Intensity Of Immunohistochemical Reaction Research Articles

Alterations in immunolocalisation of non-collagenous proteins in cartilage and bone tissue after gastrectomy, antrectomy and fundectomy in rat model

Bone, as a tissue, plays a pivotal role in maintaining whole body homeostasis, contributing to, among other things, structural support, blood cell production, and mineral storage. On the other hand, bone homeostasis depends on hormonal factors released by various tissues of the body, including the gastrointestinal tract. Thus, surgical procedures involving the removal of various parts of the stomach wall, such as, antrectomy and fundectomy or partial gastrectomy, may have profound negative effects on bone metabolism and mineralization. These effects may result from changes in the concentration of neuropeptides, such as nesfatin-1, secreted by cells located in the fundus and vestibule of the stomach. Additionally, bone morphogenetic proteins, osteocalcin, osteoprotegerin, and tissue inhibitor of metalloproteinases 2, are regulatory proteins involved in bone metabolism, playing a key role in maintaining the balance between the activity of osteoclasts and osteoblasts, cells responsible for the remodeling of bone tissue. The aim of this study was to investigate the effects of antrectomy, fundectomy, and partial gastrectomy, on bone homeostasis in a rat model. The experiment was carried out on 24 male Wistar rats randomly and equally divided into control (SHO), gastrectomy (GAST), antrectomy (ANT), and fundectomy (FUN) groups. At the beginning of the study, the rats underwent surgical removal of selected parts of the stomach. At the end of the 6-week study, the rats were sacrificed, and femurs were collected. Microscopic images of immunohistochemical reactions (IR) in trabecular and compact bone, as well as isolated articular cartilages and growth plates of femurs were analyzed. The intensity of the immunoreaction (IR) of osteocalcin (OC), osteoprotegerin (OPG), tissue inhibitor of metalloproteinases 2 (TIMP-2), bone morphogenetic protein 2 (BMP-2), and nesfatin-1 was determined, and IR-positive cells were counted in each area of the femur. The quantitative analysis of the intensity of OC IR in growth plate cartilage and trabecular bone showed the highest values after fundectomy in comparison to the other treatments. Moreover, fundectomy increased the intensity of BMP-2 IR in articular cartilage, growth plate cartilage, and trabecular bone. The intensity of OPG IR in articular cartilage and compact bone showed the highest values after antrectomy. The intensity of TIMP-2 IR in the examined parts of the femur was highest in the rats subjected to antrectomy. The intensity of expression of nesfatin-1 IR was highest in articular and growth plate cartilages for rats subjected to gastrectomy. In conclusion, surgical procedures affecting the gastrointestinal tract, such as antrectomy, fundectomy, and partial gastrectomy, can have significant effects on bone metabolism. Our study on a rat model demonstrated varying impacts on bone homeostasis, with different surgeries leading to alterations in the expression of key regulatory proteins involved in bone remodeling.

Wnt/β-catenin signaling in the adrenal glands of rats in various types of experimental hypertension.

Wnt/β-catenin signaling plays a key role in maintaining homeostasis, which is disturbed in hypertension. Taking into account the lack of literature describing changes in the Wnt/β-catenin pathway in the adrenal glands under conditions of elevated arterial pressure, here we compare the expression of WNT4, WNT10A, β-catenin, and GSK-3β in the adrenal glands of hypertensive rats of various etiologies. The studies were carried out on the adrenal glands of rats with spontaneous hypertension (SHR), renalvascular (2K1C), and deoxycorticosterone acetate (DOCA)-salt. Immunohistochemical and PCR methods were used to identify the molecular components of the canonical signaling pathway and to evaluate gene expression. Immunoreactivity and expression of WNT4, WNT10A, β-catenin, and GSK-3β in adrenals of SHR was decreased, compared to control rats. In adrenals of 2K1C rats, intensity of immunohistochemical reaction and expression of WNT4 and β-catenin was lower, while immunoreactivity and expression of WNT10A and GSK-3β were higher, compared to normotensive animals. Significantly stronger immunoreaction and expression of WNT4, β-catenin and GSK-3β but weaker immunoreactivity and expression of WNT10A were noted in adrenals in DOCA-salt rats, compared to control rats. In conclusion, our data provide new molecular information indicating that the canonical WNT pathway is disrupted in the adrenal glands of hypertensive rats. They show that the dysregulation of the WNT pathway depends on the etiology of hypertension.

