Cytochrome P450 genetic polymorphism in neonatal drug metabolism: role and practical consequences towards a new drug culture in neonatology.

The cytochrome P450 superfamily (CYP450) in humans is formed by 57 functional monooxygenases critical for the metabolism of numerous endogenous and exogenous compounds. The superfamily is organized into 18 families and 44 subfamilies. CYP nomenclature is based on the identity of amino acids. The most important functions of the CYP450 are related to metabolism of endogenous compounds, detoxification of exogenous xenobiotics and decomposition of the vast majority of currently used drugs. The expression of CYP450 enzymes in the human body is characterized by a marked substrate and tissue specificity, the most important being localized in the liver, but also present in kidney, lung, brain, breast, prostate and in the small intestine. The human cytochrome P450 3A gene family (CYP3A) accounts for the largest portion of CYP450 proteins in human liver and includes 4 genes: CYP3A4, CYP3A5, CYP3A7, CYP3A43. Multiple and complex genetic variations, marked interindividual, interethnic and gender variability have been reported regarding CYP3A isoform expression and activity. Multiple factors may affect CYP3A expression and activity, such as inducers like rifampicin, phenobarbital, 3-methylcholantrene, beta-naphtoflavone, and dexamethasone. The maturation of organ systems, paralleled by ontogeny of drug-metabolizing enzymes during fetal life and in the first months of postnatal life, surely exerts profound effects on drug disposition, probably being the predominant factor accounting for age-associated changes in drug clearance. In fact, drug dosage in the perinatal period represents a continuous challenge for neonatologists. The purpose of this article is to provide a brief review of the pharmacokinetic differences between neonates and adults, showing the peculiarities of liver CYP450-related drug metabolism in the perinatal period and at birth, and to report the toxic mechanisms of liver injury in neonates, due to the most frequently utilized drugs in NICU centers.

A new nonsense mutation of the IGHMBP2 gene responsible for the first case of SMARD1 in a Sardinian patient with giant cell hepatitis.

We describe the case of a Sardinian female child affected by SMARD1, a genetic composite heterozygote, in whom a new nonsense mutation (R788X) was found. A sister was affected by generalized muscular hypotonia and died from respiratory insufficiency at the age of 9 months, before a diagnostic definition had been formulated. Our patient died at 6 months due to respiratory insufficiency. At autopsy some differences with previous published cases were observed. In fact, our case is to the best of our knowledge the first report of giant cell hepatitis and myocarditis in SMARD1-affected patients, although this could be a chance association.

The role of immunohistochemistry in the diagnosis of hyalinizing clear cell carcinoma of the minor salivary gland: a case report.

A case of hyalinizing clear cell carcinoma (HCCC) of the minor salivary glands of the oral cavity is reported. A 52- year-old woman presented with a growing mass at the base of the tongue. The patient underwent complete resection of the tumour. The histological picture was characterized by trabeculae or solid nests of proliferating cells with a clear cytoplasm, surrounded by a hyalinizing stroma. Tumour cells were immunoreactive for Cytokeratins 5, 6, 7, 8, 14, 17 and 18. No reactivity was observed for cytokeratin 20, vimentin, S- 100 protein, smooth-muscle actin, muscle-specific actin, and calponin. These findings confirmed the diagnosis of HCCC of minor salivary glands of the oral cavity. The clinical presentation, the immunohistochemical pattern and the role of cytokeratins in the differential diagnosis of HCCC are discussed with a review of the literature.

Oesophageal tracheobronchial remnants.

SUMMARY: Congenital oesophageal stenosis due to tracheobronchial remnants is a very rare condition characterized by the presence of tracheobronchial tissue in the oesophageal wall. The most common symptoms are dysphagia, regurgitation and hypersalivation. These usually appear in early infancy when solid food is introduced into the diet. Clinical diagnosis is difficult. Conventional radiology shows a dilated oesophagus without peristalsis, incomplete expansion of the gastro-oesophageal transition zone and delayed oesophageal emptying. Lesions suggesting oesophagitis may be present at endoscopy but pH-metry may be normal. The combination of stenosis with or without oesophagitis with normal pH-metry suggests that the patient's symptoms may be due to factors other than intraluminal and that further investigation is needed. The treatment of this condition requires surgical resection of the stenosis with little morbidity or mortality. The presence of tracheobronchial remnants is confirmed upon histopathological examination of the resected bowel segment.

Thymosin beta 4 and thymosin beta 10 expression in hepatocellular carcinoma.

