Fournier's gangrene: Seven years of experience in the emergencies service of visceral surgery at Ibn Rochd University Hospital Center.

INTRODUCTION: This work aims to describe and discuss the epidemiological, clinical, therapeutic and evolution of Fournier's gangrene. MATERIALS AND METHODS: Case series with retrospective data collection of patients treated for Fournier's gangrene between January 2010 and March 2017. The main etiologies, risk factors, postoperative complications outcomes and long term follow up results were analyzed. RESULTS: Eight four (84) patients were recruited. The average age of our patients was 49 years (with limits of 20-76), the male gender dominates our series (83.33%) with a sex ratio of 5 M/1W, the most frequently found risk factor was diabetes mellitus (37%). The most common etiology was anal abscesses (32%). The average time to consultation was 8 days (limits ranges from 3 to 30 days). All patients were admitted at a necrosis stage (100%). Anemia was identified in 85% of cases. The low platelets were noticed in 44.03% of cases. Hypoalbuminemia was found in 93% of cases. All patients (100%) benefited resuscitation initially and antibiotic therapy on their admission. They received emergency surgical debridement with a cleansing stoma. The average length of hospital stay was 13 days and complications occurred in 33% of cases. The mortality rate was 7.14%. CONCLUSION: Fournier's gangrene is a medico-surgical emergency with a high morbidity and mortality rate. Early diagnosis as well as antibiotic therapy and the quality of debridement save the patients.

Surgical management of recto-vaginal fistula (about 6 cases).

INTRODUCTION: Recto-vaginal fistula (RVF) is defined as a pathological epithelialized communication between the posterior wall of the vagina and the anterior wall of the rectum through the recto-vaginal septum. RVFs are rare and represent less than 5% of rectal fistulas. Occurring after childbirth or during a proctological pathology, they create a deep distress for the patients. The aim of our work is to analyze the epidemiological particularities and the risk factors of occurrence of RVF as well as the modalities and results of our therapeutic management. MATERIALS AND METHODS: Our work is retrospective analytic and comparative concerning 6 cases operated in the department of general surgery 3 of the UHC Ibn Rochd of Casablanca for recto vaginal fistula or recidive over a period of 7 years from 2012 to 2018. RESULTS: The analysis of the results of our study allowed us to note: A frequency of occurrence of RVF of about 0.48%. The average age at diagnosis was 55 in our patients. The etiologies were dominated by post-radiation (33.33%) and post-operative (16.66%) RVFs. The predominant mode of delivery in our study was vaginal delivery (83.33%). The antecedents were dominated by pelvic irradiation in 50% of patients, and pelvic surgery for cervical cancer and/or rectal cancer (50%). The diagnosis was revealed by a vaginal stool output in all patients. Surgical treatment was performed in all our patients. The surgical technique of choice in our series was drainage by Stenon, in 83.33% of patients. A protective stoma was performed in all our patients studied, a colostomy in 66.66%, and an ileostomy in 33.33% of patients. The immediate postoperative evolution was excellent in all our patients, while the short- and medium-term evolution revealed the occurrence of recurrence in one third of the patients (33.33%). The treatment of choice for recurrence was the interposition of a pedicled fat flap of the labia majora, known as the modified Martius technique. The morbidity, represented mainly by recurrence, was 25%, with a mortality rate of 0%. DISCUSSION: The occurrence of RVF in all its etiologies seems to be infrequent. However, its real incidence remains poorly documented in the literature, it varies between 0.3% and 15.3%. RVFs are considered simple or complex depending on their size, location and etiology. The high or low location and the etiology of the RVF determine the choice of the approach during surgical management. The diagnosis is most often clinical. The examination will try to find the cause of the RVF and the associated lesions. RVF can be asymptomatic. The importance of the symptoms depends on the topography of the fistula, the diameter of the orifice, and the quality of the intestinal transit. No additional investigations are required to confirm the diagnosis of RVF, since the positive diagnosis is essentially clinical. However, in the case of a high or complex fistula, the clinician can support his or her pre-therapeutic assessment with the exploration of imaging data, especially those of the digestive opacification, MRI and pelvic CT. The causes of RVFs are multiple. However, their proportions are difficult to establish. Post-obstetrical RVFs, those due to Crohn's disease, and post-op are probably the most frequent. The literature describes a variety of surgical approaches and treatment options for RVF. However, there are no treatment recommendations. The available data are vague and do not define an optimal treatment. Medical treatment with antibiotics and sitz baths is often necessary to control the local infection. The surgical management of RVFs is complex and follows several principles. The therapeutic arsenal is very varied and constitutes a real "escalation", ranging from simple drainage by suture to the radical treatment represented by abdominal-pelvic amputation. The results of the treatment of simple VF are excellent in all studies. The healing rate varies from 75 to 100% depending on the authors. CONCLUSION: The results of this study confirm the low incidence of RVF, and show that vaginal delivery and a history of pelvic surgery (for rectal or cervical cancer) are the most frequent predictors of RVF. Thus, from a therapeutic point of view, medical treatment is always required, it allows the flow of the fistula to be reduced, which facilitates preparation for the surgical procedure.