The Role of Von Willebrand Factor in the Pathogenesis of Pulmonary Vascular Thrombosis in COVID-19.

The increased plasma levels of von Willebrand factor (VWF) in patients with COVID-19 was reported in many studies, and its correlation with disease severity and mortality suggest its important role in the pathogenesis of thrombosis in COVID-19. We performed histological and immunohistochemical studies of the lungs of 29 patients who died from COVID-19. We found a significant increase in the intensity of immunohistochemical reaction for VWF in the pulmonary vascular endothelium when the disease duration was more than 10 days. In the patients who had thrombotic complications, the VWF immunostaining in the pulmonary vascular endothelium was significantly more intense than in nonsurvivors without thrombotic complications. Duration of disease and thrombotic complications were found to be independent predictors of increased VWF immunostaining in the endothelium of pulmonary vessels. We also revealed that bacterial pneumonia was associated with increased VWF staining intensity in pulmonary arterial, arteriolar, and venular endothelium, while lung ventilation was an independent predictor of increased VWF immunostaining in arterial endothelium. The results of the study demonstrated an important role of endothelial VWF in the pathogenesis of thrombus formation in COVID-19.

Regulation of KRAS protein expression by miR-544a and KRAS-LCS6 polymorphism in wild-type KRAS sporadic colon adenocarcinoma.

Colorectal carcinoma (CRC) results from the accumulation of genetic mutations and alterations in signaling pathways. KRAS is mutated in 40% of CRC cases and is involved in increased tumor cells proliferation and survival. Although KRAS mutations are a dominant event in CRC tumorigenesis, increased wild-type KRAS expression has a similar effect on accelerated tumor growth. In this study, we investigated the KRAS status in correlation with clinicopathological features in sporadic CRC and more importantly the role of let-7a-5p and miR-544a-3p in the regulation of wild-type KRAS protein expression in the tumor center (T1) and invasive tumor front (T2). Analysis showed that 39.1% of tumor samples had KRAS mutations. In wild-type KRAS tumors, 62.0% were positive for KRAS protein expression and there was a higher percentage of KRAS-positive tumor cells and a higher intensity of immunohistochemical reaction in T2 than in T1 samples. This could not be attributed to differences in KRAS mRNA levels, suggesting regulation via miR-544a-3p expression which was significantly decreased in T2 samples. Furthermore, we demonstrated that tumor samples carrying the KRAS-LCS6 variant allele had significantly higher protein expression of the wild-type KRAS. Our results suggest the role of the KRAS-LCS6 polymorphism and miR-544a-3p expression in the regulation of wild-type KRAS protein expression in sporadic CRC.

Патогенетические механизмы нарушения эпителизации и формирования персистирующих язв трансплантата у пациентов с деструктивными процессами роговицы после сквозной кератопластики