Thymosin beta 4 (Tß4) and thymosin beta 10 (Tß10) are two members of the beta-thymosin family involved in many cellular processes such as cellular motility, angiogenesis, inflammation, cell survival and wound healing. Recently, a role for beta-thymosins has been proposed in the process of carcinogenesis as both peptides were detected in several types of cancer. The aim of the present study was to investigate the expression pattern of Tß4 and Tß10 in hepatocellular carcinoma (HCC). To this end, the expression pattern of both peptides was analyzed in liver samples obtained from 23 subjects diagnosed with HCC. Routinely formalin-fixed and paraffin-embedded liver samples were immunostained by indirect immunohistochemistry with polyclonal antibodies to Tß4 and Tß10. Immunoreactivity for Tß4 and Tß10 was detected in the liver parenchyma of the surrounding tumor area. Both peptides showed an increase in granular reactivity from the periportal to the periterminal hepatocytes. Regarding HCC, Tß4 reactivity was detected in 7/23 cases (30%) and Tß10 reactivity in 22/23 (97%) cases analyzed, adding HCC to human cancers that express these beta-thymosins. Intriguing finding was seen looking at the reactivity of both peptides in tumor cells infiltrating the surrounding liver. Where Tß10 showed a strong homogeneous expression, was Tß4 completely absent in cells undergoing stromal invasion. The current study shows expression of both beta-thymosins in HCC with marked differences in their degree of expression and frequency of immunoreactivity. The higher incidence of Tß10 expression and its higher reactivity in tumor cells involved in stromal invasion indicate a possible major role for Tß10 in HCC progression.

Modifications of the acidic soluble salivary proteome in human children from birth to the age of 48months investigated by a top-down HPLC-ESI-MS platform.

During the first year of life the infant oral environment undergoes dramatic changes. To investigate how the salivary proteome of human children evolves during infant development we have analyzed whole saliva of 88 children aged between 0 and 48months by a top-down platform based on RP-HPLC-ESI-MS. Children were divided according to their age into five groups (A, 0-6months, N=17; B, 7-12months, N=14; C, 13-24months, N=32; D, 25-36months, N=16; E, 37-48months, N=9). The proteins and peptides analyzed were histatins (histatin-1, histatin-3 1/24), acidic proline-rich proteins, statherin, P-B peptide, and salivary cystatins. Protein and peptide quantification based on the area of the RP-HPLC-ESI-MS extracted ion current peak evidenced that: (i) concentrations of the major salivary proteins/peptides showed a minimum in the 0-6-month-old group and increased with age; (ii) the level of histatin-1 reached a maximum in the 7-12-month-old group, a minimum in the 13-24-month-aged babies and it increased again in the 25-36-month-old group; (iii) S-type cystatins were almost undetectable in the 0-6-month-old group; (iv) P-B peptide concentration greatly increased with age; (v) histatin-3 1/24 and statherin concentrations did not show any age-related variation. BIOLOGICAL SIGNIFICANCE: The top-down proteomic approach undertaken in this work reveals that the salivary proteome of human children from birth to 48months of age shows important quantitative modifications. The concentrations of the major salivary proteins, with the exception of statherin and histatin-3 1/24, showed a minimum in the 0-6-month-old group when the expression in salivary glands is probably not fully activated. Concentrations of the salivary proteins slowly increased with age, with different trends. Only histatin-1 showed the highest concentration in the 7-12-month-old group, followed by a decrease in the 13-24-month-aged children. This particular trend could be related to the phenomenon of eruption of primary dentition. This study gives a contribution to the knowledge on the physiological variability occurring in human saliva during the early childhood. It could represent a strong and reliable basis for further investigation of saliva to develop diagnostic and prognostic biomarkers.

Immunoreactivity for thymosin beta 4 and thymosin beta 10 in the adult rat oro-gastro-intestinal tract.

Thymosin beta 4 (Tß4) and thymosin beta 10 (Tß10) are two members of the ß-thymosin family, involved in multiple cellular activities in different organs in multiple animal species. Here we report the expression pattern of Tß4 and Tß10 in rat tissues, in the gut and in annexed glands. The two peptide were differently expressed: Tß4 was absent in salivary glands whereas Tß10 was expressed in parotid and in submandibular glands. Tß4 was mildly expressed in the tongue and in the oesophagus, where Tß10 was absent. A similar expression was found in the stomach, ileum and colon mucosa. In pancreas Tß4 reactivity was restricted to the Langerhans islet cells; Tß4 was also detected in the exocrine cells. Both peptide were not expressed in liver cells. When the rat expression pattern in rat organs was compared to reactivity for Tß4 and Tß10 in humans, marked differences were found. Our data clearly indicate a species-specific expression of Tß4 and Tß10, characterized by the actual unpredictability of the expression of these peptides in different cells and tissues. The common high expression of Tß4 in mast cells, both in humans and in rats, represents one of the few similarities between these two species.