Optimization of morphine extraction method for the assay of its urinary 3-glucuronideconjuguate by gas chromatography-mass spectrometry.

In the field of doping, a great interest is carried for the analysis of morphine, a powerful narcotic analgesic opiate which use is prohibited during competitions. In order to confirm the abnormal analytical result in our anti-doping laboratory, a sensitive and selective gas chromatography-mass spectrometry (GC-MS) method was performed for the quantification of urinary morphine. As sample preparation is a key step for the determination of drugs in biological samples, the aim of this work consists of the optimization of the urinary human sample pretreatment conditions before quantification by GC/MS. Enzymatic hydrolysis associated with liquid-liquid extraction constitute the major pre-treatment steps. Our study has first focused on the optimization of the extraction solvents then to enzymatic hydrolysis which morphine is released from its glucuronide conjugated form. Onboard premiums, a study involving the effect of "amount of enzyme", "incubation temperature" and "duration of hydrolysis" was conducted. This univariate study has enabled us to evaluate the influence of each of these operating variables on the area ratio of morphine to the internal standard (Amorphine/AIS) response and to set the experimental fields for each one of them. Based on these results, an experimental design was established using the Box-Behnken model to determine, by multivariate analysis, the optimal operating conditions maximizing the "Amophine/AIS" response. After validation, the analysis of response surface makes it possible to set the optimum operating conditions, which the ratio "Amorphine/AIS" is maximized. The retained conditions for enzymatic hydrolysis are 160µl of Escherichia coli glucuronidase enzyme during 6hours of incubation at a temperature of 36°C. The solvent mixture Methyl-t-Butyl Ether/isopropanol (4:1, v/v) was selected since it has improved morphine extraction from the urinary matrix allowing a gain of 50% when compared to that used in our routine laboratory. Our developed extraction method can be successfully applied for our forensic anti-doping analysis of morphin in human sample urine.

Readiness factors for lean implementation in healthcare settings--a literature review.

PURPOSE: The purpose of this paper is to determine the readiness factors that are critical to the application and success of lean operating principles in healthcare organizations through a review of relevant literature. DESIGN/METHODOLOGY/APPROACH: A comprehensive review of literature focussing on lean and lean healthcare was conducted. FINDINGS: Leadership, organizational culture, communication, training, measurement, and reward systems are all commonly attributed readiness factors throughout general change management and lean literature. However, directly related to the successful implementation of lean in healthcare is that a setting is able to authorize a decentralized management style and undertake an end-to-end process view. These can be particularly difficult initiatives for complex organizations such as healthcare settings. RESEARCH LIMITATIONS/IMPLICATIONS: The readiness factors identified are based on a review of the published literature. The external validity of the findings could be enhanced if tested using an empirical study. PRACTICAL IMPLICATIONS: The readiness factors identified will enable healthcare practitioners to be better prepared as they begin their lean journeys. Sustainability of the lean initiative will be at stake if these readiness factors are not addressed. ORIGINALITY/VALUE: To the best of the knowledge, this is the first paper that provides a consolidated list of key lean readiness factors that can guide practice, as well as future theory and empirical research.

