Ascites-derived pancreatic ductal adenocarcinoma primary cell cultures as a platform for personalised medicine.

BACKGROUND: Challenges in developing drugs for pancreatic ductal adenocarcinoma (PDAC) include obtaining metastatic cancer tissue for research and validating biomarkers predicative for personalised therapeutic decisions. We have recently developed a novel therapeutic model for PDAC to address these challenges based on the isolation of viable PDAC cells derived from ascites fluid. METHODS: Ascites fluid was obtained from PDAC patients undergoing palliative paracentesis. Ascites-derived PDAC primary cells were isolated, cultured and characterised in ovo and in vitro. RESULTS: We successfully established ascites-derived primary cell cultures within 2-7 days from 92% (93 out of 101) of the ascites fluid samples obtained (from 36 different patients). Homogeneous epithelial PDAC-enriched cell cultures were identified and characterised. We observed a wide range in doubling times and migration properties among the different patient-derived cell cultures. The diverse nature of each individual patient's cell cultures was further demonstrated by differences in therapeutic susceptibility and resistance. The tumorigenicity and invasiveness of the cells were demonstrated in vivo using chicken chorioallantoic membrane grafts. CONCLUSIONS: We have developed a unique ascites-derived PDAC primary cell culture model. This model has the potential to study signalling pathways in PDAC progression and to evaluate targeted therapies for the individual patient expeditiously, thereby supporting personalised treatment decisions.

[Ticlopidine-induced neutropenia].

Ticlopidine (TCP) is a drug that inhibits platelet aggregation. Several studies have demonstrated its superiority over aspirin in preventing stroke and other thromboembolic diseases. However, neutropenia occurs in about 2% of TCP-treated patients, which therefore may advance to agranulocytosis, sepsis and death. They should be carefully followed with blood counts. We report 2 patients hospitalized with severe neutropenia while on TCP, despite having had regular blood counts. Their complications underline the need for patient selection and meticulous follow-up when ticlopidine is prescribed.

[Conservative approach in children with central line infection].

In 1994-1995, central venous lines were placed in 47 children. All except 1 were of the Broviac type, with subcutaneous tunneling via the internal or external jugular vein. Ages were between 7 days and 16 years. Indications for central venous cannulation were chemotherapy (35 cases), TPN (5), prolonged parenteral antibiotics (4), and repeated blood transfusions (3). The catheter was the source of infection in 13 children (28%), 11 of whom were immunocompromised. The commonly identified bacteria were Staphylococcus aureus (4 cases), Pseudomonas aeruginosa (4), coagulase-negative Staphylococcus (2), and various gram-negative rods (3). All cases were treated with antibiotics through the catheter. The most commonly used were oxacillin (4), ceftazidime (4), and amikacin (4). In 10, treatment succeeded without having to remove the line. In 2 others, tunnel infection developed and the catheter had to be removed. 1 child forcefully removed his catheter before treatment could be started. There were no further complications in the group treated conservatively, except for a case of superior vena cava thrombosis in a girl with recurrent infection of the tunnel. In 7 out of 13 treatment was continued and completed at home. This saved 65 days of hospitalization out of 210. We conclude that the conservative approach to treatment is feasible in most cases of infection when the source is the central venous catheter itself. However, when the tunnel is infected, conservative treatment may be ineffective. Treatment can be carried out in the home, with economy in cost and in use of hospital beds, and is preferred by patients and their parents.

Highly sensitive amperometric immunosensor for the detection of Escherichia coli.

With the aim of detecting rapidly the presence of Escherichia coli (E. coli), a disposable amperometric immunosensor was developed based on a double layered configuration at the transducer surface, consisting first of a polypyrrole-NH(2)-anti-E. coli antibody (PAE) inner layer followed by an alginate-polypyrrole (Alg-Ppy) outer packing layer. In the presence of the substrate p-aminophenyl beta-D-galactopyranoside (PAPG), the bacterial enzyme, beta-D-galactosidase produces the p-aminophenol (PAP) product, also generating an amperometric signal due to PAP electrooxidation by potentiostating the glassy carbon (GC) electrode at 0.22V. The operational procedure consists in first adding the test sample containing the bacteria, then coating it with Alg-Ppy to ensure the confinement of the released enzyme and the analyte (being generated by the enzymatic catalysis) to the electrode active surface. This procedure facilitates the diffusion of the substrate within the complex and thus creates a higher oxidation level of the PAP enabling a detection limit of 10 colony forming units (CFU)/ml. The immunosensor setup demonstrates an improved detection limit of more than 10 times less bacteria detected than other immunosensing techniques without the need for multi step pretreatments of the test sample and/or incubation as found in some of the existing methods.

