Expression of HIF and their regulators PHD and VHL from placentas of pregnancies complicated with fetal growth restriction

This project aims at revealing the possible connection between expression of Hypoxia Inducible Factors (HIF) and their pathway in pregnancies affected with Fetal Growth Restriction (FGR) together or not with preeclampsia. FGR is a complex condition in the field of current Obstetrics. The failure of a foetus to achieve its genetically determined growth potential is associated with significantly increased perinatal morbidity and mortality as well as being a major determinant of cardiovascular disease and glucose intolerance in adult life. FGR is not a disease entity with a unique pathophysiology. The term FGR is generally used for a foetus that presents reduced velocity pattern of growth (<10th centile). A variety of factors have been involved in this situation including infectious, congenital abnormalities, drug abuse or chemical substances, abnormalities of the placenta, immunological and anatomical factors. In most cases, however, incomplete placentopoiisis (placental formation) is the cause for the insufficient supply of nutritious substances and oxygen to the fetus that subsequently causes the deceleration of his growth. Hypoxia appears to constitute a common pathophysiological mechanism in almost the majority of the cases, and regardless the underlying pathology leads to the delay of fetal growth. Preeclampsia comprises a pathologic condition that, as many others, may cause foetus hypoxia. Preeclampsia affects 4-6% of pregnancies and it may have serious consequences for the maternal health as well as for the development and well-being of the foetus. This pathological entity is characterized by an increase of the arterial pressure beyond the 20th week of gestation as well as by proteinuria and/or oedema. Although a variety of conditions (including chronic hypertension, coagulopathies, immunological and genetic disorders, nutrition insufficiencies or abuses) appear to constitute risk factors and have been connected with higher risk of the disease; however, the molecular mechanisms and the precise causes that are hidden in the underlying pathophysiology remain unclear and raise a field of further investigation. Base on the studies of last decade, it has been revealed that the disturbance in the balance of circulating angiogenic factors (sFlt-1, VEGF1, PlGF) has an important role in the pathophysiology of preeclampsia that leads to endothelium abnormalities of vessels as well as to the symptoms of the disease. These factors are regulated by several other factors and conditions, but hypoxia seems to be one of the most important. One of the more important processes at the development of gestation is the physiologic formation of the fetal-placental unit that begins with the infiltration of cytotrophoblasts into the endometrium and is completed when conjunction with the spiral arteries has occurred. During this process, structural changes are required both for the endothelium of endometrium that receives cytotrophoblasts invasion and for the elastic walls of spiral arteries. This process includes the passage from a hypoxia condition into a condition of physiologic oxygenation in a cellular level; in this process the role of Hypoxia Inducible Factors (HIF) is critical. HIF-1 constitutes probably the most essential response of the cell to the hypoxia. It acts in the cell