A multiplex-PCR assay for identification of the quarantine plant pathogen Xanthomonas axonopodis pv. phaseoli.

In this study we developed an algorithm to screen for all exact molecular signatures of the quarantine pathogen Xanthomonas axonopodis pv. phaseoli (Xap), based on available data of the presence or absence of virulence-associated genes. The simultaneous presence of genes avrBsT and xopL is specific to Xap. Therefore we developed a multiplex PCR assay targeting avrBsT and xopL for the molecular identification of Xap. The specificity of this multiplex was validated by comparison to that of other molecular identification assays aimed at Xap, on a wide collection of reference strains. This multiplex was further validated on a blind collection of Xanthomonas isolates for which pathogenicity was assayed by stem wounding and by dipping leaves into calibrated inocula. This multiplex was combined to the previously described X4c/X4e molecular identification assay for Xap. Such a combination enables the molecular identification of all strains of Xanthomonas pathogenic on bean. Results also show that assay by stem wounding does not give reliable results in the case of Xap, and that pathogenicity assays by dipping should be preferred.

Synthesis, spectroscopic characterization and X-ray structure of [1,2a]benzimidazol-2-yl amidine.

[1,2a]Benzimidazol-2-yl amidine was synthesized by adding cyclopentanamine to iminoester in ethanol. The structure of amidine 1 was characterized by IR, 1H NMR, 1H-1H NOESY, 13C NMR, DEPT, XHCOR spectra, thermogravimetric analysis (TGA), differential thermal analysis (DTA), differential scanning calorimetry thermograms (DSC), elementary analysis as well as by X-ray diffraction. The single crystals suitable for X-ray measurement were obtained by recrystallization at room temperature. The amidine group of a model was found to have Z configuration in the crystal. This compound crystallizes in a P2(1)/n monoclinic unit cell with parameters a=12.679(2) Å, b=8.468(3) Å, c=13.108(2) Å, ß=96.538(2)°, V=1398.2 Å3 and Z=4.

Symbiont diversity and non-random hybridization among indigenous (Ms) and invasive (B) biotypes of Bemisia tabaci.

The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is a worldwide pest and a vector of numerous plant viruses. B. tabaci is composed of dozens of morphologically indistinguishable biotypes and its taxonomic status is still controversial. This phloem-feeder harbours the primary symbiont Portiera aleyrodidarum and potentially six secondary symbionts: Cardinium, Arsenophonus, Hamiltonella, Rickettsia, Wolbachia and Fritschea. In the southwest Indian Ocean, La Réunion hosts two biotypes of this species: B (invasive) and Ms (indigenous). A multiplex PCR was developed to study the symbiont community of B. tabaci on La Réunion. Symbiont community prevalence and composition, host mitochondrial and nuclear genetic diversity, as well as host plant and localization, were described on field populations of La Réunion for B and Ms B. tabaci biotypes and their hybrids. A clear association between symbiotypes and biotypes was shown. Cardinium, Arsenophonus and Rickettsia were found in the Ms biotype (73.6%, 64.2% and 3.3%, respectively). Hamiltonella (exclusively) and Rickettsia were found in the B biotype (78% and 91.2%, respectively). Hybrids harboured all symbiotypes found in Ms and B populations, but with a higher prevalence of Ms symbiotypes than expected under random hybridization. An unexpected majority was Cardinium mono-infected (65.6%), and a striking minority (9%) harboured Cardinium/Arsenophonus. In the hybrids only, genetic diversity was linked to symbiotype. Among the hybrids, significant links were found between symbiotypes and: (i) mitochondrial COI sequences, i.e. maternal origin; and (ii) alleles of nuclear microsatellite loci, specific to either Ms or B parental biotype. Taken together, our results suggest that Cardinium and/or Arsenophonus may manipulate the reproduction of indigenous (Ms) with invasive (B) biotypes of Bemisia tabaci.

In vitro and in vivo efficacy of photofrin and pheophorbide a, a bacteriochlorin, in photodynamic therapy of colonic cancer cells.

