A new nonlinear mixture response surface paradigm for the study of synergism: a three drug example

A flexible approach to response surface modeling for the study of the joint action of three active anticancer agents is used to model a complex pattern of synergism, additivity and antagonism in an in vitro cell growth assay. The method for determining a useful nonlinear response surface model depends upon a series of steps using appropriate scaling of drug concentrations and effects, raw data modeling, and hierarchical parameter modeling. The method is applied to a very large in vitro study of the combined effect of Trimetrexate (TMQ), LY309887 (LY), and Tomudex (TDX) on inhibition of cancer cell growth. The base model employed for modeling dose-response effect is the four parameter Hill equation [1]. In the hierarchical aspect of the final model, the base Hill model is treated as a function of the total amount of the three drug mixture and the Hill parameters, background B, dose for 50% effect D50, and slope m, are understood as functions of the three drug fractions. The parameters are modeled using the canonical mixture polynomials from the mixture experiment methodologies introduced by Scheff [2]. We label the model generated a Nonlinear Mixture Amount model with control observations, or zero amounts, an "NLMAZ" model. This modeling paradigm provides for the first time an effective statistical approach to modeling complex patterns of local synergism, additivity, and antagonism in the same data set, the possibility of including additional experimental components beyond those in the mixture, and the capability of modeling three or more drugs.     

Tumor necrosis factor-alpha (TNF-alpha) in seasonal allergic conjunctivitis and vernal keratoconjunctivitis

PURPOSE: To quantify the presence of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in allergic conjunctivitis. MATERIALS AND METHODS: Tears and peripheral blood samples were collected from patients with seasonal allergic conjunctivitis (SAC, n=6), vernal keratoconjunctivitis (VKC, n=12), and normal subjects (CT, n=12). From an additional six nonactive allergic patients, tears were collected before and after specific conjunctival allergen challenge (CAC). Upper tarsal conjunctival biopsies were obtained from five CT and five VKC patients. TNF-alpha in tears was measured by enzyme-linked immunoassay and identified in tissues by immunohistochemistry. RESULTS: Tear TNF-alpha levels in VKC patients were significantly increased compared to CT (p=0.03), and were significantly correlated with the severity of the disease. No differences were found between SAC and CT tear samples. TNF-alpha serum levels were higher in VKC than CT, however, this difference was not statistically significant. After CAC, tear TNF-alpha levels were found increased in only one of six patients. In VKC tissues, TNF-alpha positive cells were significantly increased compared to CT (p=0.03). CONCLUSIONS: TNF-alpha may have a significant role in severe forms of allergic conjunctivitis.     

Synthesis and evaluation of novel 7-trifluoromethyl-4-(4-substituted anilino)quinolines as antiparasitic and antineoplastic agents

Several novel derivatives bearing the 7-trifluoromethyl-4-(4-substituted anilino)-quinoline skeleton were synthesized and evaluated for their in vitro activity against the blood streaming form of the parasites Trypanosoma brucei rhodesiense, the trypomastigotes of Trypanosoma cruzi cultured in rat skeletal myoblasts, the amastigotes form of Leishmania donovani, Plasmodium falciparum (K1 strain) infected erythrocyte suspension, as well as their toxicity towards rat skeletal L-6 cells. In addition, three of the synthesized compounds were tested for their in vitro antitumor activity towards 60 human tumor cell lines by the National Cancer Institute (NCI). Compound 1-(4-[[7-(trifluoromethyl)quinolin-4-yl]amino]phenyl)ethan-1-one thiosemicarbazone 23 exhibited potential activity against T. b. rhodesiense, T. cruzi and P. falciparum with IC50's of 0.278, 0.85 and 0.417 microgram/ml, respectively. These values are even more valuable since this compound was clearly non-toxic (cytotoxicity IC50 > 90 micrograms/ml), leading to in vitro therapeutic indices of > 323, > 106 and > 216, respectively. Meanwhile, compound N-[4-[5-(4-chlorophenyl)-4,5-dihydroisoxazol-3-yl]phenyl]-7-(trifluoromethyl) quinolin-4-amine 21 showed broad spectrum antitumor activity with full panel median growth inhibition GI50 (MG-MID) of 1.95 mumol and total growth inhibition TGI (MG-MID) of 6.87 mumol, respectively. Compound 23 represents a very good prospective as a lead compound able to combat three tropical diseases at a time. However, the pattern for the antitumor activity did not parallel any of the antiparasitic ones, indicating that non-common mechanisms of action may exist among these activities.     

