Predictors of High Grade and Other Clinically Significant Placental Findings by Indication for Submission in Singleton Placentas From Term Births.

Indications for placental submission are variable. Established guidelines are largely based on expert opinion, and there is a need for more evidence-based criteria. A 10-year database of term placentas was used to evaluate indications significantly associated with placental pathology. Lesions in 5 categories were separated into high- and low-grade subgroups. Two additional high-grade lesions were also evaluated. Indications associated with high-grade placental lesions were chronic monitoring abnormalities, severe preeclampsia, pregestational diabetes, maternal signs of infection, postdates pregnancy, artificial reproductive technology, drug abuse, umbilical cord entanglements, selected gross placental abnormalities, stillbirth, Apgar 5 minutes <6, small-for-gestational age infant, and macrosomia. Indications for which placental findings did not differ from the population as a whole were acute monitoring abnormalities, chronic hypertension, maternal obesity, vaginal bleeding, accessory lobe/multilobed placenta, meconium-stained fluid, single umbilical artery, and borderline large-for-gestational age infant. Other indications for submission were intermediate showing significant or borderline elevations in the prevalence of low- and high-grade lesions combined. We suggest on the basis of this study that guidelines for the submission of singleton term placentas could be modified to exclude cases with clinical indications that lack a significant association with placental lesions.

Comprehensive evaluation of cytomorphologic, histologic, and molecular features of DICER1-altered thyroid lesions on FNA: A multipractice experience.

BACKGROUND: DICER1 mutations, though infrequent, are encountered on preoperative molecular testing of indeterminate adult and pediatric thyroid fine-needle aspiration (FNA) specimens. Yet, published cytomorphologic features of DICER1-altered thyroid lesions are limited. Cytomorphological features of DICER1-altered thyroid lesions were examined in a multipractice FNA cohort with clinical, radiological, and histologic data. METHODS: The cohort comprised 18 DICER1-altered thyroid FNAs, with 14 having slides available and eight having corresponding surgical resections. Smears, ThinPrep, and formalin-fixed cell block slides were reviewed and correlated with histology, when available. Clinical and radiologic data were obtained from the medical record. RESULTS: Most DICER1-altered FNAs were classified as atypia of undetermined significance (94.4%). DICER1 mutations occurred in codons 1709 (50%), 1810 (27.8%), and 1813 (22.2%). One patient had an additional DICER1 p.D1822N variant in both of their FNAs. Lesions were often hypoechoic (35.3%) and solid (47.1%) on ultrasound. Notable cytomorphologic features include mixed but prominent microfollicular or crowded component, variable colloid, and insignificant nuclear atypia. On resection (n = 10), histologic diagnoses ranged from benign follicular adenoma and low-risk follicular thyroid carcinoma to high-grade follicular-derived nonanaplastic thyroid carcinoma. Subcapsular infarct-type change was the most common histologic change. There was no evidence of recurrence or metastasis in eight patients on limited follow-up. CONCLUSION: DICER1-altered thyroid lesions occurred frequently in young females and FNAs show RAS-like cytomorphology including crowded, mixed macro-/microfollicular pattern, and bland nuclear features. On resection, DICER1-altered thyroid lesions include benign (50%), low-risk lesions (30%), or high-risk malignancies (20%).

Focal nodular hyperplasia in children, adolescents, and young adults.

BACKGROUND: Focal nodular hyperplasia (FNH) is a benign hepatic tumor that is rare in children. In order to understand whether there are differences in the etiology or appearance of FNH in children, we analyzed the clinical information and imaging of pathologically proven cases. MATERIALS AND METHODS: A pathology database was used to identify all cases of FNH diagnosed at our institution. Each patient's imaging was evaluated for the characteristics of FNH lesions. Clinical information was obtained on each patient. RESULTS: Thirteen patients with FNH were identified (7 male/6 female, mean age 14.3 years, range 1-27 years). Seven patients (5 male/2 female) had a remote history of childhood malignancy. The time interval between the diagnoses of malignancy and FNH ranged from 9 to 27 years (mean 14.4 years). On imaging, all seven cancer survivors had multiple liver lesions. In the remaining six patients (2 male/4 female), there was no history of malignancy and all but one of these patients had a solitary FNH. CONCLUSION: Half of the patients with FNH in this study were long-term cancer survivors and each of these patients had multiple masses. Recognizing the features of FNH will aid in diagnosis and appropriate management.

Interleukin-10 activates the transcription factor C/EBP and the interleukin-6 gene promoter in human intestinal epithelial cells.

