A phase II study of capecitabine and oxaliplatin combination chemotherapy in patients with inoperable adenocarcinoma of the gall bladder or biliary tract.

BACKGROUND: Advanced biliary tract carcinomas are associated with a poor prognosis, and palliative chemotherapy has only modest benefit. This multi-centre phase II study was conducted to determine the efficacy of capecitabine in combination with oxaliplatin in patients with inoperable gall bladder or biliary tract cancer. METHODS: This was a Phase II, non-randomised, two-stage Simon design, multi-centre study. Ethics approval was sought and obtained by the North West MREC, and then locally by the West Glasgow Hospitals Research Ethics Committee. Eligible patients with inoperable locally advanced or metastatic adenocarcinoma of the gall bladder or biliary tract and with adequate performance status, haematologic, renal, and hepatic function were treated with capecitabine (1000 mg/m(2) po, twice daily, days 1-14) and oxaliplatin (130 mg/m(2) i.v., day 1) every 3 weeks for up to six cycles. The primary objective of the study was to determine the objective tumour response rates (complete and partial). The secondary objectives included assessment of toxicity, progression-free survival, and overall survival. RESULTS: Forty-three patients were recruited between July 2003 and December 2005. The regimen was well tolerated with no grade 3/4 neutropenia or thrombocytopenia. Grade 3/4 sensory neuropathy was observed in six patients. Two-thirds of patients received their chemotherapy without any dose delays. Overall response rate was 23.8% (95% CI 12.05-39.5%). Stable disease was observed in a further 13 patients (31%) and progressive disease observed in 12 (28.6%) of patients. The median progression-free survival was 4.6 months (95% CI 2.8-6.4 months; Fig. 1) and the median overall survival 7.9 months (95% CI 5.3-10.4 months; Fig. 2). Fig. 1 Progression-free survival Fig. 2 Overall survival CONCLUSION: Capecitabine combined with oxaliplatin has a lower disease control and shorter overall survival than the combination of cisplatin with gemcitabine which has subsequently become the standard of care in this disease. However, capecitabine in combination with oxaliplatin does have modest activity in this disease, and can be considered as an alternative treatment option for patients in whom cisplatin and/or gemcitabine are contra-indicated.

Pancreatic cancer genomics: where can the science take us?

The incidence of pancreatic ductal adenocarcinoma (PDAC) is steadily increasing and the annual death-to-incidence ratio approaches one. This is a figure that has not changed for several decades. Surgery remains the only chance of cure; however, only less than 20% of patients are amenable to operative resection. Despite successful surgical resection, the majority of the patients still succumb to recurrent metastatic disease. Therefore, there is an urgent need to develop novel therapeutic strategies and to better select patients for current therapies. In this review, we will discuss current management by highlighting the landmark clinical trials that have shaped current care. We will then discuss the challenges of therapeutic development using the current randomized-controlled trial paradigm when confronted with the molecular heterogeneity of PDAC. Finally, we will discuss strategies that may help to shape the management of PDAC in the near future.

Metabolic activation of sulfur mustard leads to oxygen free radical formation.

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 µM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.

The skin reservoir of sulphur mustard.

Studies of the percutaneous reservoir of sulphur mustard (HD) formed during absorption carried out during WWI and WWII are inconclusive. More recent studies have indicated that a significant amount of unreacted HD remains in human epidermal membranes during percutaneous penetration studies in vitro. The present study investigated the nature and persistence of the HD reservoir formed during in vitro penetration studies using dermatomed slices of human and pig skin (0.5mm thick). Amounts of (14)C-HD that (a) penetrated, (b) remained on the surface, (c) were extractable from and (d) remained in the skin after extraction were estimated by liquid scintillation counting (confirmed using GC-MS analysis). The results demonstrated that there is a reservoir of HD in human and pig skin for up to 24 h after contamination of the skin surface in vitro with liquid agent. At least some of this reservoir could be extracted with acetonitrile, and the amounts of extracted and unextracted HD exceed the amount required to produce injury in vivo by at least 20 fold. The study demonstrated the presence of a reservoir whether the skin was covered (occluded) or left open to the air (unoccluded). The study concluded that the extractable reservoir was significant in terms of the amount of HD required to induce a vesicant response in human skin. The extractable reservoir was at least 20 times the amount required per cm(2) estimated to cause a response in all of the human population, as defined by studies carried out in human volunteers during the 1940s.

