S100B immunoreactivity: a new marker of hypoxia-related cardiac damage in newborn piglets.

OBJECTIVE: The evaluation of the expression of S100B protein, in the swine heart in an experimental model of hypoxia - reoxygenation. METHODS: Normocapnic hypoxia was induced in 40 male Landrace/Large White neonatal piglets by decreasing the inspired concentration of oxygen to 6-8%. When animals developed bradycardia or severe hypotension, reoxygenation was initiated. Piglets were allocated in four groups of 10, according to the oxygen concentration they were reoxygenated with: Group 1, 2, 3 and 4 resuscitated with 18%, 21%, 40% and 100% oxygen, respectively. The animals were further classified into 4 groups according with the time required for reoxygenation: group A (<15 min); group B (16-60 min); group C (>60 min); group D (deceased animals). RESULTS: Immunostaining for S100B protein was detected in 14 out of the 40 heart samples (35%), both inside the cytoplasm of cardiomyocytes and as globular deposits in the interstitial spaces. Significant differences were observed among groups 1-4 regarding S100B expression. Reactivity for S100B in cardiac cells was detected in 50%, 50%, 10% and 33% of animals in groups 1 and 2, 3 and 4, respectively. Marked differences were also observed among groups A-D: 75%, 33%, 12% and 22% of the animals in group 1, 2, 3 and 4, respectively, showed reactivity for S100B in the heart. CONCLUSIONS: Expression of S100B protein occurred in the heart of some of newborn piglets following severe hypoxia. S100B storage in cardiomyocytes correlates with the different oxygen concentration used during reoxygenation, being higher in piglets reoxygenated with 18% and 21%, and lower in animals reoxygenated with 40% oxygen. Intermediate levels of S100B expression were found in 100% O2-treated animals. The finding of a higher percentage of S100B-immunoreactive hearts in piglets with a fast recovery and the detection of a decreased reactivity in animals with a slow and a very slow recovery clearly indicates S100B protein as an early protective factor with a positive prognostic value in asphyxiated newborn piglets.

S100B protein expression in the heart of deceased individuals by overdose: a new forensic marker?

OBJECTIVE: The evaluation of S100B protein expression in the human heart and its correlation with drug-related death. METHOD: Left ventricular samples were collected from 74 serial forensic autopsies (15 overdose-related deaths; 59 non-overdose-related deaths) from 2007 to 2010. Tissue sections from each sample were immunostained for S100B protein by a commercial antibody. RESULTS: The S100B protein was detected in the heart samples of all 15 cases of drug-related deaths; S100B immunoreactivity was mainly observed in the cytoplasm of cardiomyocytes and as globular deposits in the interstitial spaces. No reactivity or weak reactivity was found in the cardiomyocytes of the 59 subjects who died of other causes. CONCLUSION: Our preliminary data show that the S100B protein accumulates in injured cardiomyocytes during drug-related sudden death. Given the near absence of S100B protein in the heart of subjects who died from causes other than drug overdose, S100B immunopositivity may be used as a new ancillary screening tool for the postmortem diagnosis of overdose-related cardiac death.

Hypoxia/reoxygenation-induced myocardial lesions in newborn piglets are related to interindividual variability and not to oxygen concentration.

OBJECTIVE: Evaluation of myocardial histological changes in an experimental animal model of neonatal hypoxia-reoxygenation. METHODS: Normocapnic hypoxia was induced in 40 male Landrace/Large White piglets. Reoxygenation was initiated when the animals developed bradycardia (HR <60 beats/min) or severe hypotension (MAP <15 mmHg). The animals were divided into four groups based on the oxygen (O(2)) concentration used for reoxygenation; groups 1, 2, 3, and 4 received 18%, 21%, 40%, and 100% O(2), respectively. The animals were further classified into five groups based on the time required for reoxygenation: A: fast recovery (<15 min); B: medium recovery (15-45 min); C: slow recovery (45-90 min); D: very slow recovery (>90 min), and E: nine deceased piglets. RESULTS: Histology revealed changes in all heart specimens. Interstitial edema, a wavy arrangement, hypereosinophilia and coagulative necrosis of cardiomyocytes were observed frequently. No differences in the incidence of changes were observed among groups 1-4, whereas marked differences regarding the frequency and the degree of changes were found among groups A-E. Coagulative necrosis was correlated with increased recovery time: this condition was detected post-asphyxia in 14%, 57%, and 100% of piglets with fast, medium, and slow or very slow recovery rates, respectively. CONCLUSIONS: The significant myocardial histological changes observed suggest that this experimental model might be a reliable model for investigating human neonatal cardiac hypoxia-related injury. No correlation was observed between the severity of histological changes and the fiO(2) used during reoxygenation. Severe myocardial changes correlated strictly with recovery time, suggesting an unreported individual susceptibility of myocardiocytes to hypoxia, possibly leading to death after the typical time-sequence of events.