DNA vaccination of neonate piglets in the face of maternal immunity induces humoral memory and protection against a virulent pseudorabies virus challenge

DNA vaccination represents a unique opportunity to overcome the limitations of conventional vaccine strategy in early life in the face of maternal-derived immunity. We used the model of pseudorabies virus (PRV) infection in pigs to further explore the potential of DNA vaccination in piglets born to sows repeatedly vaccinated with a PRV inactivated vaccine. A single immunisation of 8-week-old piglets with a DNA vaccine expressing secreted forms of PRV gB, gC, and gD, triggered an active serological response, confirming that DNA vaccination can over-ride significant residual maternal-derived immunity. A clear anamnestic response was evidenced when a secondary DNA vaccination was performed at 11 weeks of age, suggesting that DNA vaccination, performed in the face of passive immunity, elicited a strong humoral memory. We subsequently explored the potential of DNA vaccination in neonate piglets (5-6 days of age) in the face of very high titres of maternal antibodies and demonstrated that very high titres of passive antibodies selectively inhibited serological responses but not the establishment of potent memory responses. Finally, we demonstrated that DNA vaccination provided protection against an infectious PRV challenge at the end of the fattening period (i.e. at approximately 5 months of age). Collectively, our results pave the way for a new flexible vaccination program, which could ensure uninterrupted protection of fattening pigs over their entire economical life under field conditions.     

Prognostic assessment of gastrointestinal stromal tumor

The term "gastrointestinal stromal tumor" (GIST) has been applied to a collection of distinctive mesenchymal tumors occurring within the human gastrointestinal tract. As new drug therapy becomes available, data regarding the natural history of these unusual tumors are necessary to provide selection factors for treatment. Ninety-eight patients had light microscopy compatible with GIST at a single institution from 1989 to 2000. After immunostaining with c-kit and histopathologic review, 69 were judged to be GIST. All prognostic indicators were determined for gastric GIST, intestinal GIST, and all locations combined. The location of the GIST did not have a significant impact on survival. Clinically, tumor size, peritoneal cancer index, and completeness of cytoreduction had a significant impact on prognosis for GIST at all locations. Pathologically, cytologic atypia, necrosis, invasion and number of mitoses were significant prognostic indicators for GIST. Criteria to separate three pathologic groups of GIST according to the tumor size and the mitotic count were useful to evaluate the tumor behavior; in the borderline pathologic group invasion and cytologic atypia were statistically significant prognostic criteria. The cell phenotypes, as determined by immunostains, correlated with the prognosis of gastric GIST but not intestinal GIST. A correlation between the immunostain Ki-67 but not CD-34 or desmin and the prognosis was observed. It is possible to select clinical and pathologic parameters of GIST that impact on prognosis. Invasion and necrosis help to determine the prognosis with borderline tumors. The immunostain Ki-67 correlated with the prognosis and may be helpful to assess prognosis when dealing with small biopsy specimens.     

Ancistrotanzanine A, the first 5,3'-coupled naphthylisoquinoline alkaloid, and two further, 5,8'-linked related compounds from the newly described species Ancistrocladus tanzaniensis

The first phytochemical investigation of the recently discovered East African liana Ancistrocladus tanzaniensis is described, resulting in the isolation and structural elucidation of two new naphthylisoquinoline alkaloids, ancistrotanzanines A (5) and B (6), and the known compound ancistrotectoriline A (7). Ancistrotazanine A (5) represents a hitherto unprecedented 5,3'-coupling type between the naphthalene and isoquinoline portions, while 6 and 7 are 5,8'-coupled. The structures of the compounds were determined by spectroscopic, chemical, and chiroptical methods. Compounds 5 and 6 showed good activities against the pathogens of leishmaniasis and Chagas' disease, Leishmania donovani and Trypanosoma cruzi, while 5-7 displayed moderately potent antiplasmodial activities against Plasmodium falciparum parasites.     

The effect of hyaluronan on CD44-mediated survival of normal and hydroxyl radical-damaged chondrocytes

OBJECTIVE: To identify the CD44-receptor-mediated effects of 5-7 x 10(5)MW hyaluronan (HA, Hyalgan) on cell viability in normal and damaged human chondrocyte primary cultures isolated from articular cartilage.DESIGN: Primary cultures of human chondrocytes were established from normal articular biopsies and expanded to the second culture passage. The dose-response effects of HA on the viability of normal cultures were identified. Chondrocytes were then treated with either hypoxanthine (2 mM) and xanthine oxidase (20-60 mU), or with activated polymorphonuclear leukocytes (PMNs) to induce injury. Damaged and control cells were then treated with 5-7 x 10(5)HA in the previously identified optimal dose of 0.05 mg/ml. Viability was assessed at specific time periods for the chemically and PMN-damaged cells. To identify if HA effects were mediated by the CD44 receptor, chondrocytes were incubated with anti-CD44 antibody at saturating concentrations (5 microg/ml for 100,000 cells) to produce a maximum inhibition of HA binding. Cells were evaluated using the MTT viability assay, histology, electron microscopy and immunohistochemistry.RESULTS: Direct addition of HA (optimal dose, 0.5 mg/ml) significantly increased cell survival in normal chondrocyte primary cultures (P<0.05). Similarly, addition of this same dose of HA to cultures of free radical-damaged chondrocytes, restored the viability to baseline conditions. Cell viability rates dropped significantly (P<0.05) when CD44 receptor binding was inhibited, indicating that cell growth was mediated by the CD44 receptor.CONCLUSIONS: HA (0.5 mg/ml of 5-7 x 10(5)) significantly increased the viability of normal human chondrocytes in primary culture and restored cell viability to near normal levels after oxidative cell injury.