MUC1 marks collecting tubules, renal vesicles, comma- and S-shaped bodies in human developing kidney tubules, renal vesicles, comma- and s-shaped bodies in human kidney.

MUC1 is a transmembrane glycoprotein, apically expressed in most epithelial cells, used in the differential diagnosis of carcinomas and for discrimination of tumors of non-epithelial origin showing epithelioid features. Little attention has been paid so far though, on its possible significance in embryonic tissues. A preliminary study from our group revealed MUC1 expression in the cap mesenchymal cells during human nephrogenesis, suggesting a role for MUC1 in the process of mesenchymal-to-epithelial transition. This study aimed at investigating the expression pattern of MUC1 in various developing structures of human fetal kidney. Expression of MUC1 was examined in kidneys of 5 human fetuses. MUC1 immunoreactivity was detected in ureteric bud tips, in collecting tubules, in cap mesenchymal cells undergoing the initial phases of mesenchymal-to-epithelial transition, in renal vesicles, comma-bodies, and S-shaped bodies. Our previous preliminary report suggested a role for MUC1 in the initial phases of the process of mesenchymal-to-epithelial transition. The present data suggest that MUC1 expression characterizes multiple structures during human nephrogenesis, from the ureteric bud, to the initial phases of mesenchymal-to-epithelial transition and that MUC1 should be added to the genes activated during the process of mesenchymal-to-epithelial transition in the cap mesenchyme of human kidney.

The role of immunohistochemistry in the study of the newborn kidney.

The identification of the different cell types involved in human nephrogenesis, when solely based on morphology, may lead to errors in its interpretation, given the complexity of the histological picture of the fetal and of the newborn kidney. In this study, the most recent works utilizing immunohistochemistry for the identification of the multiple cell types involved in human nephrogenesis are reviewed. The role of WT1, MUC1, Thymosin beta 10, Thymosin beta 4, CD10 and CD44 in the different phases of glomerulogenesis and of tubulogenesis is here described, with particular emphasis on their expression in the early phases of nephrogenesis. On the basis of our data, immunohistochemistry appears to be a useful tool in the study of human nephrogenesis, giving new data on the different steps of the differentiation of metanephric mesenchyme towards the multiple cell types characterizing the mature human kidney. Moreover, allowing a better knowledge of the protein products involved in the generation of new nephrons, immunohistochemistry could open new perspectives in the field of renal regenerating medicine, evidencing the factors able to prolong nephrogenesis after birth, helping us to reach our goal: allowing newborn kidneys to restore their nephron endowment, escaping susceptibility to hypertension and renal disease in adulthood.

CD10 in the developing human kidney: immunoreactivity and possible role in renal embryogenesis.

CD10 was first identified in tumor cells of acute lymphoblastic leukemia. Most studies on CD10 expression have dealt with tumor pathology. Since no data are available for specific role in the fetal kidney, this study aimed at investigating CD10 expression during the different phases of renal embryogenesis. To this end, the expression of CD10 was evaluated in the kidney of two human fetus and in three newborns. In both fetuses, immunostaining for CD10 was compartmentalized and mainly concentrated in the mid-deep cortex. Reactivity for CD10 was stronger in the glomerular epithelium, in proximal tubules and in metanephric mesenchymal cells. At 25 weeks of gestation, CD10 was also detected in the subcapsular regions, including some pretubular aggregates of cap mesenchymal cells and renal vesicles. At 34 weeks of gestation, we observed an increased immunoreactivity for CD10 in visceral and parietal glomerular epithelium. At 39 weeks of gestation, CD10 was also expressed in the collecting tubules and in the Henle loops. Our data show a strong expression of CD10 in all stage of human kidney development, characterized by dynamic changes and support the hypothesis that CD10 plays a relevant role in renal embryogenesis.

The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions.