Thymosin ß4 expression reveals intriguing similarities between fetal and cancer cells.

Thymosin ß4 (Tß4) is highly expressed in saliva of human newborns but not in adults. Here preliminary immunohistochemical analyses on different human tissues are reported. Immunoreactivity for Tß4 in human salivary glands show high quantities of Tß4 before birth, followed by downregulation of expression in adulthood. In contrast, Tß4 is detected in tumors of salivary glands, suggesting that tumor cells might utilize fetal programs, including Tß4 synthesis. Immunohistochemical analyses in the gastrointestinal tract showed strong reactivity for Tß4 in enterocytes during development, but weak immunostaining in mature enterocytes. In colorectal cancer, the association of a high expression of Tß4 with epithelial-mesenchymal transition was observed. On the basis of these data, the process of epithelial-mesenchymal transition could represent the unifying process that explains the role of Tß4 during fetal development and in cancer progression.

Thymosin beta 10 expression in developing human salivary glands.

Thymosin beta 10 (Tß10) is a member of beta-thymosins (Tßs), a family of low molecular mass peptides abundant in many cell types. In previous studies, Tßs have been shown to play essential roles in many cellular functions, including cytokinesis, migration and endocytosis. Recently, Tß10 has been found in high quantities in the saliva of human newborns, while it disappeared in the adults. On the basis of these data, it seemed of some interest to study the influence of Tß10 during the development of the human salivary glands. To this end, we analyzed, using immunocytochemistry, the expression of Tß10 in samples of the major and minor salivary glands obtained, at autopsy, from 2 human fetuses and 4 newborns, ranging from 13 up to 33weeks of gestation. Tß10 immunoreactivity was detected in all salivary glands examined, with marked differences from one gland to the next, the parotid glands showing the highest Tß10 reactivity and minor salivary glands the lowest reactivity. Marked changes were observed in Tß10 expression and localization during embryogenesis. Tß10 was mainly localized extracellularly in the youngest human fetuses (13weeks), in the cytoplasm of immature duct cells at 20weeks, in acinar cells and in the duct lumen in 33weeks old fetuses. Our data show, for the first time, a strong expression of Tß10 in the human salivary glands during the initial phases of the physiological development, present at the 13th week of gestation, and suggesting a role for the peptide in the salivary glands' organogenesis.

Toward nephrogenesis in the pig kidney: the composite tubulo-glomerular nodule.

BACKGROUND: Significant differences regarding nephrogenesis and its completion among different animal species have been reported. Since many informations on clinical conditions (i.e. asphyxia, drugs) are extrapolated from piglets, this study aimed at analyzing nephrogenesis in piglets, in order to compare it with existing data on nephrogenesis in humans. METHODS: Six male newborn piglets were subjected to euthanasia and their kidneys were harvested. Necropsy revealed no injury and no underlying pathology in any of the animals used in the experiment. RESULTS: The analysis of the renal cortex evidenced in all the animals studied the presence of active nephrogenesis. The sequence of events identified during porcine nephrogenesis was characterized by the appearance, in the metanephric mesenchyme, of nodules undergoing mesenchymal-epithelial transition originating a specific picture that we named the tubulo-glomerular nodule. This peculiar developmental structure gives rise to the precursor of tubular and glomerular structures, till the extrusion of developing glomeruli that progressively migrate toward the mid and deep cortex. CONCLUSIONS: Nephrogenesis in pig is characterized by a peculiar morphological event, with marked differences compared with humans. These informations should be taken into account when the experimental data in piglets are extrapolated to humans, especially for clinical purposes.