nucleus, by inducing the expression of many genes that play role in angiogenesis, cell cycle and metabolism. HIF- 2 appears to have a similar function, while the data regarding HIF-3 are still insufficient. As the regulation of these factors is depending on the oxygen concentration, a clear relationship is obvious between them and the hypoxia conditions of as FGR and preeclampsia. The regulation of HIF appears to relate not only with the induction/suspension of their expression, but also post-translationally with their degradation. Factors PHD-1, 2 and 3, as well as VHL play a role of their degradation. In some pathological situations, this model is probably disturbed and hypoxia maintains the regulating action of HIF in active levels. It is obvious that all the above factors appear to possess a critical role in the process of pathogenesis of both FGR and preeclampsia. The adequate comprehension of the pathway of regulation of HIF may give an impulse to development of intervention models in those particular pathologic conditions that are related with hypoxia, common in many pregnancies. ANXA5 is also crucial for the uteroplacental unit due to its antithrombotic action. During this study the action and regulation of the above mentioned factors (HIF-1a,-2a,-3a, ARNT, PHD-1,-2,-3, VHL and ANXA5) was investigated in term placentas from 49 FGR gestations related or not to preeclampsia compared to 30 term placentas from normal pregnancies. FGR and preeclampsia were diagnosed with clinical and laboratory data and cases with particular pathological cause such as thrombophilia, chromosomal defects, infections and others were excluded. All placentas were examined by a pathologist. RT-PCR was the molecular technique of choice to determine expression profiles for each gene. Western blotting and immunohistochemistry was also performed in selected cases. Statistical analysis was processed with the use of SPSS software. The final results revealed marked downregulation of PHD-2, PHD-3 and ANXA5, slight upregulation of HIF-1a and HIF-2a, along with PHD-1, while ARNT, HIF-3a and VHL expressions didn’t produce significant differences between FGR and normal placentas. ANXA5 was expected to be downregulated, since placentas from FGR and preeclamptic pregnancies showed infarcts presence. Downregulation of PHD-2 and PHD-3, along with upregulation of HIF-1a and HIF-2a points out the inducible effect that hypoxia seems to have in HIF transcriptional pathway. Stabilization of HIF-a subunits by reduction of PHD proteins is more crucial for activation of HIF than HIF expression by itself. Moreover, with the clinical and demographic data correlation analysis along with expression profiles, a new model is introduced. Based on these results, it can be hypothesized that FGR babies with less severe disease, are able to produce a signal stimulated by the reduced placental nutrient and oxygen availability in order to overcome it, in contrast to FGR babies with a more adverse outcome that fail to produce this signal. This adaptation model is evident through the different expression pattern of the genes examined.