This study was designed to investigate the efficacy of photodynamic therapy (PDT) in treating colonic cancer in a preclinical study. Photofrin, a porphyrin mixture, and pheophorbide a (Ph a), a bacteriochlorin, were tested on HT29 human colonic tumor cells in culture and xenografted into athymic mice. Their pharmacokinetics were investigated in vitro, and the PDT efficacy at increasing concentrations was determined with proliferative, cytotoxic and apoptotic assessments. The in vivo distribution and pharmacokinetics of these dyes (30 mg/kg, intraperitoneal) were investigated on HT29 tumor-bearing nude mice. The inhibition of tumor growth after a single 100 J/cm2 PDT session was measured by the changes in tumor volume and by histological analysis of tumor necrosis. PDT inhibited HT29 cell growth in culture. The cell photodamage occurred since the time the concentrations of Ph a and Photofrin reached 5.10(-7) M (or 0.3 microg/mL) and 10 microg/mL, respectively. A photosensitizer dose-dependent DNA fragmentation was observed linked to a cleavage of poly(ADP-ribose) polymerase and associated with an increased expression of mutant-type p53 protein. PDT induced a 3-week delay in tumor growth in vivo. The tumor injury was corroborated by histological observation of necrosis 48 h after treatment, with a correlated loss of specific enzyme expression in most of the tumor cells. In conclusion, PDT has the ability to destroy human colonic tumor cells in vitro and in vivo. This tumoricidal effect is likely associated with a p53-independent apoptosis, as HT29 cells express only mutated p53. The current study suggests a preferential use of Photofrin in PDT of colonic cancer because it should be more effective in vivo than Ph a as a consequence of better tumor uptake.

Dual effect of laparoscopy on cell-mediated immunity.

Laparoscopic influence on cell-mediated immunity and tumour evolution is controversial. The objective of the present study was to assess tumour growth and immune patterns after laparoscopy on an experimental study. Lewis rats, bearing an intrapancreatic ductal carcinoma randomly underwent one of the following 2-hour procedures: anaesthesia, laparotomy or CO(2) pneumoperitoneum. Cell-mediated immunity was investigated through determination of serum IL1beta concentrations by ELISA and TNFalpha, IL6 and iNOS gene transcriptions in blood white cells and peritoneal cells by RT-PCR 1 day after operation. Tumour growth and spread patterns were assessed on anatomopathological examination 2 weeks after surgery. Tumour growth and spread were unaffected no matter what procedure was applied, but port-site seeding occurred in half of the cases undergoing laparoscopy. No significant change in acute-phase protein response, represented by IL1beta serum concentration, was found after laparoscopy. TNFalpha, IL6 and inducible NO synthase gene transcriptions were enhanced in blood white cells and depressed in peritoneal immune cells after laparoscopy. In our experimental conditions, cell-mediated immune response to CO(2) pneumoperitoneum seems to be a good systemic immune activation and a less acute peritoneal immune response as opposed to control laparoscopy. This early impairment of peritoneal macrophage immune activity, observed after a long-lasting CO(2) pneumoperitoneum, might be responsible for the high rate of port site recurrence.

Human pancreatic carcinoma cells are sensitive to photodynamic therapy in vitro and in vivo.

BACKGROUND: The aim of this study was to assess the efficiency of photodynamic therapy (PDT) on human pancreatic cancer cells in vitro and in an animal model. METHODS: Human pancreatic tumour cell lines were submitted to PDT with pheophorbide a (Ph a), a chlorophyll derivative, in culture and after grafting into athymic mice. Ph a was tested in culture (10-10-10-5 mol/l) with a 5-J/cm2 energy treatment and on tumour-bearing Nude mice (30 mg/kg intraperitoneally) with a 100-J/cm2 PDT session. The effect of PDT was assessed in vitro using proliferative, apoptotic and clonogenic tests and in vivo on tumour growth and on the induction of tumour necrosis. RESULTS: PDT inhibited tumour cell growth in culture by affecting DNA integrity. This tumour cell photodamage started at low concentration (10-7 mol/l) as corroborated by clonogenic and tumour growth tests. A strong necrosis was achieved in vivo with a single PDT session. CONCLUSION: PDT destroyed human pancreatic carcinoma after low photosensitizer supply and weak energy application. It exerted this tumoricidal effect via apoptosis induction with a gentle protocol, and apoptosis and/or necrosis with a stronger protocol.

