Blockade of Thrombopoietin Reduces Organ Damage in Experimental Endotoxemia and Polymicrobial Sepsis.

BACKGROUND AND PURPOSE: Thrombopoietin (TPO), a growth factor primarily involved in thrombopoiesis may also have a role in the pathophysiology of sepsis. In patients with sepsis, indeed, TPO levels are markedly increased, with disease severity being the major independent determinant of TPO concentrations. Moreover, TPO increases and correlates with ex vivo indices of platelet activation in patients with burn injury upon sepsis development, and may contribute to depress cardiac contractility in septic shock. Still, the role of TPO in sepsis pathophysiology remains controversial, given the protective role of TPO in other experimental disease models, for instance in doxorubicin-induced cardiotoxicity and myocardial ischemia/reperfusion injury. The aim of our study was to define the contribution of TPO in the development of organ damage induced by endotoxemia or sepsis, and to investigate the effects of inhibiting TPO in these conditions. METHODS: We synthesized a chimeric protein able to inhibit TPO, mTPOR-MBP, and studied its effect in two murine experimental models, acute endotoxemia and cecal ligation and puncture (CLP) model. RESULTS: In both models, TPO levels markedly increased, from 289.80±27.87 pg/mL to 465.60±45.92 pg/mL at 3 hours in the LPS model (P<0.01), and from 265.00±26.02 pg/mL to 373.70±26.20 pg/mL in the CLP model (P<0.05), respectively. Paralleling TPO levels, also platelet-monocyte aggregates increased, from 32.86±2.48% to 46.13±1.39% at 3 hours in the LPS model (P<0.01), and from 43.68±1.69% to 56.52±4.66% in the CLP model (P<0.05). Blockade of TPO by mTPOR-MBP administration reduced histological damage in target organs, namely lung, liver, and gut. In particular, neutrophil infiltration and lung septal thickening were reduced from a score of 1.86±0.34 to 0.60±0.27 (P<0.01) and from 1.43±0.37 to 0.40±0.16 (P<0.05), respectively, in the LPS model at 3 hours, and from a score of 1.75±0.37 to 0.38±0.18 (P<0.01) and from 1.25±0.31 to 0.13±0.13 (P<0.001), respectively, in the CLP model. Similarly, the number of hepatic microabscesses was decreased from 14.14±1.41 to 3.64±0.56 in the LPS model at 3 hours (P<0.001), and from 1.71±0.29 to 0.13±0.13 in the CLP model (P<0.001). Finally, the diameter of intestinal villi decreased from 90.69±3.95 µm to 70.74±3.60 µm in the LPS model at 3 hours (P<0.01), and from 74.29±4.29 µm to 57.50±1.89 µm in the CLP model (P<0.01). This protective effect was associated with the blunting of the increase in platelet-monocyte adhesion, and, on the contrary, with increased platelet-neutrophil aggregates in the circulation, which may be related to decreased neutrophil sequestration into the inflamed tissues. Conversely, circulating cytokine levels were not significantly changed, in both models, by mTPOR-MBP administration. CONCLUSION: Our results demonstrate that TPO participates in the development of organ damage induced by experimental endotoxemia or polymicrobial sepsis via a mechanism involving increased platelet-leukocyte adhesion, but not cytokine release, and suggest that blocking TPO may be useful in preventing organ damage in patients affected by systemic inflammatory response or sepsis.

Detection and characterization of classical and "uncommon" exon 19 Epidermal Growth Factor Receptor mutations in lung cancer by pyrosequencing.

BACKGROUND: The management of advanced stage non-small cell lung cancer is increasingly based on diagnostic and predictive analyses performed mostly on limited amounts of tumor tissue. The evaluation of Epidermal Growth Factor Receptor (EGFR) mutations have emerged as the strongest predictor of response to EGFR-tyrosine kinase inhibitors mainly in patients with adenocarcinoma. Several EGFR mutation detection techniques are available, having both sensitivity and specificity issues, being the Sanger sequencing technique the reference standard, with the limitation of a relatively high amount of mutated cells needed for the analysis. METHODS: A novel nucleotide dispensation order for pyrosequencing was established allowing the identification and characterization of EGFR mutation not definable with commercially and clinically approved kits, and validated in a consecutive series of 321 lung cancer patients (246 biopsies or cytology samples and 75 surgical specimens). RESULTS: 61/321 (19%) mutated cases were detected, 17 (27.9%) in exon 21 and 44 (72.1%) in exon 19, these latter corresponding to 32/44 (72.7%) classical and 12/44 (27.3%) uncommon mutations. Furthermore, a novel, never reported, point mutation, was found, which determined a premature stop codon in the aminoacidic sequence that resulted in a truncated protein in the tyrosine kinase domain, thus impairing the inhibitory effect of specific therapy. CONCLUSIONS: The novel dispensation order allows to detect and characterize both classical and uncommon EGFR mutations. Although several phase III studies in genotypically defined groups of patients are already available, further prospective studies assessing the role of uncommon EGFR mutations are warranted.