Expression of WT1 during normal human kidney development.

Wilms Tumor 1 (WT1) is a zinc finger protein, expressed by human podocytes in the adult kidney, which plays a relevant role in different phases of nephrogenesis in experimental animals. Since no data are available for specific role in the human fetal kidney, this study aimed at investigating the expression of WT1 during the different phases of nephrogenesis. To this end, the expression of WT1 was evaluated in the kidneys, from four human fetuses and two newborns. WT1 immunoreactivity was detected in all the examined kidneys, but not in the kidneys of the newborn at term. Immunostaining for WT1 was observed in podocytes of the glomeruli and in the subcapsular regions, in areas of active glomerulogenesis. The extent and the intensity of immunoreactivity for WT1 changed from one case to the next according to the different gestational age. This study confirms in human kidney the relevant role played by WT1 during nephrogenesis. Its expression pattern suggests a main role in the regulation of the process of Mesenchimal-Epithelial-Transition and in the development and maturation of podocytes. Further studies are needed to verify the correlation between the expression pattern of WT1 and that of other genes products involved in nephrogenesis, in order to better understand their relationship at protein level.

MUC1 in mesenchymal-to-epithelial transition during human nephrogenesis: changing the fate of renal progenitor/stem cells?

BACKGROUND: The development of the human kidney is a complex process requiring interactions between epithelial and mesenchymal cells. The condensed cap mesenchyme is hypothesized to generate a population of stem/progenitor cells that undergo mesenchymal-epithelial transition (MET) originating nephrons. Few immunohistochemical markers are available for detecting cap mesenchymal cells in the early phases of MET. METHODS: The expression of MUC1 was evaluated in the kidneys, of 4 human foetuses and 2 newborns. RESULTS: MUC1 immunoreactivity was detected in all the examined kidneys in the cap mesenchyme and in the renal vesicles. Immunostaining for MUC1 in cap mesenchymal cells changed from one nodule to the next: some mesenchymal nodules were negative, some showed MUC1 reactivity in scattered cells, whereas in others, positive cells revealed the presence of a roundish developing epithelial structure. CONCLUSIONS: Our data clearly indicates, for the first time to the best of our knowledge, immunohistochemical evidence of MUC1 expression during human kidney development. We focused on MUC1 reactivity in the cap mesenchyme. On the basis of these preliminary data, we speculate that MUC1 may be involved in human nephrogenesis and may play a relevant role in MET from the cap mesenchyme to the renal vesicle, changing the fate of renal stem/progenitor cells.

Marked interindividual variability in renal maturation of preterm infants: lessons from autopsy.

The kidney of low birthweight preterm infants is characterized by a reduced number of mature nephrons at birth. The aim of the present study was to determine whether, in preterms, active glomerulogenesis occurs in the postnatal period and whether it may compensate the reduced number of nephrons developed during the intrauterine life. Kidney samples were obtained at autopsy from 8 human fetuses, 12 premature infants, and 3 term newborns. Glomerulogenesis, as measured by radial glomerular count (RGC), was markedly decreased in all preterm infants as compared with term newborns. A marked interindividual variability was detected in the level of glomerulogenesis, which, in the vast majority of cases, did neither correlate with the gestational age at birth nor with birthweight. Active glomerulogenesis, as demonstrated by the presence of S-shaped bodies in the subcapsular region, was present in all preterm infants in the perinatal period, but it ceased in a preterm surviving for 3 months. Our data show that active glomerulogenesis continues even after birth for a short period, although it is not able to compensate a marked oligonephronia at birth. As a consequence, the incomplete nephrogenesis typical of all extremely low birthweight preterm infants possibly results in a persistent oligonephronia which should likelihood represent a major risk factors of progressive renal disease in adulthood.

Thymosin beta-10 expression in developing human kidney.