Isolated ACTH deficiency with transitory GH deficiency.

A nine-year-old girl, who presented with a severe hypoglycemic coma, proved to have isolated ACTH deficiency, a finding previously reported in only two children. On the initial evaluation, before any treatment, GH did not respond to provocative stimuli. On replacement therapy with hydrocortisone, normal linear growth was observed. Repeated testing while on glucocorticoids replacement four years after the initial attack revealed normal GH response to stimulation test. It is suggested that cortisol deficiency was responsible for the severe hypoglycemic coma and subnormal GH response. A similar mechanism is speculated for the normal growth observed in some patients with apparent deficiency of anterior pituitary hormones, including GH. The possibility of permanent ACTH deficiency and transitory GH deficiency following hypophysitis is discussed.

Complete recovery of pure red cell aplasia by intramuscular gammaglobulin therapy in a child with hypoparathyroidism.

A 10-year-old girl with multiple endocrinopathies, hypoparathyroidism, and pernicious anemia subsequently developed pure red cell aplasia, which responded only temporarily to cytotoxic therapy. Because of transfusion dependency, resistance to steroids, plasmapheresis, and Cytoxan, she was given injections of intramuscular gammaglobulin. The injections caused a prompt and sustained rise in the child's erythropoiesis.

Myeloma ascites--a favorable outcome with cyclophosphamide therapy.

A 71-year-old woman with multiple myeloma (MM) in remission was admitted for evaluation of recent abdominal distension and was diagnosed as having massive myeloma ascites. The fluid was characterized by a total nucleated cell count of 6,600/mm3 (67% plasma cells), with a plasma cell CD38+ phenotype. Chemical analysis of the fluid showed lactate dehydrogenase of 122 IU/L, total protein of 2.9 g/dL, albumin of 2.4 g/dL, diastase of 38 IU/dL, cholesterol of 46 mg/dL, and C-reactive protein of 3 g/dL. The serum-ascites albumin gradient (SAAG) was low (0.9). Electrophoresis of the ascitic fluid showed a monoclonal spike in the gamma region and immunoelectrophoresis confirmed the presence of lambda light chains similar to those seen in the urine. Further analysis of the ascitic fluid showed markedly elevated levels of beta2 microglobulin (11,161 microg/L) and interleukin-6 (146 pg/ml compared to serum level of 4.3 pg/ml). There was evidence of intraabdominal masses that completely resolved with continuous high-dose cyclophosphamide (750 mg/m2/day for four days) followed by clinical improvement and disappearance of the ascites. We stress the value of complete fluid characterization and intensive chemotherapy to achieve a favorable outcome.

Impaired immune function in hemophilia patients treated exclusively with cryoprecipitate: relation to duration of treatment.

Recently, abnormalities of cell-mediated immunity were found in hemophiliac patients receiving factor VIII concentrate therapy. Contradictory results were reported concerning cellular immune functions in hemophiliacs treated only with cryoprecipitate or fresh frozen plasma. Therefore, we evaluated the immunological status of 15 Israeli patients with severe classic hemophilia-A who were treated only with cryoprecipitate and never exposed to factor VIII concentrate whether of commercial source or blood bank prepared. As a group, only mildly depressed cellular immune functions and slight reduction in the helper to suppressor cell ratio were found. However, when patients treated more than 15 years were analyzed separately, a significant reduction in proportion of T cells, T-helper cells, helper to suppressor ratio, and proliferative response to phytohemaglutinin and pokeweed mitogen were observed compared to patients treated for less than 15 years and normal controls. Proportion of T-suppressor cells, Con A-activated suppressor activity, and IgG and IgA levels were significantly elevated in patients treated for more than 15 years. These results may support the view that derangement of immune function in hemophiliacs results from infusion of foreign proteins or an ubiquitous virus rather than contracting AIDS infectious agent.

Eosinophilic granulomatous lymphadenopathy: association with hyper-IgE and eosinophilia.

Cervical lymph node enlargement is probably the most frequently detected childhood lymphadenopathy. We report 2 cases of cervical lymphadenopathy in children associated with hyper-IgE and eosinophilia, displaying the features of necrotizing eosinophilic granulomatosis. Immunohistochemical analysis and a serological work-up failed to elucidate the underlying etiology. We would like to call the attention of physicians and pathologists to this unusual clinical picture, different from the fatal form of necrotizing eosinophilic granulomatosis, and we suggest a role for the eosinophils in the pathologic appearance of the lymph nodes.