A cutaneous full-thickness liquid sulfur mustard burn model in weanling swine: clinical pathology and urinary excretion of thiodiglycol.

Sulfur mustard (bis(2-chloroethyl)sulfide, HD) is a well-known blistering chemical warfare agent. We have developed a cutaneous full-thickness HD burn model in weanling pigs for efficacy testing of candidate treatment regimens. This report addresses clinical pathology findings and the urinary excretion profile of a major HD metabolite (thiodiglycol, TDG) in this model. Six female Yorkshire pigs were exposed to HD liquid on the ventral surface for 2 h, generating six 3-cm diameter full-thickness dermal lesions per pig. Blood samples were collected throughout a 7-day observation period for hematology and serum chemistry examinations. Urine was collected in metabolism cages. Routine urinalysis was performed and the urine analyzed for TDG using gas chromatography/mass spectrometry. Examination of clinical pathology parameters revealed subtle HD-related changes that are suggestive of a mild hemolytic episode. No other signs of clinically significant systemic toxicities were noted, including bone marrow suppression. Thiodiglycol was detected at the earliest time point tested (6-8 h post-exposure) at levels ranging from 0.66 to 4.98 microg ml(-1) with a mean of 2.14 microg ml(-1). Thiodiglycol concentrations were the highest for half of the animals at this earliest time point and at 24-48 h for the others. By the evening of day 3, the mean level had reached 50 ng ml(-1). Mean levels remained 10-40 ng ml(-1) for the remainder of the 7-day observation period, with the highest individual concentration noted during this period of 132 ng ml(-1). Our results are in general agreement with the TDG excretion profiles previously described for rodent models and humans. Urinary excretion of absorbed HD in our weanling pig wound healing model appears to follow the same pattern as is seen in other laboratory animals models. In general, urinary excretion of TDG appears to peak within the first 1-4 days following exposure, with detectable levels after 1 week. Relatively high urinary TDG levels may thus indicate agent exposure within the previous 96 h. Low levels significantly above natural background levels may indicate either exposure to low levels of agent or exposure that occurred more than 4 days prior to collection of the sample.

Detection and measurement of sulfur mustard offgassing from the weanling pig following exposure to saturated sulfur mustard vapor.

Animal models are employed to investigate mechanisms of injury and to evaluate protective measures against sulfur mustard (HD) exposure. The ability to detect and quantify HD enables the researcher to follow safe procedures in handling skin samples. We designed an experimental procedure to measure HD offgassing from animal models. A Minicams--a portable gas chromatograph equipped with a flame photometric detector and on-line sorbent collection and desorption--was used to monitor the HD concentration. Confirming measurements were made using a two-step process that trapped HD on a Tenax sorbent off-line and then transferred the sample by means of an ACEM 900 to a gas chromatograph equipped with either a flame photometric detector or a mass spectrometer. Sulfur mustard offgassing data are presented from three experiments in which weanling pigs were exposed to saturated HD vapor via vapor caps containing 10 microl of HD. The HD concentration was measured in time-weighted-average (TWA) units at a specific HD application site. The current 8-h maximum exposure limit for HD is 3-ng l(-1), (1 TWA unit). The largest TWA value measured near a 3 h time point was a Minicams measurement of 0.48 TWA at 2 h and 53 min after removal of a vapor cap containing HD from a single exposure site on an animal that had 24 concurrent dorsal exposure sites. Gas chromatography/flame photometric detection and gas chromatography/mass spectrometry were used to confirm the Minicams data and to provide greater sensitivity and selectivity down to 0.1 TWA. The gas chromatography/mass spectrometry data confirmed that HD concentrations fell below 0.1 TWA in <5 h for a specific site. These measurements of HD concentrations provided information on the expeditious and safe handling of HD-exposed tissue.