Keratoplasty in patients with recurrent destructive processes in the cornea nowadays remains one of the most serious problems in ophthalmic surgery. Purpose. The analysis of results of penetrating keratoplasty (PK) in patients with destructive processes of the cornea or the corneal graft depending on the initial level of GDNF and the activity of the MAP kinase cascade of the signaling pathways. Material and methods. The analysis of the results of PK was performed in 23 patients (34 eyes) with acute destructive processes of cornea or the corneal graft in different etiologies. The follow-up period was from 6 months to 2 years. The analysis of the results of treatment was carried out depending on the condition of the corneal graft at various times after PK. The immunohistochemical examination of the biological material (corneal disks of the recipient) was performed using antibodies to GDNF, phospho-ERK1 / 2, phospho-JNK1 / 2, Ki67, GAP43, Bcl2 and Bax. Results. The time of epithelialization of the graft after PK in destructive processes of the cornea was increased in 79% of cases. When the epithelialization of the corneal graft was completed more than 6 and 10 days after PK in the postoperative period persistent erosions of the graft (PEG) with ulceration were formed in 22% and 89% of cases, respectively. The intensity of immunohistochemical reactions with antibodies to GDNF, phospho-ERK1 / 2, phospho-JNK1 / 2 in corneal disks at the epithelialization time of up to 5, 10 days and more eythan 10 days after PK was reduced from moderately positive to weakly positive and negative, respectively. When the time of epithelialization was completed in 6-10 days and there was not PECG, the intensity of reactions with antibodies to GDNF remains moderately positive, phospho-ERK1 / 2, phospho-JNK1 / 2 was lightly positive; when there was PEG the intensity of all reactions was weakly positive. Conclusion. The severity of the destructive pathological process in the cornea and postoperative complications after PK in patients with destructive corneal processes depend on the degree of GDNF deficiency, the decrease in the activity of the MAP kinase cascade of signaling pathways; the density of nerve plexuses in the own corneal tissue or corneal graft, and the activity of apoptosis processes.

Morphological Evaluation of Oxidative Phosphorylation System in Myocardial Infarction under Conditions of Modified Vascular Endothelial Growth Factor Concentration.

The relationship between vascular endothelium growth factor (VEGF) and cardiomyocyte oxidative phosphorylation level in experimental myocardial infarction, caused by diathermocoagulation of the periconical ventricular artery, was studied by immunofluorescent microscopy. Staining showed uneven distribution of cytochrome c in the mitochondria in the focus of myocardial infarction 2 h after the operation. After 24 h uneven staining of cardiomyocytes was found in the peri-infarction zone and no staining in the zone of myocardial infarction. This indicated a significant decrease in the level of redox enzymes. This picture persisted till day 14. Intraventricular injection of VEGF to animals led to a significant increase of the immunohistochemical reaction intensity, which reached the peak by day 7. The distribution of immunohistochemical reaction products under conditions of VEGF blocking was about the same as in spontaneous postinfarction reparative restitution. Our data indicated that increase of VEGF concentration in the myocardium maintained and stimulated oxidative phosphorylation in cardiomyocytes during the postinfarction period.

Topochemistry of internuclear interneurons in the human brain stem in arterial hypertension

To study the internuclear interneurons (IN) involved in the metabolism of nitric oxide, hydrogen sulfide and carbon monoxide in the caudal brainstem in healthy people and those with lifetime hypertension. The study was performed on postmortem material of 8 healthy men and 19 men, aged 18-44 years, with lifetime arterial hypertension. Immunohistochemistry methods for nNOS, HO-2, CBS and histochemical method for NADPH-diaphorase were used to study IN groups located between the giant and small cell reticular nuclei (IN1), small cell reticular nucleus and the nucleus of the solitary tract (IN2) surrounded by the lateral reticular nucleus (IN3). IN differ in size, shape, length and structure of sprouts as well as the nature of relationships formed and intensity of immunohistochemical reaction. The intensity of MN response to the high blood pressure depends on the cell size: qualitative and quantitative changes among the most small MN are mild. In contrast, in many major MN, structural changes, a significant reduction in the proportion of these cells, concentration and reaction rate are noted. More significant changes in quantitative indicators are found among nNOS-positive neurons. The reduction in the quantitative indicators depends on the localization of cells. In IN1, where there are many large IN, changes are more pronounced than in IN3 and IN2, which do not contain so many such cells. Central mechanisms of hemodynamic control include, at least, two groups of interneurons: intranuclear and internuclear. However, among the latter, only large IN are responsive to increases in blood pressure that confirms their participation in the regulation of hemodynamics, Small IN maintain a relatively small organization and perform a stable, obviously, integrating function.