Σκοπός του προτεινόμενου ερευνητικού έργου αποτέλεσε η διερεύνηση της πιθανής συσχέτισης της έκφρασης παραγόντων σχετιζόμενων με υποξικές καταστάσεις σε πλακούντες από τελειόμηνες εγκυμοσύνες με εμβρυική υπολειπόμενη ανάπτυξη (ΕΥΑ) σε έδαφος ή μη προεκλαμψίας. Η ενδομήτρια υπολειπόμενη εμβρυϊκή ανάπτυξη (EYA) είναι ένα σύνθετο πρόβλημα στη σύγχρονη μαιευτική. Η αποτυχία του εμβρύου να φτάσει στο γενετικά καθορισμένο αναπτυξιακό του πρότυπο, συνδέεται με σημαντική νοσηρότητα και θνησιμότητα, ενώ παράλληλα αποτελεί καθοριστικό παράγοντα καρδιοαγγειακών παθήσεων και μεταβολικών διαταραχών στην ενήλικο ζωή. Πολλοί αιτιολογικοί παράγοντες έχουν ενοχοποιηθεί για την ΕΥΑ, ωστόσο στις περισσότερες περιπτώσεις είτε η πρωτοπαθής ανώμαλη πλακουντοποίηση είτε η δευτεροπαθής βλάβη του πλακούντα είναι υπεύθυνη για την ανεπαρκή προσφορά θρεπτικών ουσιών και οξυγόνου στο έμβρυο που οδηγεί στην επιβράδυνση της ανάπτυξής του. Επομένως, στον παθοφυσιολογικό μηχανισμό φαίνεται να συμμετέχει η υποξία, η οποία επίσης συσχετίζεται με μια σειρά παθολογικών καταστάσεων της κύησης συμπεριλαμβανομένης της προεκλαμψίας. Η προεκλαμψία εμφανίζεται στο 4-6% των κυήσεων και μπορεί να έχει σοβαρές επιπτώσεις στην υγεία της μητέρας και στην ανάπτυξη του εμβρύου. Πρόκειται για μία νόσο που χαρακτηρίζεται από αύξηση της αρτηριακής πίεσης μετά την 20η εβδομάδα της κύησης σε συνδυασμό με πρωτεϊνουρία ή/και οίδημα. Με βάση μελέτες της τελευταίας δεκαετίας, αποκαλύπτεται ότι σημαντική συμμετοχή στην παθοφυσιολογία της προεκλαμψίας έχει η διαταραχή στην ισορροπία των κυκλοφορούντων αγγειογενετικών παραγόντων sFlt-1 (soluble vascular endothelial growth factor receptor- 1), VEGF1 (vascular endothelial growth factor-1) και PlGF (placenta growth factor), που οδηγεί σε ανωμαλίες του ενδοθηλίου των αγγείων και σε δεύτερο χρόνο στα κλινικά συμπτώματα της νόσου (θεωρία δύο σταδίων). Οι παράγοντες αυτοί ρυθμίζονται από διάφορες παραμέτρους, σημαντική εκ των οποίων, είναι και η υποξία. Μία από τις βασικότερες διαδικασίες κατά την εξέλιξη μιας κύησης είναι η φυσιολογική δημιουργία της εμβρυοπλακουντιακής μονάδας, που ξεκινάει με τη διείσδυση της κυτταροτροφοβλάστης στο ενδομήτριο, ενώ στη συνέχεια ολοκληρώνεται με τη συνένωση με τα μητριαία σπειροειδή αγγεία. Κατά τη διάρκεια της διαδικασίας αυτής απαιτούνται δομικές αλλαγές τόσο στο ενδομήτριο που υποδέχεται την κυτταροτροφοβλάστη, όσο και στα μυϊκά κυρίως τοιχώματα των μητριαίων σπειροειδών αρτηριών με σκοπό τη δημιουργία των χοριακών λαχνών. Η διαδικασία αυτή περιλαμβάνει τη μετάβαση από συνθήκες υποξίας σε συνθήκες φυσιολογικής οξυγόνωσης σε κυτταρικό επίπεδο και επομένως απαιτεί τη σημαντική συμμετοχή των επαγόμενων από την υποξία ρυθμιστικών παραγόντων HIF. Ο παράγοντας HIF-1 κατέχει βασικό ρόλο στο μηχανισμό «απάντησης» του κυττάρου σε καταστάσεις υποξίας. Δρα στον πυρήνα του κυττάρου επάγοντας την έκφραση πολλών πρωτεϊνών που διαδραματίζουν ρόλο στην αγγειογένεση, τον κυτταρικό κύκλο και το μεταβολισμό. Παρόμοια λειτουργία φαίνεται να διαδραματίζει και o HIF-2, ενώ για τον HIF-3 τα δεδομένα είναι ελλιπή. Ο τρόπος ρύθμισης των HIF φαίνεται να μη σχετίζεται μόνο με την επαγωγή/αναστολή της έκφρασής τους, αλλά και μετα-μεταφραστικά (posttranslationally) με την αποδόμηση των HIF-a υπομονάδων τους, στην οποία ρόλο έχουν οι παράγοντες PHD-1, 2 και 3, καθώς και ο VHL. Σε υποξικές καταστάσεις όμως, η χαμηλή PO2 αναστέλλει την υδροξυλίωση της HIF-a υπομονάδας από τους PHD και την ακόλουθη πρωτεασωμική αποδόμησή της με τη συμμετοχή του VHL, με συνέπεια τη σταθεροποίησή της και τη δημιουργία του ετεροδιμερούς HIF μετά από την ένωση με τον HIF-b (ARNT) και τη μεταγραφική ενεργοποίηση. Ο καταρράκτης (cascade) γεγονότων που ακολουθεί είναι κρίσιμος, αν συνυπολογίσει κανείς τον αριθμό και το ρόλο των γονιδίων στόχων