C-Kit (CD117), the receptor for the stem cell factor, a growth factor for melanocyte migration and proliferation, has shown differential immunostaining in various benign and malignant melanocytic lesions. The purpose of this study is to compare c-Kit immunostaining in benign nevi and in primary and metastatic malignant melanomas, to determine whether c-Kit can aid in the differential diagnosis of these lesions. c-Kit immunostaining was performed in 60 cases of pigmented lesions, including 39 benign nevi (5 blue nevi, 5 intradermal nevi, 3 junctional nevi, 15 cases of primary compound nevus, 11 cases of Spitz nevus), 18 cases of primary malignant melanoma and 3 cases of metastatic melanoma. The vast majority of nevi and melanomas examined in this study were positive for c-Kit, with minimal differences between benign and malignant lesions. C-Kit cytoplasmatic immunoreactivity in the intraepidermal proliferating nevus cells, was detected in benign pigmented lesions as well as in malignant melanoma, increasing with the age of patients (P=0.007) in both groups. The patient's age at presentation appeared to be the variable able to cluster benign and malignant pigmented lesions. The percentage of c-Kit positive intraepidermal nevus cells was better associated with age despite other variables (P=0.014). The intensity and percentage of c-Kit positivity in the proliferating nevus cells in the dermis was significantly increased in malignant melanocytic lesions (P=0.015 and P=0.008) compared to benign lesions (compound melanocytic nevi, Spitz nevi, intradermal nevi, blue nevi). Immunostaning for c-Kit in metastatic melanomas was negative. Interestingly in two cases of melanoma occurring on a pre-existent nevus, the melanoma tumor cells showed strong cytoplasmatic and membranous positivity for c-kit, in contrast with the absence of any immunoreactivity in pre-existent intradermal nevus cells. C-Kit does not appear to be a strong immunohistochemical marker for distinguishing melanoma from melanocytic nevi, if we consider c-Kit expression in intraepidermal proliferating cells. The c-Kit expression in proliferating melanocytes in the dermis could help in the differential diagnosis between a superficial spreading melanoma (with dermis invasion) and a compound nevus or an intradermal nevus. Finally, c-Kit could be a good diagnostic tool for distinguishing benign compound nevi from malignant melanocytic lesions with dermis invasion and to differentiate metastatic melanoma from primary melanoma.

Expression pattern of thymosin beta 4 in the adult human liver.

Thymosin beta-4 (Tß4) is a member of beta-thymosins, a family of small peptides involved in polymerization of G-actin, and in many critical biological processes including apoptosis, cell migration, angiogenesis, and fibrosis. Previous studies in the newborn liver did not reveal any significant reactivity for Tß4 during the intrauterine life. The aim of the present study was to investigate by immunohistochemistry Tß4 expression in the adult normal liver. Thirty-five human liver samples, including 11 needle liver biopsies and 24 liver specimens obtained at autopsy, in which no pathological change was detected at the histological examination, were immunostained utilizing an anti-Tß4 commercial antibody. Tß4 was detected in the hepatocytes of all adult normal livers examined. A zonation of Tß4 expression was evident in the vast majority of cases. Immunostaining was preferentially detected in zone 3, while a minor degree of reactivity was detected in periportal hepatocytes (zone 1). At higher power, Tß4-reactive granules appeared mainly localized at the biliary pole of hepatocytes. In cases with a strong immunostaining, even perinuclear areas and the sinusoidal pole of hepatocytes appeared interested by immunoreactivity for Tß4. The current work first evidences a strong diffuse expression of Tß4 in the adult human liver, and adds hepatocytes to the list of human cells able to synthesize large amounts of Tß4 in adulthood. Moreover, Tß4 should be added to the liver proteins characterized by a zonate expression pattern, in a descending gradient from the terminal vein to the periportal areas of the liver acinus. Identifying the intimate role played by this peptide intracellularly and extracellularly, in physiology and in different liver diseases, is a major challenge for future research focusing on Tß4.

Thymosin beta 4 expression in normal skin, colon mucosa and in tumor infiltrating mast cells.

Mast cells (MCs) are metachromatic cells that originate from multipotential hemopoietic stem cells in the bone marrow. Two distinct populations of MCs have been characterized: mucosal MCs are tryptase-positive while mast cells in skin contain tryptase and chymase. We now show that a sub-population of MCs is highly immunoreactive for thymosin beta4, as revealed by immunohistochemical analyses of normal skin, normal colon mucosa and salivary gland tumors. Four consecutive serial sections from each case were immunostained for thymosin beta4 (Tbeta4), chymase, tryptase and stained for toluidine blue. In skin biopsies, MCs showed a comparable immunoreactivity for Tbeta4, chymase and tryptase. In normal colon mucosa the vast majority of mucosal MCs expressed a strong cytoplasmic immunoreactivity for tryptase and for Tbeta4, in the absence of chymase reactivity. A robust expression of Tbeta4 was detected in tumor-infiltrating and peritumoral mast cells in salivary gland tumors and breast ductal infiltrating carcinomas. Tumor-infiltrating MCs also showed a strong immunoreactivity for chymase and tryptase. In this paper, we first demonstrate that normal dermal and mucosal mast cells exhibit strong expression of thymosin beta4, which could be considered a new marker for the identification of mast cells in skin biopsies as well as in human tumors. The possible relationship between the degree of Tbeta4 expression in tumor-infiltrating mast cells and tumor behaviour warrants further consideration in future investigations.