Tumor staging but not grading is associated with adverse clinical outcome in neuroendocrine tumors of the appendix: a retrospective clinical pathologic analysis of 138 cases.

Appendiceal neuroendocrine neoplasms (NENs) are rare and usually incidentally discovered. Most cases are clinically indolent, although the rare aggressive ones are poorly predictable. The aim of this study was to test the applicability and prognostic significance of the new World Health Organization (WHO) classification and to test the several pathologic features and TNM staging systems (American Joint Committee on Cancer and European Neuroendocrine Tumor Society) in these tumors. A multi-institutional retrospective series of 138 appendiceal NENs was selected on the basis of the availability of both pathologic material and clinical information, including follow-up data. All cases were reviewed to record pathologic features and to apply year 2000 and 2010 WHO classifications, as well as European Neuroendocrine Tumor Society and American Joint Committee on Cancer TNM stages. Clinical and pathologic characteristics were compared with disease outcome by contingency, univariate, and multivariate survival analyses. Although up to one third of cases presented several malignancy-associated pathologic features, only 4 patients died of the disease. Adverse outcome was significantly associated with extramural extension (including mesoappendix), well-differentiated carcinoma diagnosis (2000 WHO classification), pT3-4 stage, older age, and presence of positive resection margins, but not with tumor size, mitotic or proliferative indexes, and, consequently, 2010 WHO grading. In the appendix, at variance with midgut/hindgut NENs, the 2000 WHO classification performs better than the grading-based 2010 WHO scheme and, together with tumor stage, is the most relevant parameter associated with clinical aggressiveness.

Selective inhibition of carbonic anhydrase IX decreases cell proliferation and induces ceramide-mediated apoptosis in human cancer cells.

Recently, carbonic anhydrase (CA) inhibitors have been proposed as a potential new class of antitumor agents. The aim of this study was to evaluate the antitumor activity of three CA inhibitors, namely acetazolamide (AZ) and two newly synthesized aromatic sulfonamides with high affinity for CA IX, 2-(4-sulfamoylphenyl-amino)-4,6-dichloro-1,3,5-triazine (TR1) and 4-[3-(N,N-dimethylaminopropyl)thioreidophenylsulfonylaminoethyl]benzenesulfonamide (GA15), against human tumor cells. The effects of AZ, TR1, and GA15 on cell proliferation and apoptosis were evaluated in CA IX-positive HeLa and 786-O cells and CA IX-negative 786-O/von Hippel-Lindau (VHL) cells. We also investigated whether the potential antitumor activity of these molecules might be mediated by an increase in ceramide production. AZ, TR1, and GA15 could significantly reduce cell proliferation and induce apoptosis in HeLa and 786-O cells. Moreover, all three inhibitors could decrease intracellular pH (pH(i)) and increase ceramide production in the same cells. Treatment with the ceramide synthase inhibitor fumonisin B1 prevented the apoptotic effects of the three CA inhibitors. In all experiments, the effects of aromatic sulfonamides were more pronounced than those of AZ. The three inhibitors did not show any antitumor activity in CA IX-negative 786-O/VHL cells and failed to lower pH(i) or increase intracellular ceramide levels in the same cells. In conclusion, CA inhibition can decrease cell proliferation and induce apoptosis in human tumor cells. The ability of CA inhibitors to decrease pH(i) might trigger cell apoptosis through mediation of ceramide synthesis. Activation of this apoptotic cascade probably is mediated by inhibition of the CA IX isoform.

Lack of phosphoinositide 3-kinase-gamma attenuates ventilator-induced lung injury.

OBJECTIVE: G protein-coupled receptors may up-regulate the inflammatory response elicited by ventilator-induced lung injury but also regulate cell survival via protein kinase B (Akt) and extracellular signal regulated kinases 1/2 (ERK1/2). The G protein-sensitive phosphoinositide-3-kinase gamma (PI3Kgamma) regulates several cellular functions including inflammation and cell survival. We explored the role of PI3Kgamma on ventilator-induced lung injury. DESIGN: Prospective, randomized, experimental study. SETTING: University animal research laboratory. SUBJECTS: Wild-type (PI3Kgamma), knock-out (PI3Kgamma ), and kinase-dead (PI3Kgamma) mice. INTERVENTIONS: Three ventilatory strategies (no stretch, low stretch, high stretch) were studied in an isolated, nonperfused model of acute lung injury (lung lavage) in PI3Kgamma, PI3Kgamma, and PI3Kgamma mice. MEASUREMENTS AND MAIN RESULTS: Reduction in lung compliance, hyaline membrane formation, and epithelial detachment with high stretch were more pronounced in PI3Kgamma than in PI3Kgamma and PI3Kgamma (p < .01). Inflammatory cytokines and IkBalpha phosphorylation with high stretch did not differ among PI3Kgamma, PI3Kgamma, and PI3Kgamma. Apoptotic index (terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling) and caspase-3 (immunohistochemistry) with high stretch were larger (p < .01) in PI3Kgamma and PI3Kgamma than in PI3Kgamma. Electron microscopy showed that high stretch caused apoptotic changes in alveolar cells of PI3Kgamma mice whereas PI3Kgamma mice showed necrosis. Phosphorylation of Akt and ERK1/2 with high stretch was more pronounced in PI3Kgamma than in PI3Kgamma and PI3Kgamma (p < .01). CONCLUSIONS: Silencing PI3Kgamma seems to attenuate functional and morphological consequences of ventilator-induced lung injury independently of inhibitory effects on cytokines release but through the enhancement of pulmonary apoptosis.

