial sequences of small subunit ribosomal RNA gene and of large subunit ribosomal RNA gene were generated, using Phyllopharyngea primers. Methacarn solution proved to be the best fixative for both histological and molecular characterisation. The morphological and molecular investigations confirmed that this ciliate belongs to the class Phyllopharyngea, order Rhynchodida. However, this organism does not belong to any known family, genus or species, therefore, a new description is necessary, following further morphological analyses. Most mussel samples containing MPX displayed mild to moderate infections, with no signs of necrosis or haemocytic response, although a single sample displayed a severe infection (∼103 ciliates per section). The localisation of this ciliate in tissues other than the digestive gland, the presence of necrosis in infected tissue of the most severely infected mussel and the binary fission of this ciliate have been observed here for the first time. We also report the first observation of the live ciliate isolated from tissue. Although MPX remains of unknown significance to the mussel industry, tools and protocols described here will be useful in further characterising these and other ciliates (subclass Rhynchodia) known as pathogens for bivalves.

Cetuximab has an inhibitory effect on cell motility in SCC-4 oral squamous cell carcinoma cell line.

Cetuximab is a chimeric monoclonal antibody that acts as a competitive antagonist, by binding to EGFR. This cell signalling pathways regulates tumor progression. The oral squamous cell carcinoma undergoes to regional spreading and distant metastasis. This study aimed to evaluate the effect of treatment with Cetuximab on cell migration and invasion in OSCC cells, by using the SCC-4 cell line. Cell migration and cell invasion assay were performed and actin cytoskeleton of control and treated with Cetuximab cells were evaluated. Differences were considered significant when p<0.05.Cetuximab inhibited the migration of SCC-4 cells at three concentrations: 1 µg/mL, 50 µg/mL and 100 µg/mL (p<0.0001) in a dose-dependent manner. The number of SCC-4 treated cells with 1 µg/mL that migrated through the membrane was statistically different from 50 µg/mL (p<0.001) and 100 µg/mL (p<0.0001), and between 50 µg/mL and 100 µg/mL (p<0.01). Cetuximab 50 µg/mL inhibited cell invasion through the MatrigelTM compared with SCC-4 control cells (p<0.01). Cetuximab 50 µg/mL affected the organization of the actin cytoskeleton. Cetuximab has an inhibitory effect on actin cytoskeleton organization, cell migration and invasion, suggesting that Cetuximab treatment can be important to avoid oral squamous cell carcinoma metastasis.

Immunohistochemical study of RhoC GTPase in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a disease with high mortality and morbidity. Metastasis is a significant prognostic factor of the OSCC patients. The Rho GTPases are signaling proteins that controls important cellular processes in various complex mechanisms involved in carcinogenesis. This study aimed to evaluate the expression pattern of RhoC in OSCC protein by immunohistochemistry in situ. Immunohistochemical reactions were performed for RhoC by the method of avitina-biotin-peroxidase activity in samples OSCC: well differentiated (BD, n=6), moderately differentiated (MD, n=24) and poorly differentiated (PD, n=13). The morphometry was taken by QuickScore (percentage and intensity of staining) and only intensity staining. There was no statistical difference (p>0.05) through none of the modes of morphometric analysis between BD, MD and PD. And the RhoC staining was not associated with the histopathologic grading (χ2 = 4.65, p>0.05). However, the morphological evaluation of immunostained for RhoC in cases BD, MD, PD OSCC, regardless of histopathologic grading. These results suggest that there is no correlation between the RhoC immunoexpression and histopathological grading of OSCC.

GTPases Rho distribution in intraepithelial and invasive neoplasias of the uterine cervix.