των HIF (αγγειογένεση, κυτταρικός κύκλος, μεταβολισμός). Φαίνεται λοιπόν ότι οι παραπάνω παράγοντες (HIF, PHD, VHL), ρυθμίζονται ανάλογα με τη μερική τάση του οξυγόνου, γεγονός που τους συνδέει άμεσα με καταστάσεις υποξίας, όπως πιθανότατα συμβαίνει στην εμβρυοπλακουντιακή κυκλοφορία σε εγκυμοσύνες με ΕΥΑ και προεκλαμψία. Η αννεξίνη V (ANXA5) είναι μία άλλη πρωτεΐνη, η έκφραση της οποίας φαίνεται να είναι σημαντική στην εμβρυοπλακουντιακή κυκλοφορία. Η αντιθρομβωτική της δράση, την καθιστά αναγκαία στη διατήρηση της καλής αιματικής κυκλοφορίας, ενώ μειωμένα επίπεδα μπορεί να αποτελέσουν παράγοντα θρομβωτικών επεισοδίων στα αγγεία του χοριακού πλέγματος. Στην παρούσα μελέτη ανιχνεύτηκε η έκφραση των παραπάνω γονιδίων (HIF-1, HIF-2, HIF-3, ARNT, PHD-1, PHD-2, PHD-3, VHL και ΑΝΧΑ5) σε πλακουντιακό ιστό από 49 κυήσεις που εμφάνισαν ΕΥΑ σε έδαφος προεκλαμψίας ή ανεξάρτητα από αυτήν και από 30 κυήσεις με ομαλή πορεία και γέννηση ικανοποιητικού βάρους για την ηλικία κύησης νεογνού. Η παρουσία προεκλαμψίας και η ΕΥΑ επιβεβαιώθηκαν με κλινικά-εργαστηριακά και υπερηχογραφικά κριτήρια αντίστοιχα, ενώ αποκλείστηκαν οι περιπτώσεις ΕΥΑ που αποδόθηκαν σε συγκεκριμένη νοσολογική οντότητα (θρομβοφιλία, ανοσολογικά και γενετικά αίτια, λοιμώξεις κ.ά). Όλοι οι πλακούντες εξετάστηκαν ιστοπαθολογικά. Για τον προσδιορισμό της έκφρασης των υπό εξέταση γονιδίων έγινε αρχικά εξαγωγή εκχυλίσματος RNA από τα δείγματα και στη συνέχεια Real Time PCR. Επίσης επιλεκτικά έγινε εξαγωγή εκχυλίσματος πρωτεϊνών από τα δείγματα και στη συνέχεια western blotting με φθορίζοντα αντισώματα. Για την ανοσοϊστοχημική ανίχνευση έγινε μονιμοποίηση των δειγμάτων σε παραφίνη και μετά ανοσοϊστοχημική χρώση με ειδικά για την πρωτεΐνη μονοκλωνικά αντισώματα. Η ανάλυση για την εξαγωγή στατιστικής σημαντικότητας πραγματοποιήθηκε με το λογισμικό SPSS και την εφαρμογή πολυπαραγοντικής ανάλυσης με τη χρήση μη παραμετρικών δοκιμασιών (Kruskal-Wallis test, Mann-Whitney test). Τα αποτελέσματα της έκφρασης των γονιδίων έδειξαν σημαντική μείωση για τα γονίδια PHD-2, PHD-3 και ANXA5, μερική αύξηση της έκφρασης των γονιδίων HIF-1a και HIF-2a, καθώς και του PHD-1, ενώ τα γονίδια ARNT, HIF-3a και VHL δεν έδειξαν ιδιαίτερα σημαντικές μεταβολές σε σχέση με τα φυσιολογικά δείγματα. Η ANXA5 έχει σαφώς μειωμένη έκφραση στην υπολειπόμενη ανάπτυξη και στην προεκλαμψία, γεγονός που δικαιολογεί την παρουσία περισσότερων θρόμβων στους πλακούντες των γυναικών με αυτές τις παθολογίες. Οι παράγοντες HIF φαίνεται να συμμετέχουν στην παθοφυσιολογία της εκδήλωσης IUGR κυρίως μέσω της μείωσης στην έκφραση του PHD-2 και του PHD-3, αλλά και με τη μικρή αύξηση των HIF-1a και HIF-2a. Η ρύθμιση των HIF είναι κυρίως μετα-μεταγραφική και η ελάττωση των παραγόντων αποδόμησής τους (PHD) αποτελεί σημαντικό κλειδί της ενεργοποίησης των HIF προς το δικό τους μεταγραφικό ρόλο. Επιπρόσθετα, από τις συσχετίσεις που προέκυψαν με διάφορες κλινικοεργαστηριακές και σωματομετρικές παραμέτρους μπορεί να υποστηριχθεί η ύπαρξη ενός μοντέλου κεντρικής απάντησης του εμβρύου μέσω παραγωγής παραγόντων από τον πλακούντα. Με βάση την παραπάνω θεωρία, στις κυήσεις με ΕΥΑ τα έμβρυα με καλύτερο προφίλ ανάπτυξης παρουσίασαν διαφορετικό πρότυπο μεταβολών στην έκφραση των μελετώμενων γονιδίων, σε σχέση με τα έμβρυα χειρότερης πρόγνωσης, εύρημα που υποδηλώνει τη δημιουργία απάντησης ή την αδυναμία απάντησης αντίστοιχα, στην υποξική αιτία που προκάλεσε ή συμμετείχε στην ενδομήτρια καθυστέρηση της ανάπτυξης

