Rossano Ambu

Uneven hepatic iron and phosphorus distribution in beta-thalassemia

BACKGROUND/AIMS Determination of hepatic iron concentration is crucial in the evaluation of iron-storage disease. Iron content is normally determined in a part of a needle liver biopsy and the value obtained is considered to be representative of the iron concentration in the whole liver. To evaluate the reliability of this procedure, we studied iron distribution in the liver of two beta-thalassemic patients. Since the transport of intracellular iron is mediated by phosphates, we also studied the hepatic phosphorus distribution. METHODS At autopsy, a liver slice extending from the left to the right lobe was divided into 51 and 49 samples, respectively. Each specimen was subdivided into two parts: one of them was paraffin-embedded and utilized for the histochemical detection of iron; the second part was analyzed for iron and phosphorus content by induced coupled plasma atomic emission spectroscopy. RESULTS The histological picture of both livers was characterized by portal and periportal fibrosis associated with iron storage of different degree, without cirrhosis. The mean iron concentration of the liver was 20,631 +/- 4903 micrograms per g of dry tissue (micrograms/g dt) and 13,901 +/- 1976 micrograms/g dt, respectively. A striking variability in iron content between samples was also found: iron concentration ranged from 11,537 to 32,347 micrograms/g dt in the first case and from 6257 to 16,493 in the second case. We even observed regional differences in iron concentration, with a preferential peripheral accumulation in both cases and a tendency of the left compartment of the liver to accumulate more iron in the first case. Histochemical analyses confirmed the uneven iron distribution even at the acinar level, showing iron mainly being stored in hepatocytes and Kupffer cells of zone 1 of the acinus, with decreasing amounts of iron in zones 2 and 3. The mean hepatic phosphorus concentration was 6662 +/- 1300 micrograms/g dt (range: 4348-9947) and 7502 +/- 986 micrograms/g dt (range: 5844-90,282), respectively. The regional distribution of phosphorus was similar to that observed for iron. A strict correlation between iron and phosphorus content was also observed. CONCLUSIONS Our data show that: 1) iron and phosphorus are unevenly distributed in the beta-thalassemic liver, even in the non-cirrhotic stages; 2) a regional pattern of iron and phosphorus distribution is evident, characterized by higher concentrations at the periphery of the liver; 3) the observed uneven distribution of iron and phosphorus implies that their content determined in a small liver sample cannot be considered as absolutely representative of the mean hepatic iron concentration. Therefore, iron concentrations determined in a part of a needle liver biopsy should be interpreted with caution in monitoring the efficacy of the iron-chelating therapy in beta-thalassemic patients.

Uneven hepatic copper distribution in Wilson's disease

BACKGROUND/AIMS Determination of hepatic copper concentration is important in the diagnosis of Wilsons disease. We studied copper distribution in the cirrhotic liver of a patient who died of Wilsons disease. METHODS A liver slice extending from the left to the right lobe was divided into 38 samples. Each sample was analyzed for copper content by Induced Coupled Plasma Atomic Emission Spectroscopy. RESULTS The mean copper concentration in the liver was 1370 micrograms/g dt. A striking variability, up to 2-3-fold, in copper levels was observed between the samples: the copper concentration ranged from 880 to 2100 micrograms/g dt, with significant differences even between adjacent samples. Lobar differences were also observed, with a tendency of the right lobe to accumulate more copper than the left lobe. Histochemical analyses confirmed the uneven distribution of copper even at the acinar level. Copper was mainly stored in periportal hepatocytes (zone 1) and at the periphery of the regenerating nodules. Moreover, we observed some nodules with the majority of hepatocytes full of copper granules, adjacent to areas of parenchyma negative for copper stains. CONCLUSIONS Our data show that: 1) copper is unevenly distributed in Wilsons disease in the cirrhotic stage; 2) a lobar pattern of copper distribution is evident in this case, characterized by a higher copper concentration in the right lobe; 3) the observed lobar pattern is different from that described in the newborn liver, characterized by a higher copper content in the left compartment of the liver; 4) copper content determined in a small liver sample cannot be considered as absolutely representative of the mean hepatic copper concentration. From a practical point of view, our data show that sampling variability deserves more consideration in the diagnosis and in the monitoring of Wilsons disease. The use of hepatic copper concentration in monitoring the efficacy of the copper-chelating therapy may be unreliable, particularly in the cirrhotic stage, because of the patchy distribution of copper, as demonstrated in this study.