Increased apoptosis in human knee osteoarthritis cartilage related to the expression of protein kinase B and protein kinase Cα in chondrocytes

Protein kinase B (Akt) and protein kinase Cα (PKCα) play important roles in the regulation of cell apoptosis. The aim of this study was to investigate the expression of Akt and PKCa in chondrocytes of human knee osteoarthritic (OA) cartilage, further evaluating their role in chondrocyte apoptosis during OA progression. Human knee OA cartilages were obtained from 38 patients undergoing knee arthroplasty, which is the medium-late stage of OA. Healthy knee cartilages were obtained from 11 amputees. The samples taken from the condyle of femur were collected routinely for morphological, immunohistochemical and Western blot detection, respectively. Light microscopy and laser-scanning confocal microscopy were used for morphological observation. The optical density with computer image analysis evaluated the intensity of immunohistochemical reaction of Akt and PKCα in OA cartilage. Western blot detected the protein expression levels. The results indicated that Akt and PKCa were involved in OA progression, along with the increase of cell apoptosis. In OA cartilage, Akt decreased (p < 0.05) and PKCα increased (p < 0.05). There was a negative correlation and interaction between Akt and PKCα (r = -0.8). These results demonstrated that both Akt and PKCα are related to increased chondrocyte apoptosis in human OA cartilage. The correlation between human OA progression, the role of Akt and PKCα, and chondrocyte apoptosis allows for new therapeutic strategies to be considered.

Increased apoptosis in human knee osteoarthritis cartilage related to the expression of protein kinase B and protein kinase Cα in chondrocytes.

Protein kinase B (Akt) and protein kinase Cα (PKCα) play important roles in the regulation of cell apoptosis. The aim of this study was to investigate the expression of Akt and PKCa in chondrocytes of human knee osteoarthritic (OA) cartilage, further evaluating their role in chondrocyte apoptosis during OA progression. Human knee OA cartilages were obtained from 38 patients undergoing knee arthroplasty, which is the medium-late stage of OA. Healthy knee cartilages were obtained from 11 amputees. The samples taken from the condyle of femur were collected routinely for morphological, immunohistochemical and Western blot detection, respectively. Light microscopy and laser-scanning confocal microscopy were used for morphological observation. The optical density with computer image analysis evaluated the intensity of immunohistochemical reaction of Akt and PKCα in OA cartilage. Western blot detected the protein expression levels. The results indicated that Akt and PKCa were involved in OA progression, along with the increase of cell apoptosis. In OA cartilage, Akt decreased (p < 0.05) and PKCα increased (p < 0.05). There was a negative correlation and interaction between Akt and PKCα (r = -0.8). These results demonstrated that both Akt and PKCα are related to increased chondrocyte apoptosis in human OA cartilage. The correlation between human OA progression, the role of Akt and PKCα, and chondrocyte apoptosis allows for new therapeutic strategies to be considered.

Immunohistochemical localization of somatostatin and pancreatic polypeptide in smokers with chronic pancreatitis

Recent studies have demonstrated a significant role of tobacco smoking in the development of chronic pancreatitis. Although there are published papers on the effects of cigarette smoking on insulin secretion in patients, no data are available on the effects of smoking on pancreatic endocrine cells secreting somatostatin and pancreatic polypeptide. The aim of the study was to evaluate the effects of cigarette smoking on endocrine pancreatic function by immunolocalization of somatostatin and pancreatic polypeptide in the pancreas from smokers and non-smoking patients with chronic pancreatitis in comparison with healthy controls. The LSAB2-HRP technique with polyclonal antibodies was used for the immunolocalization of somatostatin and pancreatic polypeptide in histological preparations of the pancreas. The intensity of immunohistochemical reaction was calculated with digital image analysis. The study demonstrated increased numbers of somatostatin (D) secreting cells and pancreatic polypeptide (PP) cells and their altered location in pancreatic islets and parenchyma of smoking patients with chronic pancreatitis, as compared to non-smoking patients and healthy controls. Smoking patients showed significantly higher immunostaining of the hormones in the pancreas compared to non-smoking patients and healthy persons. This study indicates that smoking may play a significant role in the development of endocrine disturbances in the development of chronic pancreatitis.