The effect of an exon 12 polymorphism of the human thromboxane synthase (CYP5A1) gene in stroke patients

Background: To examine the prevalence of an exon 12 polymorphism on the human Thromboxane synthase (CYP5A1) gene. Material/Methods: Using sequence-specific PCR, we examined the allelic prevalence in 237 Greek patients with ischemic strokes and in 171 controls. In addition, we compared the CYP5A1 allelic prevalence in 71 patients with stroke recurrence despite Aspirin use, in comparison with patients who have not experienced recurrent stroke while taking Aspirin. Results: The frequencies of the CYP5A1*9mutant (substitution of guanine by adenine near the heme-binding catalytic domain) and of the wild-type allele were 0.197 and 0.803, respectively; they did not differ significantly between stroke patients and controls. The wildtype allele was more frequent in the Cretan population compared to continental Greece (OR 1.80, 95% CI 1.19-2.74). The wild-type allele was more frequent among hypertensive and less frequent among diabetic stroke sufferers, respectively. The CYP5A1*9 mutant was significantly more prevalent among stroke patients with history of previous cerebrovascular attacks (p Conclusions: Allelic prevalence of the CYP5A1 exon 12 might differ between geographic areas within the same ethnic group, and is associated with particular characteristics of stroke patients. Allele mutations can abolish the enzymatic activity of thromboxane synthase, via impaired heme binding, associated with defective response to Aspirin used as secondary prevention, an effect independent from the conventional risk factors for cerebrovascular disease