P16ink4a and HPV L1 Immunohistochemistry is Helpful for Estimating the Behavior of Low-grade Dysplastic Lesions of the Cervix Uteri

As only a minority of low-grade dysplastic lesions of the cervix uteri will eventually progress to carcinoma, predicting the behavior of these lesions could be of high value in clinical practice. The aim of the study was to evaluate p16ink4a and L1 as immunohistochemical markers of the biologic potentiality of low-grade dysplasia of the uterine cervix. The study included 38 conization specimens with coexisting cervical intraepithelial neoplasia grade 1 (CIN1) and 3 (CIN3) (group A) and 28 punch biopsies from women with CIN1 and proven spontaneous regression in the follow-up (group B). In group A, all CIN3 were p16ink4a positive (p16+) and L1 negative (L1−). The CIN1 of this group were p16+L1− and p16+L1+ in 68.42% and 31.57%, respectively. No other expression pattern was found in this group. In group B, the p16+L1−, p16+L1+, p16−L1+, and p16−L1− patterns were found in 3.57%, 25%, 14.29%, and 57.14%, respectively. Overall, 96.29% p16+L1− CIN1 were found in group A, whereas all the p16−L1+ and p16−L1− CIN1 were found in group B. A significant difference between staining pattern distributions of group A and B was observed (P<0.0001). The results of the study show that p16ink4a and L1 immunohistochemistry can be helpful for estimating the biologic potentiality of low-grade squamous cervical lesions. Particularly in cases in which the grade of the lesion is morphologically difficult to assess, the p16/L1 expression pattern could be useful for planning the clinical management of these women.

Value of histochemical stains for copper in the diagnosis of Wilson's disease

Aims: The histochemical demonstration of hepatic copper is important in the diagnosis of Wilsons disease (WD). Conflicting results have been published with regard to the ability of different histochemical methods to demonstrate copper storage in the liver. Therefore, we evaluated the diagnostic value of three available histochemical methods in a large series of patients affected by WD.

Brain copper, iron, magnesium, zinc, calcium, sulfur and phosphorus storage in Wilson's disease.

PROJECT Wilsons disease (WD) is an inherited disorder of copper metabolism characterised by juvenile liver cirrhosis and by neurological symptoms. Copper levels in brain in WD have been reported to be 10 to 15 fold normal values, depending on the different brain regions. Being very few data on copper distribution in central nervous system in WD available, it seemed of interest to study the concentration of copper and of other trace elements (Zn, P, Mg, Ca, Fe and S) in the brain of a patient died for WD. PROCEDURE a 56 year old woman affected by WD was admitted to our hospital with signs of hepatic failure and died few days later. At autopsy, a brain slice extending from the left to the right hemisphere was divided in 28 samples. On each sample Copper, Iron, Magnesium, Phosphorus, Sulphur, Zinc and Calcium were determined by Induced Coupled Plasma Atomic Emission Spectroscopy. RESULTS the mean concentration of copper, ranging from 88 to 158 microg/g of dry tissue in all the brain specimens was higher than literature reference values, while that of the other tested elements was considerably lower. CONCLUSIONS 1) In the brain of WD patient examined the status of trace elements was extensively altered. Further studies are necessary to correlate the concentration of trace elements with pathological lesions and with clinical pictures. 2) The elements considered in our study showed an uneven distribution in different brain areas.

Overlapping between CYP3A4 and CYP3A7 expression in the fetal human liver during development

Abstract Objective: The cytochrome P450 (CYP450) superfamily is implicated in important life processes, including metabolism of many molecules. CYP3A account for the largest portion of CYP450 proteins in human, including CYP3A4, CYP3A5 and CYP3A7. The purpose of this study was to investigate the immunohistochemical expression of CYP3A4 and CYP3A7 in human liver at different post-conceptional (PC) ages. Methods: Human liver samples from 30 fetuses and newborns were, clustered according with the PC age, routinely processed for immunohistochemical analysis of CYP3A4 and CYP3A7. Results: CYP3A4 was positive in all but two cases, CYP3A7 was positive in all but one case, which was negative also for CYP3A4. Conclusions: Our data on immunohistochemical detection of CYP3A4 and CYP3A7 during development show that CYP3A4 expression is not restricted to the post-natal age, being the immunostaining for both CYP3A4 and CYP3A7 identical after 25 weeks of PC age, thus the relationship between these CYP450 isoforms should be considered much more complex than previous thought. A high interindividual variability was observed among subjects at all gestational age. The variable CYP3A expression suggests the existence of a marked interindividual variability in drug metabolism during the intrauterine life and in perinatal period.

Glial cell line-derived neurotrophic factor-like immunoreactivity in human trigeminal ganglion and nucleus

Glial cell line-derived neurotrophic factor (GDNF) is shown by immunohistochemistry in human trigeminal sensory system from 22 weeks of gestation to adulthood. In the trigeminal ganglion, a distinct subpopulation of GDNF-positive neurones is observed, which amounts to about 15% at early pre-term and adult ages and peaks to around 30% at perinatal ages. Labelled neurones are mostly small- and medium-sized. Occasionally, Schwann and satellite cells are stained. GDNF/substance P (SP) and GDNF/calcitonin gene-related peptide (CGRP) double stained neurones occur at all ages examined, whereas GDNF/trkA coexistence can be observed in pre- and full-term newborns only. Centrally, GDNF-immunostained fibers and terminal-like structures are mainly restricted to the spinal trigeminal nucleus, where they are codistributed with SP and CGRP. In the subnucleus caudalis, positive neurones can also be observed both in the superficial laminae and in the magnocellular part, with higher frequency in adults. These results suggest that GDNF may play a functional role in human trigeminal primary sensory neurones throughout life and provide indication for its possible involvement in the regulation of pain-related neuronal circuits in human trigeminal sensory system.