BACKGROUND: Interleukin (IL)-6 is produced by enterocytes in response to sepsis and after treatment with IL-1beta. The IL-6 promoter contains binding sites for multiple transcription factors, including nuclear factor-kappaB and C/EBP. The anti-inflammatory cytokine IL-10 downregulates nuclear factor-kappaB activity, but its effects on C/EBP activation and IL-6 production in the enterocyte are not known. METHODS: Caco-2 cells were treated with IL-1beta, IL-10, or a combination of the cytokines. C/EBP DNA binding activity was determined by electrophoretic mobility shift assay and IL-6 levels by enzyme-linked immunosorbent assay. IL-6 promoter activation was assessed by luciferase assay. RESULTS: IL-10 treatment of cultured Caco-2 cells resulted in increased C/EBP DNA binding activity. Supershift analysis revealed upregulated DNA binding activity of C/EBP-beta but not C/EBP-delta. To examine if the increased DNA binding reflected gene activation, cells were transfected with a wild-type IL-6 promoter luciferase construct or with a mutated C/EBP binding site. IL-10 potentiated IL-1 beta-induced IL-6 promoter activity. Replacing the wild-type promoter with the promoter containing a mutated C/EBP DNA binding sequence blocked the effect of IL-10. When cells were treated with 0.5 ng/mL of IL-1 beta for 24 hours, IL-6 production increased, and this response to IL-1 beta was potentiated several-fold by IL-10. CONCLUSIONS: IL-10 may activate the IL-6 gene in stimulated enterocytes by upregulating the expression and activity of C/EBP.

Hyperthermia-induced heat shock activates the transcription factor c/EBP-beta and augments IL-6 production in human intestinal epithelial cells.

BACKGROUND: Interleukin (IL)-6 production is increased in gut mucosa during sepsis and endotoxemia. The heat shock response augments IL-6 production under these conditions, but the mechanism is not known. We hypothesized that heat shock stimulates IL-6 production in enterocytes by increasing expression and activity of the transcription factor C/EBB. STUDY DESIGN: Cultured Caco-2 cells, a human intestinal epithelial cell line, underwent induction of the heat shock response by hyperthermia (43 degrees C for 1 hour). Other cells were kept at 37 degrees C. Cells were then treated with 0.5 ng/mL human recombinant IL-1beta for 4 hours. C/EBP-beta and delta DNA binding activity was determined by electrophoretic mobility shift assay and supershift analysis. In additional experiments, Caco-2 cells were transfected with expression plasmids for C/EBP-beta and delta, after which cells were subjected to hyperthermia and treatment with IL-1beta. RESULTS: C/EBP-beta, but not delta, protein levels and DNA binding activity were increased in Caco-2 cells expressing the heat shock response. Induction of the heat shock response augmented IL-6 production in IL-1beta-treated cells overexpressing C/EBP-beta, but not delta. CONCLUSIONS: Increased IL-6 production in IL-1beta-treated enterocytes expressing the heat shock response might be caused by upregulated expression and activity of CIEBP-beta. Because recent studies suggest that IL-6 might be an antiinflammatory cytokine and might exert protective effects in gut mucosa and enterocytes, understanding mechanisms by which the heat shock response augments IL-6 production might have important clinical implications.

Peripheral insertion of umbilical cord.

Numerous studies have addressed the significance of marginal and membranous umbilical cord (UC) insertion. Recent reports suggest that an eccentrically inserted UC may also be important. This case-control study assessed the potential relevance of peripheral insertion of UC (PIUC), defined as <3 cm from the nearest margin. Singleton placentas (n  =  1418) submitted to the pathology department over an 18-month period were analyzed. Each case of PIUC (n  =  119) was matched with a control placenta of the same gestational age. Placentas with marginal or membranous UC and multiple gestations were excluded. The overall prevalence of PIUC was 8.4%, but PIUC frequency was significantly increased in premature births at <28 weeks (21.4%, P < 0.001). There was no association with other adverse pregnancy outcomes. PIUC was associated with decreased placental weight Z-score (-0.69 ± 0.92 versus -0.22 ± 1.3, P  =  0.0056), but not fetal weight Z-score, suggesting increased utilization of placental reserve. PIUC was also associated with relatively elongated placentas (length minus width: 2.6 ± 3.2 versus 1.0 ± 3.1, P  =  0.006). PIUC tended to be more frequent in young primiparous mothers and was significantly less common in women with a history of prior curettage (66% vs 50%, P  =  0.013). These data, together with equivalent rates of prior cesarean section, multiparity, and advanced maternal age, support a primary developmental disorder as opposed to secondary placental migration due to underlying uterine abnormalities ("trophotropism"). Except for a borderline significant association with findings suggestive of maternal malperfusion (P  =  0.078), PIUC was not associated with other placental lesions.

