Diagnostic accuracy of des-gamma-carboxy prothrombin for hepatocellular carcinoma in a French cohort using the Lumipulse® G600 analyzer.

The increasing incidence of hepatocellular carcinoma (HCC) in Western countries requests reliable tumour markers for preclinical diagnosis. We evaluated the diagnostic accuracy of des-gamma-carboxy prothrombin (DCP), in comparison with alpha-fetoprotein (AFP) in a French cohort using a new analyser. One hundred and sixty-two patients with virus-related cirrhosis (46 HCC patients and 116 controls) were recruited in this retrospective proof-of-concept study. DCP was measured on new Lumipulse® G600 analyzer and AFP on usual Cobas e602 analyzer in serum samples that were collected at the time of HCC diagnosis for HCC patients or during follow-up for controls. DCP and AFP levels were higher in HCC patients. The area under receiver operating characteristic curve was larger for DCP than for AFP (0.89 vs 0.77, P=.03). At the cut-off value of 128 mAU/mL, sensitivity and specificity for DCP were 74% and 92%. At the cut-off value of 20 µg/L, sensitivity and specificity for AFP were 63% and 82%. NRI>0 for the association of "AFP+DCP" were 101%, P<.0001, and 23%, P=.03, compared to "AFP" or "DCP" alone, respectively. We conclude that DCP outperformed AFP for the detection of HCC.

Apoptosis and necrosis after reversible focal ischemia: an in situ DNA fragmentation analysis.

Apoptosis is one of the two forms of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alternations including membrane blebbing and nuclear and cytoplasmic condensation. Early activation of an endonuclease has been previously demonstrated after a transient focal ischemia in the rat brain Charriaut-Marlangue C, Margaill I, Plotkine M, Ben-Ari Y (1995) Early endonuclease activation following reversible focal ischemia. J Cereb Blood Flow Metab 15:385-388). We now show that a significant number of striatal and cortical neurons, exhibited chromatin condensation, nucleus segmentation, and apoptotic bodies increasing with recirculation time, as demonstrated by in situ labeling of DNA breaks in cryostat sections. Apoptotic nuclei were also detected in the horizontal limb diagonal band, accumbens nucleus and islands of Calleja. Several necrotic neurons, in which random DNA fragmentation occurs, were also shown at 6 h recirculation, in the ischemic core. Further investigation with hematoxylin/eosin staining revealed that apoptotic nuclei were present in cells with a large and swelled cytoplasm and in cells with an apparently well-preserved cytoplasm. These two types of cell death were reminiscent of those described in developmental cell death. Our data suggested that apoptosis may contribute to the expansion of the ischemic lesion.

Localization of aldolase C mRNA in brain cells.

The expression of aldolase C and aldolase A mRNA was assessed by Northern blot hybridization using RNAs purified from cultured rat and mouse brain neurons and astroglial cells. Neurons were found to contain about 4-fold more aldolase C mRNA and about twice as much aldolase A mRNA than astroglia. Analysis of the cellular localization of aldolase C mRNA by in situ hybridization to brain slices showed a predominantly neuronal labeling with an irregular distribution. A strong signal was observed in Purkinje cell somata and a weaker signal in subpopulations of neurons in cerebral cortex, striatum, hippocampus, hypothalamic nuclei and primary olfactory cortex.

First discrete autoradiographic distribution of aminopeptidase N in various structures of rat brain and spinal cord using the selective iodinated inhibitor [125I]RB 129.