DHT deficiency perturbs the integrity of the rat seminiferous epithelium by disrupting tight and adherens junctions

In rats with a DHT deficiency induced by finasteride, morphological changes in the seminiferous epithelium were observed. The structural alterations were manifested by the premature germ cells sloughing into the lumen of seminiferous tubules. The etiology of this disorder could be connected with intercellular junctions disintegration. We showed in the immunohistochemical study the changes in expression of some proteins building tight and adherens junctions. The depression of N-cadherin, β-catenin and occludin immunoexpressions could be the reason for the release of immature germ cells from the seminiferous epithelium. However, the observed increase of the immunohistochemical reaction intensity of vinculin, one of the cadherin/catenin complex regulators, could be insufficient to maintain the proper function of adherens junctions. The hormonal imbalance appears to influence the pattern of expression of junctional proteins in the seminiferous epithelium. It could lead to untimely germ cells sloughing, and ultimately could impair fertility.

Morphological and immunohistochemical comparison of three rat prostate lobes (lateral, dorsal and ventral) in experimental hyperprolactinemia.

The prolactin plays an important role in the regulation of growth and differentiation of prostate gland besides androgens. The goal of this study was to reveal the influence of elevated prolactin concentration on epithelial cells of prostate. We compared the morphology of epithelial cells of prostate dorsal, lateral and ventral lobes and expression of androgen receptors in these cells in rats with hyperprolactinemia and in control rats. We used sexually mature male Wistar rats. The experimental rats received metoclopramide; the control group received saline in the same way. The prostate dorsal, lateral and ventral lobes were collected routinely for light and electron microscopy. The intensity of immunohistochemical reaction of androgen receptor in epithelial cells of dorsal, lateral and ventral lobes was evaluated by measure of optical density with computer image analysis. The light and electron (transmission and scanning) microscopes were used for morphological observations. In experimental rats twofold increase in prolactin and twofold decrease in testosterone found. In experimental group the expression of androgen receptor was lower in columnar epithelial cells of dorsal and ventral lobes but higher in lateral one. We observed morphological abnormalities in columnar epithelial cells of lateral and dorsal lobes. The columnar epithelial cells of ventral lobes didn't show any morphological changes in hyperprolactinemia.

Renal involvement in canine leishmaniasis: a morphological and immunohistochemical study

IgG and IgM deposits in kidneys of dogs with visceral leishmaniasis (VL) were studied in 25 symptomatic dogs (case) and 15 asymptomatic dogs (control) by an immunohistochemical method. All tested dogs were positive for VL by polymerase chain reaction, enzyme-linked immunosorbent assay, and indirect immunofluorescence test. Kidney fragments were submitted to immunohistochemical reaction. Many morphological patterns of distribution of subendothelial granules were identified for IgG and IgM in glomerular capillaries: global, segmental, diffuse, or focal. Intensity of immunohistochemical reaction to IgG was not significantly different when comparing the symptomatic and the asymptomatic animal groups by Fisher's exact test. IgM reactions were significantly different between groups (P<0.01). Deposits of IgM on mesangial cells and in inflammatory interstitial infiltrate were rarely seen, although IgG reactions were frequent at these sites. This study concluded that immunohistochemical reactions for IgM were more intense than those observed for IgG in canine VL, and these reactions were characterized by distribution of subendothelial granules in glomerular capillaries.