Conformational stabilities of the rat alpha- and beta-parvalbumins.

It is widely believed that beta-parvalbumin (PV) isoforms are intrinsically less stable than alpha-parvalbumins, due to greater electrostatic repulsion and an abbreviated C-terminal helix. However, when examined by differential scanning calorimetry, the apo-form of the rat beta-PV (i.e. oncomodulin) actually displays greater thermal stability than the alpha-PV. Whereas the melting temperature of the a isoform is 45.8 degrees C at physiological pH and ionic strength, the Tm for the beta isoform is more than 7 degrees higher (53.6 degrees C). This result suggests that factors besides net charge and C-terminal helix length strongly influence parvalbumin conformational stability. Extension of the F helix in the beta-PV, by insertion of Ser-109, has a modest stabilizing effect, raising the Tm, by 1.1 degrees. Truncation of the alpha-PV F helix, by removal of Glu-108, has a more profound impact, lowering the Tm by 4.0 degrees.

Sensitivity of cross-reacting antihuman antibodies in formalin-fixed porcine skin: including antibodies to proliferation antigens and cytokeratins with specificity in the skin.

Although no animal is a perfect skin model for the study of toxicological and therapeutic agents, structurally the pig may be superior to even non-human primates. Because our work involves effects of toxicological and therapeutic agents on the skin, we wanted to identify stains which may prove useful as well as determine cross-reactivity of some newer antihuman antibodies. We performed a battery of formalin-fixed skin from weanling pigs and minipigs. The battery of antibodies included LCA, CD3, OPD-4, CD34, UCHL-1, L-26, KP-1, MAC-387, Factor XIIIa, Leu-7, S-100 protein, HMB-45, GFAP, synaptophysin, neurofilament protein, ubiquitin, vimentin, type IV collagen, laminin, fibronectin, Factor VIII related antigen, Desmin-M, smooth muscle actin, cytokeratin 7, cytokeratin 20, AEI/AE3, CAM 5.2, EMA, GCDFP, Ki-67, and PCNA. Immunohistochemical stains for CD3, Leu-7, S-100 protein, type IV collagen, laminin, Factor VIII related antigen, GFAP, synaptophysin, neurofilament protein, ubiquitin, smooth muscle actin, vimentin, Desmin-M, cytokeratin 7, cytokeratin 20, AE1/AE3, CAM 5.2, Ki-67 and PCNA showed consistent cross-reactivity. In formalin-fixed tissue, only antibodies to lymphoreticular cells showed poor cross-reactivity. A high percentage of the remaining antibodies did show good cross-reactivity but with some interesting similarities and differences in specificity.

Histopathologic and immunohistochemical features in human skin after exposure to nitrogen and sulfur mustard.

N-methyl-2,2'-dichlorodiethylamine (HN2)is a topical chemotherapeutic agent used as therapy for cutaneous T-cell lymphomas (CTCL). Di(2-chloroethyl)sulfide (SM), and less often HN2, have been used as chemical weapons, with the skin being a principle target. The mechanisms by which these chemicals produce their therapeutic and toxic effects in skin, however, are not clearly defined. We exposed human skin explants to two doses of HN2 and SM. At 18 hours after exposure, histopathologic features were compared. In addition, immunohistochemical markers to basement membrane proteins were used to evaluate the effects of both chemicals on the basement membrane zone. Gross vesication was not seen. Pyknotic nuclei with or without dyskeratotic changes within epidermal keratinocytes were present at both doses. These changes varied more between skin specimens than they did between doses. Ballooning degeneration was more marked after SM exposures. Diffuse dermal-epidermal separation was present only at high-dose exposures and did not appear to correlate with the degree of changes locally in the overlying epidermis. Antibodies to laminin-5 showed decreased immunoreactivity after exposure to HN2 and SM. Immunoreactivity for laminin- was decreased to a lesser extent, and immunoreactivity for collagen IV and VII was unchanged. HN2 and SM produce similar histopathologic and immunohistochemical features after cutaneous exposure. These features suggest that part of mechanism of action of HN2 and SM is a direct effect on the basement membrane zone. Understanding the effects of HN2 and SM separate from their effect on DNA may be important in designing therapies and in advancing our understanding of the pathophysiologic changes induced by these chemicals when delivered topically.