The selective and potent aminopeptidase N inhibitor [125I]RB 129 has been used for the radioautographic localization of this enzyme in rat brain, spinal cord and intestine. Brain microvessels and intestine brush-border cells were shown to present a high concentration of aminopeptidase N. Moreover, a labeling of various brain structures was observed. A very high level of binding occurred in the meninges, choroid plexus, pineal gland, paraventricular nucleus and pituitary gland. Moderate to high labeling was also observed in the cortex, caudate-putamen, subthalamic nucleus, central periaqueductal gray, thalamus, as well as in the dorsal and ventral horn of the spinal cord, which are known to contain a high concentration of enkephalins, opioid receptors and neutral endopeptidase. This co-localization confirms the physiological implication of aminopeptidase N in the inactivation of enkephalins accounting for the requirement of dual inhibition of neutral endopeptidase and aminopeptidase N to observe highly significant morphine-like effects induced by the protected endogenous opioid peptides. Aminopeptidase N was also visualized in moderate to high levels in other brain structures such as the hippocampus, nucleus accumbens, substantia nigra, hypothalamus (dorsomedial and ventromedial nuclei), raphe nucleus, pontine nucleus, inferior olive, and in high concentration in the granular layer of cerebellum. In summary, aminopeptidase N has been visualized for the first time in numerous brain areas using the selective inhibitor [125I]RB 129. This iodinated probe could allow the ex vivo and in vivo localization of aminopeptidase N in various tissues to be investigated and may also be used to evaluate quantitative changes in aminopeptidase N expression in pathological situations. Aminopeptidase N, which preferably removes NH2-terminal neutral amino acids from peptides, has probably a host of substrates. Nevertheless, a certain in vivo selectivity could be achieved by the presence of the enzyme in structures where the peptide effector and its receptors are also co-localized.

Dopaminergic neurons of the substantia nigra modulate preproenkephalin A gene expression in rat striatal neurons.

The messenger RNA coding for preproenkephalin A (PPA) was detected by in situ hybridization in striatal neurons in normal rats and in rats having had the right substantia nigra destroyed by an injection of 6-hydroxydopamine or by electrolysis. Animals were killed 15, 30, 45 and 70 days following the lesion. A double-stranded PPA cDNA and a single-stranded PPA cRNA labeled with 32P or 35S were used as probes to detect the PPA mRNA in brain sections. The controls demonstrated the specificity of the labeling. The darkening of X-ray film in contact with the striatum was appraised, the optical density was measured, and the density of the cells expressing the PPA gene in sections was calculated using an image analyzer. The mean number of silver grains per labeled cell (reflecting the number of PPA mRNA copies per cell) was also calculated using an image analyzer. The 6-hydroxydopamine lesion which destroyed all dopaminergic neurons in the right substantia nigra, provoked a large increase in the number of PPA mRNA copies in enkephalin neurons of the right striatum, and decreased the number of cells expressing the PPA mRNA in the left striatum. These variations substantia nigra provoked similar variations, but less intense.(ABSTRACT TRUNCATED AT 250 WORDS)

Down-regulation of striatal enkephalinergic (PPA) messenger RNA without prior apoptotic features following reversible focal ischemia in rat.

In order to determine whether striatal enkephalinergic neurons were affected by reversible focal ischemia, we have investigated the expression of the preproenkephalin (PPA) messenger by in situ hybridization (ISH) combined with TUNEL staining to display apoptosis in the same rat brain sections. Our data demonstrated a massive reduction of the number of PPA-mRNA containing neurons concomitant with the emergence of apoptotic cells. However, double-labeled neurons (ISH- and TUNEL-positive cells) were not detected, suggesting that either disruption of mRNA precedes DNA fragmentation or ischemia leads to a long lasting reduction of mRNA(s) without damage.

Effects of kainic acid-induced seizures and ischemia on c-fos-like proteins in rat brain.

We have analyzed the brain pattern and time-course of c-fos-like proteins expression in kainic acid-induced seizures in the rat. C-fos-like immunoreactivity increased initially in the hippocampus, notably in the dentate gyrus, at the time of the first limbic motor seizure (90 min after kainate). C-fos-like labelling progressively involved different structures of the limbic system when the rats manifested a permanent epileptic state (3-6 h). The labelling was still conspicuous 12 h after kainate treatment and progressively declined to reach control levels 48 h after kainate. This time-course is similar to that produced by kainic acid on 2-deoxyglucose consumption and correlates with the electrographic changes previously described, supporting the idea that c-fos-like immunostaining may provide a useful marker of neuronal activity, with a cellular resolution. Since anoxic-ischemic treatment produces a very slight and transient increase in c-fos-like immunostaining restricted to the fascia dentata, c-fos-like expression is seizure-related and not due to a local hypoxia or ischemia.