Allosteric modulation of GABAB receptor function in human frontal cortex

In the present study, the effects of different allosteric modulators on the functional activity of gamma-aminobutyric acid (GABA)B receptors in membranes of post-mortem human frontal cortex were examined. Western blot analysis indicated that the tissue preparations expressed both GABA(B1) and GABA(B2) subunits of the GABA(B) receptor heterodimer. In [35S]-GTPgammaS binding assays, Ca2+ ion (1 mM) enhanced the potency of the agonists GABA and 3-aminopropylphosphinic acid (3-APA) and that of the antagonist CGP55845, but not that of the GABA(B) receptor agonist (-)-baclofen. CGP7930 (2,6-di-t-Bu-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol), a positive allosteric modulator of GABA(B) receptors, potentiated both GABA(B) receptor-mediated stimulation of [35S]-GTPgammaS binding and inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity. Chelation of Ca2+ ion by EGTA reduced the CGP7930 enhancement of GABA potency in stimulating [35S]-GTPgammaS binding by two-fold. Fendiline, also reported to act as a positive allosteric modulator of GABA(B) receptors, failed to enhance GABA stimulation of [35S]-GTPgammaS binding but inhibited the potentiating effect of CGP7930. The inhibitory effect was mimicked by the phenothiazine antipsychotic trifluoperazine (TFP), but not by other compounds, such as verapamil or diphenydramine (DPN). These data demonstrate that the function of GABA(B) receptors of human frontal cortex is positively modulated by Ca2+ ion and CGP7930, which interact synergistically. Conversely, fendiline and trifluoperazine negatively affect the allosteric regulation by CGP7930.

Agonist activity of N-desmethylclozapine at δ-opioid receptors of human frontal cortex

The clozapine metabolite N-desmethylclozapine (NDMC) has been recently shown to act at different neurotransmitter receptors and to display both antagonist and agonist activities. We have previously reported that in cells over-expressing the recombinant delta-opioid receptor NDMC behaved as partial agonist with high intrinsic activity, but its action at the receptors naturally expressed in human brain remained to be investigated. In the present study, we examined whether NDMC was able to bind to and activate delta-opioid receptors in membranes of post-mortem human frontal cortex. In radioligand binding assays, NDMC competition curves displayed high- (K(i)=26 nM) and low-affinity (K(i)=3 microM) components, whose proportion was regulated by guanine nucleotides in an agonist-like fashion. In functional assays, NDMC stimulated [(35)S]GTPgammaS binding (EC(50)=905 nM) and inhibited cyclic AMP formation (EC(50)=590 nM) as effectively as delta-opioid agonists, whereas clozapine was much less potent and efficacious and clozapine N-oxide was completely inactive. The NDMC agonist activity was potently antagonized by the delta-opioid antagonist naltrindole, but not by the micro-opioid receptor antagonist CTAP (D-phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) or the kappa-opioid antagonist nor-binaltorphimine. Moreover, blockade of either acetylcholine muscarinic, dopamine D(2) or serotonin 5HT(1A) receptors failed to affect NDMC agonist activity. These data demonstrate that at clinically relevant concentrations NDMC behaves as an efficacious agonist at delta-opioid receptors of human frontal cortex.

Aluminum exposure and toxicity in neonates: a practical guide to halt aluminum overload in the prenatal and perinatal periods

BackgroundDuring the last years, human newborns have been overexposed to biologically reactive aluminum, with possible relevant consequences on their future health and on their susceptibility to a variety of diseases. Children, newborns and particularly preterm neonates are at an increased risk of aluminum toxicity because of their relative immaturity.Data sourcesBased on recent original publications and classical data of the literatures, we reviewed the aluminum content in mother’s food during the intrauterine life as well as in breast milk and infant formula during lactation. We also determined the possible role of aluminum in parenteral nutrition solutions, in adjuvants of vaccines and in pharmaceutical products. A special focus is placed on the relationship between aluminum overexposure and the insurgence of bone diseases.ResultsPractical points of management and prevention are suggested. Aluminum sources that infants may receive during the first 6 months of life are presented. In the context of prevention of possible adverse effects of aluminum overload in fetal tissues during development, simple suggestions to pregnant women are described. Finally, practical points of management and prevention are suggested.ConclusionsPediatricians and neonatologists must be more concerned about aluminum content in all products our newborns are exposed to, starting from monitoring aluminum concentrations in milk- and soybased formulas in which, on the basis of recent studies, there is still too much aluminum.