Increased tumor growth and spread after laparoscopy vs laparotomy: influence of tumor manipulation in a rat model.

BACKGROUND: The use of laparoscopy for assessment and treatment of malignant tumors remains controversial. The aim of this study was to evaluate the impact of tumor manipulation during laparoscopy compared with that of conventional laparotomy on growth and spread of an intraperitoneal tumor in the rat in a randomized, controlled trial. METHODS: Thirty 2-month-old male Lewis rats received a single-site intrapancreatic inoculation of a ductal adenocarcinoma. Fourteen days after cancer implanting, two groups of six animals each underwent a laparotomy (30 min 6 mmHg CO2 pneumoperitoneum). The tumor was manipulated in the one group, and exclusively visualized in the other. In two other groups, a midline laparotomy with (n = 6) or without (n = 6) tumor manipulation was performed. Animals in the control group (n = 6) underwent no procedure. Tumor volume, tumor mass, local regional invasion incidence, lymph node involvement, and liver and lung metastases were evaluated on 28-day tumors. RESULTS: No difference in tumor growth and spread was observed between laparoscopy and laparotomy when tumor manipulation was not carried out. Tumor manipulation increased tumor growth significantly in the laparotomy group, but not in the laparoscopy one. Tumor metastases were correlated to tumor growth and increased significantly after manipulation in both groups. There was no port-site or conventional wound seeding in either the surgical procedure. CONCLUSIONS: This study showed that manipulation is the main factor acting on tumor dissemination in both laparoscopy and laparotomy. Laparoscopic surgery had a beneficial effect on local tumor growth compared with laparotomy in the case of tumor manipulation. This beneficial effect of laparoscopic surgery may be related to a better preservation of immune function in the early postoperative period.

Effects of cholecystokinin octapeptide on a pancreatic acinar carcinoma in the rat.

PURPOSE: To investigate the effects of increasing concentrations of cholecystokinin octapeptide (CCK-8) on a pancreatic acinar adenocarcinoma. METHODS: Growth of the tumour was estimated in vivo on rats bearing a subcutaneous pancreatic carcinoma, and in vitro on primary cultured tumour cells. CCK receptors were characterized by binding assays. RESULTS: CCK-8, administered for 12 successive days, exerted a biphasic action on tumour growth: a dose-dependent stimulation with low doses (0.1 and 0.5 microg/kg) and inhibition with high doses (2 and 4 microg/ kg) as shown by respective increases and decreases in tumor volume, protein, RNA and amylase contents. In cell cultures, [3H]thymidine incorporation was dose-dependently increased with 10-(10) to 10(-8) M CCK-8 and inhibited with 10(-7) M. Both effects were completely suppressed by the CCK-receptor antagonists CR 1409 and L 364,718 (10(-4) M). Binding studies showed the overexpression of two classes of CCK-A receptors of low and high affinity when compared to the normal pancreas which was less sensitive to CCK-8. CONCLUSIONS: CCK-8 exerts a biphasic growth response on the acinar pancreatic carcinoma, mediated by two classes of CCK-A receptors overexpressed in the tumour.

Role of nitric oxide in pancreatic tumour growth: in vivo and in vitro studies.

