MAP2K4/MKK4 expression in pancreatic cancer: genetic validation of immunohistochemistry and relationship to disease course.

MKK4 (MAP2K4/SEK1) is a member of the mitogen-activated protein kinase family, originally identified as a kinase involved in the stress-activated protein kinase pathway by directly phosphorylating c-Jun NH2-terminal kinase. MKK4 genetic inactivation has been observed in a subset of pancreatic carcinomas, implicating deregulation of the stress-activated protein kinase pathway in pancreatic carcinogenesis. We evaluated Mkk4 protein expression patterns by immunohistochemical labeling in a series of 60 resected primary infiltrating pancreatic adenocarcinomas (24 cases with known MKK4 genetic status), and 14 different tissue arrays representing the primary carcinoma and all of the gross metastases from 26 patients that died of metastatic pancreatic cancer. Among the surgically resected carcinomas, focal or diffuse-positive immunolabeling for Mkk4 protein was found in 52 of 60 cases (86.7%). Among the eight carcinomas with negative Mkk4 immunolabeling, three harbored a homozygous deletion or intragenic mutation of the MKK4 gene, in contrast to none of the 52 cases with positive Mkk4 immunolabeling (P < 0.01). Loss of Mkk4 immunolabeling showed a trend toward shorter survival, with Mkk4-positive carcinomas having half the risk of death than Mkk4-negative carcinomas (P = 0.09). Mkk4 immunolabeling patterns were also evaluated among unresectable primary and metastatic cancer tissues from autopsy specimens, indicating intact Mkk4 immunolabeling in 88.8% of the unresectable primary carcinomas as compared with 63.3% of distant metastases (P < 0.001). Our data indicate that the loss of Mkk4 protein expression in pancreatic carcinomas may be more frequent than suggested by the rates of genetic inactivation alone and that MKK4 loss may contribute to disease progression. The correlation of MKK4 genetic status with immunolabeling patterns validate this approach for the evaluation of MKK4 status in routine histologic sections and may provide useful information regarding patient prognosis.     

The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine.

Immunotoxins are chimeric proteins consisting of a toxin coupled to an antibody. To date, several clinical trials have been conducted, and some are still ongoing, to evaluate their anti-tumor efficacy. In this view, we chemically constructed an anti-CD20 immunotoxin with the mAb Rituximab and the type 1 ribosome-inactivating protein (RIP) saporin-S6, designed for B cells non-Hodgkin's lymphoma (NHL) therapy. This immunotoxin showed a specific cytotoxicity for the CD20+ cell lines Raji and D430B, evidenced by inhibition of protein synthesis, evaluation of apoptosis and clonogenic assay. Upon conjugation, saporin-S6 increased its toxicity on target cells by at least 2 logs, with IC(50) values of 0.1-0.3 nM. The percentage of AnnexinV+ cells was over 95% in both cell lines treated with 10 nM immunotoxin. A complete elimination of Raji clones was reached with the 10 nM immunotoxin, whereas a mixture of free RIP and mAb gave about 90% of clonogenic growth. Rituximab/saporin-S6, at 10 nM concentration, also induced apoptosis in 80% of lymphoma cells from NHL patients. Moreover, sensitivity of Raji to Rituximab/saporin-S6 was augmented when cells were coincubated with Fludarabine. The synergistic toxic effect of the two drugs led to a total elimination of the neoplastic population.     

Helicobacter pylori VacA toxin up-regulates vascular endothelial growth factor expression in MKN 28 gastric cells through an epidermal growth factor receptor-, cyclooxygenase-2-dependent mechanism.