Angiogenic factors in placentas from pregnancies complicated by fetal growth restriction (Review)

The placenta is the organ that is responsible for providing the developing fetus with all the nutrients necessary for its growth and is also responsible for removing fetal waste. Placentation is a crucial process that includes angiogenesis. Angiogenesis involves not only the fetal circulation, but also placental and endometrial vascular changes. In this study, we review the literature regarding any impairment in the angiogenic process in placentas from pregnancies complicated by fetal growth restriction (FGR). Angiogenesis is regulated by a list of factors, also known as growth factors, such as the vascular endothelial growth factor (VEGF), the placental growth factor (PlGF) and the basic fibroblastic growth factor (bFGF), as well as the partial pressure of oxygen in the fetoplacental vessels. Other factors, such as transcriptional factors, also play a pivotal role, controlling the above-mentioned growth factors. Alterations in these pathways have been described in cases of growth-restricted fetuses. In this review, we provide an insight into these processes and identify the most crucial factors involved

Downregulation of notch signaling pathway in late preterm and term placentas from pregnancies complicated by preeclampsia.

Preeclampsia (PE) is a major cause of maternal mortality and morbidity, affecting 3-5% of all pregnancies. The Notch signaling pathway plays an important role during placental development, activating several target genes. Defects in the Notch pathway have adverse effect on placentation. The aim of this study was to investigate the expression of receptors NOTCH1,-2,-3,-4, ligands DLL1,-3,-4, JAG1,-2 and target genes HEY1,-2 in placental tissue samples from 20 late preterm or term pregnancies complicated by PE versus 20 normal pregnancies. mRNA levels of the studied molecules were measured by quantitative Real-Time PCR (qRT-PCR), while the protein expression of the intracellular domain of NOTCH2 (NICD2) and NOTCH3 (NICD3) was measured by Western Blot (WB). qRT-PCR analysis revealed that NOTCH1, NOTCH4 and DLL1 were not expressed in the placenta. On the contrary, NOTCH2, NOTCH3, DLL3, DLL4, JAG1, JAG2, HEY1 and HEY2 mRNA levels were downregulated in PE samples vs. controls (p<0.01). WB confirmed that NICD2 (p = 0.014) and NICD3 (p<0.001) protein levels were also lower in PE specimens. Statistical analysis revealed several significant associations: of NOTCH3 mRNA expression with smoking during pregnancy (p = 0.029), of NICD3 protein levels (p = 0.028) and DLL3 mRNA levels (p = 0.041) with birth weight centile, and of HEY2 transcript levels with parity (p = 0.034) and mode of delivery (p = 0.028). Our results suggest that Notch pathway downregulation is associated with PE. Further studies are required in order to determine the role of these molecules in PE pathogenesis and to evaluate their potential use for the early detection and treatment of PE

Topographic and quantitative relationship between prostate inflammation, proliferative inflammatory atrophy and low-grade prostate intraepithelial neoplasia: a biopsy study in chronic prostatitis patients

Inflammatory processes are important components in the pathogenesis of many human cancers. According to the 'injury and regeneration' model for prostate carcinogenesis, injury caused by pathogens or pro-inflammatory cytotoxic agents would trigger proliferation of prostatic glandular cells, leading to the appearance of epithelial lesions named 'Proliferative Inflammatory Atrophy' (PIA). Inflammatory cells infiltrating the prostate would release genotoxic reactive oxygen species, leading atrophic cells to neoplastic progression. The hypothesis pointing to PIA as risk-lesion for prostate cancer has been extensively investigated at the cellular and molecular levels, but few morphological data are available linking PIA or prostatic intraepithelial neoplasia (PIN) to inflammation or clinical prostatitis. We investigated at the morphological level 1367 prostate biopsies from 98 patients with a recent history of chronic prostatitis, and 32 patients with biopsies positive for carcinoma. Our results show that i) PIA is found more frequently in biopsy cores containing a severe or moderate inflammatory focus, compared to NON-PIA lesions (partial or cystic atrophy); ii) the PIA lesion post-atrophic hyperplasia is more frequently found in tissues showing mild or no inflammation; iii) the extent of PIA per patient correlates with the burden of moderate or severe inflammation, whereas NON-PIA lesions do not; iv) low-grade PIN is in over 90% of cases emerging from normal, non-atrophic glands and is more frequently found in biopsy cores with absent or mild inflammatory burden; v) the inverse relationship between the prevalence of low-grade PIN and the extent of PIA lesions per patient is described by a power law function, suggesting the low likelihood of the concomitant presence of these lesions in the same tissue; vi) NON-PIA lesions correlate inversely with neoplasia in patients with prostate cancer; vii) the total scores of the NIH-CPSI questionnaire correlate with both PIA and inflammation burdens at diagnosis of prostatitis but not after pharmacological intervention. These results point to a positive association between tissue inflammation, clinical prostatitis and the putative cancer risk-lesion PIA, but do not support a model whereby low-grade PIN would arise from PIA

Reduced ANXA5 mRNA and protein expression in pregnancies complicated by preeclampsia