Skewing towards neuroendocrine phenotype in high grade or high stage androgen-responsive primary prostate cancer.

OBJECTIVE: The prognostic influence of neuroendocrine (NE) differentiation in prostate cancer patients is not yet properly established. In a series of primary hormone-naive prostate cancers from a patient population that underwent radical prostatectomy, we wanted to determine the relationship between NE phenotype expression and Gleason sum, disease stage, and serum PSA concentration. METHODS: Chromogranin A (CgA) expression was scored and compared in 105 consecutive primary prostate cancers with their homologous preoperative tumor prostate biopsies. RESULTS: High grade or high stage prostate cancers expressed a significantly higher CgA score than low grade or localized diseases (p < 0.005). Both the CgA score of the surgical specimens and the PSA level in the serum increased linearly (p = 0.001). In the samples of many corresponding tumor biopsies no significant CgA staining was found. CONCLUSION: NE differentiation in primary untreated prostate cancer is closely associated with the major prognostic parameters of survival. This association cannot be shown by evaluating the CgA staining in tumor biopsies.

Ablation of phosphoinositide 3-kinase-gamma reduces the severity of acute pancreatitis.

In pancreatic acini, the G-protein-activated phosphoinositide 3-kinase-gamma (PI3K gamma) regulates several key pathological responses to cholecystokinin hyperstimulation in vitro. Thus, using mice lacking PI3K gamma, we studied the function of this enzyme in vivo in two different models of acute pancreatitis. The disease was induced by supramaximal concentrations of cerulein and by feeding mice a choline-deficient/ethionine-supplemented diet. Although the secretive function of isolated pancreatic acini was identical in mutant and control samples, in both models, genetic ablation of PI3K gamma significantly reduced the extent of acinar cell injury/necrosis. In agreement with a protective role of apoptosis in pancreatitis, PI3K gamma-deficient pancreata showed an increased number of apoptotic acinar cells, as determined by terminal dUTP nick-end labeling and caspase-3 activity. In addition, neutrophil infiltration within the pancreatic tissue was also reduced, suggesting a dual action of PI3K gamma, both in the triggering events within acinar cells and in the subsequent neutrophil recruitment and activation. Finally, the lethality of the choline-deficient/ethionine-supplemented diet-induced pancreatitis was significantly reduced in mice lacking PI3K gamma. Our results thus suggest that inhibition of PI3K gamma may be of therapeutic value in acute pancreatitis.

Endometriosis in an episiotomy scar: a case report.

Endometriosis is defined as the presence of functioning endometrial tissue in an anatomical location other than the uterine cavity. The episiotomy scar is a fairly rare site for endometriosis. The authors present the case of a 42-year-old woman referred 7 years ago for the development of a tender perianal mass at the episiotomy site associated with perianal pain and pruritus which varied in relation to menses, becoming most intense just before and immediately after the onset of menstrual bleeding. Anal endosonography performed with a B&K mechanical probe rotating through 360 degrees with a frequency of 7 and 10 Mhz showed a hypoechoic area with a diameter of 3 cm not involving the external sphincter and extending from the perianal skin to the mid third of the anal canal. Proctosigmoidoscopy findings were normal. A complete local excision was performed. Complete surgical excision of perineal endometriomas should be curative. Recurrence, supposedly due to incomplete removal, usually appears within one year.

[Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (IHCC): a double cancer in the non-cirrhotic liver]. / Carcinoma epatocellulare (HCC) e colangiocarcinoma intraepatico (IHCC): doppio tumore su fegato non cirrotico. Presentazione di un caso.

The authors report a case of a large tumour, located in the right hepatic lateral segments, (size: 15 cm), consisting of a hepatocellular carcinoma (size: 10 cm) and an intrahepatic cholangiocarcinoma (size: 5 cm). The mass was detected by ultrasonography during an examination for abdominal pain in an 80-year-old female. After tumour biopsy and histological examination, hepatic resection was performed. The postoperative course was uneventful and the patient was discharged 6 days after surgery. The rarity of this double cancer is stressed.