Immunoreactivity of thymosin beta 4 in human foetal and adult genitourinary tract.

Thymosins beta 4 (Tβ4) is a member of the beta-thymosins family, a family of peptides playing essential roles in many cellular functions. Our recent studies suggested Tβ4 plays a key role in the development of human salivary glands and the gastrointestinal tract. The aim of this study was to analyse the presence of Tβ4 in the human adult and foetal genitourinary tract. Immunolocalization of Tβ4 was studied in autoptic samples of kidney, bladder, uterus, ovary, testicle and prostate obtained from four human foetuses and four adults. Presence of the peptide was observed in cells of different origin: in surface epithelium, in gland epithelial cells and in the interstitial cells. Tβ4 was mainly found in adult and foetal bladder in the transitional epithelial cells; in the adult endometrium, glands and stromal cells were immunoreactive for the peptide; Tβ4 was mainly localized in the glands of foetal prostate while, in the adults a weak Tβ4 reactivity was restricted to the stroma. In adult and foetal kidney, Tβ4 reactivity was restricted to ducts and tubules with completely spared glomeruli; a weak positivity was observed in adult and foetal oocytes; immunoreactivity was mainly localized in the interstitial cells of foetal and adult testis. In this study, we confirm that Tβ4 could play a relevant role during human development, even in the genitourinary tract, and reveal that immunoreactivity for this peptide may change during postnatal and adult life.

Cytokeratin 20-positive hepatocellular carcinoma.

The differential diagnosis between hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and metastatic colorectal adenocarcinoma (MCA) may be difficult when only based on morphology. For this purpose immunohistochemical analyses are often required, utilizing antibodies directed against CK8-18, Hep-Par1, glypican 3, CK7, CK19, CK20. Here we report a case of a 65-year-old man who presented with a clinical picture of decompensated cirrhosis. Ultrasonography revealed two nodular areas in the right liver lobe. Liver needle biopsy revealed micro-macronodular cirrhosis associated with HCC with trabecular and pseudoglandular patterns. Immunohistochemically, tumour cells were diffusely positive for CK8-18 and also diffusely immunostained by glypican 3 and Hep-Par1. Interestingly, a diffuse and strong staining for CK20 was detected in the vast majority of tumor cells, particularly in the areas showing a pseudo-glandular pattern. No immunostaining for CK7 and CK19 was found in the tumor cells. The tumor behaved aggressively, with a rapid diffusion to the whole liver. The patient died from the disease few months after presentation. These findings underline that the interpretation of the expression of CK20 alone in the differential diagnosis among HCC, CC and MCA should be done with caution because a diffuse immunoreactivity for CK20 alone may not rule out the diagnosis of HCC.

Would a morphomolecular approach help in defining pseudosarcomatous myofibroblastic proliferations? A study of a heart polypoid lesion.

Many cardiac neoplasms lack pathognomonic clinical features, and this leads to controversial interpretations. As genomic changes may correlate with these malignancies and possibly aid in diagnosis, fluorescence in situ hybridisation (FISH) was used to study a polypoid lesion found incidentally at autopsy on the septal wall of the left ventricle of a 75-year-old man who had died from a heart attack. Histology and immunohistochemistry disclosed atypical stromal cells with irregular voluminous nuclei positive for vimentin and smooth muscle actin; these cells were reminiscent of those previously reported in a subset of nasal polyps showing aneuploidy. The scarce lymphoplasmocytic infiltrate hindering the diagnosis of inflammatory myofibroblastic tumours (IMT), and the presence of atypical cells, prompted the use of FISH: lack of ALK gene rearrangement and aneuploidy were observed in the irregular nuclei, supporting the diagnosis of a pseudosarcomatous myofibroblastic proliferation (PMP). These results stress that IMT and PMP may represent variants within a spectrum of myofibroblastic proliferations/tumours.