Nitric oxide (NO), an endogenous free radical, has been implicated in a wide range of biological functions. NO is generated enzymatically from the terminal guanidinonitrogen of L-arginine by nitric oxide synthase (NOS). Despite intensive investigations, the role of NO--either as the primary product of the L-arginine/NOS pathway or provided from the NO donor sodium nitroprusside (SNP)--in carcinogenesis and tumour cell growth remains unclear and controversial. The objective of this study was to examine the growth effects of NO on a ductal pancreatic adenocarcinoma in the rat and on a human pancreatic tumour cell line (HA-hpc2). In vivo, both SNP and endogenous induction of NO by endotoxins [lipopolysaccharide (LPS)] plus L-arginine significantly reduced the tumour growth. To investigate the mechanisms of NO anti-tumour growth action, the effects of either the SNP or L-arginine/NOS pathway were analysed on the HA-hpc2 cell line. Nitrite/nitrate production, NOS activity and iNOS expression [assessed by reverse transcription-polymerase chain reaction (RT-PCR)] were tested and related to growth (assessed by [3H]thymidine incorporation assay) and apoptosis (assessed by internucleosomal DNA cleavage). SNP exerted a dual effect on tumour cells: stimulation of the proliferation up to 1 mM and inhibition at higher concentrations. These effects were related to NO production. Both proliferative and cytostatic responses were inhibited by NO scavenger 2-phenyl-4,4,5,5-tetramethyl-hemidazoline-1-oxyl3-oxide (carboxy-PTIO). The marked apoptotic DNA fragmentation induced by SNP was also abolished by PTIO association. Unlike macrophages, the human pancreatic tumour cells did not seem to express intrinsically the L-arginine/NOS pathway. Macrophages were activated by HA-hpc2 cells as well as by LPS plus cytokines [interleukin (IL)-1beta plus tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma]. In HA-hpc2/macrophage co-cultures, NOS activity and inducible NOS (iNOS) transcription were stimulated, whereas an antiproliferative response was observed. These effects were related to both macrophage amount and NO production. Addition of LPS plus cytokines to co-cultures doubled iNOS activity, nitrite/nitrate production and tumoricidal effect. These data suggest the involvement of NO in pancreatic tumour growth and support the fact that generation of high levels of NO with potential production of endogenous reactive nitrogen intermediates may contribute to induction of apoptosis and tumour growth inhibition.

Effect of the gastrin-releasing peptide antagonist BIM 26226 and lanreotide on an acinar pancreatic carcinoma.

The effects of a potent specific gastrin-releasing peptide receptor antagonist, BIM 26226 ([D-F5 Phe6, D-Ala11] bombesin (6-13) OMe), and the long-acting somatostatin analogue, lanreotide (BIM 23014), on the growth of an acinar pancreatic adenocarcinoma growing in the rat or cultured in vitro were investigated. Lewis rats bearing a pancreatic carcinoma transplanted s.c. in the scapular region, were treated with gastrin-releasing peptide (30 microg/kg per day), BIM 26226 (30 and 100 microg/kg per day) and lanreotide (100 microg/kg per day) alone or in combination for 14 successive days. Chronic administration of BIM 26226 and lanreotide significantly inhibited the growth of pancreatic tumours stimulated or not by gastrin-releasing peptide (GRP), as shown by a reduction in tumour volume, protein, ribonucleic acid, amylase and chymotrypsin contents. This effect was more pronounced with 100 microg/kg per day BIM 26226 than with 30 microg/kg per day. However, BIM 26226 and lanreotide, given together, did not exert any additive effect on GRP-treated and -untreated tumours. In cell cultures, both BIM 26226 and lanreotide (10(-6) M) inhibited [3H]thymidine incorporation in tumour cells induced or not by GRP, but no increased effect was observed after combined treatment with both agents. Binding studies showed that BIM 26226 had a high affinity for GRP receptors in tumour cell membranes (IC50 = 6 nM). These results from in vivo and in vitro experiments suggest that BIM 26226 and lanreotide are able to reduce the growth of an experimental acinar pancreatic tumour. Thus, these agents represent interesting steps toward the development of new approaches for treatment of pancreatic carcinomas.

Inactivation of p53 in normal human cells increases G2/M arrest and sensitivity to DNA-damaging agents.

p53 mutations are found in about 70% of human cancers. In order to evaluate the role of these mutations in response to chemotherapeutic agents, it is important to distinguish between p53 response to DNA-damaging agents in normal and in tumour cells. Here, using normal human fibroblasts (NHFs), we show that cisplatin and UV radiation induce G2/M arrest which is temporally linked to p53-protein induction. To study the contribution of p53 to this G2/M arrest, we inhibited p53 induction in NHFs using p53 anti-sense oligonucleotides. Following exposure of NHFs to UV radiation, the inhibition of p53-protein induction leads to a greater accumulation of cells in the G2/M phase, but also to a decreased fraction of cells in the G1 phase. We propose that p53 does not induce G2/M arrest directly, and that the extent of this arrest may depend on the fraction of cells that do not stop at the G1 phase following exposure to DNA-damaging agents. Furthermore, inhibition of p53-protein induction leads to increased sensitivity of NHFs to UV radiation. These results suggest that inhibition of p53 protein enhances sensitivity to DNA-damaging agents in normal human cells.