Renal arteriolar C4d deposition: a novel characteristic of hematopoietic stem cell transplantation-associated thrombotic microangiopathy.

BACKGROUND: The mechanism of kidney injury in hematopoietic stem cell transplantation (HSCT)-associated thrombotic microangiopathy (TA-TMA) is not completely understood. Renal C4d staining is a marker of classic complement activation and endothelial injury and has been described in preliminary reports of HSCT recipients with TA-TMA. Our objective was to evaluate complement in the pathogenesis of small vessel injury in children receiving HSCT. We hypothesized that kidney tissue from children with TA-TMA would more frequently show C4d deposition compared with HSCT recipients without histologic TA-TMA. METHODS: We reviewed kidney specimens (biopsy or autopsy) from children who had undergone HSCT at a single center. Using histologic criteria alone, subjects were divided into TA-TMA (n = 8) and non-TA-TMA (control) groups (n = 12). C4d staining was performed by immunohistochemistry and evaluated on arterioles, peritubular capillaries, glomeruli, and tubular basement membranes. RESULTS: Diffuse or focal renal arteriolar C4d staining was more common in subjects with histologic TA-TMA (75%) compared with controls (8%). Rare peritubular capillary C4d staining was present in 50% of TA-TMA samples and was absent in controls. Glomerular C4d staining was seen at a similar frequency in cases and controls, whereas tubular basement membrane staining was less frequently observed and only in subjects with TA-TMA. CONCLUSIONS: Arteriolar C4d deposition may be a pathologic marker of TA-TMA, implicating localized complement fixation in HSCT recipients with kidney disease secondary to small vessel injury. Further studies to better characterize the preferential arteriolar C4d staining may identify a renal compartment of injury, possibly explaining the dramatic hypertension seen in TA-TMA.

Multiple transcription factors regulating the IL-6 gene are activated by cAMP in cultured Caco-2 cells.

Mucosal and enterocyte IL-6 production is increased during sepsis and endotoxemia. Recent studies suggest that cAMP potentiates IL-6 production in endotoxin- or IL-1beta-stimulated enterocytes, but the molecular mechanisms are not known. We examined the role of the transcription factors NF-kappaB, activator protein (AP)-1, CCAAT/enhancer binding protein (C/EBP), and cAMP response element-binding protein (CREB) in cAMP-induced IL-6 production in cultured Caco-2 cells, a human intestinal epithelial cell line. In addition, the role of the protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein (MAP) kinase signaling pathways was examined. Treatment of the cells with IL-1beta increased IL-6 production and activated the IL-6 promoter in cells transfected with a luciferase reporter plasmid containing a wild-type IL-6 promoter. These effects of IL-1beta were significantly potentiated by cAMP. When the binding sites for the individual transcription factors in the IL-6 promoter were mutated, results indicated that all four transcription factors may be involved in the cAMP-induced activation of the IL-6 gene. Treatment of the Caco-2 cells with cAMP increased the DNA binding activity of CREB, C/EBP, and AP-1, but not NF-kappaB. By using specific blockers, evidence was found that both PKA and p38 MAP kinase (but not PKC or p42/44 MAP kinase) may be involved in the cAMP-induced potentiation of IL-6 production. The present results suggest that cAMP activates multiple transcription factors involved in the regulation of the IL-6 gene and that the activation of these transcription factors may at least in part explain why cAMP potentiates IL-6 production in stimulated enterocytes.

Coordinate upregulation of guanylin and uroguanylin expression by hypertonicity in HT29-18-N2 cells.

Guanylin and uroguanylin are particulate guanylate cyclase-activating peptides that are secreted from the epithelia of the intestine, kidney, pancreas, and salivary gland. These peptides elicit chloride and bicarbonate secretion via the cystic fibrosis transmembrane conductance regulator. To test the hypothesis that hypertonicity mediates an increase in guanylin and uroguanylin mRNA, we subjected HT29-18-N2 to osmotic stress. Guanylin and uroguanylin RNA were increased substantially in the presence of hypertonicity but only with solutes that were relatively impermeable to the cell membrane. This hypertonicity-mediated increase was transcriptional and did not require protein synthesis. Herbimycin A and mitogen-activated protein kinase inhibitors SB-203580 and PD-98059 had no effect on basal or induced levels of guanylin or uroguanylin. Both staurosporine and prolonged exposure to phorbol ester reduced basal levels and completely blocked hypertonicity-related increases in guanylin or uroguanylin RNA. These data suggest that serine/theonine protein kinases, possibly protein kinase C (PKC), mediate the hypertonicity-associated increase in guanylin and uroguanylin RNA. We conclude that guanylin and uroguanylin are released in response to hypertonic stress and that regulation of these genes may be mediated by PKC isoforms.

Arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1 beta-stimulated Caco-2 cells independent of the heat shock response.

Recent studies suggest that sodium arsenite downregulates NF-kappaB activity by inhibiting phosphorylation and subsequent degradation of IkappaBalpha. Many effects of sodium arsenite are secondary to induction of heat shock proteins. The role of the heat shock response in arsenite-induced inhibition of NF-kappaB, however, is not known. We examined the involvement of the heat shock response in arsenite-induced inhibition of NF-kappaB activity in IL-1beta-stimulated Caco-2 cells, a human colorectal adenocarcinoma cell line with enterocytic properties. Treatment of the cells with IL-1beta resulted in increased IkappaB kinase activity, reduced levels of IkappaBalpha and increased NF-kappaB DNA binding activity. Sodium arsenite blocked all of these responses to IL-1beta without inducing changes in heat shock factor activity or heat shock protein levels. Results from additional experiments showed that the protective effect of sodium arsenite on IkappaBalpha was not influenced by the oxygen radical scavenger catalase or by inhibitors of the MAP-kinase signaling pathway. The present results suggest that sodium arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1beta-stimulated Caco-2 cells independent of the heat shock response. In addition, stabilization of IkappaBalpha by sodium arsenite does not require oxygen radical formation or activation of the MAP kinase signaling pathway.

Interleukin-6 induces thermotolerance in cultured Caco-2 cells independent of the heat shock response.

In recent studies, induction of the heat shock response increased IL-6 production in gut mucosa in vivo and in cultured Caco-2 cells in vitro. The heat shock response is associated with increased survival of cells exposed to otherwise lethal hyperthermia, so called thermotolerance, but the role of IL-6 in the induction of thermotolerance is not known. We tested the hypothesis that treatment of cultured Caco-2 cells with IL-6 results in the development of thermotolerance. Cells were treated with human recombinant IL-6 for 1h followed by 3 h recovery in cytokine-free medium whereafter cells were exposed to heat stress (48 degrees C for 2 h). In untreated cells, the heat stress resulted in an approximately 80% cell death. In cells treated with IL-6, cell viability after heat stress was significantly improved and was doubled at an IL-6 concentration of 20 ng/ml. Treatment of the cells with other cytokines (IL-4, IL-10, IL-1beta, or TNFalpha) did not induce thermotolerance, suggesting that the effect of IL-6 may be specific for this cytokine. The induction of thermotolerance by IL-6 was blocked by an IL-6 receptor antibody, suggesting that the development of thermotolerance was receptor-mediated. Treatment of cells with IL-6 did not induce an heat shock response as suggested by unaltered heat shock protein 70 and 90 levels and unaffected heat shock factor DNA binding activity. In addition, the IL-6-induced thermotolerance was not inhibited by quercetin. The present study provides the first evidence of IL-6-induced thermotolerance and suggests that this effect of IL-6 is independent of the heat shock response.

Arsenite inhibits interleukin-6 production in human intestinal epithelial cells by down-regulating nuclear factor-kappaB activity.

Previous studies have suggested that the production of interleukin-6 (IL-6) is increased in the intestinal mucosa during inflammation, and that nuclear factor-kappaB (NF-kappaB) is an important regulator of the IL-6 gene in the enterocyte. We tested the hypothesis that sodium arsenite inhibits IL-6 production in stimulated enterocytes and that this effect of arsenite is caused by down-regulation of NF-kappaB activity. Cultured Caco-2 cells were treated with sodium arsenite and were then stimulated with IL-1beta. IL-6 production and gene expression were determined by ELISA and reverse transcriptase-PCR respectively. NF-kappaB DNA binding activity was determined by electrophoretic mobility shift assay. IL-1beta increased NF-kappaB DNA binding activity, IL-6 mRNA levels and IL-6 production. These effects of IL-1beta were inhibited by treatment of the cells with sodium arsenite in a dose- and time-dependent fashion. When cells were transfected with a plasmid expressing the p65 subunit of NF-kappaB, the inhibitory effect of sodium arsenite on NF-kappaB activity and IL-6 production was blunted. These results suggest that sodium arsenite inhibits IL-6 production in enterocytes subjected to an inflammatory stimulus, and that this effect, at least in part, reflects down-regulated NF-kappaB activity.