Detection of the mRNA coding for enkephalin precursor in the rat brain and adrenal by using an 'in situ' hybridization procedure.

The messenger RNA coding for preproenkephalin A (PPA) has been detected in tissue sections of the rat brain and adrenal by using two rat PPA cDNAs labeled with 32P or 35S as probes. In the brain, neurons were labeled in areas known to correspond to sites of synthesis of enkephalins, including the caudate-putamen, the nucleus accumbens, the olfactory cortex, the hypothalamus, the brainstem and the granular layer of the cerebellum. The presence of the PPA mRNA in the normal rat adrenal medulla shows transcription of the PPA gene in such cells despite the absence of enkephalin immunoreactivity in them. These results demonstrate in situ hybridization as an efficient technique to detect the site of synthesis of PPA.

Anatomical study of enkephalin gene expression in the rat forebrain following haloperidol treatment.

The effect of haloperidol treatment on preproenkephalin A (PPA) gene expression in the rat forebrain was anatomically studied by using an in situ hybridization procedure. The PPA mRNA was detected in sections of control rats and of rats having received an i.p. injection of haloperidol for 14 or 21 days. Sections were incubated with rat PPA cDNA labeled with 32P or 35S, exposed with X-ray film and dipped in Ilford K-5 emulsion. The results showed that haloperidol treatment did not modify the number of cells expressing the PPA gene in the caudate-putamen, the nucleus accumbens, the septum and the olfactory tubercle. In contrast, the PPA mRNA content was increased in the neurons of the caudate-putamen and nucleus accumbens, but unchanged in other areas. These results demonstrate that haloperidol acts by increasing PPA mRNA content selectively in striatal neurons already expressing the PPA gene.

Distribution of CCK mRNA in particular regions (hippocampus, periaqueductal grey and thalamus) of the rat by in situ hybridization.

Cholecystokinin (CCK) mRNA was detected by in situ hybridization at high magnification in some rat brain regions where CCK octapeptide (CCK-8) is thought to produce its pharmacological effects. The labeling of the dentate gyrus and the sparse but intensively stained cells found in the CA1 layer, stratum radiatum and hilus could correspond to interneurons involved in hippocampal neural activity, in agreement with excitatory responses induced by local injection of CCK-8. The intense labeling of the Edinger-Westphal nucleus and more generally the presence of CCK mRNA in the periaqueductal gray and thalamus ventrobasal nuclei could account for the various effects of CCK in pain transmission.

Transferrin gene expression in choroid plexus of the adult rat brain.

Transferrin immunoreactivity and transferrin messenger RNA (mRNA) were recently found to be present in oligodendrocytes of the adult rat brain by using immunohistochemistry and in situ hybridization procedure. The present study demonstrates, in the same way, that epithelial cells of the choroid plexus also contain transferrin together with transferrin mRNA. Choroid plexus of the lateral and the third ventricle are rich in transferrin mRNA, while choroid plexus of the fourth ventricle contain few if any transferrin mRNA. These results demonstrate that epithelial cells of the choroid plexus as well as oligodendrocytes express the transferrin gene in the adult rat brain.

Permanent reduction of seizure threshold in post-ischemic CA3 pyramidal neurons.

The effects of ischemia were examined on CA3 pyramidal neurons recorded in hippocampal slices 2-4 mo after a global forebrain insult. With intracellular recordings, CA3 post-ischemic neurons had a more depolarized resting membrane potential but no change of the input resistance, spike threshold and amplitude, fast and slow afterhyperpolarization (AHP) or ADP, and firing properties in response to depolarizing pulses. With both field and whole-cell recordings, synaptic responses were similar in control and post-ischemic neurons. Although there were no spontaneous network-driven discharges, the post-ischemic synaptic network had a smaller threshold to generate evoked and spontaneous synchronized burst discharges. Thus lower concentrations of convulsive agents (kainate, high K(+)) triggered all-or-none network-driven synaptic events in post-ischemic neurons more readily than in control ones. Also, paired-pulse protocol generates, in post-ischemics but not controls, synchronized field burst discharges when interpulse intervals ranged from 60 to 100 ms. In conclusion, 2-4 mo after the insult, the post-ischemic CA3 pyramidal cells are permanently depolarized and have a reduced threshold to generate synchronized bursts. This may explain some neuropathological and behavioral consequences of ischemia as epileptic syndromes observed several months to several years after the ischemic insult.