PURPOSE OF INVESTIGATION: To evaluate the distribution of GTPases RhoA, RhoB, and Cdc42 in cervical intraepithelial neoplasias (CIN) and invasive neoplasias of the uterine cervix. MATERIALS AND METHODS: samples of neoplastic lesions of the uterine cervix of 44 patients were classified in: CIN I (n = 10), CIN II (n = 10), CIN III (n = 09), and invasive carcinoma (n = 15). Antibodies anti-RhoA, anti-RhoB, and anti-Cdc42 were used and staining was classified as: negative, mild, moderate, and intense positive. RESULTS: When compared with dysplastic cells, superficial cells showed: higher expression of RhoB in CIN I (p = 0.0018), and lower expression of Cdc42 in CIN I (p = 0.0225). The authors observed higher expression of RhoA (p = 0.0002) and RhoB (p = 0.0046) in CIN dysplastic cells when compared with invasive carcinoma cells. CONCLUSIONS: GTPases Rho may be involved with the regulation of biological processes, important to the progression of cervical neoplasias. Probably, RhoA is important for maintenance of cell differentiation and RhoB protects cells from malignant cervical neoplasia.

Long-term (1990-2019) monitoring of forest cover changes in the humid tropics.

Accurate characterization of tropical moist forest changes is needed to support conservation policies and to quantify their contribution to global carbon fluxes more effectively. We document, at pantropical scale, the extent and changes (degradation, deforestation, and recovery) of these forests over the past three decades. We estimate that 17% of tropical moist forests have disappeared since 1990 with a remaining area of 1071 million hectares in 2019, from which 10% are degraded. Our study underlines the importance of the degradation process in these ecosystems, in particular, as a precursor of deforestation, and in the recent increase in tropical moist forest disturbances (natural and anthropogenic degradation or deforestation). Without a reduction of the present disturbance rates, undisturbed forests will disappear entirely in large tropical humid regions by 2050. Our study suggests that reinforcing actions are needed to prevent the initial degradation that leads to forest clearance in 45% of the cases.

Laser vaporization in the management of CIN.

AIMS: To evaluate the effectiveness of laser CO2 vaporization in high-grade cervical intraepithelial neoplasias and to assess the diagnostic reliability of cytology, colposcopy, microbiology and HPV tests in predicting recurrence in a long-term outcome. METHODS: Forty-four patients affectd by high-grade cervical intraepithelial neoplasia (HG-CIN) were submitted to laser CO2 vaporization and followed-up a minimum of five years. Vaginal smears for microbiological examination were detected. HPV testing was performed by polymerase chain reaction (PCR). RESULTS: The average age of the patients was 19.5 years (range 15-24). The cure rate after a single treatment was 95%. Two cases (5%) revealed HG-CIN persistence after three months. The five year follow-up of all cases submitted to a second laser procedure revealed negative cytologic and colposcopic findings. CONCLUSIONS: A higher degree of expertise and experience from the colposcopist and long-term follow-up proves the effectiveness of laser vaporization in the management of CIN in young women. It has been suggested that HPV infection alone may not be sufficient to promote carcinogenesis and that other cofactors could be involved. Microbiological tests are important to identify and treat any inflammation which might represent a cofactor of HPV infection in the pathogenesis of cervical dysplasia. Cytocolposcopic long-term follow-up, microbiological and HPV tests can improve regression of disease.

AS601245, a c-Jun NH2-terminal kinase (JNK) inhibitor, reduces axon/dendrite damage and cognitive deficits after global cerebral ischaemia in gerbils.

BACKGROUND AND PURPOSE: Based on their proven ability, in animal models of stroke, to reduce damage to brain grey matter, many drugs have been tested in clinical trials but without success. Failure to save axons from injury and to protect functional outcome has been proposed as the major reason for this lack of success. We have previously demonstrated in two rodent models of cerebral ischaemia, that AS601245 (1,3-benzothiazol-2-yl (2-([2-(3-pyridinyl) ethyl] amino)-4 pyrimidinyl) acetonitrile), an inhibitor of the c-Jun NH(2)-terminal kinase (JNK), has neuroprotective properties. The aim of the present study was to further investigate if AS601245 in addition to its ability to protect neurons also could protect neurites and preserve memory after cerebral ischaemia, in gerbils. EXPERIMENTAL APPROACH: Using immunohistochemical techniques and a behavioural test, we studied the effect of the compound AS601245 on neurodegeneration and cognitive deficits after global cerebral ischaemia in gerbils. KEY RESULTS: At a dose of 80 mg kg(-1), i.p., AS601245 reduced damage to neurites by 67% (P<0.001 versus controls) and activation of astrocytes by 84% (P<0.001 versus controls). In addition, AS601245 (80 mg kg(-1), i.p.) prevented ischaemia-induced impairment of memory in the inhibitory avoidance task model. CONCLUSIONS AND IMPLICATIONS: The present results suggest that AS601245 reduced damage to neurites and decreased astrogliosis following global ischaemia and also improved long-term memory, supporting JNK inhibition as a promising therapeutic strategy for ischaemic insults to the CNS.