In vivo laser-induced fluorescence imaging of a rat pancreatic cancer with pheophorbide-a.

Laser-induced fluorescence (LIF) of pheophorbide-a (Ph-a) was used for imaging of a rat pancreatic tumor. Using a dimensionless function (the ratio of Ph-a fluorescence by bluish autofluorescence), the fluorescence contrasts between excised tumors and their paired pancreas were investigated up to 48 h after a 9 mg kg-1 Ph-a intravenous administration. Among five tested excitation wavelengths, 355 and 610 nm excitations gave the best distinctive contrasts, both 48 h after dye injection. The LIF imaging of six intrapancreatic tumors and six healthy pancreas was carried out in vivo using two laser excitations: 355 nm (Nd:YAG + tripling) for bluish autofluorescence and 610 nm (rhodamine 6G dye) for reddish autofluorescence and dye emission. Images were recorded through bandpass filters at 470 and 640 nm (autofluorescence) and at 680 nm (dye + autofluorescence) with an intensified charged-coupled device camera. Autofluorescence as Ph-a fluorescence images did not allow accurate LIF diagnosis of pancreatic carcinoma. An image processing, including for each pixel a computed division of Ph-a fluorescence (after subtraction of reddish autofluorescence) by bluish autofluorescence intensity generated poorly contrasted tumor images in five of six and false tumor localization in one of three of the tumor-bearing pancreas. A fitting of the digital 640 nm autofluorescence up to the mean 680 nm fluorescence intensity in pancreas prior to subtraction allowed a safe diagnosis to be made with well-contrasted tumor images. To assess automation ability of the processing, a same fitting coefficient (mean of individual values) was applied. In this way, false-negative (one of six) and false-positive (two of six) images were present in tumor-bearing animals as false-positive in one-half of the controls. A successful standardized procedure was then applied with a normalization of 640 and 680 nm pancreas intensities to a same set threshold prior processing. In opposition to thin-layered hollow organs, such as bronchial tube or digestive tract, LIF imaging of carcinoma inserted in a compact organ is exhausting. The use of a dye excitable in the red wavelength range (610 nm for Ph-a) may partly solve this problem, rendering LIF imaging more accurate and potentially automated.

Photodynamic imaging of a rat pancreatic cancer with pheophorbide a.

Laser-induced fluorescence of pheophorbide a (Ph-a) was used for in vitro photodynamic imaging (PDI) of a rat pancreatic acinar tumor. A 400 nm excitation induced a 470 nm autofluorescence and a 678 nm dye fluorescence in tumors and their surrounding pancreas 24 h after a 9 mg kg-1 body weight Ph-a intravenous administration. With lower intensities in these blood-rich tumors than in pancreas, Ph-a fluorescence signals are unable to provide tumor images. A dimensionless function (the ratio of Ph-a fluorescence by autofluorescence, called Rt for the tumor and Rp for the pancreas) was used for fluorescence contrast calculation (C = Rt/Rp) between six tumors and their paired pancreas. Among five available laser excitation wave-lengths, only the 355 nm excitation gave a distinctive contrast (C = 1.5). The PDI of six intrapancreatic tumors and their intraperitoneal metastasis and of two control normal pancreas was thus performed ex vivo using a 355 nm excitation source delivered by a tripled Nd:YAG laser and a charged-coupled device camera. Fluorescence images were recorded at 680 nm (dye), 640 nm (background) and 470 nm (autofluorescence) through three corresponding 10 nm width bandpass filters. Computed division for each pixel of Ph-a fluorescence values by autofluorescence generated false color image. In this way, contrasted tumor images were obtained. But in five out of six animals false-positive images were present due to an autofluorescence decrease in some normal pancreatic areas. A 470 nm autofluorescence imaging on the same tumors gave in all cases false-positive image and false-negative in half of the cases. These observations suggest that autofluorescence alone is unable to achieve accurate PDI of pancreatic carcinoma and that using Ph-a as a PDI dye needs strong improvements.