Identification and characterization of various cholecystokinin B receptor mRNA forms in rat brain tissue and partial determination of the cholecystokinin B receptor gene structure.

Using the powerful method of DNA amplification, we have been able to isolate several cholecystokinin B (CCKB) receptor (CCKB-R) mRNA forms from rat brain tissue, allowing the detection of a truncated mRNA species and the determination of the CCKB-R gene structure. Unspliced precursor mRNA and the mature form were identified in the cerebral cortex, hypothalamus, and hippocampus in apparently differing proportions according to the region examined, suggesting that the expression of the CCKB-R could be modulated at a posttranscriptional level. In the case of the cerebellum, only a completely unspliced mRNA form was detected, in agreement with previous studies in which CCKB ligand binding sites were not observed. In contrast, a truncated CCKB-R mRNA, lacking 250 bp, was detected in all the studied brain regions except for the cerebellum. This mRNA, for which a cellular function has not been assigned, potentially encodes a protein consisting of 168 amino acids.

Transferrin gene expression visualized in oligodendrocytes of the rat brain by using in situ hybridization and immunohistochemistry.

The presence and production of transferrin in the adult rat brain have been investigated using both immunohistochemistry and in situ hybridization in tissue sections. Indirect immunofluorescence with four distinct antisera against rat and human transferrin and one monoclonal antibody against human transferrin demonstrated labeling of the cytoplasm of oligodendrocytes (a category of glial cells) in most parts of the brain, especially in the white matter. In situ hybridization using rat transferrin 32P-labeled cDNA as a probe revealed the presence of transferrin mRNA in glial cells whose appearance, distribution, and organization exactly matched those of the cells decorated with the transferrin antibodies. These results provide evidence that the transferrin gene is expressed in the central nervous system and that transferrin is synthesized by and stored within oligodendrocytes in the adult rat brain. These data suggest that this molecule could have a specific function in nervous system activity.

In situ hybridization histochemistry for the analysis of gene expression in the endocrine and central nervous system tissues: a 3-year experience.

We report our experience in development of the in situ hybridization (ISH) procedure to detect messenger RNAs (mRNAs) coding for various molecules involved in endocrine glands and central nervous system activity, including mRNAs coding for endorphin precursors [preproenkephalin A (PPA), pro-opiocortin (POMC)], vasopressin, and transferrin. Various conditions of fixation and handling of the tissues were tested to establish optimal parameters for mRNA detection. Double-stranded DNA probes labeled by nick translation, synthetic oligonucleotides labeled at their 5' end, as well as single-stranded RNA probes were used, after incorporation of 32P- or 35S-labeled nucleotides. Specific requirements for efficient and reproducible ISH investigations are discussed. Cells expressing the PPA gene in the adrenal medulla and in the brain were detected by ISH. The results show that ISH is as sensitive as immunohistochemistry in detecting peptide-producing cells in the adrenal and that it allows detection of PPA cell bodies in brain in conditions in which they are inconstantly detected by immunohistochemistry. Unilateral destruction of substantia nigra provokes a dramatic decrease in the number of neurons expressing the PPA gene in the contralateral striatum. Cells expressing the POMC gene were detected in the pituitary of various species including man and in the rat arcuate nucleus. Neurons containing vasopressin mRNA were visualized in the supraoptic paraventricular and suprachiasmatic nucleus of the adult rat by using a synthetic oligonucleotide probe. Transferrin gene expression was shown in the central nervous system of the rat brain in two cell populations, the oligodendrocytes and the epithelial cells of the choroid plexus, by demonstration of simultaneous presence in them of transferrin immunoreactivity together with transferrin mRNA. These results show that the ISH procedure is a technique that can be routinely used to investigate gene transcription anatomically in complex heterocellular tissues such as the endocrine glands and the nervous system.