PURPOSE: Helicobacter pylori causes gastric damage and is involved in gastric carcinogenesis. Vascular endothelial growth factor (VEGF) plays a major role in gastric mucosa repair and is overexpressed in gastric cancer. We investigated: (a) whether H. pylori, and in particular H. pylori VacA toxin, affected VEGF expression in gastric epithelial cells in culture; and (b) the signal transduction pathway involved in any effect exerted by H. pylori. EXPERIMENTAL DESIGN: MKN-28 cells were incubated with uninoculated BCF (control) or with BCF obtained from VacA-producing wild-type H. pylori 60190 strain or from its isogenic mutant 60190:v1, specifically lacking vacA gene in the presence or absence of ZD 1839, a selective inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, PD098059, a selective inhibitor of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase, the kinase responsible for ERK phosphorylation, or SC-236, a selective inhibitor of cyclooxygenase (COX)-2 for 24-48 h. RESULTS: (a) Toxigenic H. pylori up-regulated VEGF mRNA and protein expression and caused a 2.5-fold increase in VEGF release compared with control, whereas nontoxigenic H. pylori did not; (b) H. pylori VacA toxin-induced up-regulation of VEGF was counteracted by selective inhibition of EGFR tyrosine kinase; (c) toxigenic H. pylori activated the ERK/MAP kinase cascade, and inhibition of MAP kinase activation counteracted H. pylori-induced VEGF up-regulation; (d) toxigenic H. pylori up-regulated COX-2 expression, and this effect was counteracted by blockade of EGFR tyrosine kinase; and (e) COX-2 selective inhibition counteracted H. pylori-induced up-regulation of VEGF. CONCLUSION: (a) H. pylori up-regulates VEGF expression in gastric epithelial cells; and (b) this effect is specifically related to VacA toxin and seems to depend on the activation of an EGFR-, MAP kinase-, and COX-2-mediated pathway.     

Informed consent, parental awareness, and reasons for participating in a randomised controlled study

BACKGROUND: The informed consent procedure plays a central role in randomised controlled trials but has only been explored in a few studies on children. AIM: To assess the quality of the informed consent process in a paediatric setting. METHODS: A questionnaire was sent to parents who volunteered their child (230 children) for a randomised, double blind, placebo controlled trial of ibuprofen syrup to prevent recurrent febrile seizures. RESULTS: 181 (79%) parents responded. On average, 73% of parents were aware of the major study characteristics. A few had difficulty understanding the information provided. Major factors in parents granting approval were the contribution to clinical science (51%) and benefit to the child (32%). Sociodemographic status did not influence initial participation but west European origin of the father was associated with willingness to participate in future trials. 89% of participants felt positive about the informed consent procedure; however, 25% stated that they felt obliged to participate. Although their reasons for granting approval and their evaluation of the informed consent procedure did not differ, relatively more were hesitant about participating in future. Parents appreciated the investigator being on call 24 hours a day (38%) and the extra medical care and information provided (37%) as advantages of participation. Disadvantages were mainly the time consuming aspects and the work involved (23%). CONCLUSIONS: Parents' understanding of trial characteristics might be improved by designing less difficult informed consent forms and by the investigator giving extra attention and information to non-west European parents. Adequate measures should be taken to avoid parents feeling obliged to participate, rather than giving true informed consent.     

Microanatomical localization of dopamine receptor protein immunoreactivity in the rat cerebellar cortex

Dopamine (DA) receptor subtype localization was investigated in rat cerebellar cortex using immunohistochemical techniques with antibodies raised against D1-D5 receptor protein. A faint D1 receptor protein immunoreactivity was developed in molecular and Purkinje neurons layers. D2 receptor protein immunoreactivity was found primarily in cerebellar white matter followed by molecular and granular layers and Purkinje neurons. Antibodies against D2S receptor protein were localized in molecular layer and to a lesser extent, in granular layer. A few Purkinje neurons displayed a faint D2S receptor protein immunoreactivity. D3 receptor protein immunoreactivity was observed primarily in molecular and in Purkinje neurons layers of lobules 9 and 10. A faint D3 receptor protein immunoreactivity was also localized in Purkinje neurons and to a lesser extent, in molecular and granular layers of cerebellar lobules 1-8. D4 receptor protein immunoreactivity was found in cerebellar white matter. A pale immunostaining was also visualized in molecular layer. D5 receptor protein immunoreactivity was localized primarily in molecular and Purkinje neurons layers and to a lesser extent, in granular layer and in white matter. The above results indicate that rat cerebellar cortex expresses the DA receptor subtypes so far identified. Purkinje neurons, which are the only efferent neurons of cerebellum, are richest in DA receptor protein immunoreactivity. This suggests that dopaminergic neurotransmission may modulate efferent inputs from cerebellum. The localization of the majority of D2 and D4 and of a faint D5 protein receptor immunoreactivity in cerebellar white matter suggests that these receptors may be presynaptic and transported axonally.     