Expression of a novel oncofetal mRNA-binding protein IMP3 in endometrial carcinomas: diagnostic significance and clinicopathologic correlations

Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) is a newly identified oncofetal mRNA-binding protein that is involved in embryogenesis and carcinogenesis of some malignant neoplasms. To investigate the diagnostic and clinicopathologic significance of this protein in endometrial carcinomas, we evaluated immunohistochemical expression of IMP3 in the two most common forms of endometrial malignancies, endometrioid adenocarcinoma and serous carcinoma. We selected 167 endometrial adenocarcinoma cases including 122 cases of endometrioid adenocarcinoma and 45 cases of serous carcinoma. Twenty samples of benign endometrium obtained from 20 patients with nonmalignant uterine lesions were used as controls. Positive immunohistochemical stain for IMP3 was identified in all serous carcinoma cases, among which, 39 (86%) and 3 (7%) cases showed IMP3 immunoreactivity in >50%, and 21–50, or 6–20% of tumor cells, respectively. Immunohistochemical reaction intensity for IMP3 was identified to be strong in 38 (84%) and intermediate in 7 (16%) cases of serous carcinoma. Fifty-four (44%) cases of endometrioid adenocarcinoma were negative for IMP3. Thirty (25%), 20 (16%), 10 (8%), and 8 (7%) cases of endometrioid adenocarcinoma demonstrated positive immunoreactivity for IMP3 in 1–5, 6–20, 21–50, and >50% of the tumor cells. Strong IMP3-staining intensity was noted in 34 (28%), intermediate in 26 (21%), and weak in 8 (7%) cases of endometrioid adenocarcinoma. All 20 control cases were negative for IMP3. To compare p53 with IMP3 expressions, we found that 35 (78%) of the serous carcinoma cases showed strong p53 immunohistochemical activity in >50% of the tumor cell nuclei. In contrast, 11 of 112 (10%) endometrioid adenocarcinoma cases demonstrated strong p53 positivity in >50% of the tumor cell nuclei. In conclusion, our findings demonstrate significant expression of IMP3 in serous carcinoma as compared to endometrioid adenocarcinoma (P<0.0001). Expression of IMP3 and p53 may be helpful biomarkers in the distinction of endometrial serous carcinoma from endometrioid adenocarcinoma. In addition, expression of IMP3 in endometrioid adenocarcinoma correlates with higher nuclear and architecture grades of the tumor (P=0.0000 and P=0.0002, respectively).

IMP3 is a novel biomarker for adenocarcinoma in situ of the uterine cervix: an immunohistochemical study in comparison with p16INK4a expression

AbstractAdenocarcinoma in situ of the uterine cervix remains a diagnostic challenge in a small proportion of cases. This suggests a need for biomarker that may be of help in establishing the diagnosis. The aim of this study was to evaluate the potential of insulin-like growth factor-II mRNA-binding protein 3 and cyclin-dependent kinase inhibitor p16INK4a as biomarkers for adenocarcinoma in situ. Forty-four samples of adenocarcinoma in situ from 40 patients and 23 control cases of benign uterine cervix were included in this study. In addition to benign endocervical epithelium, 19 of these 23 control cases also showed focal tubal metaplasia. Cytoplasmic immunoreactivity for insulin-like growth factor-II mRNA-binding protein 3 was identified in 41 (93%) adenocarcinoma in situ samples, among which, 29 (71%), 10 (24%), and 2 (5%) samples showed insulin-like growth factor-II mRNA-binding protein 3 positive staining in 50% or more, >5 to <50 and <5% of adenocarcinoma in situ lesional cells, respectively. Immunohistochemical reaction intensity for insulin-like growth factor-II mRNA-binding protein 3 was found to be strong in 34 adenocarcinoma in situ samples, intermediate in five, and weak in two. All 23 control cases were negative for insulin-like growth factor-II mRNA-binding protein 3. p16INK4a expression was identified in all of the adenocarcinoma in situ samples with intermediate staining intensity seen in seven samples and strong in the remainder. Fourteen of 19 (74%) tubal metaplasia cases showed p16INK4a immunoreactivity in >50% of the tubal metaplastic epithelium with staining intensity ranging from weak to strong. Our findings demonstrate significant expression of insulin-like growth factor-II mRNA-binding protein 3 and p16INK4a in adenocarcinoma in situ as compared to benign endocervical glands, suggesting that expression of these biomarkers may be helpful in the distinction of adenocarcinoma in situ from benign endocervical glands, particularly in difficult borderline cases.