Increased smooth muscle actin, factor XIIIa, and vimentin-positive cells in the papillary dermis of carbon dioxide laser-debrided porcine skin.

BACKGROUND: Pulsed carbon dioxide (CO2) laser debridement is now being used as therapy for photodamaged skin. It has been proposed that the long duration of erythema and a tissue scaffold, which results from tightening of the collagen helix induced by the laser heat, may lead to tightening of sagging skin and skin creases of lesser magnitude. METHODS: Weanling pigs exposed to mild and moderate erythema producing doses of sulfur mustard (bis-2-chloroethyl sulfide; HD) were treated with the CO2 laser (Tru-Pulse) at 6, 24, and 48 hours after exposure. In addition to histologic examination of laser-debrided and nondebrided biopsy specimens obtained at 14 days after exposure, immunohistochemical staining with antibodies to smooth muscle actin, Factor XIIIa, vimentin, and CD3 was performed. RESULTS: CO2 laser debridement of the HD-exposed skin resulted in clearing of the cytologic atypia induced by this chemical carcinogen and reduced the inflammatory infiltrate. In addition laser debridement resulted in increased numbers of stromal cells within the papillary dermis, which showed immunohistochemical staining for smooth muscle actin, Factor XIIIa, and vimentin. CONCLUSIONS: CO2 laser debridement is effective in clearing the epidermis of cytologically damage cells in HD as well as solar-damaged skin. In addition CO2 laser debridement may result in tightening of sagging skin and produce a decrease in skin creases initially, by inducing increased stromal cells within the papillary dermis, with prominent contractile actin filaments. The collagen produced by these stromal cells may subsequently maintain these improvements in the photoaged skin.

Immunohistochemical studies of basement membrane proteins and proliferation and apoptosis markers in sulfur mustard induced cutaneous lesions in weanling pigs.

Sulfur mustard (2,2-dichlorodiethyl sulfide, HD) is a chemical warfare agent that is a threat to both troops and civilians. The focus of HD research has been on intracellular adduct formation leading to apoptosis and/or necrosis in cutaneous lesions. However, there is work which suggests that HD may have a more direct effect on the basement membrane zone. Immunohistochemical staining to desmosomal proteins, cellular fibronectin, laminin 1, laminin 5, collagen IV, collagen VII, p53, Bcl-2, and PCNA was performed on weanling pig skin exposed to vesicating doses of HD, GB3, an antibody to laminin 5, showed a progressive decrease with loss of expression during the time period of clinical vesiculation. The other basement membrane proteins showed no change or inconsistent changes. PCNA, and p53 staining increased in the overlying epidermis in areas of vesiculation without significant necrosis. Bcl-2 positive cells were decreased or absent after exposure. This study implicates laminin 5 as the main basement membrane protein affected acutely by HD exposure. The patterns of staining of PCNA, Bcl-2, and p53 within the epidermis suggest that apoptosis and cellular necrosis both may play a role in cell death secondary to HD.

Depth of morphologic skin damage and viability after one, two, and three passes of a high-energy, short-pulse CO2 laser (Tru-Pulse) in pig skin.