Correlation between squamous intraepithelial lesions (SILs) and bacterial vaginosis.

Bacterial vaginosis (BV) is a condition that seldom occurs in prepuberal girls or postmenopausal women, suggesting a hormonal component in its aetiology. The precise mechanisms by which BV arises are not fully understood. One proposed mechanism suggests that carcinogenic nitrosamines act either independently or via human papilloma virus (HPV). Human papillomavirus is known to be associated with the development of squamous intraepithelial lesion (SIL). Still today the relationship between BV and SIL is debated. Many confounding factors regarding the relationship between BV and SIL include the presence of HPV and/or other sexually transmitted diseases. In a case-controlled study the correlation between BV, SIL and the presence of HPV was evaluated. BV was diagnosed according to standard criteria: vaginal pH > 4.5; positive amine test or 'whiff' test; presence of clue cells and abnormal discharge. High risk-HPV testing by PCR was performed. X2 Pearson analysis was applied for statistical evaluation of data. The results of the study have shown that BV is not associated with SIL.

Glutamate-induced increase of extracellular glutamate through N-methyl-D-aspartate receptors in ethanol withdrawal.

Ethanol withdrawal is a physiopathological state associated with increased number and function of N-methyl-D-aspartate glutamate receptors. We assessed the effect of N-methyl-D-aspartate receptor stimulation on the extracellular levels of glutamate in vivo by the focal application of N-methyl-D-aspartate in the striatum of dependent rats following withdrawal from chronic treatment with ethanol. In control, chronic sucrose-treated rats, 800 microM N-methyl-D-aspartate increased glutamate levels to 268% of baseline values. In ethanol-withdrawn animals, 12 h after interruption of the chronic treatment, the application of N-methyl-D-aspartate increased glutamate levels to 598% of baseline values. In ethanol-intoxicated rats N-methyl-D-aspartate was ineffective. Concentration-response curves showed that in ethanol withdrawn animals N-methyl-D-aspartate was five-fold more potent than in controls. In withdrawn animals, the non-competitive N-methyl-D-aspartate receptor antagonist dizocilpine (1.0 mg/kg i.p.) or ethanol (5 g/kg i.g.) markedly reduced the N-methyl-D-aspartate-induced increase in glutamate levels. These results are consistent with the up-regulation of N-methyl-D-aspartate receptors by chronic ethanol and add biochemical evidence for the presence of N-methyl-D-aspartate receptors facilitating glutamate release through a positive feedback mechanism. The glutamate-induced, N-methyl-D-aspartate receptor-mediated elevations of extracellular glutamate may constitute a neurochemical substrate for the neuropathological alterations associated with alcoholism.

Increased extracellular glutamate in rat prefrontal cortex: movement artifact contribution.

Extracellular glutamate levels were measured by microdialysis in the prefrontal cortex (PFC) of anaesthetised rats in response to a short, experimenter-provoked mechanical movement of the animal head. Movement caused significant, nerve impulse-independent elevations of glutamate levels (maximum increase, 300+/-30% of baseline). This study reveals a possible artifact in the measurement of extracellular glutamate concentrations by microdialysis and suggests that, in awake animals, treatments associated with stimulation of motor activity can cause non-specific efflux of glutamate in the PFC.

Ethanol withdrawal is associated with increased extracellular glutamate in the rat striatum.