Introduction The placental anticoagulant protein Annexin A5 (ANXA5) is a multifunctional protein that is highly expressed on the apical surfaces of syncytiotrophoblasts, and plays an important role in haemostatic regulations, maintaining blood fluidity of the placenta. The aim of this study was to investigate the expression of ANXA5 in pregnancies complicated by preeclampsia (PE). Materials and Methods Placental tissue samples were collected from 23 pregnancies with PE and 34 normal pregnancies. ANXA5 mRNA levels were measured by quantitative Real-Time PCR (qPCR), while ANXA5 protein expression was measured by Western Blot (WB) and immunohistochemistry. Results ANXA5 mRNA expression in PE samples was lower than 1% of its expression in normal samples (mean ± SD: 0.002 ± 0.004 vs. 0.55 ± 0.38, p &lt; 0.001), while ANXA5 protein levels in PE samples were approximately at 65% of the average normal expression (mean ± SD: 0.53 ± 0.30 vs. 0.81 ± 0.25, p = 0.001). Immunohistochemical analysis also verified the above results, since PE placentas tended to have low labelling indexes (LIs), in contrast to controls which demonstrated high LIs (p = 0.020). Statistical analysis of the WB data revealed that ANXA5 protein expression was increased in PE smokers vs. PE non-smokers (mean ± SD: 0.64 ± 0.23 vs. 0.41 ± 0.33, p = 0.027). Conclusions These results suggest that ANXA5 downregulation could be part of the pathophysiology of PE and the possible impairment in coagulation processes, which are seen in pregnancies that demonstrate PE. Further studies may investigate whether ANXA5 could be used as a biomarker for the early detection of PE and for the prediction of its severity. © 2013 Elsevier Ltd

Subcutaneous Fat Necrosis and Hypercalcemia with Nephrocalcinosis in Infancy: Case Report and Review of the Literature

Subcutaneous fat necrosis is an uncommon benign panniculitis affecting more commonly full-term newborns. It has been associated with birth asphyxia and meconium aspiration, as well as therapeutic hypothermia. Although the prognosis is generally favorable, complications such as hypercalcemia, thrombocytopenia, hypoglycemia and hypertriglyceridemia may complicate its course. The most serious complication is hypercalcemia that may reach life threatening levels and can be associated with nephrocalcinosis. We thereby describe a case of subcutaneous fat necrosis after therapeutic hypothermia, which presented with late-onset refractory severe hypercalcemia and persistent nephrocalcinosis during the follow up of the patient. Due to the risk of the development of chronic kidney disease, we highlight the importance of careful monitoring of hypercalcemia and review the literature of subcutaneous fat necrosis related to nephrocalcinosis

High prevalence of Human Herpes Virus 8 (HHV-8) in patients with Warthin\u27s tumors of the salivary gland

Background: Warthin\u27s tumor is a common benign neoplasm of the salivary gland. Human Herpes Virus 8 (HHV-8) is the etiologic agent for all forms of Kaposi\u27s sarcoma (KS), and HHV-8 DNA is present in saliva, suggesting that non-sexual transmission is associated with latent infection of the salivary gland. Objectives: To provide insights into the HHV-8 cell tropism, the presence of HHV-8 was investigated in a series of Warthin\u27s tumors of the salivary gland and corresponding adjacent normal tissue. Study design: Forty-three patients with Warthin\u27s tumors (cystadenolymphoma) were tested for the presence of HHV-8 DNA, and corresponding adjacent normal tissue samples were obtained from 15 patients. DNA was extracted from the paraffin-embedded tissues. A nested polymerase chain reaction (PCR) assay was applied, and the positive samples were confirmed by direct sequencing. Results: HHV-8 DNA was detected in 19 out of 43 (44%) salivary gland tumor samples. Among the 15 cases with paired samples, 9 were HHV-8-positive for both samples, 4 were HHV-8-negative for both samples while in two cases HHV-8 was detected only in the tumor specimens. Conclusions: HHV-8 is frequently detected in adenoid salivary neoplasms, suggesting a significant role of the virus in the etiopathogenesis of the disease. Larger studies are required to investigate the role of HHV-8 in the development or progression of Warthin\u27s tumors