Thymosin beta-10 (Tß10) is a member of beta-thymosins (Tßs), a family of low molecular mass peptides, which play essential roles in many cellular functions, including apoptosis, cell proliferation, cell migration, and endocytosis. The report that the Tß10 gene is expressed at high levels in embryonic human brain as well in human kidney induced us to study Tß10 reactivity in the preterm kidney in order to verify, at tissue level, the expression of this peptide during renal embryogenesis. To this end, we analyzed, using immunocytochemistry, the expression of Tß10 in samples of human kidney obtained, at autopsy, from 8 fetuses, 12 preterm infants, ranging from 25 to 36 weeks of gestation and 3 at term newborns. Tß10 immunoreactivity was detected in 20 out of 22 kidneys examined, and was mainly localized in proximal and distal tubular structures, in the cytoplasm and occasionally in the nuclei of ductal cells. In 11 cases, we also detected a focal and mild reactivity for the peptide in glomeruli. In 13 kidneys, we also observed immunostaining for Tß10 inside the "comma-shaped bodies" and the "S-shaped bodies" during active glomerulogenesis. Our data show, for the first time, the expression of Tß10 in the human kidney during the initial phases of its physiological development, mainly restricted in the proximal and the distal tubuli. Further studies are needed in order to better characterize the role of Tß10 in kidney embryogenesis.

S100B protein expression in the heart of deceased individuals by overdose: a new forensic marker?

OBJECTIVE: The evaluation of S100B protein expression in the human heart and its correlation with drug-related death. METHOD: Left ventricular samples were collected from 74 serial forensic autopsies (15 overdose-related deaths; 59 non-overdose-related deaths) from 2007 to 2010. Tissue sections from each sample were immunostained for S100B protein by a commercial antibody. RESULTS: The S100B protein was detected in the heart samples of all 15 cases of drug-related deaths; S100B immunoreactivity was mainly observed in the cytoplasm of cardiomyocytes and as globular deposits in the interstitial spaces. No reactivity or weak reactivity was found in the cardiomyocytes of the 59 subjects who died of other causes. CONCLUSION: Our preliminary data show that the S100B protein accumulates in injured cardiomyocytes during drug-related sudden death. Given the near absence of S100B protein in the heart of subjects who died from causes other than drug overdose, S100B immunopositivity may be used as a new ancillary screening tool for the postmortem diagnosis of overdose-related cardiac death.

Hypoxia/reoxygenation-induced myocardial lesions in newborn piglets are related to interindividual variability and not to oxygen concentration

OBJECTIVE: Evaluation of myocardial histological changes in an experimental animal model of neonatal hypoxiareoxygenation. METHODS: Normocapnic hypoxia was induced in 40 male Landrace/Large White piglets. Reoxygenation was initiated when the animals developed bradycardia (HR <60 beats/min) or severe hypotension (MAP <15 mmHg). The animals were divided into four groups based on the oxygen (O2) concentration used for reoxygenation; groups 1, 2, 3, and 4 received 18%, 21%, 40%, and 100% O2, respectively. The animals were further classified into five groups based on the time required for reoxygenation: A: fast recovery (<15 min); B: medium recovery (15-45 min); C: slow recovery (45-90 min); D: very slow recovery (>90 min), and E: nine deceased piglets. RESULTS: Histology revealed changes in all heart specimens. Interstitial edema, a wavy arrangement, hypereosinophilia and coagulative necrosis of cardiomyocytes were observed frequently. No differences in the incidence of changes were observed among groups 1-4, whereas marked differences regarding the frequency and the degree of changes were found among groups A-E. Coagulative necrosis was correlated with increased recovery time: this condition was detected post-asphyxia in 14%, 57%, and 100% of piglets with fast, medium, and slow or very slow recovery rates, respectively. CONCLUSIONS: The significant myocardial histological changes observed suggest that this experimental model might be a reliable model for investigating human neonatal cardiac hypoxia-related injury. No correlation was observed between the severity of histological changes and the fiO2 used during reoxygenation. Severe myocardial changes correlated strictly with recovery time, suggesting an unreported individual susceptibility of myocardiocytes to hypoxia, possibly leading to death after the typical time-sequence of events.