Extracellular glutamate was measured by microdialysis in the striatum of ethanol-dependent, freely behaving rats following withdrawal from chronic ethanol treatment. Within 12 h from withdrawal, extracellular glutamate rose to 255% of that in control, chronic sucrose-treated rats. Glutamate output remained elevated for the subsequent 12 h and returned to control levels within 36 h from the interruption of the treatment. The changes in glutamate were time-locked to the overt physical signs of withdrawal. In 12-h ethanol-withdrawn rats an ethanol challenge suppressed the withdrawal signs and reduced the extracellular glutamate. The NMDA receptor antagonist, dizocilpine, reduced both the physical signs of withdrawal and glutamate output. In contrast, diazepam reduced the withdrawal signs but failed to change the glutamate levels. These findings suggest that the increased extraneuronal glutamate reflects overactivity of excitatory neurotransmission during withdrawal. Furthermore, they provide a biochemical rationale for the use of NMDA receptor antagonists and ethanol itself in the treatment of ethanol withdrawal syndrome.

Brain dialysis and dopamine: does the extracellular concentration of dopamine reflect synaptic release?

We considered the drug-induced circling behaviour of rats monolaterally lesioned with kainic acid (KA) as a marker of the dopamine (DA) concentration in the synaptic space. D-Amphetamine produced a dose-related increase in the DA concentration of the dialysate from an intact striatum and a proportional number of ipsilateral circlings. Pargyline or L-dihydroxyphenylalanine (L-Dopa), alone or in combination with benserazide, increased the concentration of DA to a similar or even higher level than d-amphetamine, but failed to elicit a circling response. Apomorphine given after these drugs at the peak of DA accumulation always induced circling behaviour. The results suggest that released DA may undergo different inactivation processes before reaching the dialysis probe, and that these processes may be differentially affected by drug treatments. Alternatively, it may be suggested that DA can be released into the synaptic space, in a functional manner, or into the interstitial fluid, from where it cannot reach the synaptic receptors.

Stress increases noradrenaline release in the rat frontal cortex: prevention by diazepam.

Foot-shock produced a more than 2-fold increase in noradrenaline (NA) release from the frontal cortex of freely moving rats. The effect of acute stress was almost completely prevented by the administration of diazepam (5 mg/kg i.p.). Diazepam alone inhibited cortical NA release, the maximal inhibition (-57%) being observed 90 min after the injection. Cortical NA release therefore appears to be a reliable index of central noradrenergic activity in response to stressful conditions.

Ornithine decarboxylase inhibition or NMDA receptor antagonism reduce cortical polyamine efflux associated with dialysis probe implantation.

Dialysis probe implantation in the rat parietal cortex results in delayed, prolonged and biphasic increases in the efflux of putrescine and spermidine with primary and secondary efflux peaks 6-8 h and 20-24 h after implantation. Putrescine and spermidine efflux remain elevated for at least 30 h after implantation. The primary efflux peak is attenuated by the continual infusion via the dialysis probe of either the ornithine decarboxylase inhibitor difluoromethylornithine or by the NMDA antagonist 2-APV. The secondary peak is resistant to either of these treatments. These changes in polyamine outflow are likely related to the traumatic brain damage associated with dialysis probe implantation which may be a useful model to study the effects of local brain trauma.

Ethanol prevents the glutamate release induced by N-methyl-D-aspartate in the rat striatum.

The administration of ethanol (2 g/kg, i.p.) or of the non-competitive antagonist(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloepten-5,1 0-imine maleate (MK-801; 1 mg/kg, i.p.) induced a decrease in the extracellular concentrations of glutamate, as studied by microdialysis in the striatum of awake rats. Moreover, ethanol and MK-801 completely prevented the increase in extraneuronal glutamate concentration induced by the focal application of N-methyl-D-aspartate (NMDA). The present results suggest that ethanol suppresses glutamate release through an inhibition of NMDA glutamate receptors in the rat striatum.

The non-competitive NMDA-receptor antagonist MK-801 prevents the massive release of glutamate and aspartate from rat striatum induced by 1-methyl-4-phenylpyridinium (MPP+).