Tentorial Meningiomas. Report on Twenty-Seven Cases

Summary ? Objective. Report our experience with 27 tentorial meningiomas (TM) surgically treated between 1985 and 1998.  Methods. The records of 27 patients with TMs were retrospectively reviewed for clinical presentation, neuroradiological evaluation, surgical treatment and long-term outcome. The extent of tumor resection was scored according to the Simpson's grading for tumor removal. Long-term results were evaluated according to the Glasgow Outcome Score (GOS).  Results. The average age was 53 years. Female predominance was 74%. The most common complaints at presentation were headaches (51%), gait ataxia (33%), memory disturbances (30%) and hypo-acousia (30%). A classification of TMs into 5 subgroups according to tumor site is proposed on the basis of imaging studies. A cerebrospinal fluid shunt was established prior to direct approach in 7 patients and as the sole procedure in one inoperable patient. Twenty-seven direct approaches were undertaken in 26 patients, including 17 infratentorial and 10 supratentorial approaches. Total tumor removal was achieved in 20 patients (77%) and subtotal removal in 6 (23%). Fifteen patients (55%) experienced 22 postoperative complications. One patient died three months after a subtotal resection (mortality=3,7%). With a mean follow-up of 54 months, all 26 survivors are currently alive with 23 having resumed their normal activities and 3 needing assistance. Five of 6 patients with subtotal resection survived and were followed for a period ranging from 72 to 132 months: none showed residual tumor progression and no re-operation was considered. An additional patient experienced a ?true? recurrence 6 years after total removal, with no tumor progression 2 years after his recurrence was recognized.  Discussion. The best surgical approach to TMs is still a controversial matter. The advantages and drawbacks of conventional versus transbasal approaches are reviewed. Our experience suggests that subtotal removal can be associated with long recurrence-free intervals and preserved quality of life. TMs located at the tentorial edge carried a definitely worse prognosis than peripheral forms.     

Piracetam in acute stroke: a systematic review

We studied whether the administration of piracetam in acute, presumed ischemic stroke affects case fatality and functional outcome. The Cochrane Stroke Group strategy was used to evaluate all randomized controlled trials of patients with presumed ischemic stroke examined within 48 h; death and (when available) functional outcome were used as end points. Three studies were included; the most recent one contributed more than 97% of the data. There were 501 patients treated with piracetam and 501 controls. Piracetam was associated with a nonsignificant 31% increase in the odds of death (95% CI –5% to 81%). This result was due almost completely to the effect of the larger trial, which, however, reported that the difference in case fatality rate between piracetam and control disappeared after correcting for the imbalance in stroke severity between the two groups. Data on functional outcome were available only for the largest study, and no difference was reported. Data obtained from the manufacturer suggested a nonsignificant trend (–10%) towards reduction in dependency with piracetam (CI –33% to 20%); the proportions of patients dead or dependent in the two groups were the same. Relevant adverse effects were not reported. The evidence from this review does not support routine administration of piracetam in patients with acute ischemic stroke; however, since a possible beneficial effect cannot completely be ruled out, further controlled trials are warranted. Received: 31 August 1999/Received in revised form: 3 November 1999/Accepted: 25 November 1999     

Study of vestibular evoked myogenic potentials in unilateral vestibulopathy: Otolithic versus canal function testing.

OBJECTIVE: The study provides a qualitative evaluation of unilateral vestibulopathy by comparing otolithic and canal function, to establish possible relationships between the type of dysfunction observed and the evolving clinical pictures associated with it. STUDY DESIGN: Retrospective study of a series of cases. SETTING: Department of Medical-Surgical Specialization, Otolaryngology and Cervicofacial Surgery Division, University of Perugia, Perugia, Italy. PATIENTS: Twenty patients whose medical history showed at least one episode corresponding to the clinical parameters of acute vestibulopathy. INTERVENTIONS: Study of vestibular function by recording VEMPs and repeating canal function testing at least 6 months after the first episode of vertigo. MAIN OUTCOME MEASURES: Relationship between the type of vestibulopathy (canal and otolithic) and the clinical pictures observed. RESULTS: Paroxysmal positional vertigo, observed in 4 patients, was correlated with the presence of vestibular evoked myogenic potentials (VEMPs) and the absence of an ipsilateral canal response in all cases (100%). Persistent dizziness was observed in nine patients, and VEMPs were absent in all of them (100%); three (33.3%) showed the recovery of previously absent canal function. Comparison of responses in six patients with recurrent acute vestibulopathy showed persistent and complete loss of canal function in five cases (83.3%), whereas impairment of otolithic response was less constant (40%). CONCLUSION: The combined VEMPs-canal test study shows predictive value regarding certain evolving clinical pictures of vestibulopathy. The absence of VEMPs confirms the role of otolithic dysfunction in the onset of dizziness. Likewise, it suggests that a vestibular origin of these disorders should be considered in cases that have shown aspecific symptoms since onset, without frank vertigo and with normal vestibular response to canal function testing.