Expression and characterization of gastrin-releasing peptide receptor in normal and cancerous pancreas.

The biochemical and pharmacological characteristics of specific binding sites for gastrin-releasing peptide (GRP) were investigated in normal exocrine pancreas and in an azaserine-induced pancreatic carcinoma in the rat, under similar experimental conditions. Cells from both types of tissues contained rapid, reversible, temperature-dependent, and highly specific binding sites for GRP. Scatchard analysis of equilibrium data obtained with normal and tumor plasma membranes indicated a single class of high-affinity sites (KD = 0.42 +/- 0.06 and 0.35 +/- 0.05 nM, respectively), but the number of GRP receptors was significantly different (Bmax = 31 +/- 4.5 and 189 +/- 20 fmol/mg protein, respectively). Binding of 125I-GRP1-27 was sensitive to GTP analogues, suggesting that the GRP receptor is functionally linked to a guanyl regulatory protein; however, the wheat germ agglutinin-agarose purified receptor had lost this G-protein activity. Cross-linking of 125I-GRP1-27 either to normal and neoplastic cells or to crude membranes, solubilized membrane proteins, and partially purified receptors revealed the presence of a specific MW 75-kDa polypeptide. N-Glycanase treatment reduced this apparent MW to about 45 kDa. Together, these data suggest that normal and tumor pancreatic cells contain a specific GRP receptor that is expressed more on malignant pancreatic tissues.

Experimental pancreatic cancer in the rat treated by photodynamic therapy.

Selective histological necrosis of experimental pancreatic carcinoma by photodynamic therapy (PDT) has been successful with haematoporphyrin derivatives and phthalocyanine as photosensitizers. This report describes the feasibility of PDT with pheophorbide A as the photosensitizer to treat azaserine-induced pancreatic rat carcinoma and analyses survival of the animals. An organ distribution study 24 h after pheophorbide A administration (9 mg/kg intravenously) gave a selectivity ratio of 13.5:1 between tumour and surrounding tissue. Light of 660 nm and 100 J/cm2 induced selective necrosis of the tumour. Six of nine rats were cured in 120 days whereas all 36 control animals died within 35 days (P < 0.01). The pancrease and hepatic pedicle were relatively unaffected by PDT, but the duodenum was injured.

Inhibition of growth of a transplanted rat pancreatic acinar carcinoma with CCK-8.

Gastrointestinal hormones and neuropeptides are known to regulate growth of various normal gastrointestinal tissues and many cancers. Since cholecystokinin (CCK) is considered the most potent trophic factor for the exocrine pancreas, we studied its effect on growth of an acinar cell tumor, initially induced by azaserine and transplanted to the rat, in comparison with the normal pancreas. When tumors became palpable, rats were treated three times daily for 12 or 14 days with CCK-8 or NaCl 0.9% (controls) alone or in combination with the CCK receptor antagonist CR1409 (10 mg/kg) administered subcutaneously twice daily. Then tumors and pancreata were analyzed for their size, composition, and CCK receptors. Tumor volume, weight, and protein content, RNA, DNA, and enzymes decreased after CCK-8 treatment in a dose-dependent manner, the maximal effect being observed with 4-micrograms/kg treatment. This inhibitory effect was partially suppressed by CR1409, which by itself also reduced tumor growth, but to a lesser degree. CCK-8 exerted a stimulating effect on growth of the normal pancreas with low doses (1 and 2 micrograms/kg) and an inhibitory effect or no effect with a higher dose (4 micrograms/kg). CR1409 prevented this latter effect, but did not affect by itself the normal pancreas. These findings suggest that CCK-8 inhibits growth of an acinar cell tumor grafted to the rat; this effect is mediated by the occupation of specific CCK receptors present in high density on these cells. In contrast, CCK-8 exerts a biphasic effect on the normal pancreas as a function of its dose.