The concentrations of dopamine (DA) and of the excitatory amino acids (EAAs) glutamate (Glu) and aspartate (Asp) were measured in dialysates from the striatum of awake rats in order to study the link between the release of DA and of EAAs induced by the infusion of 1-methyl-4-phenylpyridinium ion (MPP+). DA and EAAs were detected simultaneously by HPLC-EC. The infusion of MPP+ at the concentration of 1 mM elevated DA levels in the perfusates, but did not affect EAA release. However, MPP+ at 10 mM maximally stimulated Glu and Asp release to 230- and 68-fold of baseline, respectively. In this condition, pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (5 mg/kg, i.p.) prevented the MPP(+)-induced EAA release. In contrast, MK-801 had no effect on DA release induced either by 1 or 10 mM MPP+. These results suggest that MPP(+)-induced DA and EAA release are independently regulated processes. In addition, the finding that MK-801 inhibits MPP(+)-induced EAA release suggests that EAAs may act on NMDA receptors to stimulate their own release through a positive-feedback mechanism.

Release of spermidine from the rat cortex following permanent middle cerebral artery occlusion.

We have studied the effects of middle cerebral artery (MCA) occlusion in rats on polyamine efflux in the parietal cortex using the microdialysis technique. Dialysis probe implantation itself provoked a delayed, prolonged and vigorous release of spermidine and putrescine. Spermidine release returned to stable baseline levels within 48 hours. Putrescine release also returned to lower levels within this time period but putrescine levels in the dialysate fluctuated dramatically in individual animals. Because of the underlying effects of the dialysis probe (likely a reflection of traumatic cerebral damage and stimulation of polyamine metabolism and release within the immediate vicinity of the dialysis probe), MCA occlusion was performed 48 hours after probe implantation. MCA occlusion persistently (5/5 animals) resulted in a significant increase in cortical spermidine efflux, although the onset, magnitude and duration of this increased release was variable. Putrescine efflux was significantly increased in 2/5 animals with MCA occlusion but the increase in release was similar to the spontaneous fluctuations observed in control animals. Spermine was not detectable in cortical dialysates of control or MCA occluded groups. Spermidine, but not spermine or putrescine is consistently released from the parietal cortex following permanent focal ischaemia and may contribute to ischaemic neuropathology either through its effects at the N-methyl-D-aspartate (NMDA) receptor or via direct, and as yet uncharacterised, neurotoxic effects.

Macrophages and liposomes in inflammatory disease: friends or foes?

Liposome-encapsulated corticosteroids have shown to exert strong beneficial effects in inflammatory diseases, such as arthritis and cancer. To extend the clinical applicability of these potent nanomedicines, the therapeutic effect of dexamethasone phosphate loaded long-circulating liposomes (LCL-DXP) was evaluated in animal models of multiple sclerosis (MS) and Crohn's disease (CD). In mice with experimental autoimmune encephalitis (EAE), a model for MS, treatment with LCL-DXP, but not free DXP, resulted in a decrease in disease activity when compared to PBS treated mice. In contrast, in mice with chronic DSS-induced colitis, a model for CD, treatment with LCL-DXP did not induce an improvement, but in fact worsened the fecal blood loss after treatment, indicating an aggravation of the disease. It is hypothesized that modulation of macrophage polarization towards a M2 phenotype underlies the efficacy of corticosteroid-based drug delivery systems, which is supported by the presented data. On the one hand, M1 polarized macrophages are part of the pathogenesis of MS; the modulation to M2-polarization by LCL-DXP is therefore beneficial. On the other hand, M1-polarized intestinal macrophages fulfill a protective and inflammation-suppressing role in intestinal homeostasis; changing their phenotype to M2 causes reduced protection to invading microorganisms, leading to a more severe intestinal inflammation. These findings therefore indicate that the interplay between the specific phenotype of macrophages and the specific inflammatory context of the inflammatory disease in question may be an important determining factor in the therapeutic applicability of liposomal corticosteroids in inflammatory disease.