BACKGROUND: CO2 laser energy is absorbed by water, which is present in all tissue. The depth of penetration of CO2 lasers is narrow with minimal reflection, scatter, or transmission. However, thermal damage has limited the usefulness of conventional, continuous-wave CO2 lasers for debridement as demonstrated by wound healing studies. The development of high-energy CO2 lasers, with pulse durations that are less than the thermal relaxation time of tissue, have made vaporization of skin for resurfacing and wound debridement possible because of the decreased risk of thermal damage. OBJECTIVE: This study was performed to evaluate thermal damage produced by a CO2 laser. METHODS: Routine histopathologic examination and nitroblue-tetrazolium chloride (NBTC) staining were used to evaluate the depth of tissue damage and viability in weanling pig skin after one, two, and three passes of the laser. RESULTS: At a pulse energy of 300 mJ, with a pulse duration of 60 microseconds, one pass of the laser produced vaporization of the epidermis with minimal thermal damage. Two passes produced areas of denatured collagen with loss of viable cells in the superficial papillary dermis. Three passes extended the damage into the papillary dermis. CONCLUSION: Hyalinization of collagen appears to correspond well with the level of thermal damage as measured by NBTC staining. Our findings suggest that the energy necessary to vaporize the dermis may be greater than that needed to vaporize epidermis.

Construction and characterization of the soybean leaf metalloproteinase cDNA.

The cloning and analysis of a cDNA clone encoding the soybean metalloproteinase obtained by polymerase chain reaction (PCR) and the rapid amplification of cDNA ends (RACE) reaction are described. The cDNA was constructed from poly(A)+ RNA isolated from 15-17 day old leaves. The deduced amino acid sequence of the cDNA reveals that the plant metalloproteinase is synthesized as a preproenzyme and the proenzyme form shares a structural motif, responsible for maintenance of inactive zymogen, with the matrix metalloproteinase (e.g. collagenase) family of enzymes from vertebrate origin. Northern and Western blot analysis demonstrated that the metalloproteinase transcript and protein are under a strict developmental program in that both are expressed only in leaf tissue and in a temporal fashion. The physiological function of the metalloproteinase still remains unclear although the data suggest that the enzyme is extracellular and a portion of the mature form of the enzyme is tightly bound to the cell wall.

Evaluation of cross-reacting anti-human antibodies in the euthymic hairless guinea pig model (HGP) suggests that the HGP may be a model for the study of proliferative skin disease.

Animal models have an important role in cutaneous research. The guinea pig has proven to be a useful model in a wide spectrum of these cutaneous studies; however, its usefulness is often compromised by the need for depilation. A euthymic hairless guinea pig (HGP) model avoids the problems associated with depilation. Morphologically, as in human skin, these animals have a multi-cell-layer epidermis. Proliferation kinetic studies, as well as documentation of the degree of immunologic cross-reactivity between available antibodies to human cutaneous antigens, could extend the usefulness of this animal model. We performed a battery of anti-human antibodies on formalin fixed tissue, to a variety of antigens present within the skin and on inflammatory cells. These included CD3, UCHL-1, OPD4, L-26, KP-1, Factor XIIIa, S-100 protein, cytokeratin (AE1, AE3 and CK1), CAM 5.2, vimentin, CD 34, Factor VIII, fibronectin, SM actin, collagen IV, laminin, Bcl-2, p53, Ki-67, and PCNA. Cross-reacting antibodies included: CD3, S-100 protein, cytokeratin (AE1, AE3 and CK1), vimentin, Factor VIII, SM actin, collagen IV, p53, Ki-67, and PCNA. Although this battery of antibodies is limited, the markedly increased staining of Ki-67 and PCNA within keratinocytes in the epidermis as compared to normal human skin reflects a high proliferative rate. In addition, positive staining for p53, Ki-67, and PCNA may be useful in studying effects on cell cycle kinetics and apoptosis.

Purification and molecular analysis of an extracellular gamma-glutamyl hydrolase present in young tissues of the soybean plant.