Proteasome inhibitors activate the transcription factors C/EBP-beta and delta in human intestinal epithelial cells.

In recent studies, induction of the heat shock response by hyperthermia upregulated the expression and DNA binding activity of the transcription factor C/EBP. This is an important observation because it may at least in part explain why the heat shock response upregulates IL-6 production in the intestinal mucosa and in the enterocyte. A novel method to induce the heat shock response is proteasome inhibition. The influence of this treatment on the expression and DNA binding activity of C/EBP is not known. We treated cultured Caco-2 cells, a human intestinal epithelial cell line, with one of the proteasome inhibitors, MG-132 or lactacystin, and measured C/EBP-beta and delta DNA binding activity by electrophoretic mobility shift assay and supershift analysis. In addition, nuclear levels of C/EBP-beta and delta protein were determined by Western blot analysis. Treatment of the cells with the proteasome inhibitors resulted in increased cellular levels of heat shock protein 72, consistent with induction of the heat shock response. Treatment also resulted in increased DNA binding activity and nuclear protein levels of C/EBP-beta and delta. The effects of the proteasome inhibitors on C/EBP were inhibited by treating the cells with quercetin, a substance known to block the heat shock response. The results suggest that proteasome inhibition activates the transcription factors C/EBP-beta and delta in human intestinal epithelial cells and that this response, at least in part, is caused by induction of the heat shock response. The observations are important because they provide support for a novel method to influence gene activation in the enterocyte.

Transcription factors C/EBP-beta and -delta regulate IL-6 production in IL-1beta-stimulated human enterocytes.

In recent studies, treatment with IL-1beta of cultured Caco-2 cells, a human intestinal epithelial cell line, resulted in transcriptional upregulation of IL-6 production. The role of C/EBP-beta and -delta in enterocyte IL-6 production is not known. Stimulation with IL-1beta of Caco-2 cells transiently transfected with a luciferase reporter plasmid containing a wild-type IL-6 promoter resulted in an approximately 3.5-fold increase in luciferase activity. This effect of IL-1beta was reduced by approximately 30% when the C/EBP binding site in the IL-6 promoter was mutated, supporting a role of C/EBP in the regulation of IL-6 production. When Caco-2 cells were treated with IL-1beta in the presence of the MAPK inhibitor, PD-98059, IL-6 mRNA and protein levels were reduced by the same concentrations of PD-98059 that inhibited C/EBP DNA binding activity in previous studies. Finally, overexpression of C/EBP-beta and -delta in IL-1beta-treated Caco-2 cells resulted in a 10-12-fold increase in IL-6 production. The results suggest that the beta and delta isoforms of the C/EBP family of transcription factors at least in part regulate IL-6 production in human intestinal epithelial cells.

Proteasome inhibitors induce heat shock response and increase IL-6 expression in human intestinal epithelial cells.

In previous studies, the heat shock response, induced by hyperthermia or sodium arsenite, increased interleukin (IL)-6 production in intestinal mucosa and cultured human enterocytes. A novel way to induce the heat shock response, documented in other cell types, is treatment with proteasome inhibitors. It is not known if proteasome inhibition induces heat shock in enterocytes or influences IL-6 production. Here we tested the hypothesis that treatment of cultured Caco-2 cells, a human intestinal epithelial cell line, with proteasome inhibitors induces the heat shock response and stimulates IL-6 production. Treatment of Caco-2 cells with one of the proteasome inhibitors MG-132 or lactacystin activated the transcription factor heat shock factors (HSF)-1 and -2 and upregulated cellular levels of the 72-kDa heat shock protein HSP-72. The same treatment resulted in increased gene and protein expression of IL-6, a response that was blocked by quercetin. Additional experiments revealed that the IL-6 gene promoter contains a HSF-responsive element and that the IL-6 gene may be regulated by the heat shock response. The present results suggest that proteasome inhibition induces heat shock response and IL-6 production in enterocytes and that IL-6 may be a heat shock-responsive gene, at least under certain circumstances. The observations are important considering the multiple biological roles of IL-6, both locally in the gut mucosa and systemically, and considering recent proposals in the literature to use proteasome inhibitors in the clinical setting to induce the heat shock response.