Dysfunction of the Pancreas in Healthy Smoking Persons and Patients With Chronic Pancreatitis

The aim of the study was to assess the effect of cigarette smoking on the endocrine pancreatic function by determining the levels of serum glucose and plasma insulin as well as by defining immunohistochemical localization of insulin and glucagon in tissue specimens of the pancreata derived from healthy persons and smoking and nonsmoking patients with diagnosed chronic pancreatitis (CP). The oxidative method was used to measure fasting glycemia in blood plasma and the method enzyme-linked immunoassay to determine the level of insulin in plasma. Immunohistochemical localization of hormones in paraffin tissue specimens of the pancreas was performed using the LSAB2-HRP visual test with polyclonal insulin and glucagon antibodies. The intensity of immunohistochemical reaction was calculated with digital imaging methodology. The study revealed a substantially higher level of serum glucose in smoking CP patients and in healthy persons compared with nonsmoking patients and healthy persons, whereas insulin concentration in smoking patients was statistically lower than in nonsmokers. Smoking patients showed significantly lower expression of insulin and glucagon in the pancreas compared with nonsmoking patients and healthy persons. Impairment of the endocrine function of beta and alpha cells in the pancreatic islets is frequently manifested by complications in pancreatitis resulting among others from long-term smoking.

Expression of Estrogen Receptor-α, Estrogen Receptor-β and Placental Endothelial and Inducible NO Synthase in Intrauterine Growth-Restricted and Normal Placentas

Nitric oxide seems to play important roles in the physiology of placental blood circulation, although their expression in pathological placentas and their role is still unclear. Therefore, the aim of this study was to investigate the expression of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta) and the endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) in intrauterine growth-restricted (IUGR) placentas, hemolysis, elevated liver enzymes, low platelets (HELLP) placentas and in normal healthy control placentas. Slides of paraffin-embedded placental tissue were obtained after delivery from patients diagnosed with IUGR, HELLP and normal term placentas and analyzed for eNOS and iNOS, as well as ERalpha/beta expression. Intensity of immunohistochemical reaction was analyzed using a semi-quantitative score, and statistical analysis was performed. In addition, Western blot experiments were performed for comparison of staining intensities obtained by immunohistochemistry and Western blot. Expression of eNOS and iNOS is significantly reduced in trophoblast cells of placentas with HELLP. However, ERbeta expression in HELLP placentas demonstrated a significantly elevated expression intensity compared to normal controls. ERalpha expression was not significantly different in all three pathologies investigated. We speculate that the increased ERbeta expression in both syncytiotrophoblast and extravillous trophoblast cells represents accelerated proliferation of placental tissue or can be seen as a compensatory effect in HELLP placentas.

Expression of the Proliferation Marker Ki-67 and of p53 Tumor Protein in Trophoblastic Tissue of Preeclamptic, HELLP, and Intrauterine Growth-Restricted Pregnancies