A polypeptide present in intercellular wash fluids of young leaves of Glycine max has been purified to electrophoretic homogeneity. The protein has been identified as gamma-glutamyl hydrolase (GGH) based on the shared homology with a recently cloned cDNA from rat. The enzyme is present within the extracellular space of young leaves and a portion is bound to the cell wall. Northern and Western analysis confirm that this polypeptide is expressed only in young (1-15 d old) leaf, stem and root tissue and is therefore expressed under a strict developmental program. The primary sequence of gamma-glutamyl hydrolase shares amino acid identity with a cDNA clone from rat and two partially sequenced cDNAs from Arabidopsis. Although the complete in vivo function of gamma-glutamyl hydrolase in plants is unclear, it is known that the protein plays a critical role in folate metabolism and therefore likely in meeting the physiological demands of growing plant tissues.

Percutaneous versus open tracheostomy: a retrospective cohort outcome study.

Percutaneous tracheostomy has been advocated as a faster, safer, and less invasive method of placing tracheostomy tubes in ventilated patients. To compare outcome differences, as measured by complication rates, between percutaneous and open tracheostomy, a retrospective cohort study was performed. All procedures were performed in the intensive care unit of a university-affiliated hospital. The minor complication rates did not differ significantly between percutaneous and open tracheostomy (12/31 vs. 12/29, respectively; p > 0.05), nor did there appear to be a difference in rates of major complications between the two groups (7/31 vs. 5/29; p > 0.05). This study identified a trend towards an increased risk of delayed airway loss in the percutaneous tracheostomy group.

A review of computed tomography in the diagnosis of intestinal and mesenteric injury in pediatric blunt abdominal trauma.

OBJECTIVE: To determine the sensitivity, specificity, and positive and negative predictive values of the computed tomography (CT) scan in the diagnosis of clinically significant intestinal and mesenteric injury in pediatric blunt abdominal trauma. PATIENTS: The records of 145 children who presented to a tertiary care pediatric hospital between 1987 and 1994 were reviewed retrospectively. All had experienced single or multiple injuries and underwent CT as part of the trauma assessment. METHODS: The patients were divided into two cohorts, based on the results of the initial CT scan: either positive (n = 20) or negative (n = 152) for evidence of intestinal or mesenteric injury. The two cohorts were similar with respect to age, trauma score, and timing of CT scan. The outcome of surgical (n = 23) and conservative management (n = 122) was compared with the initial CT scan results. (Some of the laparotomies were for solid-organ injury only.) RESULTS: The sensitivity of the CT scan in the diagnosis of clinically significant intestinal and mesenteric injury is 0.93. The specificity and positive and negative predictive values are 0.95, 0.65, and 0.99, respectively. CONCLUSION: The CT scan is an excellent test to screen for clinically significant intestinal and mesenteric injury in pediatric patients with blunt abdominal trauma. Because of the lower positive value, other clinical and diagnostic imaging information may help to improve diagnostic accuracy. Most importantly, CT rarely misses a significant intestinal or mesenteric injury.

Microtubule-associated protein 2 (MAP-2): a sensitive marker of seizure-related brain damage.

We have assessed the efficacy of MAP-2 immunohistochemistry as a marker of seizure-related brain damage and its suitability for quantitation of the damage using densitometric and morphometric image analysis. Seizures were produced in rats by administration of 1.5 LD50 soman, an irreversible AChE inhibitor. Our results demonstrate that neuronal damage, assessed using hematoxylin and eosin, and cresyl violet staining, was colocalized on adjacent serial sections with clearly demarcated reductions in MAP-2 staining. The most severely damaged brain regions were devoid of MAP-2 staining. Reductions in MAP-2 immunostaining were found to be exceptionally well suited for quantitation using densitometric and morphometric image analysis. This study represents the first demonstration of seizure-induced excitotoxic alterations in MAP-2.