The human placenta owns the biochemical machinery to proliferate throughout gestation. The aim of this study was to investigate the expression of the proliferation marker Ki-67 in trophoblastic tissue of intrauterine growth retarded (IUGR) placentas, preeclamptic, HELLP, and in normal trophoblastic tissue. Slides of paraffin-embedded trophoblastic tissue of patients with IUGR, preeclamptic patients, HELLP patients, and normal term placentas were incubated with monoclonal antibodies against Ki-67 and p53. Staining reaction was performed with the ABC reagent. Intensity of immunohistochemical reaction on the slides was analyzed using a semiquantitative score. Identification of Ki-67-expressing cells was done by immunofluorescence double staining with Ki-67 and cytokeratin antibodies. Expression of Ki-67 and p53 are significantly elevated in cytotrophoblastic cells of placentas with HELLP as investigated by immunohistochemistry and double immunofluorescence. However, preeclamptic cytotrophoblastic tissue on the other hand showed no significantly different expression intensity of Ki-67 compared with normal placental tissue controls and no changes in p53 expression compared with controls. In IUGR cytotrophoblastic cells, we found no statistically significant change in Ki-67 expression but a statistically significant down-regulation of p53. An elevated proliferation of cytotrophoblastic cells seems to be related to HELLP, and this enhanced proliferation seems to be controlled by p53.

High Thyroglobulin (Tg) Concentrations in Fatal Traumatic Brain Injuries

It is well known that under physiological conditions, the Tg molecule is the substrate for the hormones triiodothyronine and thyroxine. Its function outside the thyroid gland is unknown. Under certain pathologic conditions, an increase in Tg concentrations in the blood can occur, a phenomenon that is used in the clinical diagnosis of certain thyroid disorders. However, fatal traumatic brain injuries (TBI) can also produce a raised Tg concentration in blood. In such cases, mean (n=151) Tg concentrations of 405.8+/-353.3 ng/mL have been observed, with the values in 57% of these cases exceeding 200 ng/mL. Since it was possible to rule out specifically any thyroid disorders in these cases, the raised values may be explained by the effect of TSH in the spectrum of the "hypothalamic-pituitary-thyroid axis." This assumption has been substantiated by immunohistochemical tests for TSH and Tg. Compared with the controls (sudden unnatural deaths: Tg 23.3+/-27.6 ng/mL), the raised Tg values in the TBI cases were usually accompanied by a reduction in the intensity of immunohistochemical reactions relating to TSH and Tg. The hypophysis showed evidence of significant damage with morphologic changes such as edema, hemorrhages, and focal necrosis in association with reduced TSH levels.

Expression of Inhibin/activin Subunits alpha (-α), beta A (-β A) and beta B (-β B) in Placental Tissue of Normal and Intrauterine Growth Restricted (IUGR) Pregnancies

During human pregnancy the placenta produces a variety of proteins like steroid hormones and their receptors that are responsible for the establishment and ongoing of the feto-placental unit. Inhibins are dimeric glycoproteins, composed of an alpha-subunit and one of two possible beta-subunits (beta (A) or beta (B)). Aims of the present study were the determination of the frequency and tissue distribution patterns of the inhibin/activin subunits in human placental tissue of normal pregnancies and pregnancies complicated with fetal growth restriction (IUGR). Slides of paraffin embedded placental tissue were obtained after delivery from patients diagnosed with IUGR (n = 6) and normal term placentas (n = 8). Tissue samples were fixed and incubated with monoclonal antibodies inhibin/activin-subunits -alpha, -beta (A), -beta (B). Intensity of immunohistochemical reaction on the slides was analysed using a semi-quantitative score and statistical analysis was performed (P<0.05). A significant lower expression of the inhibin-alpha subunit in IUGR extravillous trophoblast compared to normal pregnancies was observed, while the inhibin-alpha immunostaining was significantly upregulated in syncytiotrophoblast. Additionally, a significant down-regulation of inhibin-beta (B) subunit in extravillous trophoblast cells in IUGR syncytiotrophoblast cells was demonstrated. A co-localisation of inhibin-alpha and the beta-subunits was also observed, suggesting a production and secretion of intact inhibin A and inhibin B. Although the precise role of these inhibin/activin subunits in human placenta and IUGR pregnancies is still unclear, they could be involved in autocrine/paracrine signalling, contributing to several aspects like angiogenesis and tissue remodelling.